DPhil in Cancer Science

University of Oxford
2021 Intake Project Book
 DPhil in Cancer Science
2021 Intake Project Book

Introduction
This booklet provides an overview for prospective students looking to study for a DPhil in
Cancer Science at Oxford University, starting in 2021. The Programme provides research
based doctoral training for cancer researchers from clinical, biological, engineering,
mathematics and statistics backgrounds. Students will receive a world-leading research
training experience that integrates an education initiative spanning cancer patient care,
tumour biology and research impact; on- and post-programme mentorship; and a specialised,
fundamental, subject-specific training tailored to individual research needs. Students
participating in the scheme will be offered:

• a choice of interdisciplinary cutting-edge cancer research projects.

• the ability to gain a working in-depth knowledge of the fundamentals of cancer biology
and cancer patient care through advanced level seminars.
• a world-renowned research environment that encourages the student’s originality
and creativity in their research.
• opportunities to develop skills in making and testing hypotheses, in developing new
theories, and in planning and conducting experiments.
• an environment in which to develop skills in written work, oral presentation and
publishing the results of their research in high-profile scientific journals, through
constructive feedback of written work and oral presentations.

At the end of their DPhil course, students should:

• have a thorough knowledge of the basic principles of cancer research including the
relevant literature and a comprehensive understanding of scientific methods and
techniques applicable to their research.
• be able to demonstrate originality in the application of knowledge, together with a
practical understanding of how research and enquiry are used to create and interpret
knowledge in their field.
• have developed the ability to critically evaluate current research and research
techniques and methodologies.
• be able to act autonomously in the planning and implementation of research.
• have the grounding for an influential cancer researcher of the future.

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Selection Criteria & Eligibility
There are three tracks in the programme as described below, meaning that non-clinicians,
undergraduate medical students and post-graduate medical trainees are all eligible to apply
for the fully funded (at home rate) studentships.

Application Track 1 – Clinical Trainees. Qualified doctors at all stages of training from the
foundation training to higher specialist training.

Application Track 2 – Medical Undergraduates. Medical students who are currently

undertaking a primary medical qualification (MBBS, MBChB or equivalent)

Application Track 3A – Non-Clinical/Fundamental Scientist. Science graduates that hold (or

be predicted to achieve) the equivalent of a first-class or strong upper second-class
undergraduate degree with honours in a biological, medical, or chemical science, as
appropriate for the projects offered.

Application Track 3B – Non-Clinical/Fundamental Scientist. Science graduates that hold (or

be predicted to achieve) the equivalent of a first-class or strong upper second-class
undergraduate degree with honours in a engineering, mathematical/data, or physical science,
as appropriate for the projects offered.

All applicants will be judged on the following:

• commitment and passion to a career in cancer research
• evidence of motivation for and understanding of the proposed area of study
• commitment to the subject, beyond the requirements of the degree course
• preliminary knowledge of relevant research techniques
• capacity for sustained and intense work
• reasoning ability and academic curiosity.

Funding
All offered places are fully funded at the home rate. This includes salary/stipend,
University/College fees, and a research consumables budget of £13k p.a.. Salary and stipend
provisions are summarised below:
• Application Track 1 – 3 years of salary at Grade E63 or E64 Clinical Researcher rate.
• Application Track 2 – 3 years of stipend at the flat rate of £19,000 per annum.
• Application Track 3A & 3B – 4 years of stipend at the flat rate of £19,000 per
annum.

International applicants are eligible, however funding is limited to the Home level for this
programme and therefore international applicants would need to either source further
funding or support themselves financially for the remaining fees.

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How to Apply
A detailed summary on how to apply can be found here. In brief, prospective students apply
with a prioritised list of three projects selected from this booklet by Friday January 8th 2021.
Shortlisted students will be invited to interview in February. If successful, students will be
allocated a project on the basis of their ranking during the review process. It is strongly
suggested that students contact supervisors of projects they are interested in applying for
prior to application.

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Projects at a Glance
Projects are listed below in the following structure “Title – Main Supervisor Eligible Application Tracks”

Targeting DNA repair mechanisms in precision cancer therapies – Prof. Lakin1,2,3A 7

Molecular understanding of patient responses to immunotherapy in gastrointestinal
tract cancers – Prof. Lu1,2,3A 9
Mechanisms Of Cure in Acute Myeloid Leukaemia Treated with Intensive
Chemotherapy – Prof. Vyas1,2,3A 11
Evaluating the roles of the AHR in melanoma: melanin sensing and regulating the
melanoma microenvironment – Dr. Alves1,2,3A 13
Improving immunotherapy by defining the interaction between the bacterial vaccine
BCG and the host epigenomic and immune response – Prof. McShane1,2,3A 15
Targeting epigenetic regulators to improve tumour responses to immunotherapy –
Prof. Shi1,2,3A 17
Elucidating the Rad51-independent pathway of recombination-dependent replication
– Prof. Whitby1,2,3A 19
Analysis of spindle assembly checkpoint function in health and disease – Prof.
Gruneberg2,3A,3B 21
Identifying novel regulators of cancer stem cells in pancreatic ductal
adenocarcinoma– Dr. Pauklin 1,2,3A 23
Understanding The Therapeutic Efficacy Of Chemotherapy In Pancreatic Cancer at
the single cell level – Dr. Sivakumar1,2,3A,3B 25
Heterogeneity of macrophages in colorectal cancer: the role of IRF5 – Prof.
Udalova1,2,3A,3B 27
Modelling cancer stem cell dormancy using organoids and advanced 3D culture
models – Dr. Boccellato1,2,3A,3B 29
Decoding immune surveillance within the tumour microenvironment: how the
extracellular matrix enables escape from immunity – Prof. Midwood1,2,3A 31
UGT8 and microcarrier signalling in the development of breast cancer – Prof.
Wilson1,2,3A 33
Studying the role of a chromatin remodelling factor (ATRX) in normal gene expression
and in malignancy – Prof. Higgs1,2,3A 35
ARH3/ADPRHL2 as a biomarker for PARP inhibitor sensitivity/resistance – Prof.
Ahmed1,2,3A,3B 37
Discovery and mechanistic elucidation of small molecule inducers of myeloblast
differentiation for ALL – Prof. Russell3A 39
Single-cell analysis of haematopoietic stem cells in SF3B1 mutant MDS: identification
of new therapeutic targets/treatments – Dr. Pellagatti1,2,3A 41

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 Dissecting the biological basis of HLA variation to susceptibility and disease severity
in Crohn’s disease & ulcerative colitis as risk factors for the development of colorectal
cancer – Prof. Satsangi1 43
The Artemis DNA Repair nucleases – from mechanism to therapeutic inhibition – Prof.
Schofield3A 45
Investigating IGFs as immunosuppressive cytokines and cancer risk factors– Prof.
Macaulay1,2,3A 47
Genetic and functional characterisation of novel immune escape mutations in
mismatch repair deficient endometrial cancer – Dr. Church1,2,3A 49
Mass spectrometric probes for intraoperative brain cancer diagnosis – Prof
Vallance1,2,3A,3B 51
Developing an on chip screen for PDAC in high risk groups – Prof Davis1,2,3A 53
Understanding STING regulation in cancer and the crucial role of ubiquitination at the
ER – Dr. Christianson1,2,3A 55
Spatial mapping of the bone marrow for improved leukaemia diagnosis using
machine learning/artificial intelligence – Dr Royston4 57
Bioengineered gastrointestinal tissues to study neural signalling in cancer
development and metastasis – Prof. Bayley1,2,3A,3B 59
Discovery of potent wild-type and surrogate agonist peptides for anti-tumour T Cells
– Dr. Fernandes1,2,3A 61
In vitro and in silico models of human induced pluripotent stem cell to investigate the
effects of doxorubicin – induced cardiotoxicity – Prof. Zaccolo1,2,3A 63
Genome-wide screening to identify factors impacting on cellular survival upon acute
depletion of BRCA2 or PALB2 – Prof. Esashi1,2,3A,3B 65
Galectin-3 promotes glioblastoma emergence from the subventricular zone stem cell
niche – Prof. Szele1,2,3A,3B 67
Effects of androgen deprivation on multimodality prostate cancer therapy – Prof.
Bryant1,2,3A 69
Characterising the developmental origins in the pathogenesis of mesenchymal
tumours of the central nervous system – Prof. Sauka-Spengler1 71
Investigating pathological crosstalk between mature tumour cells and
haematopoietic progenitor cells in chronic myelomonocytic leukaemia – Prof. Mead1 73
Mathematics of Lymphoma Immunotherapies: Application of Mechanistic Models to
Accelerate and De-Risk Therapeutic Development for Blood Cancers – Prof. Coles1,2,3A,3B 75
Pre-clinical testing of a novel mitochondrial inhibitor (NBS037) in combination with
radiotherapy and immunotherapy – Prof. Higgins 1,2,3A 77
Mechanisms of therapeutic response and resistance to BCMA-directed therapy in
multiple myeloma- a systems biology approach – Prof. Oppermann1,2,3A,3B 79
Personalised monitoring intervals for cancer surveillance – Dr Oke1,2,3A,3B 81

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 Presentation, Diagnosis and Outcomes of Hodgkin Lymphoma: the role of Primary
Care – Prof. Bankhead1,2,3A,3B 83
The impact of microenvironmental components on cellular plasticity in colorectal
cancer – Prof. Buczacki1,2,3A 85
The impact of microenvironmental components on cellular plasticity in colorectal
cancer – Dr. Jiang1,2,3A 87
The Epitope Abundance-Avidity-Efficacy Axis In Cancer – Prof. Elliot1,2,3A 89
Cell stress and the premalignant niche in ovarian – Prof. Blagden1,2,3A 92
The immune landscape of the pancreas during neoplastic transformation – Prof.
O’Neill1,2,3A, 3B 94

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 Targeting DNA repair mechanisms in precision cancer therapies –
Prof. Lakin1,2,3A
Primary Supervisor: Nicolas Lakin
Additional Supervisors: Peter McHugh
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.
Project Summary
Inhibitors of DNA repair have emerged as powerful agents in cancer therapy, either as
monotherapies that exploit synthetic lethal interactions between DNA repair pathways, or by
increasing the efficacy of chemo- and radiotherapies1. Principal in this strategy is inhibition of
Poly(ADP-ribose)-polymerases (PARPs), enzymes that regulate DNA strand break repair, and
PARP inhibitors (PARPi) are being used to treat tumours with defects in homologous
recombination (HR). However, these strategies are restricted to treating HR-defective ovarian
cancers, with limited information on additional synthetic lethal interactions that will broaden
the use of PARPi to treat other tumours. By combining our expertise in PARP biology and DNA
repair2-6 with cutting edge genome editing, proteomics and cell biology, this research will
address this fundamentally important question by characterising novel cancer-related genes
that are synthetic lethal with PARP dysfunction. To this end, through a genome-wide CRISPR-
Cas9 based screen we identified a novel gene (PASL9) that is synthetic lethal with PARPi and
critical to resolve replication-associated DNA damage through a pathway that is often
mutated in colorectal cancers. This multidisciplinary hypothesis-driven research will define
how PASL9 regulates replication-associated DNA repair and the mechanistic basis of synthetic
lethality with PARPi. Our long-term vision is to exploit these findings by developing novel
strategies to exploit PARPi to treat a variety of tumours, including digestive cancers.

Research Objectives and Proposed Outcomes

Background
We performed a genome-wide CRISPR-Cas9 screen to identify synthetic lethal interactions
with the principal DNA repair PARPs (PARP1/PARP2). A top hit was a gene of unknown
function that we named PASL9 (PARP and ATR Synthetic Lethal 9). Independent pasl9Δ cell
lines are sensitive to PARP or ATR inhibitors, phenotypes associated with replication-repair
defects7, 8. Consistent with PASL9 functioning in replication-associated repair, it fulfils 3 well-
established criteria for a role in these pathways: A) PASL9 is recruited to chromatin in
response to replication stress induced by hydroxyurea (HU); B) pasl9∆ cells display elevated
levels of γH2AX following replication stress; C) pasl9∆ cells are sensitive to HU. Moreover, our
data indicate that PASL9 interacts with and functions in the same pathway as HUWE1, a
replication-associated repair gene9 that is mutated in colorectal cancers10. Together, these
data identify PASL9 as a novel DDR gene that functions with HUWE1 to maintain genome
stability. We will define the role of PASL9 in combating replication stress and assess its
potential as a target to treat colorectal cancer by pursuing the following objectives:

Define the nature of the synthetic lethal interaction between PARP and PASL9
We will define the PARP-dependent pathway that is synthetic lethal with PASL9. Our data
indicate that in addition to single strand break repair, PARP1/2 can regulate Rad51-dependent
and -independent replication fork recovery pathways6. We will establish which of these
pathways is synthetic lethal with PASL9 by assessing cell survival after depleting SSB repair,
or replication associated repair (HR; break-induced replication factors in pasl9∆ cells. We will

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look at the requirements for PASL9 in various aspects of replication dynamics using DNA fibre
analysis (replication fork speed, restart and stability) and markers of genome stability
associated with defective replication-associated repair (increased chromosome
fragmentation, ultrafine anaphase bridges, micronuclei formation etc.). We will assess
whether any phenotypes observed are exacerbated by treating pasl9∆ cells with PARPi or
ATRi.

Define the replication repair mechanisms regulated by PASL9

Whilst PASL9 functions with HUWE1, an E3 ubiquitin ligase that accumulates at stalled
replication forks9, the mechanistic basis of this regulation is unknown. PASL9 is synthetic
lethal with ATRi and PARPi, suggesting it functions in a repair mechanism that is synthetic
lethal with both these agents such as the HR, Fanconi Anaemia, ATM and ATR pathways. We
will IP PASL9 protein complexes before or after replication stress and identify PASL9
interactors by mass spectrometry to further inform on which pathways it regulates. We will
assess whether HU sensitivity and markers of genome instability of pasl9∆ /huwe1∆ cells (see
above) are epistatic with these pathways, in addition to assessing whether biomarkers of
pathway activation (ATM/ATR/Chk1 phosphorylation; FANCD2 ubiquitination etc.) and/or
assembly of pathway components at DNA damage is defective in pasl9∆/huwe1∆ cells.
Mechanistically, we will establish where in the pathway PASL9/HUWE1 function by defining
the factors required to assemble PASL9/HUWE1 at stalled/damaged replication forks.

Targeting PASL9 defective cancer cells with PARPi

We will compare the synthetic lethal interaction of PASL9/HUWE1 with PARPi/ATRi in non-
cancer (RPE1; MRC5) and cancer cells (e.g. U2OS; HCT116). Given the increased reliance on
certain DNA repair mechanisms in transformed cells11, 12, we will test whether oncogene
activation (e.g. cyclin E over-expression) influences the synthetic lethal interaction. Given
HUWE1 is synthetic lethal with PARP1/2 and is often mutated in colorectal cancers10, we will
assess whether colorectal cancer cells are sensitive to PARPi and if this correlates with HUWE1
mutations.

Translational Potential
This work will provide fundamental advances in our understanding of how cells tolerate DNA
damage. Characterising novel synthetic lethal interactions with genes that are mutated in
colorectal cancers will facilitate the use of PARPi to treat tumours beyond those defective in
HR, including digestive cancers.

References: 1. M. J. O'Connor. Mol Cell 60: 547-60 (2015). 2. C. A. Couto, et al. J Cell Biol 194: 367-75
(2011). 3. A. R. Gunn, et al. J Cell Sci 129: 3845-3858 (2016). 4. A. L. Kolb, et al. Nucleic Acids Res 45: 10056-
10067 (2017). 5. A. Rakhimova, et al. Sci Rep 7: 43750 (2017). 6. G. E. Ronson, et al. Nat Commun 9: 746
(2018). 7. N. Hustedt, et al. BioRxiv (2019). 8. M. Zimmermann, et al. Nature 559: 285-289 (2018). 9. K. N.
Choe, et al. EMBO Rep 17: 874-86 (2016). 10. K. B. Myant, et al. EMBO Mol Med 9: 181-197 (2017). 11. L.
Costantino, et al. Science 343: 88-91 (2014). 12. S. K. Sotiriou, et al. Mol Cell 64: 1127-1134 (2016).

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 Molecular understanding of patient responses to immunotherapy in
gastrointestinal tract cancers – Prof. Lu1,2,3A
Primary Supervisor: Xin Lu
Additional Supervisors: Alison Simmons
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Oesophageal cancer is the 6th most common cancer: in 2012 it caused ~400,000 deaths
worldwide and incidence is rising rapidly. Notably, it has been identified by CRUK as an area
of unmet need and it is a strategic priority for the CRUK Oxford Centre. Standard care for
oesophageal cancer is chemo- or chemo/radiotherapy and surgery. Mortality has remained
closely related to oesophageal cancer incidence, with a 5 year survival of <15%, indicating
that many oesophageal cancer cells are resistant to existing therapies. Therefore a major
clinical challenge is to develop novel and effective therapies for oesophageal cancer.

Immunotherapy is now entering standard clinical care for several cancer types, but only some
tumours respond to this mode of treatment. To inform future clinical decision making, we
need to identify the molecular differences underlying diverse responses to immunotherapy;
this is essential to guide the most effective care for patients. This project presents a unique
opportunity to tap into the power of rich, deep-phenotyping datasets from an experimental
medicine clinical trial of immune checkpoint therapy in oesophageal cancer, and to follow-up
insights with frontier technologies. Blood and tissue samples have been collected at multiple
time-points and from several tissue sites from patients throughout the trial. Analysis of these
clinical samples is on-going, using state-of-the-art genomic, transcriptomic, epigenomic,
proteomic and immunological analysis technologies to enable dissection of the molecular
basis for different responses to therapy. Our current work is revealing tantalising initial
insights into features in patients who do or do not experience clinical benefit from the
treatment. This project will build on our preliminary findings, testing hypotheses using a
combination of cutting-edge molecular assays, organoids, co-culture techniques and single-
cell sequencing. Analysis of liquid biopsies may also reveal novel biomarkers, such as cell free
DNA or autoantibodies. This work will form an essential component of our overall aim to
improve future treatment of gastrointestinal cancers and inform for the broader
implementation of immunotherapy.

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Research objectives and proposed outcomes

Background: Reinvigoration of host immune systems to eliminate tumours is one of the most
exciting developments in cancer therapy. Therapies are being developed to inhibit pathways
that tumours use to evade immune surveillance. Antagonists of the CTLA-4 and PD-1/PD-L1
immune checkpoint pathways (i.e. antibodies to CTLA-4, PD-1 or PD-L1) unleash previously
suppressed T-cells to eliminate tumour cells. This strategy - termed immune checkpoint
targeting therapy (ICT) - has achieved durable overall survival in patients with highly
metastatic tumours. However, only a subset of tumours responds to ICT and understanding
why many are resistant to ICT and ICT-related combination therapies is a major scientific
challenge. Examining cellular responses before and after interventions is the key to address
this problem.

Objectives: The overall aim is to identify molecular differences before and after immune
checkpoint therapy and between responders and non-responders. The project will be testing
hypotheses arising from analysis of data and samples from patients who have completed
treatment in an experimental medicine clinical trial of immune checkpoint therapy in
oesophageal cancer, aiming to identify the molecular basis of different responses

Approaches: Key approaches include co-culture techniques and organoid technology to

explore interactions between tumour cells and immune cells. Single-cell sequencing may be
used to dissect the cellular-level response to altered interactions. Liquid biopsies from
patients will be analysed to explore biomarkers, with potential for analysis of the cell free
DNA, including the epigenome, antibodies, and other molecular markers.

Mentoring, training and collaboration: The overall strategy for the project and the laboratory
research will be supervised by Prof. Xin Lu, Director of the Ludwig Institute for Cancer
Research in Oxford, who has extensive experience of mentoring clinical and non-clinical DPhil
students. The student will be given training in the necessary cell and molecular biology
techniques including single cell genomics and organoids. Prof Simmons also has extensive
mentoring experience and will provide expertise in gastroenterology, immunology, and
organoid culture. This project will enable the student to benefit from expertise and
technologies at both the Ludwig Institute for Cancer Research and Weatherall Institute of
Molecular Medicine. The student will have opportunities to integrate with the wider scientific
and clinical communities in Oxford through established collaborative networks, and with the
national and international communities at conferences. The student will benefit from the
training and career development programme at the Ludwig, which includes: regular oral,
journal clubs, and skills development in writing, data management and public engagement.

Translational potential of the project: This project is poised to have major implications for
guiding future clinical decision making for patients with oesophageal cancer. Specifically, the
connections made by the student between molecular characteristics and responses to
therapy in the trial of immunotherapy in oesophageal cancer will be vital for developing new
clinical stratification models.

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 Mechanisms Of Cure in Acute Myeloid Leukaemia Treated with
Intensive Chemotherapy – Prof. Vyas1,2,3A
Primary Supervisor: Paresh Vyas
Additional Supervisors: Bilyana Stoilova, Claus Nerlov, Supat Thongjuea, Ryan Beveridge
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Unmet clinical need: Acute Myeloid Leukaemia (AML) is the most common, aggressive human
leukemia. There are ~42 000 new cases of AML in USA and EU/year[1, 2]. Only ~ 15% of patients survive.
Most patients die within 12 months of diagnosis. Thus, there is a significant unmet clinical need to
improve therapy. Fundamental scientific premise: Within the whole group of AML patients there is a
subset of patients (35%), typically younger (less than 65 years of age) who receive intensive
conventional combination cytotoxic chemotherapy (anthracyclines and nucleoside analogues), who
have a higher cure rate (~65%)[3]. Despite these cytotoxic drugs being in routine clinical use since the
1970’s, the field surprisingly still does not understand why these patients are cured. Conventional
wisdom is that these patients are cured, because this intensive cytotoxic therapy kills all AML cells.
However, this has never been rigorously proven and alternative hypotheses have not been tested.
This proposal aims to understand how these patients are cured. In particular, we will test the
hypothesis that patients are cured because there is a “reset” of the patient’s own innate, and acquired,
immune system that allows life-long autologous immune-based control of disease after successful
reduction of disease burden by intensive cytotoxic chemotherapy. Furthermore, from the science that
is proposed here, we aim to understand fundamental general principles that could deliver novel
immune therapies for all cancers.

Research Objectives
Overall Objectives
Identify mechanism of cure in AML patients treated with intensive chemotherapy. Cure could result
from:
1) increased AML “kill” cells in cured patients versus those that relapse
2) autologous innate
3) acquired immune anti-AML cell response.

Specific Aims
1) Contrast amount of AML cells left after treatment (measurable residual disease, MRD), between
cured patients compared to those who relapse. Measure MRD in bone marrow (BM) samples, in
patients uniformly treated with standard intensive chemotherapy, after all patients have received
exactly the same amount of treatment (after cycle 2 and at end of treatment).
2) If residual disease is detected in any of the samples, characterise the single cell (sc) transcriptome
and stage of differentiation (leukemic stem/progenitor cell or more mature cell). (Aims 1 and 2 will
specifically measure if there are differences in amount of AML cells left after treatment in cured
patients versus those who later relapse).
3) Perform an unbiased sc transcriptomic analysis of innate and acquired immune cells in BM, and
peripheral blood (PB), in uniformly treated patients, tested at the same points during treatment (after
2 cycles of chemotherapy and end of treatment), between cured patients compared to those who
relapse.
4) If differences are detected in comparable immune cells populations between cured patients versus
those that relapse, we will conduct functional experiments. (Aims 3 and 4 will test if there are
difference in PB and BM immune cell composition between cured patients versus those that later
relapse).

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Strategy, Methods, Analyses
1) Patient Cohort: 30 uniformly treated, age matched (<70 years old), well-clinically annotated,
intermediate and good risk AML patients; 20 cured (>5 years from end of treatment) and 10 relapsed.
3-4 viably frozen sequential BM and PB samples are available from each patient (i) from diagnosis; (ii)
after two courses of chemotherapy; (iii) at the end of treatment: (iv) and in those who relapse, at
relapse. Collaboration: Haembio (Oxford haematology biobank and UK AML trial group).
2) MRD assessment: two independent orthogonal methods will be employed: next generation
sequencing detection of AML-specific mutations using our 108 gene panel (sequencing at 500X)[4] (and
Vyas unpublished data) at diagnosis and patient-specific panels[5]. at complete remission and flow
cytometry that quantitively detect AML cells at a sensitivity of 1 in 104 to 106 cells)[6, 7] and leukaemic
stem cell (LSC)[4, 8-10]. If we detect AML cells we will purify them and assay for combined scRNA-seq
and AML mutation a single cell level using the most appropriate method[11, 12]. These
methods/computational pipelines are routine the laboratory. This will determine if there are specific
transcriptional programs that allow cells to resist therapy. Collaboration: With UK AML AML MRD
group and the international European LeukaemiaNet (ELN) MRD group of which Vyas is a founder
member.
3) Sc analysis of innate and acquired immune cells. We will perform combined sc 5’
transcriptomics and T-cell receptor and B-cell receptor sequencing and sc CITE-Seq and scATAC-Seq
on post treatment sample and end of treatment using the 10X Genomics platform. All these methods
and computational pipelines are in routine use in the laboratory. This will be complemented by high-
dimensional flow cytometry-based analysis (using 3 30-colour Aurora panels) of the relative frequency
and phenotype of effector and regulatory CD4 and CD8 T cell subsets and subpopulations of NK cells,
antigen presenting cells and B cells. Further functional experiments will depend on the outcome of
the initial analysis. Collaborations: Prof Burrow, Prof Nerlov, Drs Stoilova Thonjuea and Beveridge.

Proposed outcomes and translational potential of the project

1) Identify biomarkers of cure that will identify cured patients. For example, it could be that a
combination of both MRD and immune markers together, better identify patients who will cured,
and those who will relapse. These biomarkers would then have to be validated in a larger
independent cohort.
2) Generate proof of concept data, either supporting, or refuting, the importance of
autologous immune mechanisms to cure patients. This data would then support further functional
studies to identify approaches to manipulate the immune response to create an immune
environment seen in cured patients.

References
1. Miranda-Filho, A., et al., Epidemiological patterns of leukaemia in 184 countries: a population-based study. Lancet
Haematol, 2018. 5(1): p. e14-e24. 2. Society, A.C., Key Statistics for Acute Myeloid Leukemia (AML).
https://www.cancer.org/cancer/acute-myeloid-leukemia/about/key-statistics.html. 3.Dohner, H., et al., Diagnosis and
management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood, 2017. 129(4): p. 424-
447. 4. Quek, L., et al., Clonal heterogeneity of acute myeloid leukemia treated with the IDH2 inhibitor enasidenib. Nat Med,
2018. 24(8): p. 1167-1177. 5. Hourigan, C.S., et al., Impact of Conditioning Intensity of Allogeneic Transplantation for Acute
Myeloid Leukemia With Genomic Evidence of Residual Disease. J Clin Oncol, 2020. 38(12): p. 1273-1283. 6. Freeman, S.D., et
al., Measurable Residual Disease at Induction Redefines Partial Response in Acute Myeloid Leukemia and Stratifies Outcomes
in Patients at Standard Risk Without NPM1 Mutations. J Clin Oncol, 2018. 36(15): p. 1486-1497. 7.Freeman, S.D., et al.,
Prognostic relevance of treatment response measured by flow cytometric residual disease detection in older patients with
acute myeloid leukemia. J Clin Oncol, 2013. 31(32): p. 4123-31. 8. Goardon, N., et al., Coexistence of LMPP-like and GMP-like
leukemia stem cells in acute myeloid leukemia. Cancer Cell, 2011. 19(1): p. 138-52. 9. Quek, L., et al., Genetically distinct
leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. J Exp Med,
2016. 213(8): p. 1513-35. 10. Craddock, C., et al., Azacitidine fails to eradicate leukemic stem/progenitor cell populations in
patients with acute myeloid leukemia and myelodysplasia. Leukemia, 2013. 27(5): p. 1028-36. 11.Nam, A.S., et al., Somatic
mutations and cell identity linked by Genotyping of Transcriptomes. Nature, 2019. 571(7765): p. 355-360. 12. Rodriguez-
Meira, A., et al., Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel
RNA Sequencing. Mol Cell, 2019. 73(6): p. 1292-1305 e8.

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 Evaluating the roles of the AHR in melanoma: melanin sensing and
regulating the melanoma microenvironment – Dr. Alves1,2,3A
Primary Supervisor: Pedro Alves
Additional Supervisors: Colin Goding
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
In cancer, it is now recognized that the intra-tumour microenvironment, combined with cell plasticity, leads
cells to mount an adaptive survival response that drives metastatic dissemination and contributes to
resistance to targeted and immunotherapies. The adaptive response to stress represents a potential key
therapeutic vulnerability. Although in melanoma multiple phenotypic states have been identified, how they
are established and maintained remains a key issue. One critical microenvironmental factor, is the level
and genetically determined types of melanin produced. How melanin influences melanoma progression
and therapy resistance, and how interactions between keratinocytes and melanocytes in the skin dictate
melanoma susceptibility are poorly understood. We have found that the Aryl hydrocarbon receptor (AHR),
a critical hub for sensing environmental signals that plays a crucial role in melanoma initiation, progression,
and responses to treatment, binds and responds to melanin. We will therefore investigate how the AHR
senses the melanoma microenvironment and shapes anti-tumour responses. In brief, we will assess the
AHR as a melanin sensor, and whether ligands in the microenvironment serve as cues for the AHR to sense
melanoma status and modulate the immune responses to this cancer. This work will illuminate molecular
sensing and signalling pathways underlying melanoma susceptibilities and microenvironmental dynamics,
with implications for treatment strategies.
This project arises from new insights made from Dr. Moura Alves
showing the AHR is able to sense diverse pigments1-3 (Fig.1). This
project aims to extend this observation, validating the preliminary
data showing the AHR as a sensor of melanin, and also evaluating
its role in sensing and shaping the melanoma microenvironment. A
role for the AHR in pigmentation has been described4-6, and links
have been made to skin cancers and cancer drug therapy7-10, but
this project breaks new ground in examining this receptor as a
sensor of pigmentation, as a sensor of the melanoma
Fig. 1- AHR sensing
microenvironment and a mediator melanocyte/keratinocyte melanin. A) Melanin (cyan)
crosstalk. This project involves establishing zebrafish xenograft and docked to the homology
model of human AHR
11
avatar models at the LICR. Such models will allow us to: i) further (grey). B, C) Luciferase
activity of AHR reporters
dissect the role of the AHR in sensing melanoma, its exposed to B) melanin or
microenvironment and the elicited responses (e.g. immune cell C) B16-F10 melanoma
cells conditioned medium.
1-3,12
recruitment and angiogenesis ) in an in vivo setting, using both
melanoma cell lines and patient derived xenografts; ii) implement an in vivo pipeline for drug screening-
initial focus on candidate molecules targeting the AHR, arising from an unbiased drug screen currently
being performed at Moura Alves laboratory; and iii) evaluate the impact of AHR targeting therapies in
melanoma, tailored to not only the genetically determined pigmentation type (e.g.
eumelanin/pheomelanin ratio) or its levels (e.g. total melanin content), but also to the cellular phenotype
(e.g. proliferative versus invasive).

Research Objectives And Proposed Outcomes: Melanoma is a highly aggressive type of cancer that arises
from melanocytes, and is responsible for most skin cancer deaths14. Transformation of melanocytes is
triggered by genetic and environmental factors such as UV damage15. As well as the external environment,
melanoma initiation and progression are also influenced by interactions in the local cellular environment15.
To improve understanding of differing susceptibility to melanoma and responses to targeted and
immune therapies, we need better knowledge of the molecular pathways that mediate crosstalk
between melanocytes, external cues, and other cells in their microenvironment, such as keratinocytes.
The AHR is a highly conserved ligand-dependent transcription factor that can bind a vast set of endogenous
and exogenous ligands from diverse cellular and tissue microenvironments16. Upon ligand binding, the AHR

13
regulates different cellular and tissue functions, including cell proliferation and differentiation, apoptosis,
expression of inflammatory mediators and recruitment of immune cells16. Recent reports suggest that the
AHR is important in tumorigenesis and maintenance of various skin cancers, and is a prognostic factor for
melanoma9,17. The carcinogenic effects of UV and several chemical carcinogens are at least partly mediated
by the AHR and it has also been implicated in resistance to BRAF inhibitors8,9 and suppression of anti-
tumour immune responses9,17. The overarching aim of this work is to investigate the role of the AHR in
sensing the melanoma microenvironment and shaping anti-tumour responses.
Dr. Moura Alves recently showed that the AHR senses diverse bacterial molecules, including pigments from
Pseudomonas aeruginosa and Mycobacterium tuberculosis1,2 and have unpublished data demonstrating
that the AHR is able to sense Aspergillus fumigatus melanin. Consequently, we hypothesized that the AHR
is able to detect human melanins and we have preliminary data suggesting that human and mouse
melanin fits into the AHR binding pocket and increases AHR activation in reporter cells (Fig. 1).
Importantly, it has been previously shown that the AHR is able to regulate diverse functions and responses
in both melanocytes and keratinocytes3,4,7,18,19. For example, FICZ, which is a photooxidation product of
tryptophan produced by exposure to UV, is a high affinity ligand of the AHR that is able to modulate the
pigmentation status of melanocytes and the expression of inflammatory mediators in keratinocytes4,7,18,19.
Therefore, emerging evidence is pointing towards the AHR as a crucial sensor in the melanoma
microenvironment and in shaping anti-tumour responses. Elucidation of how the AHR acts in melanoma
will advance knowledge of melanoma biology and provide avenues for developing therapeutic
interventions.
The main objectives of this PhD project are to:
I) Evaluate the role of the AHR as a melanin sensor;
II) Identify AHR-elicited responses in melanocytes and keratinocytes;
III) Dissect roles of the AHR in melanocyte-keratinocyte interplay and in the melanoma
microenvironment;
IV) Evaluate the impact of AHR modulation on melanoma immune cell recruitment;
V) Assess potential crosstalk between the AHR and other signalling pathways in melanoma, with a focus
on MITF20.

Translational Potential: There is extensive current interest in targeting the AHR in cancer 9,10,17. Due to its
capacity to bind a set of diverse ligands, the druggability of the AHR is not a limitation. However, it is critical
to better evaluate and understand AHR functions in the tumour microenvironment before it can be
considered as a rational therapeutic target. The AHR modulates both pro- and anti-tumour responses, by
mechanisms that are not fully understood. Thus, to design therapeutic strategies that specifically activate
or inhibit the AHR in a particular tumour type/stage, deeper understanding of how the AHR senses the
tumour microenvironment is needed. Therefore, our elucidation of how the AHR acts in melanoma will
advance knowledge of melanoma biology, shining light on potential underlying mechanisms involved in
melanoma susceptibility, such as those linked to melanin type and abundance, and will also provide
avenues for prospective development of therapeutic interventions. Dr. Moura Alves was awarded a John
Fell Fund to perform an unbiased screen to identify clinically approved drugs with AHR modulatory
properties, feeding into this project to test potential drug candidates in the melanoma context.
REFERENCES: 1 Moura-Alves, P. et al. Nature, 512, (2014). 2 Moura-Alves, P. et al. Science, 366, (2019). 3 Lozza, L. et al. Sci Rep, 9, (2019). 4
Luecke, S. et al. Pigment Cell Melanoma Res, 23, (2010). 5 Abbas, S. et al. Chem Res Toxicol, 30, (2017). 6 Jux, B. et al. J Invest Dermatol, 131,
(2011). 7 Contador-Troca, M. et al. Carcinogenesis, 34, (2013). 8 Corre, S. et al. Nat Commun, 9, (2018). 9 Hidaka, T. et al. Front Med
(Lausanne), 6, (2019). 10 Kolluri, S. K. et al. Arch Toxicol, 91, (2017). 11 Costa, B. E., M.F.; Mendes, R.V.; Fior, R. Cells, 9, (2020). 12 Roman, A.
C. et al. J Biol Chem, 284, (2009). 13 Puyskens, A. et al. Cell Host Microbe, 27, (2020). 14 Siegel, R. L. et al. CA Cancer J Clin, 67, (2017). 15
Shain, A. H. et al. Nat Rev Cancer, 16, (2016). 16 Stockinger, B. et al. Annu Rev Immunol, 32, (2014). 17 Xue, P. et al. Front Immunol, 9, (2018).
18 Di Meglio, P. et al. Immunity, 40, (2014). 19 Fritsche, E. et al. Proc Natl Acad Sci U S A, 104, (2007). 20 Goding, C. R. et al. Genes Dev, 33,
(2019).

14
 Improving immunotherapy by defining the interaction between the
bacterial vaccine BCG and the host epigenomic and immune
response – Prof. McShane1,2,3A
Primary Supervisor: Helen McShane
Additional Supervisors: Xin Lu
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Abstract
The first FDA approved cancer immunotherapy is the only licensed vaccine against
tuberculosis, Bacille Calmette Guerin (BCG). In the past 4 decades, BCG has been established
as one of the most effective treatment for non-muscle invasive bladder cancer (NMIBC). BCG
treatment reduces recurrent rate and prevents progression in around 60% of treated
patients. However the non-responsive patients have poor prognosis and high morbidity.
Hence there is an unmet clinical need to stratify patients for BCG treatment as well as to
understand why the remaining 40% of bladder cancer patients fail to benefit from BCG
treatment. Additionally an intriguing question is whether BCG based therapy can be adopted
to treat cancer types other than NMIBC. Here we propose to address these important
challenges through a multi-disciplinary approach by combining Xin Lu’s molecular and cell
biology expertise together with Helen McShane’s biological and clinical understanding of BCG
vaccine biology. We propose to study how BCG can alter our immune function in vivo using
recently developed state of the art technologies such as ATAC-seq and TAPs-seq to investigate
the impact of BCG on the epigenome of collected samples derived from two BCG trials and
BCG treated bladder cancer patients.

Background and plan of investigation

BCG is a live attenuated strain of Mycobacterium bovis and is best known for its ability to
protect us against tuberculosis. However the protective efficacy of BCG against tuberculosis
is highly variable and there is an urgent need for an improved vaccine. Additionally there has
been a resurgence of interest in BCG itself as a protective and therapeutic vaccine. The impact
of vaccine delivery routes on the efficacy of BCG is also a subject of intensive study. In
particular studies in non-human primates suggest that aerosol or intravenous delivery of BCG
is more protective than the licensed intradermal route (2-5). In addition to the specific
protective effect of BCG against mycobacterial infection, it has been clear for many decades
that BCG may have therapeutic utility beyond tuberculosis. There is an increasing body of
evidence suggesting that BCG may confer non-specific protection against all-cause mortality
in infants, particularly in low and middle income settings (6). These findings led us to
hypothesis that BCG may have a long lasting impact on our immune system. Consistent with
this, recent studies suggest that at least some of the non-specific effects are mediated by
epigenetic changes and so called ‘training’ of the innate immune system (9). Defining the
molecular mechanism(s) and durability of these non-specific protective effects would allow
us to stratify patients with bladder cancer who are most likely to respond to BCG, develop
more specific interventions and minimise the side effects of BCG.
We will utilise a unique collection of samples, taken from healthy volunteers who have
received BCG vaccination either by intradermal injection or by aerosol delivery, to interrogate
the epigenetic and immunological effects of BCG, both in peripheral blood and in broncho-
alveolar lavage fluid (10). Epigenetic regulation is mainly controlled by chromatin remodelling

15
and DNA methylation. Thus we will use ATAC-seq to examine the openness of chromatin and
to use a state-of-the-art technologies such as TAPs developed in the Ludwig Institute to
interrogate DNA methylation. We will complement these data with flow cytometry and
functional assays using PBMCs. The outcome of this study will enable us to comprehensively
interrogate whether the observed epigenetic changes are associated with the observed non-
specific effects of BCG in the examined immune cells. Having defined potential mechanisms
of effect, we will then evaluate blood and bladder biopsy samples taken from patients who
have received BCG intravesically as a treatment for their bladder cancer.

Samples available:
1. Healthy volunteers, vaccinated with intradermal BCG. PBMC and serum samples taken
at baseline (prior to vaccination) and at regular early and late time points after BCG
vaccination (Figure 1A). Numbers available vary by time point but 20-40 available.
2. Healthy volunteers, vaccinated with aerosol BCG. PBMC and serum samples taken at
baseline (prior to vaccination) and at regular early and late time points after BCG vaccination.
Bronchalveolar lavage (BAL) and lung biopsy (in some cases) samples taken at various time
points after vaccination (Figure 1B). Numbers available vary by time point but 20-40 for
PBMC/Serum and ~3-5 for BAL and lung biopsy
3. Patients with non-invasive bladder cancer, PBMC and serum samples taken at baseline
and at regular time points after intravesical BCG installation. Bladder biopsies available at
selected time points. Numbers: 5-10.

Figure 1: Schedule for intradermal and aerosol BCG studies

A: Intradermal B: Aerosol

Research objectives and proposed outcomes

1. How does BCG impact on the host epigenomic and immune response?
2. How do these epigenomic and immunological effects impact on cancer response
rates?
This interdisciplinary project will define the precise mechanisms of therapeutic effect of
intravesical BCG, which would allow stratification of patients and improve treatment
outcomes. This knowledge will facilitate the development of improved therapies. These
results will be of interest for both fields of cancer immunology and vaccinology, and address
the increasingly evident link between infectious diseases and cancer.

References: 1. Nemes et al, NEJM 2018, 2. Dijkman et al, NM 2019, 3. Sharpe et al, Pharmaceutics
2020, 4. Darrah et al, Nature 2020, 5. Sharpe et al, Tuberculosis, 2016, 6. Higgins et al BMJ 2016, 7.
https://www.nice.org.uk/guidance/ng2/resources/bladder-cancer-diagnosis-and-management-of-
bladder-cancer-51036766405, 8. Zlotta et al, Can Urol Assoc J, 2009, 9. Kleinnijenhuis et al PNAS 2012,
10. ClinicalTrials.gov Identifiers: NCT02380508; NCT03912207

16
 Targeting epigenetic regulators to improve tumour responses to
immunotherapy – Prof. Shi1,2,3A
Primary Supervisor: Yang Shi
Additional Supervisors: Benoit van den Eynde
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Cancer immunotherapy, in particular PD-(L)1-directed immune checkpoint blockade (ICB) therapy, has
revolutionized cancer treatment. PD-(L)1 inhibitors are FDA-approved to treat a wide range of
cancers1, but a majority of cancer patients show only partial, temporary or no response. Thus, there
is a clear unmet clinical need to extend the benefits to non-responders and to achieve a long-lasting
therapeutic effect. Multiple mechanisms operative in tumors and/or T cells can contribute to the
unresponsiveness or “coldness” of tumors, including low antigenic mutations, defects in antigen
processing and presentation, lack of T cell infiltration or recognition, and epigenetic factors2. A better
understanding of how tumors’ response to T cell immunity and PD-(L)1 blockade therapy is regulated
at both genetic and epigenetic levels will reveal new therapeutic strategies for improving ICB.
In this project, we will investigate the role of epigenetic regulators in modulating antitumor immunity
and tumor responses to immunotherapy. While we and others have identified isolated examples of
epigenetic factors that regulate ICB efficacy, the whole network of epigenetic regulators (~1,000
genes) remains to be fully explored. We will carry out in vivo CRISPR/Cas9 screens using an epigenetic
factor-focused library, paying particular attention to identifying novel epigenetic targets for turning
“cold” tumors “hot”. All hits will be validated in multiple tumor models and mechanistic studies will
be conducted to elucidate the underlying epigenetic programs and immunologic basis. These
proposed studies will not only uncover new individual epigenetic targets but also possibly reveal a
network of epigenetic regulators important for anti-tumor immunity and ICB, which is crucial for
future development of combination cancer therapy.

Research objectives and proposed outcomes

Cancer immunotherapy has shown great success in the treatment of many cancer types, however its
potential has been limited by the low or temporary responses of many tumors. One of the great
challenges in the field is how to induce a long-lasting response in previous non-responders.
Mis-regulation of chromatin modifications has emerged as a main contributor to tumorigenesis3-5, but
their roles in the overall response of tumors to the immune system is largely underexplored. Previous
work suggests that epigenetic regulators can impact tumor immunogenicity6-16. In addition, we have
shown that LSD1, which we discovered as the first histone demethylase in 200417, is a potent inhibitor
of antitumor T cell immunity and tumor responsiveness to anti-PD-1 therapy18. LSD1 ablation in “cold”
tumors enhances tumor immunogenicity and T cell infiltration, and elicits significant responses of ICB-
refractory mouse melanoma to anti-PD-1 therapy.
Although a few recent studies have reported genome-wide in vitro CRISPR/Cas9 screens, an unbiased
in vivo screen of all ~1000 epigenetic regulators for their ability to modulate tumor response to host
immunity and ICB therapy is required. Preliminary data from our lab using a mouse melanoma model
and syngeneic mice indicates that our screening set-up is functional; a pilot screen uncovered the
enrichment (Ifngr1 and Stat1) and depletion (SWI/SNF chromatin remodeller) controls, whose
ablation cause tumor cell resistance and sensitivity to T cell immunity, respectively19,20. We are thus
primed to further explore the role of epigenetic regulators in immunity and ICB therapy.

In this studentship project, we aim to understand how manipulating epigenetic regulators can impact
tumor immunogenicity and overcome resistance of “cold” tumors to ICB by:
1. Performing in vivo CRISPR/Cas9 screens of epigenetic regulators to generate a statistically robust
list of hits that impact tumor response to host immunity and anti-PD-1 therapy. A stable mouse

17
 melanoma B16 cell line expressing Cas9 will be transduced with a lentiviral library containing
~4,000 gRNAs targeting ~1,000 genes encoding epigenetic regulators. After 10 days, we transplant
these gene-edited B16 cells into three groups of syngeneic mice (TCRα KO, WT and WT plus anti-
PD-1). At day 14, whole tumor mass will be harvested to identify significantly depleted gRNAs in
WT+anti-PD-1 versus WT mice.
2. Validating the screen hits in multiple tumor models, including an additional mouse melanoma
model with defined genetic mutations (BrafV600E/pten-/-) and breast cancer 4T1. For hits that only
score in the anti-tumor immunity assay but do not synergize with anti-PD-1, we will determine if
they may collaborate with other checkpoint inhibitors, such as anti-CTLA4 and anti-TIM3.
3. Investigating the mechanistic basis of the validated hits. For example, if the hit is an enzyme, we
will determine if catalytic activity is required for its role in antitumor immunity. The student will
investigate the immunologic and cellular basis of the hit ablation-induced antitumor immunity,
including determining CD8+ T cell involvement and functional state by measuring cell proliferation
markers, apoptosis indicators, cytokine production levels and cytotoxic molecules. We will
characterize the underlying molecular mechanisms using a range of epigenetic tools, including
ChIP-seq and ATAC-seq, possibly at the single-cell level.
This will have academic value in furthering our mechanistic understanding of how epigenetic
regulators modulate antitumor T cell immunity and ICB and translational potential to identify
druggable candidates for drug development and combination therapy in cancer treatment.
Collaborations: This award will enable a collaboration for the first time between the Shi and Van den
Eynde labs, combining world-leading expertise in cancer epigenetics and tumor immunology
respectively.

Training: The student will receive training in the necessary cellular, molecular, epigenomic and mouse
biology techniques for this project, including CRISPR/Cas9 screening, epigenomic profiling (ChIP-seq,
ATAC-seq, single-cell transcriptomic analysis), and FACS. Outside the lab, the Ludwig Institute for
Cancer Research runs a bespoke graduate training program, including regular seminars with high-
profile external speakers, journal clubs, and training in presentation skills, scientific writing, and data
management.

Translational potential of the project

Increasing tumor responsiveness to immunotherapy would be hugely beneficial to improve the
efficacy of cancer treatments. This project will uncover novel epigenetic regulators of antitumor
immunity and ICB and their mechanisms of action, and provide insights into how tumor
responsiveness or refractoriness to PD-(L)1 blockade is determined by chromatin landscapes. With
this knowledge, we hope to identify targets for future drug development to enable combination
therapy with ICB to improve patient survival.

References: 1 Sharpe AH & Pauken KE Nat Rev Immunol 18, 153-167 (2018). 2 Sharma P et al. Cell 168,
707-723 (2017). 3 Greer EL & Shi Y. Nat Rev Genet 13, 343-357 (2012). 4 Dawson MA & Kouzarides T.
Cell 150, 12-27 (2012). 5 Baylin SB & Jones PA. Nat Rev Cancer 11, 726-734 (2011). 6 Chiappinelli KB
et al. Cell 162, 974-986 (2015). 7 Roulois D et al. Cell 162, 961-973 (2015). 8 Topper MJ et al. Cell 171,
1284-1300 e1221 (2017). 9 Ghoneim HE et al. Cell 170, 142-157 e119 (2017). 10 Pan W et al. Immunity
47, 284-297 e285 (2017). 11 Fraietta JA et al. Nature 558, 307-312 (2018). 12 Peng D et al. Nature 527,
249-253 (2015). 13 Canadas I et al. Nat Med 24, 1143-1150 (2018). 14 Adeegbe DO et al. Cancer Discov
7, 852-867 (2017). 15 Miao D et al. Science 359, 801-806 (2018). 16 Pan D et al. Science 359, 770-775
(2018). 17 Shi, Y. et al. Cell 119, 941-953 (2004). 18 Sheng W et al. Cell 174, 549-563 e519 (2018). 19
Gao J et al. Cell 167, 397-404 e399 (2016). 20 Manguso RT et al. Nature 547, 413-418 (2017).

18
 Elucidating the Rad51-independent pathway of recombination-
dependent replication – Prof. Whitby1,2,3A
Primary Supervisor: Matthew Whitby
Additional Supervisors: Benoit Kornmann
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
The course to complete genome duplication is littered with obstacles, including DNA lesions
and DNA binding proteins, that waylay the replication machinery1. These “replication fork
barriers” threaten the successful completion of DNA replication by causing replication fork
“collapse”. Fork collapse involves the disassembly and/or remodelling of the replisome, which
Elucidating the Rad51-independent pathway of recombination-dependent replication
renders it unable to continue DNA synthesis. The consequent loss of replicative capacity can
lead to mitotic catastrophe through
Professors Matthew Whitbythe attempted segregation
and Benoît Kornmann, Department ofofincompletely
Biochemistry replicated
chromosomes. To avoid this, cells
i. Abstract deploy
(including homologous
project importance, recombination
aims and relevance to repairresearch)
to cancer collapsed forks
and restart DNA replication in
The course a
to process termed
complete genome recombination-dependent
duplication is littered with obstacles, includingreplication
DNA lesions and
binding proteins, that waylay the replication machinery . These “replication fork barriers” threaten the
1
(RDR)
DNA

(also known as break-induced replication

successful completion [BIR] when
of DNA replication by causing initiated from
replication fork a DNA
“collapse”. Fork double strand
collapse involves
disassembly and/or remodelling of the replisome, which renders it unable to continue DNA synthesis. The
the
2,3
break) . consequent loss of replicative capacity can lead to mitotic catastrophe through the attempted
incompletely replicated chromosomes. To avoid this, cells deploy homologous recombination to repair
segregation of

collapsed forks and restart DNA replication in a process termed recombination-dependent replication (RDR)
(also known as break-induced replication [BIR] when
Two pathways of RDR have been identified in eukaryotes,
initiated from a DNA double strand break)2,3.

which can be distinguished

Two pathwaysby their differing
of RDR have reliance
been identified on
in eukaryotes,
which can be distinguished by their differing reliance on
the recombinase Rad51 the (Figure
recombinase 1).Rad51
Rad51-dependent RDR
(Figure 1). Rad51-dependent
has been a focus of RDR has been a focus of research for over two decades.
research for over two decades.
In contrast, Rad51-independent RDR, which was thought In
contrast, Rad51-independent
relatively littleRDR, which waspoorly
thought
to be a minor and inefficient pathway, has received
attention and remains to .
understood 4,5

be a minor and inefficient

However,pathway, has received relativelywas
interest in Rad51-independent RDR
recently re-awakened by discoveries of its importance in
little attention andhumans, remains poorly understood4,5.
where it appears to be especially key for cancer
cells to cope with oncogene-induced replication stress
However, interest in Rad51-independent
and survive RDR alternative
without telomerase by driving
lengthening of telomeres . These findings highlight the
6-8
was
recently re-awakenedRad51-independent
by discoveries RDR of its
pathwayimportance in
as a potential target
for novel anticancer therapies. Indeed, a key component
humans, where it appears to be Rad52,
of this pathway, especially key
is currently forinvestigated
being canceras
a target for synthetic lethality-based anticancer
cells to cope with oncogene-induced
therapies . 9 replication stress and
survive without telomerase
To fully explore by driving
the potential alternative
of targeting the Rad51-
independent
6-8 RDR pathway in cancer treatment, we must
lengthening of telomeres . These
first determine findings
how the highlight
pathway works. The aimtheof this
project is to elucidate the molecular mechanism of
Rad51-independent RDR pathway asRDR
Rad51-independent a potential target forand
through the identification
characterisation of its component parts. To achieve this goal, the student will take advantage of unique
novel anticancer therapies.
experimentalIndeed,
systems anda key
geneticcomponent of this
approaches developed by thepathway, Rad52,
Whitby and Kornmann labsis(see
currently
below).
9
being investigated as a target for synthetic lethality-based anticancer therapies .
ii. Overview of research objectives, methodology and outcomes
To fully explore the potential of galvanize
This project will targeting the Rad51-independent
a collaboration between the Whitby and KornmannRDR pathwaylabs, bringingin cancer
together the
expertise of the Whitby lab in studying DNA repair and recombination, and the Kornmann lab’s pioneering
treatment, we must first
work indetermine
developing the how the
Saturated pathway
Transposon works.
Analysis in YeastThe
(SATAY)aim of this
technique project
for rapid is to
genome-wide
genetic screening . 10
elucidate the molecular mechanism of Rad51-independent RDR through the identification
As the key components of Rad51-independent RDR are likely to be well conserved from lower eukaryotes to
and characterisation of itsthecomponent
human, parts. To achieve
fission yeast Schizosaccharomyces pombe willthis goal,asthe
be exploited student
a tractable will
system for take
expediting
our understanding of this pathway. The Whitby lab has pioneered approaches to investigate RDR in this
advantage of unique experimental
organism, including systems and genetic
the use of site-specific approaches
replication developed
fork barriers to precisely by thefork
track replication Whitby
collapse
and restart at specific genomic sites through a combination of genetic, live cell imaging and in vivo
and Kornmann labs (see below).approaches . Using these tools, the student will be able to perform a more detailed and
biochemistry 11-17

accurate in vivo analysis of Rad51-independent RDR than would be possible using other more commonly
used approaches in the field, which typically elicit fork collapse using drugs and genotoxins that have
Overview of research objectives, methodology and outcomes
pleiotropic and genome-wide effects. Indeed, fission yeast is currently the only organism in which a system
for studying Rad51-independent RDR from a site-specific replication fork barrier has been established and
This project will galvanize a collaboration between the Whitby and Kornmann labs, bringing
validated (Whitby lab unpublished data).
To identify novel components of the Rad51-independent RDR pathway, the student will develop a powerful
together the expertisenew
of genome-wide
the Whitby labgenetic
forward in studying DNA
screen based repair
on the and recombination,
SATAY technique and the
pioneered by the Kornmann lab

19
Kornmann lab’s pioneering work in developing the Saturated Transposon Analysis in Yeast
(SATAY) technique for rapid genome-wide genetic screening10.
As the key components of Rad51-independent RDR are likely to be well conserved from lower
eukaryotes to human, the fission yeast Schizosaccharomyces pombe will be exploited as a
tractable system for expediting our understanding of this pathway. The Whitby lab has
pioneered approaches to investigate RDR in this organism, including the use of site-specific
replication fork barriers to precisely track replication fork collapse and restart at specific
genomic sites through a combination of genetic, live cell imaging and in vivo biochemistry
approaches11-17. Using these tools, the student will be able to perform a more detailed and
accurate in vivo analysis of Rad51-independent RDR than would be possible using other more
commonly used approaches in the field, which typically elicit fork collapse using drugs and
genotoxins that have pleiotropic and genome-wide effects. Indeed, fission yeast is currently
the only organism in which a system for studying Rad51-independent RDR from a site-specific
replication fork barrier has been established and validated (Whitby lab unpublished data).
To identify novel components of the Rad51-independent RDR pathway, the student will
develop a powerful new genome-wide forward genetic screen based on the SATAY technique
pioneered by the Kornmann lab (Figure 2)10. Using this screen, the student will be able to
identify both loss- and gain-of-function mutants as well as functional domains within proteins
(including those
(Figure 2) . Using
10 proteins
this screen, whose
the student will complete
be able to identify inactivation
both loss- and would
gain-of-function mutants as render a cell inviable).
well as functional domains within proteins (including those proteins whose complete inactivation would
render a cell inviable).

The SATAY screen will

generate a catalogue of
putative RDR genes, which will
be validated and prioritized
for more detailed follow-up
studies by a combination of
bioinformatic analysis and
established genetic assays.
This work will provide a
valuable resource of
information that will be vital
for fully elucidating the
Rad51-independent RDR
pathway and identifying
potential novel targets for anti-cancer therapies. In addition
The SATAY screen will generate a catalogue of putative RDR genes, which will be validated and prioritized
for more detailed follow-up studies by a combination of bioinformatic analysis and established genetic to producing a comprehensive
assays. This work will provide a valuable resource of information that will be vital for fully elucidating the
catalogue
Rad51-independent ofRDR
RDR components,
pathway it is envisaged
and identifying potential that the
novel targets for anti-cancer student
therapies. In will be able to perform a more
addition to producing a comprehensive catalogue of RDR components, it is envisaged that the student will
detailed
be able to characterisation of at least one novel component of the Rad51- independent RDR
perform a more detailed characterisation of at least one novel component of the
independent RDR pathway in fission yeast, as well as preliminary genetic analysis of its homologue in
Rad51-

pathway
human cells.
in fission yeast, as well as preliminary genetic analysis of its homologue in human
cells.
iii. Justification for support
This project aligns squarely with CRUK and Oxford Centre’s research strategy focusing on genes and
epigenetics. It will deliver an important new data resource from which potential targets for novel anti-cancer
References: 1) Lambert
therapies will be identified. & Carr
It will also provide (2013)
a unique Chromosoma
and diverse 122,
training opportunity for the33-45; 2) Anand et al (2013) Cold Spring Harb
DPhil student
incorporating method development, high throughput screening, big data analysis, and yeast and human
Perspect
genetics. TheBiol 5,willa010397;
student benefit from the 3)support
Ait Saada et al (2018).
of two well-resourced labs ledDNA
by PIs Repair (Amst)
with excellent track 71, 135-147; 4) Kramara et al (2018)
records both in their research and postgraduate student supervision.
Trends Genet 34,518-531; 5) Lambert et al. (2010) Mol Cell 39, 346-359; 6) Min et al (2017) Mol Cell Biol
37; 7) Bhowmick
References (hyperlinked et al (2016) Mol Cell 64, 1117-1126; 8) Sotiriou et al. (2016) Mol Cell 64, 1127-1134; 9)
to PubMed)
Toma et al (2019) Cancers
1) Lambert & Carr (2013) Chromosoma 122, (Basel) 11; 10)
33-45; 2) Anand Michel
et al (2013) et al.
Cold Spring (2017)
Harb Elife
Perspect Biol 5, 6; 11) Jalan et al (2019). Elife 8; 12)
Morrow
34, 518-531;et al. (2017)
5) Lambert ElifeMol
et al. (2010) 6; Cell
13)39,Nguyen
346-359; et al (2015)
6) Min et al (2017)Elife 4, e04539;
a010397; 3) Ait Saada et al (2018). DNA Repair (Amst) 71, 135-147; 4) Kramara et al (2018) Trends Genet
Mol Cell Biol 37; 7) 14) Sun et al. (2008) Mol Cell 32, 118-
128; 15) Tamang et al. (2019) Elife 8; 16) Ahn et al (2005) EMBO J 24, 2011-2023; 17) Osman & Whitby
Bhowmick et al (2016) Mol Cell 64, 1117-1126; 8) Sotiriou et al. (2016) Mol Cell 64, 1127-1134; 9) Toma et
al (2019) Cancers (Basel) 11; 10) Michel et al. (2017) Elife 6; 11) Jalan et al (2019). Elife 8; 12) Morrow et
(2009) Methods Mol Biol 521, 535-552.
al. (2017) Elife 6; 13) Nguyen et al (2015) Elife 4, e04539; 14) Sun et al. (2008) Mol Cell 32, 118-128; 15)
Tamang et al. (2019) Elife 8; 16) Ahn et al (2005) EMBO J 24, 2011-2023; 17) Osman & Whitby (2009)
Methods Mol Biol 521, 535-552.
2

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 Analysis of spindle assembly checkpoint function in health and
disease – Prof. Gruneberg2,3A,3B
Primary Supervisor: Ulrike Gruneberg
Additional Supervisors: Bela Novak
Eligibility: Track 2, 3A and 3B students only are eligible to apply for this project.
Project Summary
Human bodies consist of millions and millions of cells which are produced through the process of cell
division. To avoid disease, it is essential that each cell division is carried out correctly. Most importantly,
before cells divide, the genetic material, DNA, has to be duplicated and then shared equally between the
two daughter cells. If anything goes wrong during this process, DNA may be lost or gained in the daughter
cells, resulting in a condition called aneuploidy, strongly associated with cancer [1]. A critical cellular
safeguarding mechanism, the spindle assembly checkpoint (SAC), monitors the process of chromosome
segregation in healthy cells and prevents aneuploidy from occurring [2, 3]. We propose to use a
combination of cutting-edge live cell imaging of normal and tumour cells, and computational analysis and
mathematical modelling to analyse the functioning of the
spindle assembly checkpoint in tumour and non-tumour cells.
This will help us to understand which aspects of spindle
assembly checkpoint signaling, and consequently the
chromosome segregation process, are altered in cancer cells.

During mammalian cell division, it is critical that chromosome

segregation and anaphase onset are executed only once all
chromosomes have been stably tethered to microtubules via
their kinetochores. The success of this process is ensured by the
spindle assembly checkpoint. While many molecular details
about spindle assembly checkpoint functioning have been
elucidated in the past decade [3], key aspects of how this
checkpoint functions are still poorly understood. We now know
that the spindle assembly checkpoint checks that all
chromosomes are attached via their kinetochores to
microtubules before segregation of the chromosomes is
initiated and that if attachment errors are detected, cell cycle progression is arrested and anaphase entry
delayed [3]. However, it is still unclear how the spindle assembly checkpoint sustains a robust cell cycle
arrest in the presence of just a few, or indeed just one, unattached kinetochore, yet is promptly silenced
and cell cycle progression resumed once the last kinetochore has been attached. We have previously
successfully combined experimental work and computational modelling to identify critical concepts and
parameters for the functioning of the spindle checkpoint [4, 5]. However, further progress on rate-limiting
factors in spindle checkpoint signalling has been hampered by the absence of experimental data on the
concentration of proteins involved in spindle checkpoint function and the absence of knowledge of the
biological consequences of changing these concentrations. Hence, our aim is now to bring together state-
of-the-art genetic manipulation and live cell imaging of human cells with computational modelling to
identify which parts of the spindle checkpoint pathway are critical for the robust yet sensitive behaviour of
the spindle checkpoint, and how this is altered in cancer cells.

Research objectives and proposed outcomes

Spindle assembly checkpoint gene expression is frequently altered in cancer cells, with both up- and down
regulation having been observed [6]. While in yeast cells it is well understood which spindle assembly
checkpoint genes are rate-limiting for spindle checkpoint functionality, the distinct physiology of yeast cells
precludes a simple translation of yeast data to human cells, and the lack of quantitative data on the human
spindle checkpoint limits our understanding of the consequences of such alterations in gene expression for
chromosome segregation and cell cycle progression. The aims of this proposal are therefore to close this
knowledge gap by analysing the effects of SAC protein downregulation using a combination of cell biology
and mathematical modelling.

21
1.) Cell biological analysis of the effects of spindle checkpoint protein downregulation on cell cycle
progression in transformed and non-transformed cells
We propose to systematically analyse the effect of downregulating known spindle checkpoint components
BUBR1, BUB1, BUB3, MAD2, MAD1, CDC20, MPS1, ZW10 and p31comet in the background of transformed
or non-transformed cell lines (aneuploid, HPV-transformed HeLa cells versus telomerase immortalised
diploid RPE-1 cells;
immortalised non-
transformed breast epithelial
MCF10A cells in comparison
to engineered MCF10 cells
expressing constitutive or
inducible RasV12 Ras-mutant
and matched Ras-WT tumour
cell lines) [7, 8]. To enable us
to analyse these cell lines in
live cell imaging we will use CRISPR/Cas9-mediated genetical engineering of the cells to express
fluorescently tagged cyclin B1 as well as a fluorescently tagged version of the spindle checkpoint protein of
interest. Since cyclin B is one of the key proteins that is degraded at anaphase onset (Figure 1), this double
tagging will allow us to modulate the levels of the tagged spindle checkpoint protein of interest by RNAi,
determine the precise level of residual protein by calibrated fluorescence measurements, and then
evaluate quantitatively by live cell measurements of cyclin B-GFP the ability of these cells to arrest cell cycle
progression and stop cyclin B degradation in response to spindle poisons.
2.) Computational analysis of spindle checkpoint functionality
In cells expressing fluorescently tagged cyclin B1 and SAC protein of interest, we can quantitatively measure
the amounts of the proteins-of-interest, as well as cyclin B degradation kinetics (a proxy for cell cycle
progression and SAC proficiency) for single cells under conditions when the checkpoint should be on.
Together, these measurements allow us to calculate threshold levels of key spindle checkpoint activities
[4]. When combined with modelling, these data help us understand which components become rate-
limiting for spindle checkpoint maintenance in vivo in situations when levels of checkpoint proteins are
reduced. We will carry out this analysis in parallel for transformed and untransformed cells with the aim of
understanding which parameters change in cancer cells and how that affects the faithfulness of
chromosome segregation in normal and transformed cells.

Translational potential
The failure of chromosome segregation can lead to aneuploidy and chromosomal instability, associated
with cellular transformation, cancer and resistance to chemotherapy. A detailed understanding of the
regulation of the chromosome segregation process is therefore an important goal and may enable us to
design targeted therapies to modulate cell division in disease situations such as cancer. Furthermore, given
that many cancer cells downregulate the expression of spindle checkpoint proteins [6] and our analysis will
enable us to predict the cellular consequences, this information could be pertinent for personalised tumour
therapies.

References 1. Sansregret, L., and Swanton, C. (2017). The Role of Aneuploidy in Cancer Evolution. Cold Spring Harb Perspect
Med 7. 2. Kops, G.J., Weaver, B.A., and Cleveland, D.W. (2005). On the road to cancer: aneuploidy and the mitotic checkpoint.
Nat Rev Cancer 5, 773-785. 3. Musacchio, A. (2015). The Molecular Biology of Spindle Assembly Checkpoint Signaling
Dynamics. Curr Biol 25, R1002-1018. 4. Hayward, D., Alfonso-Perez, T., Cundell, M.J., Hopkins, M., Holder, J., Bancroft, J.,
Hutter, L.H., Novak, B., Barr, F.A., and Gruneberg, U. (2019). CDK1-CCNB1 creates a spindle checkpoint-permissive state by
enabling MPS1 kinetochore localization. J Cell Biol 218, 1182-1199. 5. He, E., Kapuy, O., Oliveira, R.A., Uhlmann, F., Tyson,
J.J., and Novak, B. (2011). System-level feedbacks make the anaphase switch irreversible. Proc Natl Acad Sci U S A 108, 10016-
10021. 6. Weaver, B.A., and Cleveland, D.W. (2005). Decoding the links between mitosis, cancer, and chemotherapy: The
mitotic checkpoint, adaptation, and cell death. Cancer Cell 8, 7-12. 7. Molina-Arcas, M., Hancock, D.C., Sheridan, C., Kumar,
M.S., and Downward, J. (2013). Coordinate direct input of both KRAS and IGF1 receptor to activation of PI3 kinase in KRAS-
mutant lung cancer. Cancer Discov 3, 548-563. 8. Matthews, H.K., Ganguli, S., Plak, K., Taubenberger, A.V., Win, Z.,
Williamson, M., Piel, M., Guck, J., and Baum, B. (2020). Oncogenic Signaling Alters Cell Shape and Mechanics to Facilitate Cell
Division under Confinement. Dev Cell 52, 563-573 e563.

22
 Identifying novel regulators of cancer stem cells in pancreatic ductal
adenocarcinoma– Dr. Pauklin 1,2,3A
Primary Supervisor: Siim Pauklin
Additional Supervisors: John Christianson
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Pancreatic cancer is one of the most lethal malignancies in human due to its highly metastatic
characteristics and the poor responsiveness to current therapeutics. Pancreatic
tumorigenesis involves a dedifferentiation process of cellular identity and the acquisition of a
stem cell-like state of a subpopulation of cells known as cancer stem cells (CSCs). These cells
resemble partly to naturally occurring stem cells and are exceptionally important because
their developmental plasticity allows them to metastasize and give rise to whole tumours in
the organism (1-4). Currently it remains unclear, which transcription factors and epigenetic
machineries control the expression of stem cell genes and the stem cell-like identity of
pancreatic CSCs. This knowledge would be valuable for developing more efficient pancreatic
cancer therapeutics in the future. The research objective of the project is to identify and
characterize novel epigenetic machineries and transcriptional regulators which govern gene
expression, proliferation and stem cell-like characteristics of pancreatic CSCs.
The DPhil project will apply a broad range of cutting-edge research techniques covering
human cell culture systems, genome-wide, proteomic, genetic and biochemical methods (4-
8). These include human cancer stem cell spheres and pancreatic ductal adenocarcinoma
cultures, genome-wide studies (RNA-seq, ChIP-seq, ATAC-seq, ChIA-PET), functional studies
(CRISPR/Cas9-mediated gene editing; tumour sphere assays), proteomics (Co-IP / mass-
spectrometry), and mechanistic studies (confocal microscopy, flow cytometry, cell sorting,
real-time PCR, western blotting, CyTOF).
Collectively, this research will provide key insight to the signalling pathways and molecular
mechanisms essential for the formation and maintenance of pancreatic CSCs, helping to
better understand the tumorigenic process, and to uncover novel ways for diagnosing and
treating this lethal cancer.

23
Research objectives and proposed outcomes.
The research will uncover key epigenetic mechanisms that control gene expression and
thereby the stem cell-like state of pancreatic cancer stem cells. The project has three sub-
aims:

1. Performing small molecule screening experiments with compound sets targeting kinases
and/or epigenetic mechanisms. The screening library will contain a unique collection of small
molecule compounds with biological activity against a range of candidates including a broad
range of epigenetic regulators that target distinct enzymes involved in regulating gene
expression, as well as proteasome and kinase inhibitors. Our preliminary data has already
uncovered interesting candidates for in-depth experiments.
2. Studying the molecular mechanisms of selected candidates from the screen in more detail.
This includes functional and mechanistic analysis of candidates by loss-of-function and gain-
of-function experiments via CRISPR/Cas9 mediated editing for gene knockdown and
overexpression effects on tumour sphere formation and marker expression by flow
cytometry, western blotting and confocal microscopy; RNA-sequencing for transcriptomic
changes upon candidate knockdown and inhibition by small molecules; ATAC-sequencing for
chromatin accessibility analyses; ChIP-sequencing for indentifying candidate binding sites;
and ChIA-PET for promoter-enhancer interaction analyses.
3. Using patient samples and animal studies for candidate functional effects on tumorigenesis.
We will perform CyTOF and single cell RNA-sequencing data of tumours from primary
pancreatic cancer patients upon the treatment of epigenetic inhibitors; mouse tumorigenesis
studies by orthotopic transplantation of pancreatic cancer cells with a knockout of candidates.

Collaborators from different disciplines. The award will help build a collaboration between
researchers with expertise in pancreatic cancer and cancer stem cells (Siim Pauklin);
tumorigenesis and cancer therapeutics (John Christianson); epigenetic mechanisms, NGS
technologies and computational biology (Udo Oppermann), as well as proteomic specialists
at TDI mass-spectrometry facility, and clinical oncologists at NHS Oxford University Hospitals
with access to primary pancreatic tumour samples.

Translational potential of the project

Relevance of the project to cancer. Our research will identify novel therapeutic targets that
control epigenetic mechanisms responsible for regulating the stem cell-like characteristics of
cancer stem cells in pancreatic ductal adenocarcinoma. Our preliminary experiments from
candidate screening have already identified novel compounds that impact pancreatic cancer
cell survival and cancer stem cell marker expression. Since we know the target enzymes of
these compounds the project will provide valuable information on potential therapeutic
candidates that could ultimately be tested in pancreatic ductal adenocarcinoma patients.

REFERENCES 1. French, R., Feng, Y., and Pauklin, S. (2020). Targeting TGFβ signalling in cancer: toward context-
specific strategies. Trends in Cancer 7, 538-540. 2. Feng, Y., and Pauklin, S. (2020). Two sides of the same coin:
the roles of TGF-β in colorectal carcinogenesis. Gastroenterology 20, 30395-4. 3. Stoica, A., Chang C-H, and
Pauklin, S. Molecular therapeutics of pancreatic ductal adenocarcinoma: targeted pathways and the role of
cancer stem cells. Trends in Pharmacological Sciences (in press). 4. Feng, Y., and Pauklin, S. (2020). Revisiting 3D
chromatin architecture in cancer development and progression. Nucleic Acids Res. 2020 Sep 17:gkaa747. doi:
10.1093/nar/gkaa747.

24
 Understanding The Therapeutic Efficacy Of Chemotherapy In
Pancreatic Cancer at the single cell level – Dr. Sivakumar1,2,3A,3B
Primary Supervisor: Shivan Sivakumar
Additional Supervisors: Rachel Bashford-Rogers, Michael Dustin
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
Pancreatic cancer has the worst survival of any human cancer1. Breakthroughs in treating this
disease have been modest. Our best treatment is chemotherapy with a regime called
FOLFIRINOX that extends survival by 5 months in the metastatic setting and cures an extra
10-15% of patients with resectable disease. We have made substantial efforts in the last few
years to understand the single cell composition of pancreatic cancer. This project will entail
to understand the effects of chemotherapy on the single cell composition of pancreatic cancer
and how it effects important cell populations such as the cancer cells, immune cells and
fibroblasts. The project will have available a large single cell dataset we have generated of
untreated pancreatic cancer including immune cells, cancer cells and fibroblasts to act as a
comparator. The fellow’s project will be to elucidate the effect of chemotherapy on the single
cell changes in pancreatic cancer.

Research Objectives and Proposed Outcomes

Objectives:
- To characterise the immune cell subset changes due to chemotherapy of human
pancreatic cancer cases
- To characterise the cancer cell transcriptional changes due to chemotherapy of
human pancreatic cancer cases
- To characterise the fibroblast population changes due to chemotherapy of human
pancreatic cancer cases
- To determine the interactions between cell types that form positive and negative
feedback loops resulting tumour microenvironmental changes
The proposed outcome is to generate and analyse single cells from patients with resectable
pancreatic cancer who have been treated with chemotherapy before their operation. They
will generate data from 20 patients who will be a mix of responders and non-responders. They
will process tumour samples into single cells immediately after their operation and sort into
immune cells, cancer cells and fibroblasts. These cells will be single cell sequenced and the
fellow analysing this and using a previously generated dataset to make their observations. As
the tumour tissue will be collected, any interesting cell populations will be validated on these
samples.
The applicant will be trained in fundamental immunology, in depth analysis of samples from
human tissue with techniques such as flow cytometry, single cell sequencing,
immunofluorescence as well as transcriptomic and image analysis. They will also appreciate
how to frame clinical problems as tractable research questions and translational medicine
experimental design. The fellow would also be expected to present the results in national and
international meetings. The findings from the study would inform us on how chemotherapy
is working in pancreatic cancer and make help us select patients better and help us design
better trials. The research itself is highly novel and urgently needed in a cancer of unmet need.

25
This project will show the biological effect of using chemotherapy in pancreatic cancer. The
project is a highly collaborative project with the student needing to interface with many
departments in the hospital and within the university. Expertise in genetics will be learned
from the genomics centre in the Wellcome Trust Centre for human genetics and imaging from
the translational pathology laboratory in the university oncology department.
Translational potential of the project
This work will help identify which subset of pancreatic cancer patients benefit from
chemotherapy and help us understand how we could augment different therapeutic agents
to gain a better response. This study will give us a real insight into which patients respond and
which do not and why this is the case. We envisage that if there is a clear group of responders,
we can develop a stratification system so this can feed into national trial platforms such as
Precisionpanc to guide how we should treat chemotherapy responsive and unresponsive
patients on the national arena.

References:
1) Ilic M, Ilic I. Epidemiology of pancreatic cancer. World J Gastroenterol.
2016;22(44):9694-705. doi:10.3748/wjg.v22.i44.9694.
2) De Santiago, I., Yau, C., Heij, L., Middleton, M.R., Markowetz, F., Grabsch, H.I.,
Dustin, M.L. and Sivakumar, S., 2019. Immunophenotypes of pancreatic ductal
adenocarcinoma: Meta-analysis of transcriptional subtypes. International journal of
cancer.
3) Sivakumar, S., Abu-Shah, E., Ahern, D., Arbe-Barnes, E.H., Mangal, N., Reddy, S.,
Rendek, A., Easton, A., Kurz, E., Silva, M. and Heij, L.R., 2020. Immune responses in
pancreatic cancer may be restricted by prevalence of activated regulatory T-cells,
dysfunctional CD8+ T-cells, and senescent T-cells. bioRxiv.
4) Conroy, T., Desseigne, F., Ychou, M., Bouché, O., Guimbaud, R., Bécouarn, Y., Adenis,
A., Raoul, J.L., Gourgou-Bourgade, S., de la Fouchardière, C. and Bennouna, J., 2011.
FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. New England
Journal of Medicine, 364(19), pp.1817-1825.
5) Conroy, T., Hammel, P., Hebbar, M., Ben Abdelghani, M., Wei, A.C., Raoul, J.L.,
Choné, L., Francois, E., Artru, P., Biagi, J.J. and Lecomte, T., 2018. FOLFIRINOX or
gemcitabine as adjuvant therapy for pancreatic cancer. New England Journal of
Medicine, 379(25), pp.2395-2406.

26
 Heterogeneity of macrophages in colorectal cancer: the role of
IRF5 – Prof. Udalova1,2,3A,3B
Primary Supervisor: Irina Udalova
Additional Supervisors: Fiona Powrie
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
The intestinal immune system is a delicately balanced system between tolerance to
commensal and food particles and raising an immune response upon infection. Dysregulation
of the intestinal immune system can lead to inflammation, which may progress to colorectal
cancer (CRC) 1. Macrophages are playing a central role in maintenance of homeostasis,
initiation of inflammation, restoration of tissue upon injury and mediation of
chemoresistance in tumours. Integrating cues of their immediate tissue microenvironment,
macrophages can adapt their functions according to tissue-specific needs and adapt upon
change 2. Depending on the polarisation of the tumour-associated macrophages (TAMs),
cancer progression and initiation can be hindered or helped 3. Various subsets of
macrophages have recently been identified in different tissues using single-cell RNA
sequencing (scRNA-seq), highlighting the heterogeneity and plasticity of macrophages 4–7.
Furthermore, a role for Interferon Regulatory Factor 5 (IRF5), a master transcription factor
involved in regulating the transcription of pro-inflammatory mediators in shaping
macrophage polarisation was identified 5,8. IRF5 controls both acute and chronic inflammation
and is protective in pathogen clearance 5,9,10. IRF5 has also been identified as a DNA-damage
sensor, highlighting a potential beneficial role in CRC 11. Therefore, dissecting the molecular
mechanisms involving macrophage polarisation and function is crucial for identification of
treatment options for both inflammatory bowel disease (IBD) and CRC.

Research objectives and proposed outcomes

The aim of this project is to firstly assess the role of IRF5 in macrophages in resolution of
inflammation using the Helicobacter hepaticus and anti-IL10R colitis model 12. Previous work
in the lab has profiled IRF5-dependent inflammatory CD11c+ macrophages at peak of
inflammation using scRNA-seq 5. Based on this work, we aim to profile the heterogeneity of
macrophages in resolution of inflammation using targeted mouse models (CX3CR1-IRF5 fl/fl
and CCR2-mKate ER2 IRF5 fl/fl). We hypothesize that lack of IRF5 is beneficial for resolution
of inflammation as macrophages are being polarised towards a tissue-regenerating
phenotype.
Furthermore, comparison of macrophage heterogeneity in resolution to CRC will help identify
molecular targets in shaping macrophage phenotype and directing towards resolution rather
than progression of inflammation into cancer development. It also was suggested that
localisation of macrophages within the tumour microenvironment might be a crucial
determinant of their function 3. Therefore, assessing the localisation of different macrophage
subsets and their interaction cell-cell contacts could also provide further information about
their function and potential targeting. The role of TAMs in CRC is yet unclear with various
studies suggesting both detrimental and beneficial effects. Thus, in addition to inhibition of
IRF5 (as above) we would also consider stimulating IRF5 specifically at tumour sites might
improve anti-cancer immunity 6,11. This could be achieved by targeted delivery of adenoviral

27
vector expressing IRF5 (overexpression) or inhibition of IRF5 through phosphorylating kinase
inhibition 13.
We have an established collaboration with the group of Prof Fiona Powrie, Kennedy Institute,
who have developed a number of CRC models and will be able to guide us through.
Translational potential of the project
T cell immunity, which is beneficial in tumours, is undermined by immunosuppressive myeloid
cells, of which a subset of TREM2+ macrophages have been identified as a potential target in
tumours 14. Understanding the role of macrophages as pivotal cells in the resolution of
inflammation as well as progression of inflammation into CRC will help shaping specific
therapies targeting macrophages. IRF5 plays a crucial role in mediating differentiation of
infiltrating monocytes into pro-inflammatory macrophages during intestinal inflammation
and may therefore be central during resolution and cancer development.

References :
1. Mantovani, A., Allavena, P., Sica, A. & Balkwill, F. Cancer-related inflammation. Nature (2008).
doi:10.1038/nature07205
2. Lavin, Y. et al. Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local
Microenvironment. Cell 159, 1312–1326 (2014).
3. Caprara, G., Allavena, P. & Erreni, M. Intestinal Macrophages at the Crossroad between Diet ,
Inflammation , and Cancer. Int. J. Mol. Sci. (2020).
4. Chakarov, S. et al. Two distinct interstitial macrophage populations coexist across tissues in
specific subtissular niches. Science (80-. ). 363, eaau0964 (2019).
5. Corbin, A. L. et al. IRF5 guides monocytes toward an inflammatory CD11c + macrophage
phenotype and promotes intestinal inflammation. Sci. Immunol. 6085, 1–16 (2020).
6. Xiong, Y. et al. Profiles of immune infiltration in colorectal cancer and their clinical significant:
A gene expression-based study. Cancer Med. (2018). doi:10.1002/cam4.1745
7. Norton, S. E., Dunn, E. T., Mccall, J. L., Munro, F. & Kemp, R. A. Gut macrophage phenotype is
dependent on the tumor microenvironment in colorectal cancer. Clin. Transl. Immunol. 5, 76
(2016).
8. Krausgruber, T. et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17
responses. Nat Immunol 12, 231–238 (2011).
9. Weiss, M. et al. IRF5 controls both acute and chronic inflammation. Proc. Natl. Acad. Sci. U. S.
A. 112, 11001–11006 (2015).
10. Pandey, S. P., Yan, J., Turner, J. R. & Abraham, C. Reducing IRF5 expression attenuates colitis in
mice, but impairs the clearance of intestinal pathogens. Mucosal Immunol. (2019).
doi:10.1038/s41385-019-0165-1
11. Hu, G., Mancl, M. E. & Barnes, B. J. Signaling through IFN regulatory factor-5 sensitizes p53-
deficient tumors to DNA damage-induced apoptosis and cell death. Cancer Res. 65, 7403–7412
(2005).
12. Arnold, I. C. et al. CD11c+monocyte/macrophages promote chronic Helicobacter hepaticus-
induced intestinal inflammation through the production of IL-23. Mucosal Immunol. 9, 352–
363 (2016).
13. Byrne, A. J. et al. A critical role for IRF5 in regulating allergic airway inflammation. Mucosal
Immunol. 10, 716–726 (2017).
14. Molgora, M. et al. TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-
PD-1 Immunotherapy. Cell 1–15 (2020). doi:10.1016/j.cell.2020.07.013

28
 Modelling cancer stem cell dormancy using organoids and
advanced 3D culture models – Dr. Boccellato1,2,3A,3B
Primary Supervisor: Francesco Boccellato
Additional Supervisors: , Colin Goding, Ahmed Ahmed
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
Over recent years substantial advances have been made in our understanding of cancer and the
development of a range of more effective therapies. Nevertheless, after apparently successful anti-cancer
therapy, disease may recur even after many years owing to the presence of therapy-resistant cells. One of
the principle causes of relapse is cancer cell dormancy. Some cancer cells stop dividing and enter a dormant
state resembling that used by many physiological stem cells that divide to regenerate damaged tissue or
replace cells that are naturally turned over. Why and how cells enter or emerge from a state of dormancy
is unclear, but understanding how cells become dormant may offer opportunities for therapies designed
to reduce relapse. Investigating human dormant cancer stem cells has been difficult due to the lack of
appropriate in vitro models, and our current knowledge is extrapolated from experiments in mouse models
or steady state analysis of human tumour masses. In this project we aim to use a newly identified biomarker
for dormancy to detect and study this cell state ex vivo by using patient-derived biopsies. Further we will
use organoids and other advanced cell culture models (the mucosoids) to identify and isolate dormant cells
in vitro. The specific aims are to: 1. Characterise the hallmarks of dormant cells; 2. Decipher the signals
regulating the generation, maintenance and elimination of dormant cancer cells. This study promises to
identify potential therapeutic vulnerabilities in cancer dormancy. To achieve this, we will use single cell
RNA-seq, label retention and lineage tracing, 3D culture systems, and live cell fluorescent reporter assays
to compare dormant cancer cells with their physiological counterparts.

Research objectives and proposed outcomes

Tumours contain two kinds of non-proliferating cancer cells that are either quiescent or dormant. Most
studies on dormancy do not distinguish between these two states. By examining physiological stem cells
we have found that a hallmark of dormant, but not quiescent, cells is that they turn off expression of the
majority of genes by shutting down transcription, evidenced by absence of an RNA polymerase II (PolII)
modification associated with active transcription (Figure 1A). Since low transcription would reduce protein
translation and suppress cellular metabolic activity, transcriptional silencing would allow cells to remain
inactive for long periods. Importantly, such ‘PolII-negative’ cells are also found in human melanomas,
mouse patient derived xenografts (PDX) (Figure 1B) and in 3D models of melanoma (not shown), but are
not observed in 2D culture. The Goding lab has also
developed a novel lentivirus-delivered ratiometric
fluorescent reporter designed to enable detection of
dormant PolII-low cells (Figure 1C). Using this reporter in
intestinal organoids revealed that PolII-low cells at the base
of the crypt (Figure 1C, open arrows) exhibited both red
and green fluorescence, whereas PolII high cells (eg. white
closed arrows) were red only. Although still to be tested
widely, this reporter potentially enables live dormant cells
Figure 1. Detecting PolII-low dormant cells. A. to be isolated and characterised. The Boccellato lab has
Immunofluorescence using PolII-specific antibody developed a homeostatic, long-lived (>1 year) stem cell
(Green) together with lineage markers (red) and DNA based human primary cell culture, called mucosoids, for
(DAPI, blue). B. Detection of PolII-low cells in a
the stomach (Figure 2A,B) and is currently using this
melanoma PDX mouse tumour. C. Activity of a
ratiometric fluorescent reporter to detect dormant technology to cultivate cells from fallopian tubes (Figure
cells. PolII-Low cells (open arrows) marked by absence 2C,D).
of blue fluorescence are Green and red, but PolII-High
cells are red only.

29
Objectives
1. Characterise the hallmarks of dormant cells. Since PolII-low dormant cells can readily be detected in
organoids, 3D culture and in tumours we will use the PolII marker in co-immunofluorescence assays to
identify characteristics of dormant cells together with antibodies directed against candidate markers of
interest. These include epigenetic marks, cell surface markers associated with minimal residual disease (eg
aquaporins, CD36), and markers of stem cell populations identified by lineage tracing experiments in gastric
and other organoid systems. In parallel, we will undertake laser-capture microdissection of human and PDX
model tumour sections followed by RNA-seq to
identify gene expression patterns associated with
the PolII-low population, and by using the live cell
fluorescent reporter assay in organoids or
mucosoids, isolate candidate dormant cells for
single cell-RNA-seq. We anticipate that we will
identify biomarkers of dormant cells that will be
useful for monitoring their generation and response
to microenvironmental cues and to therapies, and a
gene expression program that will reveal
mechanisms underpinning the generation and
maintenance of dormant cells. Figure 2. A. Schematic of a mucosoid culture. B. Gastric
2. Decipher the signals regulating dormancy. Using mucosoid culture infected with H.pylori. C. A fallopian tube
mucosoid culture stained for the stem cell marker Pax8. D.
the biomarkers identified, including low PolII, we the same culture stained for CA125 and with visible cilia
will then vary culture conditions for physiological
and cancer organoids and 2D cultures to identify signalling pathways that increase or decrease the
proportion of dormant cells. These conditions include hypoxia, nutrient limitation, infection and signalling
molecules such as TGF or TNF that are important in generating physiological stem cells, as well as
targeted and chemotherapies. RNA-seq data will be mined for clues to key signalling pathways that may
represent targetable vulnerabilities in dormant cells. The results obtained will generate novel insights into
the origins of cancer stem cells, their relationship to physiological stem cells and should identify potential
therapeutic vulnerabilities in dormant cells.
Collaborations involved:- The project brings together three laboratories (one clinical, two academic) that
have not previously collaborated to identify a therapeutic vulnerability in cancer cell dormancy. The
preliminary work is based on observations from the Goding lab, which has a long-standing interest in how
the microenvironment generates phenotypic heterogeneity in cancer (Garcia Jimenez and Goding, 2019),
primarily using melanoma as a model (Rambow et al , 2019). They recently identified PolII-low stem cells
in multiple tissues, including in melanomas grown in 3D and PDX models, and have generated of a potential
fluorescent reporter for dormancy. Ahmed’s lab has recently identified 4 different cell populations,
including stem cells, in the fallopian tube using single cell RNA-seq, and has shown that these populations
are also found in ovarian cancer (Hu et al 2020). The Boccellato lab is new to Oxford, but has developed a
unique stem cell-dependent mucosoid culture system that accurately recapitulates the multi-lineage,
highly polarized gastric epithelium that can be used as an in vitro model for gastric cancer (Boccellato et al
2018). The strength of this proposal comes from the shared interests and complementary expertise of each
group in comparing physiological stem cells to those present within cancers, and especially in using the
low-PolII status of dormant cells to identify and characterize dormant cells and their relationship to minimal
residual disease.

Translational potential of the project - The project addresses a critical question in cancer using novel
technologies and preliminary observations: what is the nature of cancer cell dormancy? We anticipate that
the characterization of dormant cells may enable therapeutic vulnerabilities to be identified, with the
potential to prevent and eradicate dormant cancer cells.

References: Boccellato, F. et al., 2018, GUT. García-Jimenéz, C., and Goding, C.R. 2019, Cell Metabolism. Hu et al., 2020
Cancer Cell. Rambow, F., Marine, J.C., and Goding, C.R. 2019. Genes & Dev

30
 Decoding immune surveillance within the tumour
microenvironment: how the extracellular matrix enables escape
from immunity – Prof. Midwood1,2,3A
Primary Supervisor: Kim Midwood
Additional Supervisors: Francesca Buffa, Adrian Harris
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Our immune system detects tumors and activates inflammation to destroy them. However, tumors
develop effective strategies to evade elimination, for example switching off cytotoxic T cells that are
mobilized to kill them. Re-activating these cells after they have been disarmed by tumors has
revolutionized the treatment of people with cancer. However, this approach does not work for all
patients, nor all types of tumor, and can be associated with severe autoimmune side effects. The
importance of the interplay between tumor cells, stromal cells and immune cells in determining
whether a tumor will be eliminated, or will thrive, provided a landmark shift in disease philosophy,
opening therapeutic avenues beyond targeting the tumor cell. However, in most solid tumors these
cell populations do not exist in isolation, but assemble together within a tumor specific extracellular
matrix (T-ECM), which creates the framework of the tumor microenvironment (TME)[1]. Building on
our work which revealed that matrix molecules can trigger inflammation, and shape the resultant
immune response, we discovered how tumors exploit the immuno-modulatory properties of the
matrix to escape immune surveillance. We showed that the T-ECM creates distinct sub-tumoral niches
which control immune cell infiltration, localization and phenotype, and we identified new
subpopulations of tumor-associated myeloid cells whose behaviour is programmed by
microenvironmental cues. We also developed therapeutic antibodies targeting matrix molecules,
which prevent tumor growth and spread, by enhancing the infiltration of cytotoxic T cells and
macrophages into the tumor, and by driving macrophage polarization towards a tumoricidal
phenotype [2, 3]. This project will further investigate how crosstalk between the T-ECM and tumor,
stromal and immune cells dictates the balance between an immunogenic and a tolerogenic TME.
Using an interdisciplinary approach incorporating state of the art computational genomics, multiplex
and high resolution tissue imaging, and therapeutic manipulation of the extracellular matrix, our aims
are: 1) to understand the precise immunological roles of distinct components, and compartments, of
the T-ECM, and 2) to determine whether targeting molecules within the T-ECM is a viable strategy
with which to treat human disease.

Research objectives and proposed outcomes

Tenascin-C (TNC), an extracellular matrix protein not expressed in healthy adult tissues, but up-
regulated in the stroma of many solid cancers, protects tumors from destruction by re-programming
inflammation in the TME. In a syngeneic, immunocompetent grafting model of breast cancer, TNC-
rich tumors contain high numbers of immune cells that support tumor growth including reparative
macrophages and Th17 cells, and low numbers of CD8 cells, compared to tumors lacking TNC (Figure
1A). Analysis of the tumor myeloid compartment at the single cell level revealed previously
undiscovered cellular heterogeneity, highlighting subpopulations with discreet functional
specialization (B). The balance of myeloid subsets was altered in TNC high vs TNC null tumors; and
specific subsets correlated differently with survival in people with breast cancer (C). In mice, tumor-
associated macrophages (TAMs) were confined almost exclusively within niches defined by expression
of TNC (D), which induced a localized phenotypic switch, downregulating M1-macrophage associated
genes and co-stimulatory markers, and upregulating M2-associated genes, yielding TAMs incapable of
T cell activation [2]. Treatment with antibodies that block TNC activation of toll-like receptor 4 (TLR4)
(anti-FBG or C3) prevented autochthonous tumor growth (E) and spread [2], showing comparable

31
efficacy to checkpoint inhibition alone (anti-PDL1)[2], whilst combination treatment significantly
enhanced, and sustained, inhibition of tumor growth (E). Anti-FBG treatment increased TAM
infiltration into the tumor compared to isotype treated mice where a significant proportion of TAMs
were excluded from the tumor [2], simultaneously upregulating co-stimulatory markers (F).

This project will: 1) further

investigate the location,
function and ontogeny of novel
tumor-associated myeloid
subsets using cluster specific
markers to image, isolate and
deplete subpopulations during
murine models of breast
cancer, 2) better define the
cellular and molecular basis of
TNC-mediated tumor growth
and spread by examining i)
changes in myeloid
Figure 1. Tenascin-C (TNC) protects tumors from destruction by re-programming subpopulation localization and
inflammation in the TME. (A, C-F, and all other data cited but not shown are published in
[2]; B is our unpublished data).
phenotype following treatment
with anti-FBG antibodies, and ii)
signalling pathways activated downstream of TNC ligation of TLR4 that contribute to cell re-
programming, and 3) identify whether myeloid subpopulations identified in murine breast tumors are
conserved in human disease, by i) interrogation of publicly available human breast tumor RNA seq
data sets (bulk; single cell (sc) from total cells) and sc data sets generated from sorted CD45+ cells
(Harris/Buffa) and ii) imaging biopsy tissue sections (Harris) to map cell-matrix interaction networks in
human disease. Together these data will provide a better understanding at a high resolution of how
distinct myeloid cell subpopulations drive tumor growth and spread, and how cell-matrix interactions
within discreet sub-tumoral niches impact the immune axis in breast cancer.

Translational potential of the project and relevance to cancer research or patient care.
The move towards treatments that allow the immune system to attack tumor cells has generated
much excitement, offering the advantages of a more natural tumor killing mechanism than simply
poisoning the tumor, as well as the possibility of generating long lasting immunity against the cancer.
The first wave of immune-oncology therapies focused on blocking the tumors ability to de-activate
cytotoxic lymphocytes already infiltrating the cancer, allowing existing effector cells to kill tumor cells.
The aim of this proposal is to bring forward treatments that allow more efficient generation of effector
cells within the tumor, and at metastatic sites, by targeting microenvironmental cues from the T-ECM,
to prevent cancer growth and spread. This mode of action is distinct from current and emerging
immune-oncology therapies, thereby providing an additional treatment option that may work
synergistically with other approaches. Moreover, reducing primary tumor burden as well as control of
tumor spread would be a significant step forward in treatment.

References: [1] Pickup, M.W., J.K. Mouw, and V.M. Weaver, The extracellular matrix modulates the hallmarks
of cancer. EMBO Rep, 2014. 15(12): p. 1243-53). [2] Deligne C, Murdamoothoo D, Gammage AN, Gschwandtner
M, Erne W, Loustau T, Marzeda AM, Carapito R, Paul N, Velazquez-Quesada I, Mazzier I, Sun Z, Orend G,
Midwood KS. Matrix-Targeting Immunotherapy Controls Tumor Growth and Spread by Switching Macrophage
Phenotype. Cancer Immunol Res. 2020 Mar;8(3):368-382. [3] Immobilization of infiltrating cytotoxic T
lymphocytes by tenascin-C and CXCL12 enhances lung metastasis in breast cancer. Murdamoothoo, D., Sun, Z.,
Yilmaz, A., Deligne, C., Velazquez-Quesada, I., Erne, W., Mörgelin, M., Midwood, K.S., Orend, G. Under review
EMBO J.

32
 UGT8 and microcarrier signalling in the development of breast
cancer – Prof. Wilson1,2,3A
Primary Supervisor: Clive Wilson
Additional Supervisors: Adrian Harris
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.
Project Summary
Intercellular signals and signalling pathways promote the development of all cancers. Traditionally,
such signals are considered to be discrete molecular entities like growth factors, but recently, more
complex multimolecular complexes, such as extracellular vesicles (EVs), which can simultaneously
reprogramme many aspects of cell behaviour, have emerged as key mediators. Using a Drosophila
prostate-like genetic model, we have recently identified a novel EV subtype called Rab11a-exosomes,
which we showed mediate growth and angiogenic effects in human cancer models (see also recent
News and Views article)1. Employing the same model, we have discovered other new signalling
complexes called microcarriers (Fig. 1A), which have a neutral lipid core, store signals at their
surfacethat also control their fusigenic properties, and can then rapidly release them when the
extracellular microenvironment changes2. We hypothesise that these too will provide novel insights
into cancer biology and have therapy implications.
Genetic analysis of microcarrier biogenesis has revealed that their release is controlled by UDP
Glycosyltransferase 8 (UGT8), a ceramide galactosyltransferase, which is the most evolutionarily
conserved lipid glycosyltransferase in the animal kingdom3,4. UGT8 knockdown in Drosophila prostate-
like cells leads to all microcarriers remaining attached by projections to these cells (Fig. 1B). Even
though these flies can transfer sperm to females, they are completely sterile, because microcarriers
are not transferred, illustrating the importance of these intercellular signalling structures.
UGT8 is highly expressed in several different, more aggressive cancers and particularly in breast
cancer5-7. It promotes proliferation, survival and metastasis via mechanisms that are poorly
characterised. Interestingly, breast epithelial cells secrete neutral lipid droplets, which carry
galactosylceramide at their surface8, using mechanisms that are also not well characterised. Our
central hypothesis is that breast cancer cells up-regulate UGT8 to secrete galactosylceramide-coated,
neutral lipid-containing, microcarrier-like structures and that these play important intercellular
signalling roles.
To test this hypothesis, we will combine the expertise of Prof Wilson’s group, which has discovered
microcarriers and the mechanisms by which they are formed using the Drosophila model, with the
extensive experience of Prof Harris in basic and clinical breast cancer biology. They have a strong and
long-standing track record of collaboration in other areas of tumour cell biology, where Drosophila
can inform cancer studies, eg [1,9]. The student will investigate how breast cancer microcarriers and
lipid secretion are regulated by UGT8, define microcarrier cargos and determine their functions. This
work has the potential to open up a new field in cancer biology, relevant not only to breast cancer,
but to other cancers where high level UGT8 expression is a marker for tumour metastasis and therapy
resistance7,10.

33
Research objectives and proposed outcomes
The project will have the following four research objectives and proposed outcomes, and will fully test
our central hypothesis that breast cancer cells secrete neutral lipid in structures equivalent to
microcarriers and that UGT8 controls release of these structures and the resulting intercellular
signalling:
1. Optimise assays for microcarriers and lipid droplets secreted from breast cancer cell lines: Initially
use neutral lipid dyes (eg LipidTox) to stain small volumes of conditioned medium from 2D culture of
breast cancer cell lines with high UGT8 expression5,6. These dyes are highly specific and do not stain
other secreted multimolecular signalling structures like extracellular vesicles. Also use organoids
generated from UGT8-expressing cell lines, eg. PMC42, MCF1011, staining lumen of fixed tissue (and
extend to breast carcinoma organoids, if successful). In secreting cells, we will express a GFP protein
carrying a glycosylphosphatidylinositol anchor (GFP-GPI), which associates with secreted
phospholipid-coated structures and inserts into the outer coat of microcarriers in flies, to test as a
vital marker for microcarriers. Confirm microcarrier identity by absence of transmembrane protein
cargos, failure to stain with lipid bilayer dyes, immuno-EM and UGT8-dependency (2). Develop assays
for measuring size and number of microcarriers. Proposed outcome: Identification of microcarrier-
secreting cells and development of microcarrier assays.
2. Characterise the role of UGT8 in microcarrier biogenesis: Block ceramide synthesis, eg with
zoledronic acid6, or knockdown UGT8 to test roles in microcarrier biogenesis, measuring microcarrier
numbers and size in conditioned medium, and analysing secreting cells and organoids for stalled
microcarrier secretion. Proposed outcome: Determine the roles of UGT8 and ceramide in microcarrier
biogenesis.
3. Identify cargos carried by microcarriers: Pull-down GFP-GPI-labelled microcarriers or isolate by
density gradients on basis of low density, and undertake proteomics analysis of cargos using several
cell lines (with Dr Roman Fisher [Target Discovery Institute, with whom we work on exosome analysis).
Screen for cargos previously identified in Drosophila, which are already implicated in UGT8 function,
eg. Contactin2,12. Start to screen for specific cargos in patient samples, eg. organoids and blood, as the
basis for grant applications to CRUK. Proposed outcome: Identification of signalling molecules and
other cargos on microcarriers in vitro and in patients.
4. Determine functions of microcarriers: Reduce microcarrier secretion by UGT8 knockdown (or
knockdown of cargos from 3) and test effects of resulting conditioned medium and control medium
on breast cancer cell growth and migration, blood vessel network formation, etc, with assays guided
by cargos found in 3. If time allows, undertake preliminary xenograft experiments to test effects on
microcarrier secretion, tumour signalling, growth and metastasis. Proposed outcome: Determine the
functions of breast cancer microcarriers.
References: 1. Fan S-J et al. (2020) Glutamine deprivation alters the origin and function of cancer cell exosomes. EMBO J.
2020, e1030093 and N&V, van Niel G, Théry C. (2020) Extracellular vesicles: eat glutamine and spit acidic bubbles. EMBO J.
2020, 39:e105119; 2. Wainwright, S.M. et al. (2020) Drosophila Sex Peptide Controls the Assembly of Lipid Microcarriers in
Seminal Fluid. BioRxiv, https://doi.org/10.1101/2020.04.24.059238, PNAS, under revision; 3. Ahn SJ, et al. (2011)
Comparative analysis of the UDP-glycosyltransferase multigene family in insects. Insect Biochem Mol Biol. 42:133-47; 4.
Meech R, et al. (2019) The UDP-Glycosyltransferase (UGT) Superfamily: New Members, New Functions, and Novel Paradigms.
Physiol Rev. 99:1153-1222; 5. Owczarek TB, et al. (2013) Galactosylceramide affects tumorigenic and metastatic properties
of breast cancer cells as an anti-apoptotic molecule. PLoS One 8:e84191; 6. Cao Q, et al. (2018) Inhibition of UGT8 suppresses
basal-like breast cancer progression by attenuating sulfatide-αVβ5 axis. J Exp Med. 215:1679-92; 7. Dzięgiel P, et al. (2010)
Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases. Br J Cancer
103:524-31; 8. Bouhours JF, Bouhours D. (1979) Galactosylceramide is the major cerebroside of human milk fat globule
membrane. Biochem Biophys Res Commun. 88, 1217-1222; 9. Fan, S-J et al. (2016) PAT4 Levels Control Amino Acid Sensitivity
of Rapamycin-Resistant mTORC1 from the Golgi and Affect Clinical Outcome in Colorectal Cancer. Oncogene 35, 3004-15;
10. Al-Mahrouki A, et al. (2017) Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide
metabolism. PLoS One 12:e0181951; 11. Marella NV et al. (2009) Cytogenetic and cDNA microarray expression analysis of
MCF10 human breast cancer progression cell lines. Cancer Res. 69:5946-53; 12. Traka M, et al. (2002) The neuronal adhesion
protein TAG-1 is expressed by Schwann cells and oligodendrocytes and is localized to the juxtaparanodal region of myelinated
fibers. J Neurosci. 22:3016-3024.

34
 Studying the role of a chromatin remodelling factor (ATRX) in
normal gene expression and in malignancy – Prof. Higgs1,2,3A
Primary Supervisor: Douglas Higgs
Additional Supervisors: Richard Gibbons
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Thalassaemia is the most common form of inherited anaemia throughout the world. In all
cases, it results from an imbalance in the production of the a-like and b-like globin chains of
haemoglobin, leading to a-thalassaemia and b-thalassaemia respectively. The aim of our
laboratory is to understand how the globin gene clusters are normally regulated during
development and differentiation and how this is perturbed in patients with thalassaemia. By
approaching these questions, we are also developing a general understanding of the
principles by which mammalian genes are normally switched on and off and how these
processes go awry in acquired and inherited human genetic diseases, including cancer.
During the course of this work, we have identified about 130 patients who have a rare form
of a-thalassaemia which occurs in the context of a pre-malignant condition called the
myelodysplastic syndrome (MDS). Many of these patients go on to develop acute myeloid
leukaemia. These patients have no pre-existing forms of a-thalassaemia (AT) and so this
condition is acquired specifically in the pre-malignant clones of cells in MDS: hence the
condition is referred to as the ATMDS syndrome. When we analyse the bone marrow cells of
patients with ATMDS we find a distinct constellation of mutations in epigenetic readers,
writers and erasers which are also found in other patients with MDS but, importantly, in
addition, most patients with ATMDS have mutations in a chromatin remodelling factor called
ATRX. This protein was discovered in our laboratory in 1995 as a cause of X-linked a-
thalassaemia associated with developmental abnormalities (ATR-X syndrome) and ATRX has
more recently been recognised as a tumour suppressor gene in a wide variety of malignant
tumours including glioblastoma, melanoma, pancreatic neuroendocrine tumours and a wide
range of sarcomas.

By understanding the role of ATRX in vivo, we hope to understand its normal role in gene
expression and how this is perturbed in malignancy. Analysis of naturally occurring mutations
often provide important clues to mechanism of disease. The key scientific question in this
project is how do mutations in ATRX down regulate a-globin gene expression in ATMDS
syndrome and why is the effect so much greater in ATMDS compared with ATR-X syndrome.
We have been studying both primary cells from patients with ATMDS or ATR-X syndrome and

35
developing much needed erythroid cell models of these diseases. This has been challenging
for a variety of reasons but recently we have shown that using single cell analysis we are now
able to identify a sub-population of erythroid cells that appear to be more affected by ATRX
mutations than others and we are currently investigating why this should be so. Some clues
to this will come from analysing the impact of other genes that are mutated in ATMDS
syndrome: appropriate cell lines in which such genes have been mutated individually and in
combination are now edited and available for further studies. The aims of this project will
therefore be to further characterise primary cells and the recently established erythroid cell
models of ATMDS syndrome using transcriptional, epigenetic and chromosome conformation
studies to analyse how a-globin expression is perturbed in this severe, acquired form of a
thalassaemia. All of such experimental approaches are well established in our laboratory. This
project will contribute to our understanding of globin gene regulation, the general
mechanism(s) by which chromatin remodelling factors normally work and how they may
contribute to malignant diseases when mutated.
Research objectives and proposed outcomes The role of chromatin remodelling factors is at
the forefront of research into gene regulation and cancer. We contribute to many aspects of
gene regulation both transcriptional and epigenetic aspects via publication in high impact
journals and engagement in international meetings. We have expertise on a wide range of
approaches to transcriptional and epigenetic biology in our laboratories. We also provide
training in computational biology. We have well established long-standing collaborations with
clinical haematologists in the UK (Oxford Haematology and Quek, KCH), EU (Hellstrom,
Karolinska) and US (Steensma and Ebert, Harvard) who have been contributing to this
programme of work and we have an established network of collaborators in the
transcriptional and epigenetic areas of research.
Translational potential of the project and relevance to cancer Since we originally identified
the ATRX gene we have also shown that the protein is part of a protein complex together with
a histone chaperone DAXX and the histone variant H3.3. Mutations in all three components
of this complex have now been associated with a wide variety of malignant tumours. Almost
all of such tumours maintain their telomeres via the so-called alternative (ALT) pathway of
telomere maintenance. This aspect of ATRX is also being studied independently by a member
of Oncology (Dr David Clynes) in collaboration with Professor Richard Gibbons (Co-
supervisor). The current proposal aims to study the role of ATRX in gene expression. There
seems little doubt that understanding the normal biological role of this complex will be of
importance in understanding its role in the development of cancer.

Relevant Publications 1) Gibbons RJ, Picketts DJ, Villard L & Higgs DR (1995) Mutations in a putative global
transcriptional regulator cause X-linked mental retardation with a-thalassemia (ATR-X Syndrome). Cell, 80, 837-
845. 2) Gibbons RJ, Pellagatti A, Garrick D, Wood WG, Malik N, Ayyub H, Langford C, Boultwood J, Wainscoat JS
& Higgs DR (2003) Identification of acquired somatic mutations in the gene encoding chromatin-remodelling
factor ATRX in the a thalassaemia myelodysplasia syndrome (ATMDS). Nat Genet, 34, 1-4. 3) Steensma DP,
Gibbons RJ & Higgs DR (2005) Acquired -Thalassemia in Association with Myelodysplastic Syndrome and Other
Hematologic Malignancies. Blood, 105, 443-452. 4) Hanssen LLP, Kassouf MT, Oudelaar AM, Biggs D, Preece C,
Downes DJ, Gosden M, Sharpe JA, Sloane-Stanley JA, Hughes JR, Davies B & Higgs DR (2017) Tissue-specific CTCF-
cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo. Nat Cell Biol 19:
952-961. 5) Hughes JR, Roberts N, McGowan S, Hay D, Giannoulatou E, Lynch M, de Gobbi M, Taylor S, Gibbons
R & Higgs DR (2014) Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-
throughput experiment. Nat Genet, 46: 205-212. 6) Hay D, Hughes JR, Babbs C, Davies JOJ, Graham BJ, Hanssen
L, Kassouf MT, Oudelaar AM, Sharpe JA, Suciu M, Telenius J, Williams R, Rode C, Li P-S, Pennacchio LA, Sauka-
Spengler T, Sloane-Stanley JA, Ayyub H, Butler S, Gibbons RJ, Smith AJH, Wood WG & Higgs DR (2016) Testing
the super-enhancer concept by in-vivo dissection. Nat Genet, 48, 895-903.

36
 ARH3/ADPRHL2 as a biomarker for PARP inhibitor
sensitivity/resistance – Prof. Ahmed1,2,3A,3B
Primary Supervisor: Ahmed Ahmed
Additional Supervisors: Ivan Ahel
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.
Project Summary
To protect the genome from damage organisms have evolved a cellular defence mechanisms
termed the DNA damage response (DDR). The DDR includes a diverse set of signal
transduction pathways and effector proteins that act to sense DNA lesions and effectively
repair the damage, limiting the propagation of genomic instability. Exploiting DDR pathways
to specifically target and kill cancer cells has become an attractive therapeutic avenue within
cancer research. This is exemplified by the synthetic lethal interaction between PARP
inhibition and BRCA1 or BRCA2-deficient tumours1. Ivan Ahel (co-supervisor on this project)
laboratory recently identified HPF1 protein as a novel interactor and critical regulator of
PARP1 ADP-ribosylation activity upon DNA damage2. Functionally, HPF1 suppresses DNA
damage-induced hyper auto-modification of PARP1 and promotes in trans ADP-ribosylation
of histones and many other proteins involved in regulation of genome stability. They further
demonstrated that HPF1 is a critical specificity factor that allows modification of target
proteins by PARP1 on serine residues (Ser-ADPr)3,4. Crucially, the work also identified ARH3
as a hydrolase which specifically removes Ser-ADPr5 and further showed that Ser-ADPr is the
major form of ADP-ribosylation following DNA damage6. Taken together, the insights
surrounding Ser-ADPr open a large, exciting, and novel area of research into the fundamental
understanding of the pathways regulated by this modification. Strikingly, our unpublished
data show that ARH3 knockout in model cell lines associates with PARP inhibitor (PARPi)
resistance, while ARH3 overexpression is associated with PARPi sensitivity. Based on these
results, we hypothesize that ARH3 activity and protein levels affect sensitivity to PARPi, thus
representing; i) a predictor for the success of these therapies and, ii) a novel target for further
drug development. Currently, PARP inhibitors are used to treat ovarian cancer and several
other cancers, and we therefore propose to test the hypothesis that ARH3 expression might
be a useful diagnostic tool with which to stratify cancer patients into sub-groups that will be
sensitive/resistant to PARPi treatment with a particular focus on ovarian cancer. The
mechanism of sensitivity/resistance of cells with deregulated ARH3 expression cells to PARPi
is unknown, and elucidating this mechanism will be another goal of this proposed work.

Research objectives and proposed outcomes

Objective 1. Characterise the effect of ARH3 under- and overexpression in a series of model
and primary cancer cell lines on PARP inhibitor sensitivity/resistance. We will collect and test
a variety of ovarian cancer cell lines, profiling them for ARH3 protein expression levels and
then treating with several different PARPi of varying PARP-trapping capabilities (olaparib,
talazoparib, veliparib). To determine the impact of ARH3 protein levels on PARPi vulnerability,
we will not only assess drug sensitivity and levels of PARP1, PARG, and ARH3 across a panel
of ovarian cancer cell lines, but also assess the impact of systemically varying ARH3 by
knockdown, knock out and inducible overexpression in HGSOC lines of defined genotype,
including Ovcar8 (BRCA1/2 wt, PARPi resistant), PE01 (BRCA2-mutant, PARPi sensitive),
Kuramochi (BRCA2-mutant, PARPi partially sensitive) and COV362 (BRCA1-mutant, PARPi

37
sensitive). Rescue experiments with wild type vs. catalytically inactive ARH3 will assess the
suitability of ARH3 as a target for the development of inhibitors.
Objective 2. To determine the frequency of ARH3 gene alterations in a larger set of HGSOC
samples, we will: i) interrogate data of an ongoing whole exome sequencing study of 504
ovarian cancers searching for ARH3 and PARG copy number alterations and mutations; and ii)
perform semi-quantitative detection of ARH3, as well as of PARG, PARP1 and PAR, by
immunohistochemistry (IHC) on two independent sets of tissue microarrays (TMAs)
containing a total of 1200 ovarian cancers. To augment these analyses, which will be limited
by the small number of tumors treated with PARPi, we will also evaluate levels of ARH3, PARG,
PARP1 and PAR in patient-derived xenograft (PDX) models that have been assayed for
response to single-agent PARPi, including ones that have a high HRD score but did not
respond.
Objective 3. Elucidating the mechanistic basis for the sensitivity/resistance of cells with
deregulated ARH3 expression cells to PARPi (modulation of the PARP-trapping, regulation of
DNA repair pathway choice, regulation of the chromatin structure/epigenetic marks). For
these studies we will use largely cell biology/biochemical and genomics approaches. This
objective will be performed in co-supervisor (Dr Ivan Ahel) laboratory at the Sir William Dunn
School of Pathology, University of Oxford.

4. Translational potential of the project

Our data suggest that ARH3 protein expression levels in cancer patients might be a marker
that confers sensitivity/resistance of the tumour to PARPi, providing a rationale for using
PARPi for certain patients. In longer term, understanding the mechanisms of DNA repair and
PARPi resistance through studies of ARH3 protein, may reveal new, unexpected avenues for
treatments in the future.

References
1. Bryant et al (2005) Specific killing of BRCA2-deficient tumours with inhibitors of
poly(ADP-ribose) polymerase. Nature 434, 913-917.
2. Gibbs-Seymour, I., Fontana, P., Rack, J.G., and Ahel, I. (2016) HPF1/C4orf27 Is a PARP-
1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity. Mol Cell 62,
432-442.
3. Bonfiglio, J.J., Fontana, P., Zhang, Q., Colby, T., Gibbs-Seymour, I., Atanassov, I.,
Bartlett, E.J., Zaja, R., Ahel, I.*, and Matic, I.* (2017) Serine ADP-ribosylation depends
on HPF1. Mol Cell 65, 932-940. (*Corresponding authors)
4. Suskiewicz, M.J., Zobel, F., Ogden, T.E., Fontana, P., Ariza, A., Yang, J., Zhu, K., Bracken,
L., Hawthorne, W.J., Ahel, D., Neuhaus, D., and Ahel, I. (2020) HPF1 completes the
PARP active site for DNA-damage induced ADP-ribosylation. Nature 579, 598-602.
5. Fontana, P., Bonfiglio, J.J., Palazzo, .L, Bartlett, E., Matic, I., and Ahel, I. (2017) Serine
ADP-ribosylation reversal by the hydrolase ARH3. Elife Jun 26;6. pii: e28533.
6. Palazzo, L., Leidecker, O., Prokhorova, E., Dauben, H., Matic, I., and Ahel, I. (2018)
Serine is the major residue for ADP-ribosylation upon DNA damage. Elife Feb 26;7. pii:
e34334.

38
 Discovery and mechanistic elucidation of small molecule inducers
of myeloblast differentiation for ALL – Prof. Russell3A
Primary Supervisor: Angela Russell
Additional Supervisors: Thomas Milne
Eligibility: Track 3A students only are eligible to apply for this project.
Project Summary
The most common childhood cancer is acute lymphoblastic leukaemia (ALL), a disease which leads to
the accumulation of immature lymphoid cells in the bone marrow. This is thought to be caused in part
by a block in normal lymphocyte differentiation. There has been amazing progress in treating
childhood ALL, but unfortunately a subset of childhood ALL continues to be refractory to treatment,
especially in those patients that harbour a rearrangement (r) of the Mixed Lineage Leukaemia (MLL)
gene. The most common MLLr results in a fusion protein, MLL-AF4, that is responsible for many of the
poor prognosis ALL patients. In addition, even for children who are cured, conventional therapies are
often toxic and can cause long lasting life-altering effects. Current treatments typically aim to kill
abnormal cells via chemotherapy, but our goal is to establish a new paradigm in the treatment of ALL,
that is to induce differentiation of ALL blasts.

Our inspiration comes in part from the wave of new small molecule therapies for acute myeloid
leukaemia (AML) that have been shown to have reduced toxicity compared to conventional therapy
and function by causing AML cells to differentiate. Our hope is that by applying the concept of
differentiation therapy to ALL we will be able to i) provide novel treatments for refractory ALL such as
MLLr leukaemias and ii) develop novel therapies that have fewer toxic side effects than current
conventional therapies. Previously, we established an in vitro screen to detect differentiation of AML
cells using flow cytometry and used this to identify multiple classes of small molecules which can block
proliferation and overcome the differentiation block in AML blasts. Our leading examples are orally
bioavailable in mice and are being progressed into in vivo trials to determine efficacy. We have
performed some preliminary time-course studies and global RNA-seq analyses to better understand
the compounds’ effects at a cellular level. From these data we have shown that our compounds are
distinct from other known inducers of differentiation in AML cells. However, we have not yet defined
their direct cellular target(s). Preliminary data also suggests that treatment of ALL cells with these
compounds impairs their growth in vitro. Our goal in this project is to apply these same compounds,
alongside other classes of AML drug candidates which also induce differentiation, to ALL blast cells
from a novel MLL-AF4 humanized model (see Figure 1) as well as ALL patient samples to determine i)
which novel compounds can disrupt ALL growth; ii) if ALL blasts can be induced to differentiate; iii)
how the compound(s) impact the function of the target(s), and (iv) what downstream cellular
pathways are impacted by target engagement.

Research objectives and proposed outcomes: In this project we aim to use a combination of chemical
and biological techniques to address questions (ii)-(iv) for one of these series of molecules. Two
parallel approaches will be developed using an integrative approach combining existing cutting-edge
expertise in Milne/Russell groups and collaborators Dr Anindita Roy and Prof Irene Roberts:
1. Analyze differentiation of ALL cells using a novel MLL-AF4 ALL model, with a combination of
tools as well as novel compounds. Counterscreen for toxicity and/or B-lineage differentiation potential
of normal cells (e.g. cord blood) to exclude compounds which exhibit non-specific effects.
2. Identifying compound binding partners in ALL cells through a combination of affinity and
photoaffinity proteomics, candidate screening, native intact mass analysis and follow up target
validation.
3. Identify key pathways controlled/impacted by compound treatment through a combination of
nascent and RNA-seq, CRISPR/CAS9 screening and proteomic analysis.

39
The overall workflow is depicted in Fig. 1.
In the environment of the chemistry research
laboratories (CRL), training will be provided in
chemical synthesis, analytical methods (e.g. NMR,
mass spectrometry), medicinal chemistry, drug
design, photoaffinity labelling, chemical biology,
affinity and photoaffinity protein profiling and
proteomics. These techniques are well established
in the Russell group, and have been successfully
mastered by several DPhil students in recent years
(for example Wilkinson et al, 2020). As well as all of
these techniques, training will also be provided in
how to ask and answer scientific questions in
medicinal chemistry and chemical biology.
Similarly, a wide range of technical training will be
provided in the RDM/WIMM environment
including basic cell biology techniques, analytical
methods (e.g. FACS), drug screening, RNA-seq and
other next generation sequencing techniques,
CRISPR/CAS9 screening, basic bioinformatics and
dataset analysis. Training will also be provided in
answering questions about biological impact and
efficacy of compounds. Fig. 1: Proposed project workflow.

Professor Russell and Professor Milne already collaborate and hold biweekly joint meetings to discuss
and monitor research progress. It would be expected that the student would attend these meetings
as a primary means to monitor progression in the presence of both academic supervisors so as to
ensure coherence and consistency of messaging – a critical factor when students have joint
supervision arrangements. It would not be expected that the student would present at every one of
these meetings – most likely every other one. These meetings will be critical to set overall objectives
and project direction. Together, these distinct environments will provide the unique opportunity for
the candidate to bring together two interdisciplinary areas of research to address and important area
of unmet medical need in oncology.

Translational potential of the project: Accomplishing the goals of this project could potentially impact
four specific areas: 1) pharmacodynamic biomarker discovery, 2) yield insights into the basic biology
underpinning ALL blast cell differentiation, 3) identifying patient subsets to target in clinical trials, and
4) revealing new molecular targets for future drug discovery endeavours. As the intention with this
project is to focus on a mechanistic evaluation of tool compounds this means there would be no
commercial restriction or delay to publish the outcomes of the research. The sharing of new data with
the scientific community in a timely fashion we anticipate will further the overall goal of identifying
and developing drugs that will go into clinical trials to one day impact patient health.

References: Godfrey, L.; Milne, T. A.* DOT1L inhibition reveals a distinct subset of enhancers dependent on
H3K79 methylation. Nat. Commun. 2019; DOI: 10.1038/s41467-019-10844-3.
Wilkinson, I.V.L.; Russell, A.J.* Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon
Receptor as a Molecular Target of the Utrophin Modulator Ezutromid. Angew. Chem. Int. Ed. 2020; DOI:
10.1002/anie.201912392.

40
 Single-cell analysis of haematopoietic stem cells in SF3B1 mutant
MDS: identification of new therapeutic targets/treatments – Dr.
Pellagatti1,2,3A
Primary Supervisor: Andrea Pellagatti
Additional Supervisors: Adam Mead, Supat Thongjuea
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.
Project Summary
The myelodysplastic syndromes (MDS) are common myeloid malignancies.1 There are few
effective treatments for MDS and the vast majority of patients will die as a result of their
disease. The MDS originate in bone marrow haematopoietic stem cells (HSCs) that are
essential for disease initiation and progression, and the eradication of the malignant HSCs is
necessary to achieve cure. Splicing factor mutations are the most common mutations found
in MDS. The splicing factor gene SF3B1 is the most frequently mutated gene in MDS, and we
and others have shown that SF3B1 mutations result in aberrant pre-mRNA splicing.1-3 We
recently showed that SF3B1 mutant MDS is a distinct nosologic entity.4 Interestingly we
demonstrated that increased R-loops and DNA damage occur in SF3B1 mutant MDS patient
bone marrow cells.5 SF3B1 mutations have been identified in other cancers, including chronic
lymphocytic leukemia, uveal melanoma, breast cancer and pancreatic cancer, suggesting that
somatic mutations in spliceosome genes have an important role in tumorigenesis.1 We will
use a single-cell method for high-sensitivity mutation detection with parallel RNA sequencing
analysis (TARGET-seq)6 to determine the genetic and transcriptomic heterogeneity of single
HSCs from individual MDS patients harbouring SF3B1 mutations. Our approach allows the
dection of changes in gene expression levels and also aberrantly spliced transcripts.
Dysregulated genes/pathways will be identified using appropriate bioinformatics analyses7 in
mutation-defined HSC subpopulations in each MDS patient (Figure 1) and used as input to
identify drugs that can target the different HSC subpopulations in an individual patient. The
identified drugs will be tested in vitro using long-term culture-initiating cell (LTC-IC) assays to
determine the drugs that are cytotoxic to MDS HSCs (with the relevant mutation profile) while
sparing the HSCs from healthy controls. Importantly, the drugs identified may also have
efficacy in other myeloid malignancies with similar mutation profiles. This approach will lead
to the identification of new treatments for SF3B1 mutant MDS and has broad applicability to
other haematological malignancies and other cancers.

Figure 1. Sequential mutation acquisition in

MDS. An example of clonal hierarchy in an MDS
patient harbouring SF3B1, TET2 and CBL
mutations is shown. Using TARGET-seq, we will
profile the single-cell transcriptome for
individual MDS patients and identify different
HSC subpopulations on the basis of their
mutation status. In a case with SF3B1 as a
founder mutation, with TET2 as a subsequent
mutation, and CBL as a late mutation, the HSC
subpopulations could comprise cells with SF3B1
mutation only, cells with both SF3B1 and TET2
mutations, cells with all three mutations, as well as wildtype cells (non-clonal).

41
Research objectives and proposed outcomes
The aim of this project is to identify new therapeutic targets/treatments from the
transcriptomic analysis of single HSCs (i.e. the disease-propagating cell population in MDS)
from MDS cases with SF3B1 mutation. Our objectives are:
1- To perform single-cell mutational analysis with parallel transcriptomic (RNA-seq) analysis
on the HSCs of a group of MDS patients harbouring SF3B1 gene mutations
2- To perform bioinformatics analysis on the single-cell data in order to identify molecular
signatures and druggable targets, and drugs known to target them, in each mutation-defined
HSC subpopulation in the MDS patients
3- To test the identified drugs in vitro to determine the drugs which are cytotoxic to MDS HSCs
(with the relevant mutation profile) while sparing the HSCs from healthy controls
We will identify dysregulated genes (both at the expression and splicing levels) and pathways
in the HSCs of SF3B1 mutant MDS patients, with the potential to discover novel pathways
involved in MDS pathogenesis. This will broaden our knowledge of MDS pathophysiology.
We will identify drugs and drug combinations that can specifically target all HSC subsets
within each MDS patient, enabling treatment tailored both to the individual MDS patient
(precision medicine), as well as to whole groups of patients with SF3B1 mutations and
mutation combinations. Toxicology and safety data may be already available for some of the
drugs identified and this will accelerate their introduction into the clinic to treat MDS as a
new disease indication for these drugs. This project will benefit from collaboration with
Professor Amit Verma (New York), an expert in MDS HSC biology and drug target identification
from gene expression data in myeloid malignancy.

Translational potential
We will identify new drugs and drug combinations for MDS patients with SF3B1 mutations.
The drugs identified may also have efficacy in other myeloid malignancies (e.g. acute myeloid
leukaemia) and other cancers with SF3B1 mutations. This approach will lead to the
identification of new treatments for SF3B1 mutant MDS and has broad applicability to other
haematological malignancies and other cancers.

References 1. Pellagatti A, Boultwood J. Splicing factor gene mutations in the myelodysplastic

syndromes: impact on disease phenotype and therapeutic applications. Adv Biol Regul 2017;
63:59-70. 2. Dolatshad H, Pellagatti A, et al, Smith CW, Boultwood J. Cryptic splicing events in
the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic
syndromes. Leukemia 2016; 30:2322-2331. 3. Pellagatti A, Armstrong RN, et al, Smith S,
Boultwood J. Impact of spliceosome mutations on RNA splicing in myelodysplasia:
dysregulated genes/pathways and clinical associations. Blood 2018; 132:1225-1240. 4.
Malcovati L, Stevenson K, et al, Pellagatti A, et al, Cazzola M. SF3B1-mutant MDS as a distinct
disease subtype: a proposal from the International Working Group for the Prognosis of MDS.
Blood 2020; 136:157-170. 5. Singh S, Ahmed D, et al, Pellagatti A, Boultwood J. SF3B1
mutations induce R-loop accumulation and DNA damage in MDS and leukemia cells with
therapeutic implications. Leukemia 2020; 34:2525-2530. 6. Rodriguez-Meira A, Buck G, et al,
Thongjuea S, Mead AJ. Unravelling Intratumoral Heterogeneity through High-Sensitivity
Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell 2019; 73:1292-1305. 7.
Giustacchini A, Thongjuea S, et al, Jacobsen SEW, Mead AJ. Single-cell transcriptomics
uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia. Nat Med
2017; 23:692-702.

42
 Dissecting the biological basis of HLA variation to susceptibility and
disease severity in Crohn’s disease & ulcerative colitis as risk factors
for the development of colorectal cancer – Prof. Satsangi1
Primary Supervisor: Jack Satsangi
Additional Supervisors: Nicola Ternette Simon Leedham, Rebecca Powell Doherty
Eligibility: Track 1 students only are eligible to apply for this project.
Project Summary
IBD is a chronic, inflammatory disorder of the gastrointestinal tract, driven by dysregulated immune responses
to environmental triggers in a genetically susceptible individual. It is well recognised that patients with IBD are
at an increased risk of developing colorectal cancer (CRC), the excess CRC risk is estimated at 2.4 [SIR] in UC
alone [1]. Moreover, the risk of colitis-associated CRC (CA-CRC) is closely linked with disease extent, duration
and severity of inflammation[2, 3]. Understanding the complex genetic-environmental interactions is key to
elucidating the pathogenesis of IBD and its complications. HLA class II polymorphisms have now been implicated
as potentially causal or contributory to disease progression and severity[4]. Specifically, high-impact genome
wide association studies provide unequivocal evidence that the class II variant HLA-DRA/DRB1*01:03 has a
primary role in both CD and UC[5]. Early work also linked HLA-DRA/DRB1*01:03 to extensive UC and increased
risk of colectomy [6]. Given the role of HLA molecules in antigen presentation for subsequent T-cell stimulation,
the strength of this association suggests investigation into potential mechanisms underlying it may provide
critical insight into the identity and source of antigens that may play a role in initiating pathogenesis and disease
severity, and that lay the basis for the clinical onset of CRC.
We hypothesise that HLA-DRA/DRB1*01:03 plays a causal role in colonic IBD via the presentation of specific
peptides to aberrant T-cell populations. Using single allele transfected cell lines and mass spectrometry to
identify HLA-associated peptide ligands, we will characterise the sequence requirements for binding to the
indicated HLA allele and those closely related but unassociated with disease. Having deciphered the antigenic
sequence nuances required for presentation, we will proceed to identify peptide antigen candidates implicated
in disease progression. We will directly interrogate inflamed and non-inflamed patient tissue and identify the
DRA/DRB1*01:03-associated peptide antigens with our immunopeptidomics workflow. We will validate
antigens by testing the response to the candidate sequences utilising T-cells from patient PBMCs and colonic
biopsies in cultured ELISpots, tetramer binding assays, and flow cytometry for T-cell population characterisation.
Using our novel immunopeptidome driven approach and focusing on biopsies from individual patients and
healthy volunteers, we will be able to explore these mechanisms in a patient specific manner, identifying
antigens unique to a given individual, and potentially allowing the development of personalized
immunotherapies. Such potential for immunotherapeutic disease amelioration may serve to also reduce the
occurrence of CA-CRC in this population, which remains a significant cause of morbidity and mortality.

Objectives and Outcomes

Objective 1: Characterisation of the preferred peptide binding motif for HLA-DRA/DRB1*01:03 with comparative
analysis between APCs and other cell types
We will use CRISPR modified THP-1 cell lines and DLD-1 cells to generate single allele transfected lines, including
HLA-DRA/DRB1*01:03 and two closely related controls unassociated with disease (HLA-DRA/DRB1*01:01 and
*01:07). We will use HLA-DR specific immunoprecipitation to collect HLA molecule:peptide complexes and
subsequently elute bound peptides. We will then analyse the resulting peptide arrays via mass spectrometry to
characterise the preferred peptide motif for each allele and to determine if there is any difference in HLA variant
peptide motifs based on the cells in which the HLA molecules are expressed. This comparison has particular
implications for the link between CD/UC and significantly increased risk of colorectal carcinoma.
Outcome: Full Characterisation of Preferred Peptide Binding Motif for CD/UC associated HLA-DRA/DRB1*01:03
in multiple biologically relevant cell types in comparison to closely related alleles.

Objective 2: Identification of patient specific host candidate antigen targets causal to IBD and early CRC
In order to understand the HLA-DRA/DRB1*01:03-associated antigen landscapes directly in affected patient
tissues, we will proceed to analyse biopsy samples from 5 patients expressing the relevant haplotypes and
experiencing active colonic inflammatory disease, and 5 patients with confirmed, early-stage colitis-associated
CRC. We will purify HLA-DR complexes from patient tissue using a DR-specific antibody, and proceed to identify
the presented peptide antigens using our LC-MS workflow. In parallel, we will analyse non-inflamed gut tissue,
and cancer-free gut tissue from the same individuals as matched controls. We will use the results of Objective 1

43
to shortlist peptide antigens likely indicated in disease. We will further refine the list of candidate antigens by
confirming their absence in non-inflamed/cancer-free tissues. Finally, we will interrogate the list of resulting
antigens and the proteins from which these antigens derive for their relationship through specific pathways, and
prioritise those antigens that have commonalities in their relationship networks. The final list of HLA-
DRA/DRB1*01:03-associated antigens that are likely to be directly associated to the progression of the disease
will be further evaluated in T-cell validation studies.
Outcome: Identification of patient specific candidate antigens associated with IBD.

Objective 3: Validation of candidate antigens by elucidating their T cell immunogenicity in patient PBMC
We will generate synthetic peptide candidates on the basis of our findings from Objective 2 and determine the
capacity each candidate peptide has for stimulating downstream T-cell activity. To do this, we will collect PBMC
T-cell isolates from the same patients from which matched biopsy were collected. We will first use these patient
PBMC in cultured ELISPOT assays to determine whether specific recognition can be established by IFN-γ, TNF,
IL-17 and IL-4 secretion, in comparison to suitable controls. For those antigens that can be confirmed to be
immunogenic in the patient PBMC, we will then synthesise tetramers for further evaluation. We will enrich CD4+
and CD154+ T-cells using magnetic beads, and characterise the antigen specific T-cells regarding their expression
of integrin-β7 and CCR9, both of which are markers for gut tissue homing. These assays will then be expanded
to larger patient cohorts, in order to validate whether the identified antigens are recognised in a wider group of
IBD patients.
Outcome: Complete workflow that produces full characterisation of individual patient T-cell populations in the
context of patient specific IBD antigen candidates, and therefore identification of patient specific targets for
tolerogenic immunotherapeutics.

Collaborations: The project is a joint venture between Professors Jack Satsangi, Nicola Ternette, Paul Klenerman
and Simon Leedham. Professors Satsangi (applicant), Klenerman and Leedham are integrated into the
Translational Gastroenterology Unit in the NDM Experimental Medicine department, focussing on
gastrointestinal disease and inflammation, immunology, and colorectal carcinoma, respectively. Assoc.
Professor Ternette is the Head of the Antigen Discovery Group at the Centre for Cellular and Molecular
Physiology, one of the few labs across the world highly specialised in HLA molecular analysis via mass
spectrometry. This unique collaboration is thus extremely well-placed to guide the project from multiple
perspectives.

Academic value: The methods and techniques we propose would be of benefit to the wider scientific
community. The Ternette group is specialised in the analysis of HLA molecules, utilising state of the art mass
spectrometry technologies. Continued development of well validated, characterised, robust workflows will allow
us to contribute to the growing body of HLA class II data required to advance the immunopeptidomics field.
Defining an immunopeptidomic approach to characterise the pathogenic relevance of this variant of interest
may be extended to other HLA class II gene associations in IBD or furthermore, in other immune-mediated
conditions.

Translational Potential: By characterising individual patient T-cell populations in the context of patient specific
IBD antigen candidates, we will create avenues for the development of innovative, personalised, tolerogenic
immunotherapeutic treatments for IBD. Modifying disease severity and progression with ultimately reduce the
risk of IBD-associated complications including malignancy.
References 1. Annese, V., et al., European Evidence-based Consensus: Inflammatory Bowel Disease and Malignancies. J Crohns Colitis, 2015.
9(11): p. 945-65. 2. Eaden, J.A., K.R. Abrams, and J.F. Mayberry, The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut, 2001.
48(4): p. 526-35. 3. Rutter, M., et al., Severity of inflammation is a risk factor for colorectal neoplasia in ulcerative colitis. Gastroenterology,
2004. 126(2): p. 451-9. 4. Ahmad, T., S.E. Marshall, and D. Jewell, Genetics of inflammatory bowel disease: the role of the HLA complex. World
J Gastroenterol, 2006. 12(23): p. 3628-35. 5. Goyette, P., et al., High-density mapping of the MHC identifies a shared role for HLA-DRB1*01:03
in inflammatory bowel diseases and heterozygous advantage in ulcerative colitis. Nat Genet, 2015. 47(2): p. 172-9. 6. Satsangi, J., et al.,
Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotype in inflammatory bowel disease. Lancet,
1996. 347(9010): p. 1212-7.

44
 The Artemis DNA Repair nucleases – from mechanism to
therapeutic inhibition – Prof. Schofield3A
Primary Supervisor: Christopher Schofield
Additional Supervisors: Opher Gileadi, Peter McHugh
Eligibility: Track 3A students only are eligible to apply for this project.

Project Summary
Many cancer treatments rely on inducing DNA damage including interstrand cross-links (ICLs)
and double-strand breaks (DSBs). A conserved family of human DNA repair factors, the
metallo ß-lactamase MBL-fold enzymes SNM1A, SNM1B/Apollo and SNM1C/Artemis,
counteract several crucial forms of therapeutic damage. This project will focus on a DNA
repair nuclease known as Artemis. Artemis is an excellent target for sensitising tumours to
radiation and increasing the efficacy of the very common form of cancer treatment.
Moreover, Artemis also is required for cell survival in tumours that lack the BRCA genes, which
control and important DNA repair pathway, meaning that Artemis Inhibitors could act as an
effective stand-alone treatment, particularly in some breast and ovarian cancers.

Justification for support, Research Objectives and Outcomes.

The Schofield, Gileadi, and McHugh labs are already collaborating to determine the
mechanistic basis for DNA-PKcs stimulation of the DNA repair endonuclease Artemis. Artemis
is required for VDJ recombination during immune system development, but also plays a key
role in processing the ‘dirty’ ends (containing damaged bases) present at the termini of DNA
double-strand breaks generated by ionising radiation. Therefore, inhibition of Artemis is a
compelling strategy to sensitise human tumour cells to the site-directed DNA damage
introduced during radiotherapy. Moreover, Artemis is required for cell survival in tumours
lacking the BRCA genes, which control and important DNA repair pathway, meaning that
Artemis Inhibitors could act as an effective stand-alone treatment, particularly in some breast
and ovarian cancers.

The work will be underpinned by our extensive preliminary development work on assays and
structural studies of metallo- -lactamase (MBL) fold enzymes and (metallo)-enzyme
inhibition (see references). Inhibiting this type of target (intracellular metallo DNA repair
enzymes) is unprecedented, but we are confident in our chemical starting points. We expect
that this project will involve substantial basic research effort to develop assays and define
mechanisms of Artemis, leading to the generation of small molecules with novel modes of
inhibition. The work will address basic questions surrounding the mechanisms of genome
maintenance and their relevance to cancer cell survival.

In the first phase of work, the student would become familiar with a collection of expertise
required to produce and characterise nucleases. These involve protein expression and
purification, mass-spectrometry-based DNA analysis assays, structural biology and medicinal
chemistry platforms and biophysical analysis platforms (X-ray crystallography, MS, ITC, SPR,
fluorescence polarisation) that are employed across our groups to characterise DNA
modifying enzymes and their ligand interactions. We envisage the project will be tailored to
the specific interests and expertise of the student; we outline one project plan below.

45
Using the purified Artemis protein, the student will devise and validate a fluorescence-based
screening assay. The assay takes advantage of the experience of our groups in screening for
nuclease inhibitors on fluorescence-based platforms. The student would initially use this
assay to probe an important, but very poorly understood feature of Artemis biochemistry.
Specifically, Artemis contains an auto-inhibitory domain in its C-terminal. This inhibition of
enzymatic activity is relieved by phosphorylation targeting this C-terminal domain. Using a
combination of mutant forms of the protein generated by the student and reconstituted
protein phosphorylation reactions, we will probe the consequences of Artemis
phosphorylation on its activity and aim to provide a detailed explanation for the molecular
basis of this. This will require the collection of techniques listed above to determine the effect
of phosphorylation on the interaction of Artemis with its DNA substrate, how phosphorylation
affects key catalytic steps in DNA hydrolysis and also whether phosphorylation induces
changes in Artemis structural conformation. Together, this would arm the student with broad
expertise in nucleic acid chemistry, enzymology and an introduction to the exciting potential
of chemical biology in cancer research.

Armed with a strong knowledge of Artemis mechanism and a robust screening assay the
student would then search for Artemis inhibitors. We will explore this by undertaking high
throughput screens as well as using a panel of several hundred candidate nuclease inhibitors
and their relatives from the same chemotypic families. These studies are likely to generate
new leads (Kd <100 nM with a 5-fold selectivity for Artemis/SNM1C over related nucleases
that can de be further developed in through subsequent medicinal chemistry efforts. The
inhibitors will be characterized by the candidate using a broad range of functional biochemical
and cell biology assays. For example, we will employ DSB repair assays using Artemis
disrupted cells and those harbouring engineered catalytically inactivating mutations to
perform functional reporter-based V(D)J recombination assays to validate the cellular activity
of candidate Artemis inhibitors, especially in the context of BRCA loss.

Translational Impact
Cancer cells experience greatly increased stress and damage to their genomes, relative to
non-malignant cells. Consequently, tumours are reliant on DNA repair pathways for their
propagation. Most tumours, therefore, are sensitive to inhibition of one or more of the
pathways that maintain genome stability. We propose to develop small molecule inhibitors
to the Artemis DNA repair factor. This will provide new options for cancer treatment since
cells that are BRCA defective will be sensitive to these inhibitors. Moreover, Artemis inhibition
is an excellent candidate for radiosensitising tumours. Our approach will provide new
avenues for treating and sensitising tumours to radiation, but is also likely to reveal previously
unanticipated, novel therapeutic anti-cancer strategies.

References: 1. Allerston CK et al. The Structures of the SNM1A and SNM1B/Apollo nuclease domains reveal
a potential basis for their distinct DNA processing activities. Nucl. Acids Res., 2015, 15;43(22):11047-60 2.
Wang AT et al. Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.
Genes and Dev. 2011; 25 (17):1859-70. 3. Sengerová B et al. Characterization of the human SNM1A and
SNM1B/Apollo DNA repair exonucleases. J Biol Chem. 2012; 287:26254-67

46
 Investigating IGFs as immunosuppressive cytokines and cancer risk
factors– Prof. Macaulay1,2,3A
Primary Supervisor: Valentine Macaulay
Additional Supervisors: Len Seymour, Clare Verrill, Sarah Blagden
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
This project for a Clinical Research Fellow (CRF) will provide research training that complements our
investigation of insulin-like growth factors (IGFs) as cancer risk factors. The project plan is informed by 3 recent
findings. First, serum IGF-1 associates with altered expression of IGF receptor (IGF-1R) in malignant prostatic
epithelium. Second, IGFs have unanticipated ability to regulate the function of ribonucleotide reductase (RNR)
and dNTP supply, effects that are potentially mutagenic. Thirdly, we find that IGFs deregulate proteins and
pathways that influence anti-tumour immune responses in prostate cancer cells, complementing reports that
IGFs enhance Treg function and suppress autoimmunity in preclinical models. These data lead us to speculate
that increased IGF supply drives a pro-mutagenic tumour profile while suppressing the ability to mount an anti-
tumour immune response. The CRF will investigate the latter aspect of this hypothesis, using in vitro models and
clinical tissues including samples from subjects recruited to an academic trial of IGF blockade. Techniques may
include in vitro co-culture, patient-derived explants, IHC and transcriptional profiling, to investigate the
relationship between IGF supply and immune cell function. Training in research methods will be provided by
Macaulay, Seymour, Verrill and Buffa and Early Phase Trials practice by Blagden. This is an exciting opportunity
for training in basic, translational and clinical research. The results will shed light on the contribution of the
tumour immune microenvironment to cancer risk and progression, with the goal of developing novel approaches
to risk reduction.

Research objectives and proposed outcomes

Background: Subjects with low serum IGF-1 are strongly protected from cancer (1, 2), while those with high IGF-
1 are at increased risk of breast, prostate and colorectal (CRC) cancer (3-5). IGFs are implicated in the association
of height with incidence of many solid and haematological cancers, and with aggressive, lethal prostate cancer
(6, 7). There is compelling preclinical and clinical evidence that IGF-1 is not just associated with risk, but is
causative (2, 8, 9). Our long-term goal is to identify mediators of high IGF-1 in order to develop novel approaches
to risk reduction. This aim is supported by Cancer Research UK Early Detection project grant, PCUK Research
Innovation Award and Human Immune Discovery Initiative (HIDI) grants to the Macaulay group.
Preliminary data: Of relevance to the current project, we recently identified 3 novel effects of varying IGF supply
in prostate, breast and CRC models and clinical cases (Fig 1). First, we identified an association between serum
IGF-1 and IGF-1R content of malignant epithelium in men with early prostate cancer (Fig 1A-B). This is the first
clinical tissue-level change linked to serum IGF-1. IGF-induced IGF-1R upregulation is also reported in infiltrating
immune cells in murine CRC (10), representing an initial step to understand how IGF-1 affects cancer risk.
Secondly, we find that IGF-1R depletion or IGF neutralizing antibody xentuzumab cause in vitro and in vivo
downregulation of the RRM2 subunit of ribonucleotide reductase (RNR), the rate-limiting step for dNTP
production. This reduces dNTP supply and induces hallmarks of replication stress including delayed replication
fork progression (Fig 1B-D) (11). Conversely, increased IGF supply upregulates RRM2 leading to dNTP pool
imbalance (Fig 1E-F). Comparable dNTP imbalance is reported to impair proof-reading by the replicative
polymerases, resulting in increased mutation load in other models (12).
Finally, we find that IGF-1 upregulates expression of immunosuppressive cytokines and cell surface checkpoints
(Fig 1G-H). We are currently investigating whether these latter changes are direct consequences of cell signaling
or of cytoplasmic DNA release secondary to perturbation of DNA replication. IGFs are also reported to suppress
Class I expression, enhance function of T regulatory (Treg) cells and secretion of immunosuppressive cytokines,
with clinical evidence of low immune cell infiltration in high IGF-1R prostate cancer bone metastases (13-17).
Furthermore, recent data implicate IGFs as mediators of resistance to anti-PD-1 antibody therapy (18). These
data underpin our hypothesis that IGF-1 drives a pro-mutagenic tumour profile while suppressing the ability to
mount an anti-tumour immune response.
Research objective: to investigate the hypothesis that immune sequelae of high IGF-1 supply contribute to the
risk of cancer development and progression.

47
Figure 1. IGF axis regulates regulates dNTP supply, DNA replication and expression of immune markers A. IGF-1R IHC (a, b) in upper: benign, lower: cancer areas of
prostatectomy; IGF1R in situ hybridization (RNAScope; c, d) in cancers with low (c) and high (d) IGF-1R IHC signal, confirming altered regulation at mRNA level. Table: serum
IGF-1 associates with IGF-1R in malignant but not benign prostate. B, C. IGF-1R depletion downregulates RRM2 in MCF7 breast cancer cells (B, also prostate cancer, CRC,
sarcoma cells, not shown) and delays replication fork progression (DNA fiber assay, C). D. RRM2 downregulation in SKCO-1 CRC xenografts by 3 weeks’ xentuzumab
treatment. E, F. IGF-1 upregulates RRM2 and dNTP content. G, H. IGF-treatment of prostate cancer cells upregulates: G, PD-L1 at level of mRNA (left) and cell surface (flow
cytometry, right); H, mRNA expression of immunosuppressive cytokines.

Experimental plan: The CRF will be supervised day-to-day by PCUK-funded Research Assistant and CRUK-funded
Post-doctoral scientist and Bioinformatician, the latter co-supervised with Prof Buffa.
1) Testing effects of manipulating IGF supply on phenotype and function of immune cells. Approaches will
include immunoprofiling by flow cytometry and xCELLigence killing assays, quantifying killing as loss of
adherence of breast, prostate or CRC cells when killed by non-adherent effector CD8+ T cells as (19). Initial assays
will use cancer cell lines and fresh human T cells or cell line TALL-104 co-cultured ± IGF-1 in vitro. These assays
will be extended to fresh clinical tumour explants and immune cells sorted from blood and murine Myc-CaP
prostate cancers established in wild-type vs high IGF-1 mice (20) being used in our CRUK project. The CRF will
also benefit from methods in use in our HIDI project, in which we are collaborating with Prof Seymour to
characterise tumour-infiltrating immune cells in prostate cancer.
2) Contribution to an academic clinical trial of IGF blockade. This trial, planned to open in early 2021, will test
4 weeks’ treatment with IGF neutralizing antibody xentuzumab pre-prostatectomy (PI Macaulay, supported by
PCUK and Boehringer Ingelheim). Data in Fig 1D and (10) suggest 3-4 weeks treatment is sufficient to influence
RRM2 expression and the profile of tumour-infiltrating immune cells. The CRF will use methods developed by
Verrill including IHC (21) and 3’-RNAseq on macro-dissected tumour from formalin-fixed paraffin-embedded
(FFPE) prostate biopsies. Applying this method to trial subjects’ biopsies and prostatectomies will enable the
CRF to assess transcriptional responses to IGF blockade, inferring effects on immune populations as in (22). In
preparation for analysis of trial tissues, the CRF will use this method on paired FFPE tissues of 10 non-trial
subjects, allowing assessment of transcriptional profiles in the absence of intervention. The results will inform
interpretation of trial samples, which will be batch analysed on completion of trial recruitment, predicted Q4
2022.
Academic value of the research IGF effects on malignant epithelium are relatively well-characterised. In contrast
very little is known about pro-tumorigenic effects of IGFs in the TME. This project has potential to identify
proteins and pathways that are novel mediators of high IGF-1 on cancer risk and progression.

Translational potential. The overarching aim of this research is to identify and block the key mediators of high
circulating IGF-1, which may be novel stromal biomarkers of risk, and targets for risk reduction.

References: 1.Guevara-Aguirre et al. Sci Transl Med, 2011. 3: 70ra13. 2.Laron. Endocr Pract, 2015. 21: 1395-402. 3.Group et al. Lancet Oncol,
2010. 11: 530-42. 4.Travis et al. Cancer Res, 2016. 76: 2288-300. 5.Ma et al. J Natl Cancer Inst, 1999. 91: 620-5. 6.Nunney. Proc Biol Sci, 2018.
285. 7.Perez-Cornago et al. BMC Med, 2017. 15: 115. 8.Wu et al. Cancer Res, 2003. 63: 4384-8. 9.Murphy et al. Ann Oncol, 2020. 31: 641-
49. 10.Rayes et al. Oncotarget, 2018. 9: 15691-704. 11.Rieunier et al. https://biorxiv.org/cgi/content/short/2020.08.11.245886v1, 2020.
12.Kumar et al. Nucleic Acids Res, 2011. 39: 1360-71. 13.Trojan et al. Science, 1993. 259: 94-7. 14.Pan et al. PLoS One, 2013. 8: e58428.
15.Kooijman and Coppens. J Leukoc Biol, 2004. 76: 862-7. 16.Bilbao et al. EMBO Mol Med, 2014. 6: 1423-35. 17.Nordstrand et al. Clin Exp
Metastasis, 2017. 34: 261-71. 18.Ajona et al. Nature Cancer, 2020. 1: 75-85. 19.Cerignoli et al. PLoS One, 2018. 13: e0193498. 20.Cannata
et al. Endocrinology, 2010. 151: 5751-61. 21.Rao et al. J Mol Diagn, 2020. 22: 652-69. 22.Steen et al. Methods Mol Biol, 2020. 2117: 135-57.

48
 Genetic and functional characterisation of novel immune
escape mutations in mismatch repair deficient endometrial
cancer – Dr. Church1,2,3A
Primary Supervisor: David Church
Additional Supervisors: Nicola Ternette, Marco de Bruyn, Tjalling Bosse, Viktor Koelzer
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Endometrial cancer (EC) is the most common gynaecological malignancy in developed nations and affects
100,000 women each year in Europe alone. ~25% of ECs carry substantially increased tumour mutation burden
(TMB) as a consequence of DNA mismatch repair deficiency (MMRd); a defect which leads to hypermutation
and instability at DNA microsatellites (MSI). Interestingly, in contrast to early-stage colorectal cancer (CRC)
where MMRd confers a favourable prognosis (presumed due to a T cell response against mutated (non-self)
peptides), in EC MMRd is associated with poor outcome. The reason for this discordance has remained
unexplained; however, we have recently shown that MMRd elicits a weaker T cell response in EC than it does in
CRC. Furthermore, our unpublished analysis of whole genome sequencing (WGS) from >700 ECs in the Genomics
England (GEL) 100KGP has identified novel and common loss of function driver mutations in genes required for
MHC class I presentation in MMRd cases, providing a possible explanation for this observation. Given that
immune checkpoint blockade (ICB) is established therapy in metastatic MMRd EC (and being trialled in early-
stage disease), and given that defects in antigen presentation predict resistance to ICB, there is a compelling
rationale to define the mechanistic and clinical consequences of these, and other candidate predictors of the
immune response in EC. This proposal seeks to do this by combining genetic and immunological analysis of
human ECs, with functional analysis of antigen presentation and the immunopeptidome in cell and animal
models. The student will gain training and expertise in state-of-the-art experimental methods and bioinformatic
analysis, and benefit from a highly collaborative project environment with scope for a period of research
abroad.
The project sits at the interface of basic and clinical research under the themes of big data, genes and immunity.
The work is novel (the candidate immune escape variants we have identified are previously unreported). Our
clinical cohorts are world-class (largest collection of ECs with WGS worldwide, practice-changing EC clinical trial
cohorts), and the experimental methods we will use cutting edge (immunopeptidomics, multispectral co-
immunofluorescence etc). The supervisors and collaborators bring expertise in immunopeptidomics,
immunology, pathology, translational science and clinical translation. The proposal has potential for near-term
clinical impact through the TransPORTEC group (leading EC trial collaborative). This project represents an
outstanding opportunity for a basic scientist, medical student or clinician to gain expertise in experimental and
analytical methods and clinically-applied science.

WP1. Novel immune escape mutations as determinants of immune response and clinical outcome in MMRd
EC
Preliminary data: MMRd predicts favourable prognosis in CRC but not EC1,2; unpublished analysis of these
tumour types has revealed attenuated T cell response in ECs (Fig. 1A). Correlation with JAK1 mutation status
(known immune escape variant) has shown this
may be partly explained by loss of IFN-γ signalling
(Fig. 1B). Unpublished analysis of 710 ECs from
Genomics England EC domain (lead Church) by
IntOgen has identified two novel driver genes
mutations in which , like JAK1, are strongly enriched
in the MMRd subgroup (190 tumours). Both
function in MHC class I presentation and are thus
plausible immune escape variants in this
hypermutated, immunogenic tumour subgroup.
Proposed work: The relationship between
mutation of novel immune escape genes and other
genomic factors (e.g. TMB, neoantigen burden,
clonality etc) and transcriptome will be defined in
the Genomics England (lead Church) and TCGA

49
(access approved) cohorts. The type, density, and localisation of Intratumoral immune infiltrate will be
determined by multispectral co-IF (GE Cell Dive) on FFPE tumour slides in a minimum of 100 MMRd ECs from
the Genomics England cohort. Digital pathological analysis of images will be performed in collaboration with
Viktor Koelzer (Univ Zurich) in an extension of an existing collaboration3. Correlation of immune infiltrate with
novel immune escape mutations and other candidate genomic predictors (eg, TMB, neoantigen burden, JAK1
mutation etc) will be performed by the student (after training) using unsupervised (e.g. random forests) and
supervised methods with penalization given high-dimensionality of data. The student may also have the
opportunity to travel to Leiden, Groningen or Zurich to contribute to this work or the corresponding analysis of
the PORTEC3 trial (450 cases with tumour material). Correlations with clinicopathological variables and clinical
outcome (eg Cox PH models) will be performed by the student with all required training provided. Outputs:
Genomic, immunological and clinical correlates of novel immune escape mutations in MMRd EC.

WP2. Impact of novel immune escape mutations on the immunopeptidome in cell lines and human cancers
Preliminary data: The Ternette group have established reliable experimental workflows for the purification of
MHC class I and II molecules from cells and the elution and characterisation of the immunopeptidome by mass
spectrometry4. In unpublished work, they have extended this to characterise the immunopeptidome in renal cell
carcinoma. Exome sequencing of 25 EC cell lines in the Church laboratory reveals several carry the novel immune
escape mutations we have identified. Proposed work: To define the impact of novel immune escape mutations
on MHC class I presentation and the immunopeptidome we will perform both: (i) re-introduction of candidate
genes by stable re-expression (e.g. transduction) in EC cell lines with LOF mutations; (ii) CRISPR-Cas9 knockout
of each gene in cells with normal expression. MHC class I pathway components will be interrogated by in-situ
methods including live cell imaging where informative. Definition of the impact of genetic re-introduction/loss
will be performed by the student under the supervision of a postdoc from the Ternette lab. If successful,
experiments will be extended to human cancers (~100 frozen ECs available at present; opportunity for
prospective collection). Outputs: Demonstration of the impact of novel immune escape mutations on the MHC
class I pathway and antigen presentation.

WP3. Impact of novel immune escape mutations on intratumoral immune infiltrate, immunopeptidome and
ICB sensitivity in MMRd EC models
Preliminary data: Previous work within the Church group has established a relevant preclinical model of EC by
deletion of Pten in the murine endometrium. In related work, we have shown that inducing MMRd by Mlh1 loss
strongly potentiates tumorigenesis in the context of Apc loss in the mouse intestine (immunophenotyping in
progress). We are currently breeding mice to define the impact of MMRd on EC development and immune
infiltrate in the context of Pten loss, and the potential modification of this by concomitant Jak1 loss (all alleles
already in house). Proposed analyses: We will either import or generate (using the WHG transgenics facility –
lead Ben Davies), mice with conditional (‘floxed’) alleles of novel immune escape genes. These will be crossed
with Ltf-Cre (endometrial-specific recombinase), and conditional Pten and Mlh1 mice to generate animals with
endometrial Pten and Mlh1 loss with or without concomitant loss of novel immune escape genes. In addition
to standard tumour analyses, we will perform detailed immunophenotyping by flow cytometry and multispectral
co-IF, and define the immunopeptidome using similar methods to those in WP2. The impact of loss of these
novel genes on response of MMRd EC to ICB will be examined by therapeutic administration of anti-PD1, with
either sacrifice at defined timepoints, or longitudinal imaging. The student will be assisted in mouse husbandry
and genotyping by a dedicated Research Assistant in the Church lab, and in experimental analyses by
postdoctoral support from the Church and Ternette labs. Outputs: Generation of clinically-relevant models of
MMRd EC and functional validation of the impact of novel immune escape mutations on MMRd EC growth,
immune response and ICB response. Academic value and collaborations: The novel mouse models and the data
they generate will be of substantial value to the academic community, and will help underpin patient
stratification for ICB in MMRd EC. Comparison of the murine and human data will extend the existing
collaboration between the Church group and the Ternette group as well as with the Bosse/Nijman groups.

References: 1. Domingo, E. et al. Lancet. Gastro & Hepatol 1, 207-216 (2016). 2. Stelloo, E. et al. Clin Cancer Res 22, 4215-4224, (2016). 3.
Horeweg N et al. Can Immunol Res (accepted manuscript);4. Paes, W. et al. Proc Natl Acad Sci U S A 116, 24748-24759 (2019)

50
 Mass spectrometric probes for intraoperative brain cancer
diagnosis – Prof Vallance1,2,3A,3B
Primary Supervisor: Claire Vallance
Additional Supervisors: Olaf Ansorge
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
‘Oncometabolomics’ links metabolic cancer signatures to tumour genetic subtype, e.g. glioma
subtypes may be associated with raised levels of 2-hydroxyglutarate, cystathionine, fumarate, or
succinate. Together with detection of specific peptide signatures, highly precise intraoperative real-
time diagnostics becomes a reality, and has the potential to replace subjective intraoperative
histology. This project aims to develop the technology for this approach and validate it across primary
and metastatic brain cancers, including matched primaries such as renal cell carcinoma. This project
is multidisciplinary and will provide training in practical and theoretical aspects of both physical
chemistry and neuropathology. Two types of mass spectrometry will be employed, one chosen for
ease of use and speed of measurement, and one chosen for the high level of detail it can provide on
the spatially-resolved chemical composition of the sample. Data from both types of measurement will
be analysed with the aid of a variety of machine learning techniques in order to classify samples
according to tumour type and to identify important biomarkers associated with tumour type. We aim
to develop a robust approach to tumour classification for use by neurosurgeons and
neuropathologists, as well as to investigate biomarkers associated with different tumour regions. Our
approach can in principle be applied to any cancer, and is therefore of high translational value. To our
knowledge, no other UK research group examines complementary rapid-diagnostics and spatially
resolved tools for this purpose.
Background
The diagnosis of a malignant primary or metastatic brain tumour is devastating for patients and
relatives, with median survival of around 14 months even with full medical and surgical treatment.
Brain tumours have been identified by Cancer Research UK as a cancer of unmet need, and there are
a number of ways in which treatments can potentially be improved. Genome-wide sequencing efforts
have revealed that many cancers are associated with mutations affecting metabolic pathways that
determine specific nutrient dependency of the cancer cells, thus providing an “Achilles heel” for novel
targeted treatments1,2. We postulate that these genomic insights are translatable to rapid and low-
cost measurements that can be used as a surrogate for genetic sequencing. Mass spectrometric
approaches have the potential to fill this gap.
From a surgical point of view, one of the major challenges lies in the identification of tumour
margins during resection. The surgeon aims to achieve maximal tumour resection, which is associated
with prolonged survival3, with minimum patient morbidity and no permanent post-operative
neurological deficit. However, tumour cells tend to penetrate extensively into the normal brain tissue
surrounding the main tumour bulk, and even with fluorescence-guided surgery4,5 it can be difficult to
achieve optimal resection. Furthermore, fluorescence-guided surgery cannot currently be used for
low-grade tumours, for which optimal resections can in some cases effectively be curative. Mass
spectrometry has the potential to provide a sensitive, label-free, almost real-time measurement of
tumour (and ‘normal brain’) biomarkers near the tumour margins, offering a powerful and perhaps
more sensitive alternative to fluorescence-guided approaches.
We will investigate two types of mass spectrometry for the above purposes. The first uses an
atmospheric solids analysis probe (ASAP) coupled to a quadrupole mass spectrometer for rapid single-
point measurements on tissue samples, with no need for any special sample preparation. The second
employs imaging mass spectrometry to map the chemical composition across the surface of the
sample. The two approaches represent opposite ends of the spectrum from the point of view of cost,
ease of use, and speed of data acquisition. The former offers ease of use and rapid analysis at

51
relatively low cost, while the latter provides extremely detailed information with high spatial and mass
resolution across a large sample area, though requires extremely expensive instrumentation (to which
we have access), long acquisition times, and generates very large data sets which need significant
processing and analysis to make them tractable. The data from both approaches will be analysed
using supervised and unsupervised machine learning techniques in order to determine the extent to
which classification of the mass spectra (and therefore the tissue samples) according to tumour type
and tumour region (e.g. tumour core, tumour margin, metastasis vs. primary, or normal tissue) can be
automated. This process will also provide information on which mass peaks are important for the
purposes of classification. We will attempt to assign as many of these individual mass peaks as
possible in order to identify biomarkers of interest. For the ASAP measurements we expect to detect
primarily lipids and metabolites, while the wider accessible mass range in the imaging mass
spectrometry measurements will also allow us to probe peptides and proteins.

Research objectives and proposed outcomes

The key objectives are:
1. Employ ASAP-quadrupole mass spectrometry to record mass spectra of primary and
metastatic brain tumours, including premetastatic samples.
2. Employ matrix-assisted laser-desorption ionisation time-of-flight (MALDI-ToF) imaging mass
spectrometry to record spatially resolved mass spectra for a suitably chosen subset of the
samples.
3. Investigate a variety of unsupervised and supervised classification and machine learning
algorithms (e.g. k-means/hierarchical clustering, linear discriminant analysis, support vector
machines, k nearest neighbours, and others) for automated classification of the mass spectra
according to tumour type and location within the tumour.
4. Identify mass peaks that are important for distinguishing between the various classifications,
and assign the peaks to the relevant chemical constituents of the sample.
5. Correlate the results with data from whole genome sequencing of tumours.
Outcomes:
1. A robust method that combines mass spectrometry with machine learning to classify tumours
according to their genetic subtype.
2. An understanding of the important biomarkers involved in this classification.
3. Technology that could be deployed for rapid intraoperative diagnostics.

Translational potential of the project

The project will develop a mass spectrometric approach to the genetic and metabolomic classification
of primary and metastatic brain cancers, providing a novel, rapid diagnostic tool that will provide the
blueprint for the development of intraoperative probes and deliver unique insights into fundamental
human cancer biology concerning the spatial relationship of genotype and metabolic and peptidomic
phenotype. Our technology will eventually be applicable to all cancer types.

References: 1.C. Yong, G. D. Stewart, and C. Frezza, Oncometabolites in renal cancer, Nat. Rev. Nephrol. (2019). 2.J. Bi, S.
Chowdhry, S. Wu, W. Zhang, K. Masui, and P. S. Mischel. Altered cellular metabolism in gliomas - an emerging landscape of
actionable co-dependency targets, Nat. Rev. Cancer, 20(1), 57-70 (2020). 3.N. Sanai, M. Y. Polley, M. W. McDermott, A. T.
Parsa, and M. S. Berger, An extent of resection threshold for newly diagnosed glioblastomas. J Neurosurg 115, 3-8
(2011). 4.W. Stummer et al., Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a
randomised controlled multicentre phase III trial, Lancet Oncol., 7, 392-401 (2006). 5.W. Stummer et al., Extent of resection
and survival in glioblastoma multiforme: identification of and adjustment for bias, Neurosurgery, 62, 564-576 (2008).

52
 Developing an on chip screen for PDAC in high risk groups – Prof
Davis1,2,3A
Primary Supervisor: Jason Davis
Additional Supervisors: Prof Michael A Silva
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
There is a long held view that there are likely to be detectable changes in the composition of bodily fluids,
perhaps several years before the symptomatic onset of disease. This brings with it the possibility of detection,
definitive diagnosis and intervention much earlier than is often currently possible. Many of the important
markers of disease onset are circulating proteins (the “liquid biopsy”).Their reliable and selective detection in
bodily fluids remains, however, challenging, particularly when one seeks to detect multiple molecules
simultaneously (a requirement if diagnosis is to be definitive). Currently available methods of protein molecule
detection are typically expensive laborious, multi-step, slow, report on only one marker at a time, and are subject
to significant user error, nonspecific interference (potentially false positives) and lab-to-lab variation (meaning
conflicting clinical results). Pancreatic cancer remains one of the most distressing diseases largely due to late
diagnosis and difficulties in effectively treating disease that has spread to distant organs. Its diagnosis occurs,
more often than not, when the disease is at a very late stage. In preliminary work this team has identified a
panel of serum marker proteins which, in combination, present an exciting opportunity to detect PDAC early in
patients at risk (individuals over 50 years of age with new-onset diabetes) and thus enable intervention at a
stage where it can truly enable survival. This project specifically seeks to integrate this marker panel into a new
reagentless electrochemical detection platform. This is based on following the response of antibody and
antibody film capacitors to specific target binding and is designed to support the extremely sensitive, cheap,
high dynamic range and multiplexed detection of protein markers in a manner which, ultimately, will require
minimal user intervention (a test much akin to the glucose skin prick tests). This proposal, then, tightly integrates
state of the art molecular detection protocols, molecular films, polymer interfaces, biophysics etc with the “coal
face” of clinical need. It seeks to build on substantial preliminary work in developing a disruptive capability in
our ability screen for PDAC.

Research Objectives and Proposed Outcomes:

The Need - Pancreatic Cancer; The incidence of PDAC is on the rise. In 2013, there were 9,408 new cases in the
UK: 4,716 (50%) in males, an incidence rate of 15 per 100,000. For all stages of PDAC combined, the one-year
survival rate is 20%, with only 7% of patients surviving five years. Earlier detection greatly improves outcome;
the five-year survival for those patients who undergo potentially curative surgery and chemotherapy is now
close to 30%.1 Currently the diagnosis of PDAC is mainly established by EUS, CT or MRI scans on presentation
with symptoms. Biomarkers capable of identifying PDAC prior to onset of overt symptoms and/or increasing the
proportion of patients eligible for surgery could significantly enhance overall survival. An ability to conveniently
and effectively screen groups at high risk and enable stratification for risk is badly needed.
The Vision: High Risk groups; Approximately 10% of PDAC cases occur in individuals with a family history. An
estimated 40%-80% of PDAC patients have new-onset diabetes mellitus (DM) or glucose intolerance at the time
of diagnosis of cancer,2 3 making new-onset DM the highest risk group for sporadic PDAC.3 Research suggests
that these individuals had early-stage PDAC at the time they were diagnosed with DM. In effect, diabetes was
an early warning sign of the presence of cancer. The average time between the diagnosis of DM and the
subsequent diagnosis of PDAC is 13 months.4 This provides a significant window for earlier detection of PDAC.5
The incidence of DM in the general population is rising and current clinical modalities for the detection of PDAC
(EUS, MRI, CT) are insufficiently accurate (incurring too many false positives) for PDAC screening. To this end,
the applicants have developed a panel of biomarkers (below) to facilitate the detection of PDAC in individuals
with new-onset DM. This will be integrated into a developed highly-sensitive, low background, reagentless, on-
chip electroanalytical assay in producing a convenient platform that screens for up to 4 markers simultaneously.
Of assaying methods which are scaleable across a general population, non-lab based and enable the highly
sensitive quantification of multiple markers with little user input, electrochemical methods stand out. One of
the most sensitive and powerful means of doing this is by electrochemical impedance (EIS).6 We have
successfully shown that translation of this approach to clinical samples is viable.7-11 Here we propose to develop
new derived reagentless detection tools, receptive and responsive molecular films and an on-chip detection to
facilite early, pre-symptomatic, PDAC diagnosis. During the past five years, the PI’s team have been engaged

53
with collaborating clinical teams in establishing electroanalytical platforms capable of the sensitive detection of
antibodies in Parkinson’s patient cerebrospinal fluid (CSF) and plasma and protein markers in serum analysis.7, 9,
11
We have demonstrated the dual electronic detection of markers in serum,7 the viability of a triple marker
detection chip and sample-normalised exosomal marker detection.12 We have also demonstrated a reliable
electroanalytical assay of CA19-9 that reports within minutes. Prior work has identified a number of serum
proteins differentially regulated in PDAC.13, 14 15-17 including adiponectin, thrombospondin-1 (TSP-1)18 and
interleukin-1 receptor agonist (IL-1Ra). CA19-9 is the only biomarker used in the routine management of PDAC
patients. It is known that combining CA19-9 with TSP-1 (AUC 0.86) significantly enhances the performance of
CA19-9 (AUC 0.77) in distinguishing PDAC cases from controls up to 2 years prior to diagnosis.18 Moreover,
combining adiponectin, IL-1Ra and CA19-9 (AUC 0.96) significantly enhanced the performance of CA19-9 (AUC
0.87) in distinguishing individuals with PDAC from those with long-standing T2DM (p=0.037). Few studies have
explored changes occuring in serum in the months prior to PDAC diagnosis, making our data particularly
powerful. Developing these markers for the detection of PDAC in patients newly diagnosed with DM could
enable a step change in our ability to detect PDAC at a time when intervention would significantly improve
outcome for patients.

Specific aims:
1. To develop new on-chip antibody-supporting high-surface area polymer films supporting highly
selective marker recruitment.
2. To develop and utilise new label free molecular detection protocols based on antibody capacitor films.
3. To increase detection selectivity using slow off rate selectivity protocols.
4. To increase marker detection dynamic range using arrays.
5. To further validate the specificity of CA19-9, Adiponectin, IL1-Ra and TSP-1 in samples from new-onset
DM.
6. To establish an ultrasensitive biomarker chip to simultaneously assay CA19-9, Adiponectin, IL1-Ra and
TSP-1 in human serum.
7. To correlate EIS-derived sensitivities/specificities with those obtained through conventional methods.

The project truly spans cutting edge clinical and physical-analytical science. The investigators have strong track
records in the effective execution of multi-disciplinary projects and it will be under this umbrella that the
graduate will work. They will be trained in polymer chemistry, biological assays, microfluidics, electroanalysis,
sensor development, statistical analysis etc, equipping them for careers in academic or industrial life sciences
research. A positive project outcome would be associated with numerous high profile publications and plans to
extend the study to new problems/collaborating teams and additional patient cohorts. Translational routes,
aided significantly by both a long standing relationship with both industrial (Roche, Somologic and BioRad) and
clinical partners, will be through the technology transfer arm of Oxford University. The project lead has relevant
IP and is the founder of Osler Diagnostics; based in the centre of Oxford and employing close to 100 people,
Osler are now one of the leading diagnostic companies in Europe and currently translating sensor platforms
directly to market with the vision of directly empowering patients.

References: 1.The Lancet 2017, 389 (10073), 1011-1024. 2.The American Journal of Surgery 1993, 165 (1), 61-67. 3.Gastroenterology 2008,
134 (4), 981-987. 4.Nature Reviews Gastroenterology and Hepatology 2013, 10 (7), 423-433. 5.Diabetes 2017, 66 (5), 1103-1110. 6.Chemical
Society Reviews 2013, 42 (13), 5944-5962. 7.Analytical Chemistry 2014, 86 (11), 5553-5558. 8.Biosensors and Bioelectronics 2013, 39 (1), 94-
98. 9.Chemical Science 2012, 3 (12), 3468-3473. 10.Bioanalysis 2015, 7 (6), 725-742. 11.RSC Advances 2014, 4 (102), 58773-
58777. 12.Analytical Chemistry, 2017, 89 (5), 3184-3190. 13.Journal of Proteomics 2009, 73 (2), 352-356. 14.Methods in molecular biology
(Clifton, N.J.) 2010, 658, 281-291. 15.Molecular Cancer 2014, 13 (1), 1-13. 16.Clinical Cancer Research 2016, 22 (7), 1734-1743. 17.Journal of
Proteomics 2015, 113, 400-402. 18.Clinical Cancer Research 2016, 22 (7), 1734-43.

54
 Understanding STING regulation in cancer and the crucial role of
ubiquitination at the ER – Dr. Christianson1,2,3A
Primary Supervisor: John Christianson
Additional Supervisors: Eileen Parkes
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Cancers interact with their surrounding environment (the tumour microenvironment) by remodelling
it to contain cells promoting tumour invasion and spread, and resistance to anti-cancer therapies.
Innate immune pathways, typically used to defend cells from infection by viral and bacterial
pathogens, are hijacked in cancer. The mechanisms by which cancer cells modify innate immunity are
currently not well understood. A key pathway is the cGAS-STING pathway – the cytoplasmic sensor
cGAS recognises non-self or mislocalised DNA and activates STING (the STimulator of Interferon
Genes). STING is embedded in the endoplasmic reticulum (ER) – activation of the STING-mediated
interferon response requires oligomerisation and efflux from the ER (1). Fine tuning of this response
is paramount, and ubiquitination of STING has emerged as an important post-translational
modification capable of modulating these signalling events. Importantly, evidence is emerging of
important interferon-independent effects of cGAS-STING signalling which may drive tumour
progression. Establishing how ubiquitination and its conjugating machinery impact the cGAS-STING
pathway is key to understanding how cancers subvert this pathway to their own ends. This project will
biochemically and functionally characterise ER-resident ubiquitination machinery that modulates
STING signalling in order to its regulation of the interferon response.
While immune checkpoint blockade has transformed outcomes in the treatment of metastatic
cancers, the ability of the tumour to prevent an interferon response is an important and unaddressed
mechanism of resistance to this therapy. The cGAS-STING pathway is a crucial component of the
interferon response, and as such cancers have developed many mechanisms for preventing activation
of this pathway. This includes prevention of mobilisation of STING from the ER, the regulation of which
is the focus of this project. This project is oriented towards translation and can be partnered with
clinical samples from ongoing and imminent trials of STING agonists and other immune therapies in
advanced cancer. The supervisors are experts in the fields of protein homeostasis, ubiquitination,
cGAS-STING regulation and immunotherapy. Professor Christianson has recently discovered a novel
mechanism regulating STING, and Dr Parkes is the principal investigation for STING-targeting trials in
the early phase unit. As such, this project aligns closely to the Oncology theme of immunology. Centre
funds used to support this project will therefore support the generation of robust pre-clinical data,
with a focus on translation to the clinical setting. Funds for this project will include training in novel
techniques, access to required equipment, consumables required and ability to present findings at
national and international conferences.

Research Objectives and Outcomes:

Recently, our lab identified a multi-subunit complex organised around ER-resident ubiquitin ligase (E3)
RNF26, whose constituents modulate signalling through STING to scale the magnitude of the
interferon response (2). We are now investigating how each component of this RNF26 complex
impacts STING to contribute to the response. Understanding STING regulation will identify
mechanisms of resistance to immune targeting agents (immune checkpoint blockade and STING
agonists) in advanced cancers.

Objective 1: Molecular dissection of ubiquitin conjugating machinery competing to modify STING in

the ER. Genomic editing, gene silencing and dominant negatives will establish relative contributions
of ER-resident E3s (RNF26, RNF5, gp78) to STING properties including its; stability/degradation,
ubiquitination profile, oligomerisation, trafficking, and activation of the downstream interferon

55
response, in model cell lines. Both mass spectrometry and linkage-specific deubiquitinase sensitivity
will aim to reveal the diversity and dynamic nature of ubiquitin chain linkages modifying STING.
Outcome: Establishment of key ubiquitination events governing STING in the ER and consequently the
magnitude of the interferon response

Objective 2: Define how ER-resident cofactors differentially contribute to E3 recognition and

ubiquitination of STING. Beginning with RNF26 cofactors (extended to other E3s), IP and proximity-
labelling strategies will define E3 complexes’ spatiotemporal organisation and their interaction/s with
STING. Bioinformatics will identify key regulatory domains within cofactors while genomic editing,
site-directed mutagenesis, and truncations/deletions will and interrogate their impact on STING
activity. Interaction/recognition of STING by ER-E3s and their cofactors will be pharmacologically
probed for changes using STING agonists and antagonists (currently being developed for clinical
applications).
Outcome: Characterisation of ER-resident modulators of STING activation to uncover novel factors
impacting the interferon response

Objective 3: Preclinical validation of STING modulating factors. Identified STING regulating factors will
be modified using gene editing and CRISPR-cas9 approaches in syngeneic tumour models. These will
be studied in vivo to determine the effect of modulating STING regulating factors on the tumour
microenvironment. Using clinically relevant immunotherapeutic agents (for example, anti-PD-1) the
role of STING-modulating E3s and co-factors in response to immune checkpoint blockade will be
investigated. Response to STING agonists and other novel immunotherapies will be determined.
Immune analysis of the tumour microenvironment using flow cytometry and immunohistochemistry
will determine the effect of STING modulating on specific immune cell populations.
Outcome: Characterisation of targetable mechanisms of STING suppression determining response to
cancer immunotherapy.

Collectively this research will develop insights into the fundamental cellular controls of immune
signalling using both in vitro to in vivo models. Along with ongoing work in the lab, it will form part of
our broad effort to define ubiquitination events and mechanisms at the ER responsible for essential
cellular homeostatic functions and their relationship to cancer.

Translational relevance of the project: This project will address important fundamental and clinical
questions relevant to personalising immunotherapy treatment in cancer. Tailoring immune targeting
approaches and understanding resistance mechanisms (such as STING repression) has potential to
improving clinical responses. In this study novel STING regulating mechanisms will be characterised as
potential biomarkers and/or targets for further clinical study.

References: (1) Hopfner K and Hornung V (2020) Molecular mechanisms and cellular functions of cGAS–STING
signalling. Nature Reviews Molecular Cell Biology. 197: 1-21 (2) Fenech EJ, Lari F, Charles PD, Fischer R, Thezenas
ML, Bagola K, Paton AW, Paton JC, Gyrd-Hansen M, Kessler BM, Christianson JC (2020) Interaction mapping of
endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signaling. eLife 2020;9:e57306
DOI: 10.7554/eLife.573 (3) Parkes EE, Walker SM, Taggart LE, et al. Activation of STING-Dependent Innate
Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer. J Natl Cancer Inst . 2017;109(1).
doi:10.1093/jnci/djw199.

56
 Spatial mapping of the bone marrow for improved leukaemia
diagnosis using machine learning/artificial intelligence – Dr Royston4
Primary Supervisor: Daniel Royston
Additional Supervisors: Jens Rittscher, Bethan Psaila
Eligibility: Track 4 students only are eligible to apply for this project.

Project Summary
Leukaemias are blood cancers arising from haematopoietic stem / progenitor cells in the bone marrow.
Their diagnosis and classification requires integration of clinical, morphological and genetic findings.
Despite major advances in our understanding of the molecular and genetic basis of leukaemias, the
complex interactions between neoplastic cells, bone marrow stroma and immune cell infiltrates remain
incompletely understood. Improved quantitative analysis of the marrow microenvironment using routinely
prepared and widely available biopsy samples has huge potential to improve the diagnosis and monitoring
of leukaemia patients and will advance the translation or novel laboratory-based therapeutic strategies
into effective treatments. This will require detailed, objective analysis of biopsy samples that is beyond the
scope of conventional morphological descriptions provided by pathologists. To this end, we have
developed the first machine learning approaches for the automated identification, quantitative analysis,
and abstract representation of megakaryocytes that are important features of several leukaemia subtypes.
This strategy has established the potential of automated tissue analysis to improve the morphological
assessment of bone marrow biopsies in leukaemia patients and offers new opportunities to build
sophisticated morphomolecular tools that are ripe for translation into the clinical investigation and
management of patients. Recent work from our group has highlighted the potential of computational
analysis to augment the diagnosis and classification of certain leukaemias using tissue sections prepared
as part of the routine assessment of patients (Figure 1)1. Our quantitative comparisons of sequential tissue
samples are beyond conventional pathological assessment and compliments current genomic and
cytogenetic approaches for disease diagnosis and monitoring. The aim of this proposal is to expand the
computational description of the bone marrow microenvironment to incorporate multiple neoplastic,
stromal and immune cell populations along with key signalling pathways implicated in normal and
neoplastic haematopoiesis. Automated analysis of these constituent marrow elements in the context of
annotated sample cohorts that have already been collected in Oxford will greatly accelerate the translation
of the advanced cellular and molecular techniques that are well established for leukaemias by our close
collaborators in Oxford.

Figure 1. Overview of the computational pipeline for the disease cohort analysis of MPNs. To effectively build an annotated
library of megakaryocytes, assisted annotation tools for identification (A) and delineation (B) have been developed. (C) A
library of annotated megakaryocytes from reactive and MPN samples was generated and validated by a hematopathologist.
(D) Clustering analysis performed on the library of megakaryocytes identified candidate phenotypes. (E) The phenotypic and
topographical profile of megakaryocytes was extracted and used to create abstract representations of each trephine sample.
(F) Based on these abstract representations, the analyzed samples can be represented in 2-dimensional space with new
samples indexed to annotated disease cohorts.

57
Recent work from our group has highlighted the potential of computational analysis to augment the
diagnosis and classification of certain leukaemias using tissue sections prepared as part of the routine
assessment of patients (Figure 1). Our quantitative comparisons of sequential tissue samples are beyond
conventional pathological assessment and compliments current genomic and cytogenetic approaches for
disease diagnosis and monitoring. The aim of this proposal is to expand the computational description of
the bone marrow microenvironment to incorporate multiple neoplastic, stromal and immune cell
populations along with key signalling pathways implicated in normal and neoplastic haematopoiesis.
Automated analysis of these constituent marrow elements in the context of annotated sample cohorts that
have already been collected in Oxford will greatly accelerate the translation of the advanced cellular and
molecular techniques

Research objectives
Integrating morphological and topographical features from multiple stromal and cellular components of
the bone marrow into unifying disease classifications
We have already demonstrated the potential of applying machine learning to biopsy samples by analysing
megakaryocytes in the context of myeloproliferative neoplasms, a subset of leukaemias. We now wish to
expand and modify this strategy to other stromal and cellular constituents of the marrow including other
haematopoietic lineages (erythoid, myeloid etc.) and immune cell subsets to allow a more comprehensive
description of the morphological features in diverse leukaemic conditions. This has the potential to rapidly
impact on the accurate diagnosis, classification and prognostication of these diseases with the potential to
establish accurate disease monitoring using sequential trephine samples taken before and after treatment.
The infrastructure and expertise for the development of such analytical pipelines is already established
within our group.
Analysis and validation of novel therapeutic targets identified from state-of-the-art sequencing and
imaging modalities using clinical samples
The need to integrate diverse molecular, genomic and immunophenotypic data with tissue morphology
using routine diagnostic material is critical to increasing our understanding of leukaemia and evaluating
future therapeutics. As part of existing collaborations with groups employing state-of-the art equipment in
single cell genomics and multiplex imaging (e.g. iCyTOF, CODEX) this project will comprise an important
‘translational bridge’ by establishing robust, automated pipelines for the analysis and validation of newly
identified cellular and molecular targets using clinical bone marrow samples. We will utilise samples
obtained and stored locally in addition to material obtained through established collaborations with major
trial cohorts (e.g. MAJIC, PHAZAR and PT1) and international cancer centres including Mayo clinic and
MSKCC in the United States.

Translational potential of the project: The project outlined in this proposal represents a structured
approach towards the integration of established in-situ single cell profiling, genomics and transcriptomics
facilities and the morphological analysis of clinical tissue samples. The tools developed and validated as
part of this project and the subsequent publications will be directly applied to the diagnostic reporting of
samples from leukaemia patients. The existing collaborations with leading UK and international haemato-
oncology departments, along with prominent clinical trial co-ordinators, have been established with the
specific purpose of bringing such computational approaches direct to the clinic. Within the time period of
the studentship we plan to have taken the first steps towards the implementation of these platforms and
algorithms in clinical care within the local NHS Trust (OUHFT).

References: AI-Based Morphological Fingerprinting of Megakaryocytes: a New Tool for Assessing Disease in MPN Patients.
Korsuk Sirinukunwattana, Alan Aberdeen, Helen Theissen, Nikolaos Sousos , Bethan Psaila, Adam J. Mead, Gareth D.H.
Turner, Gabrielle Rees, Jens Rittscher and Daniel Royston. Blood Adv. 2020 Jul 28;4(14):3284-3294. Doi:
10.1182/bloodadvances.202000230. 2. Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis
and Identify Mutant Clone-Specific Targets.Psaila B, Wang G, Rodriguez-Meira A, Li R, Heuston EF, Murphy L, Yee D, Hitchcock
IS, Sousos N, O'Sullivan J, Anderson S, Senis YA, Weinberg OK, Calicchio ML; NIH Intramural Sequencing Center, Iskander
D, Royston D, Milojkovic D, Roberts I, Bodine DM, Thongjuea S, Mead AJ. Mol Cell. 2020 May 7;78(3):477-492.e8. doi:
10.1016/j.molcel.2020.04.008.

58
 Bioengineered gastrointestinal tissues to study neural signalling in
cancer development and metastasis – Prof. Bayley1,2,3A,3B
Primary Supervisor: Hagan Bayley
Additional Supervisors: Xin Lu
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.
Project Summary
Metastasis is responsible for the majority of deaths from cancer. During the past few years,
the importance of nervous system in cancer development and metastasis has been
recognised. Nerve fibers have been found in some tumor tissues with possible synaptic
contact between neural and cancer cells. Further, the nervous system produces neural-
related factors to modulate cancer metastatic processes, including cancer cell invasion,
migration and colonisation. Understanding complex nervous system-cancer cell interactions
could lead to the development of new strategies for the treatment of cancer metastasis.
Previously, the Bayley Lab established a droplet-based 3D printing technique for the
construction of functional 3D neural tissues. Joint efforts between the Bayley and Lu labs have
led to a versatile technique for bioengineering tubular gastrointestinal (GI) tissues. Here we
propose a bioengineering approach to fabricate tubular GI tissues for the investigation of
neural signalling in cancer progression. The engineered GI tract will have defined layers: an
epithelial layer derived from epithelial GI organoids and a sub-epithelial layer containing
neural cells with or without fibroblasts and immune cells. These structurally defined tissues
will recapitulate the cellular architecture of natural GI tracts. Importantly cancer cells, with
specific genetic mutations, and neural cells will be patterned at specific locations in the
fabricated GI tissues. An understanding of the role of neural cells in cancer development will
aid the development of new strategies for the treatment of cancer metastasis.

Figure 1. Bioengineering techniques developed in Bayley and Lu labs.1,2 A, Printed droplet network
with membrane protein as synthetic tissues. B, Gallery of printed 3D droplet network with different
patterns. C, Bioengineered GI tract with complex shape (left) and Images from sectioned samples

59
demonstrate the layered structure of the GI tract: H&E staining (right) and immunostaining
(bottom).

Our approach: We propose to use bioengineered gastrointestinal (GI) tissues to study neural
signalling in cancer development and metastasis. We will use our tissue engineering
technique2, including 3D droplet-printing1,12 , to generate layered tubular GI tissues (Figure
1). These GI tubes will contain an epithelial layer derived from GI organoids and a subepithelial
layer containing neurons with or without fibroblasts and immune cells. Both healthy and
cancerous epithelial cells will be used to investigate how cancer develops under the influence
of neural cells. Importantly cancer cells with specific genetic mutations will be patterned at
different densities at specific locations of the fabricated GI tissues. This will allow us to
monitor the subsequent cancer cell proliferation and migration. Further, drug treatments and
environment factors, such as acid reflux and infection by pathogens, will be evaluated.

Research objectives, proposed outcomes and translational potential of the project:

1) Optimise our tissue engineering techniques to incorporate neural cells in the sub-
epithelium layer of the fabricated GI tissues.
2) Incorporate cells, from dissociated normal and diseased duodenum and gastric organoids,
to the epithelial layer of the GI tissues.
3) Investigate the effect of neural cells on the proliferation and migration of cancer cells in
the tissues. Use single cell sequencing to identify gene expression changes in cancer cells
after interaction with neural cells.
4) Develop potential therapeutic strategies, e.g. by drug screening, to block the neural cell-
cancer cell communication and stop cancer progress.

References: 1. Villar, G., Graham, A. D. & Bayley, H. A tissue-like printed material. Science 340, 48-52,
doi:10.1126/science.1229495 (2013). 2. Linna Zhou, C. R. P., Brittany-Amber Jacobs, Xiaoyue Han, Richard
Lisle, Hagan Bayley, Xin Lu. Bioengineered tubular gastrointestinal tissues from arrayed droplet networks.
Submitted (2020). 3. Reiche, E. M., Nunes, S. O. & Morimoto, H. K. Stress, depression, the immune system,
and cancer. Lancet Oncol 5, 617-625, doi:10.1016/S1470-2045(04)01597-9 (2004). 4. Lu, S. H., Zhou, Y.,
Que, H. P. & Liu, S. J. Peptidergic innervation of human esophageal and cardiac carcinoma. World J
Gastroentero 9, 399-403 (2003). 5. Seifert, P. & Spitznas, M. Tumours may be innervated. Virchows Arch
438, 228-231, doi:DOI 10.1007/s004280000306 (2001). 6. Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H. &
Albo, D. Perineural Invasion in Cancer A Review of the Literature. Cancer-Am Cancer Soc 115, 3379-3391,
doi:10.1002/cncr.24396 (2009). 7. Li, S., Sun, Y. L. & Gao, D. W. Role of the nervous system in cancer
metastasis (Review). Oncol Lett 5, 1101-1111, doi:10.3892/ol.2013.1168 (2013). 8. van Zijl, F., Krupitza, G.
& Mikulits, W. Initial steps of metastasis: Cell invasion and endothelial transmigration. Mutat Res-Rev
Mutat 728, 23-34, doi:10.1016/j.mrrev.2011.05.002 (2011). 9. Yang, E. V. et al. Norepinephrine up-
regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and
MMP-9 in nasopharyngeal carcinoma tumor cells. Cancer Res 66, 10357-10364, doi:10.1158/0008-
5472.Can-06-2496 (2006). 10. Okugawa, Y. et al. Brain-derived neurotrophic factor/tropomyosin-related
kinase B pathway in gastric cancer. Brit J Cancer 108, 121-130, doi:10.1038/bjc.2012.499 (2013). 11. Drell,
T. L. et al. Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast
carcinoma cells. Breast Cancer Res Tr 80, 63-70, doi:Doi 10.1023/A:1024491219366 (2003). 12. Zhou, L. et
al. Lipid-Bilayer-Supported 3D Printing of Human Cerebral Cortex Cells Reveals Developmental Interactions.
Adv Mater, e2002183, doi:10.1002/adma.202002183 (2020).

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 Discovery of potent wild-type and surrogate agonist peptides for
anti-tumour T Cells – Dr. Fernandes1,2,3A
Primary Supervisor: Ricardo Fernandes
Additional Supervisors: Tao Dong, Benedikt Kessler
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
T cells probe the surrounding environment using the T-cell receptor (TCR) to scan peptides
presented by the major histocompatibility complex. The nature and potency of the T cell
response towards pathogens or tumor cells is determined by the signaling output from two
distinct classes of immune receptors: the TCR and co-receptors, which includes activating and
inhibitory checkpoint receptors such as CD28 or PD-1 and CTLA-4, respectively. The latest
advances in single-cell sequencing have facilitated the identification of TCRs from clonally
expanded, tumor-infiltrating T cells. However, the identification of strong agonist peptides is
still notoriously challenging. The aim of this project is to establish a framework to identify
potent agonist peptides recognized by effector T cells , with a strong focus in identifying
peptides recognized by TCRs from expanded tumor infiltrating lymphocytes.
The identification of antigens recognized by the TCR has been challenging given the extreme
diversity of the three individual components involved: peptide antigens, TCR and MHC. Our
aim is to identify peptides, neoantigens and mimotopes, recognized by the TCR of clonally
expanded CD8+ effector and CD4+ T cells in tumor settings. To this end, we will engineer large
(> 109) peptide-MHC libraries to be displayed at the surface of yeast cells after which we will
use an affinity-based screen to identify peptides recognized by TCRs of interest. This affinity-
based approach will be complemented by a functional screen using an engineered system in
mammalian cells, whereby the peptide-MHC library is fused to a CAR-like signaling module
displayed by T cells. This functional-based selection hijacks the unique sensitivity and
specificity of the CD28/CD3 signaling modules to report on a productive TCR/pMHC
interaction. Sorting of cells based on the upregulation of activation markers such as CD69 and
CD25 will be used to isolate agonist peptides of different potency. The combination of affinity-
and activity-based selections will guide the identification of potent agonist mimotopes and
the discovery of self-peptides or neoantigens using custom built algorithms to identify closely
related wild-type peptides. The identification of peptides recognized by T cells of interest will
further enable the production of tumor-specific peptide-MHC tetramers to be used in T cell
isolation for detailed phenotypic characterization using a wide-range of techniques such as
single-cell transcriptomics and proteomics. Agonist peptide identification combined with
single-cell sequencing and quantitative proteomic analysis of relevant T cells will expand our
current understanding of the role of diverse T cell subsets during an anti-tumor immune
response. The outcome of this research is expected to contribute towards a better
understanding of T cell function and to the development of relevant immunotherapies in
cancer settings. Furthermore, the discovery of disease-related agonist peptides opens the
possibility to modulate T cell activity by peptide immunization, an important first step towards
achieving in vivo expansion and activation of tumor-reactive T cells.

61
 Research objectives and
proposed outcomes
The objective of this
research plan is to develop a
framework for the
identification of peptides
recognized by T cells of
interest with a particular
focus in tumor-reactive T
cells. We anticipate two
main outcomes. First, the
engineered peptide-MHC
library displayed by yeast
and mammalian cells will be
made readily available to the
scientific community.
Second, using the approach
described we expect to
identify potent peptide agonists which will facilitate the identification, isolation and
expansion/activation of tumor-reactive T cells. These reagents will also be made available to
the scientific community working in this field.

Translational potential of the project.

The deorphanization of TCRs is notoriously challenging and has limited the possibility of
expanding (in vivo) tumor-reactive T cells. Checkpoint inhibition blockade using antibodies
against PD-1 and CTLA-4 to enhance T cell activity has shown great promise in the clinic, but
it is also clear that most patients fail to respond. We anticipate that the next stage of
immunotherapy development will involve a combination of checkpoint blockade therapy –
which broadly enhances T cell responses and is thus largely unspecific - with peptide antigens
specific to tumor-reactive T cells. The research plan we propose will establish a rapid and
facile method to discover peptide antigens for tumor-reactive T cells. This project has
therefore a significant translational potential by aiming to fill a current gap in the
development of effective anti-tumor immunotherapies.

References: Gee MH, Han A, Lofgren SM, Beausang JF, Mendoza JL, Birnbaum ME, Bethune MT, Fisher S, Yang
X, Bingham DB, Sibener LV, Fernandes RA, Velasco A, Baltimore, D, Schumacher TN, Khatri P, Quake SR, Davis
MM, Garcia KC. Antigen identification for orphan T cell receptors expressed on tumor-infiltrating lymphocytes.
(2018) Cell. Jan 25;172(3):549-563.e16. Sibener LV, Fernandes RA, Kolawole EM, Carbone CB, Liu F, McAffee D,
Yang D, Su DF, Yu D, Dong S, Gee MG, Jude KM, Birnbaum ME, Goddard WA, Davis MM, Groves JT, Heath JR,
Evavold BD, Vale RD, Garcia KC. Isolation of a structural trigger required for TCR signaling from analysis of non-
stimulatory peptide-MHC ligands. (2018) Cell. Jul; 174 (3), 672-687. e27. Saligrama N, Zhao F, Sikora MJ, Serratelli
W, Fernandes RA, Louis DM, Yao W, Chien YH, Garcia KC, Davis MM. Opposing T Cell Responses in Experimental
Autoimmune Encephalomyelitis. (2019) Nature. Aug; 572(7770):481-487. Fernandes RA*, Li C*, Wang G, Yang
X, Savvides CS, Glassman CR, Dong S, Luxemberg E, Sibener LV, Birnbaum ME, Benoist C, Mathis D, Garcia KC.
Discovery of surrogate agonists for visceral fat Treg cells that modulate metabolic indices in vivo. (2020) eLife.
Aug; 9:e58463

62
 In vitro and in silico models of human induced pluripotent stem
cell to investigate the effects of doxorubicin – induced cardiotoxicity
– Prof. Zaccolo1,2,3A
Primary Supervisor: Manuela Zaccolo
Additional Supervisors: Christopher Toepfer, Blanca Rodriguez
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.
Project Summary
Despite continuous progress in the field of cardio-oncology, cardiotoxicity is still one of the major complications
of anti-cancer therapy. Recent advances suggest that adrenergic signalling disfunction may contribute to the
cardiotoxicity associated with anthracyclines treatment. This multidisciplinary project combines cutting-edge
approaches in real-time imaging, human inducible pluripotent stem cell derived cardiac myocytes and
computational models of human cardiac myocytes to investigate the involvement of adrenergic signalling in the
cardiotoxic effects of doxorubicin.

Anthracyclines, including doxorubicin (DXR), are widely used as antineoplastic agents but their use is
compromised by the occurrence of severe cardiomyopathy1. The cardiotoxic effects of anthracyclines are
becoming a major health issue due to the increased amount of cancer patient survivors. Notably, the underlying
molecular mechanisms of anthracyclines-induced cardiotoxicity remain obscure2, 3. Most of the implicated
potential targets, including reactive oxygen species (ROS), and topoisomerases3, appear as double-edge swords,
as they also exhibit a vital role in doxorubicin anticancer activity4. Therefore, the design of cardioprotective
interventions counteracting doxorubicin cardiomyopathy that would not hinder the anti-tumor activity of the
drug represents a great challenge5. Published literature so far has focused mostly on mitochondria dysfunction
with consequent oxidative stress, Ca2+ overload, and cardiomyocyte death, leading to heart dysfunction.
However, more recent evidence suggest that the mechanisms of cardiotoxicity may be more complex than just
mitochondria dysfunction and involve alterations of specific signaling pathways that may be directly targeted by
the anticancer drugs itself. Although the underlying mechanisms remain to be elucidated, significant evidence
points to maladaptive b-adrenergic stimulation occurring during the acute phase of DXR-induced cardiotoxicity,
primarily through the cAMP-PKA pathway, as one of the most critical contributing pathways in the ongoing
cardiac damage3, 8-10. cAMP-PKA signaling is now known to be organized at the subcellular level in distinct
nanocompartments with tight cross-compartment coordination to maintain control over the complex and often
divergent functions of the pathway11. No information is currently available on the effects of DXR on local
cAMP/PKA signaling. While most studies addressing DXR-induced cardiotoxicity use animal models, human
inducible pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) are emerging as potent models for
assessing cardiotoxicity and strategies for cardioprotection12 with obvious advantages for translational
applications; similarly, human computational models are powerful tools both for hypothesis generation in
mechanistic studies and for in silico drug testing13. This interdisciplinary project combines cellular/molecular
approaches and computational modelling to investigate the effects of DXR on local adrenergic signalling, using
available hiPSC-CM models that recapitulate both healthy and diseased human cardiac phenotypes14, 15.

Research Objectives
Cutting edge technologies for local detection of cAMP in living cells using FRET-targeted reporters16 will be used
to study how cAMP/PKA signals are affected, at the subcellular level, by DXR treatment and to identify potential
targets involved in cardiotoxicity with high spatial resolution. For this, healthy or diseased (genetic models of
cardiomyopathy) iPSC-CMs in 3D cultures will be studied alongside simultaneous assessment of calcium
transients and cellular contractility using SarcTrack and CalTrack algorithms (see below). Experimental work will
be carried out in
combination with in silico
modelling and simulation
for hypothesis testing and
mechanistic investigations.
Experimental
characterisation will be
integrated into a novel
mathematical model of
human cardiomyocyte

63
electromechanical activity that will incorporate the local subcellular handling of cAMP/PKA signalling. In detail
the funds will be used to pursue the following 3 specific aims:

Aim 1: Profiling cAMP in subcellular compartments in iPSC-CMs (collaboration with Professor Mummery,
Leiden University). A central component of this proposal is to establish a 3D culture system with highly
differentiated CM and amenable to single cell analysis. These protocols will be developed with the assistance of
our collaborator Prof C. Mummery who has recently developed a protocol to generate 3D microtissues (MTs) by
co-culturing isogenic iPSC-derived CM, endothelial cells and fibroblasts17. In these MTs, CM achieve a superior
degree of maturation and have the additional advantages of being easy to set up and are low cost, making them
amenable to high-throughput production. Critically, 3D MTs can be easily dissociated to yield high quality single
cells. Once the 3D MTs are established, we will use viral transduction of genetically encoded fluorescent
reporters of cAMP developed in the Zaccolo laboratory that are targeted to distinct subcellular compartments
(cytosol, myofilament, plasma membrane sarcoplasmic reticulum, mitochondria)16 (Fig 1) to study how DXR
affects local cAMP signalling in iPSC-CM. The iPSC are tagged with GFP15 as a sarcomeric reporter of contractility.
3D MTs expressing cAMP sensors will be established to test the effects of adrenergic agonists, inhibitors of cAMP
hydrolysis (phospodiesterases
A C Figure 2 – A) Representation of inhibitors) and anticancer drugs in
wavelet pair used by SarcTrack to fit
sarcomeres in the iPSC-CM GFP-TTN different subcellular compartments.
line. B) Simulation of wavelet pairs
B with degrees of freedom for fitting
Our preliminary experiments show
which are used to fit sarcomeres in that the cAMP reporters are
each frame of an image stack. C)
Representative TTN-GFP iPSC-CM expressed in human iPS-CM18 and
that has been fit by the wavelet pairs
that these cells respond to
D E
to identify sarcomeres. Colour coding
defines distance spread of sarcomere sympathetic stimulation and PDE
pairs across the image. D) Mean
sarcomere shortening of each iPSC- inhibition (Fig 1). This aim will
CM cell measured by SarcTrack. provide information of how DXR
MyBPCt/+ is a heterozygous myosin
binding protein C truncating variant. affects cAMP handling in key
E) Mean relaxation time of each iPSC-
CM cell line (WT or MyBPCt/+
subcellular compartments and how
measured by SarcTrack. these effects can be attenuated by
manipulation of adrenergic
signalling.

Aim 2: Impact of DXR on CM contractility, calcium transients and cAMP. To link cAMP changes to CM function,
here we will develop protocols for simultaneous measurements of cellular contractility, calcium transients and
cAMP signals in iPSC-CMs isolated from 3D MTs. cAMP and Ca2+ signals will be assessed using compartment
specific fluorescent reporters and fluorescent dyes, respectively. This will be combined with sarcomere length
measurements using hiPSC-CM with fluorescent tags on sarcomere proteins (TTN-GFP) and the SarcTrack
algorithm developed by Toepfer19. Using this approach, the activity of hundreds of sarcomeres per cell can be
quantified, with high statistical power and high fidelity, providing information on sarcomere content, and
contractile parameters (Fig 2). This will allow the correlation of cellular calcium transients and contractility to be
assayed with cAMP activity. These studies will elucidate how DXR-dependent alterations in local cAMP signals
and PKA-dependent phosphorylation impact CM cellular function.
Aim 3: Incorporate b-adrenergic signalling changes into human computational models. Incorporation of b-
adrenergic signalling changes in CM to human computational models of cardiovascular function and disease will
be used for refining hypothesis, testing and validation of experimental findings. This will be performed under
the guidance of Blanca Rodriguez, Computer Science, building on modelling and simulation techniques already
available (University of Oxford). The Rodriguez group with collaborators have already shown the power of
combining iPSC-CM data with computational models to reproduce experimental recordings in silico (13). This
work will be extended to include calcium recordings, contractile behaviours and subcellular cAMP signalling.

References: 1. Schimmel KJ et al. Cancer Treat Rev, 30:181-91 (2004). 2. Li DLet al. Circulation, 133:1668–1687 (2006). 3. Zhang Set al. Nat
Med, 18:1639–1642 (2012). 4. Mordente Aet al, Curr Med Chem, 24:1607–1626 (2017). 5. Octavia Yet al. J Mol Cell Cardiol, 52:1213–1225
(2012). 6. Sala V et al Antiod Red Sig., 32, (2020) 7. Xu L. et al, Cardiovascular Toxicology20:11–19 (2020). 8. Shah P. Am J Cardiol,
124:789−794 (2019). 9. Efentakis P et al Cardiovascular Research 116, 576–591, (2020). 9. Zhang Y et al Circulation. 138:1988–2002 (2018).
10. Roca-Alonso L, et al Cell Death Dis 6: e1754, (2015). 11. D. M. Bers, Y. K. Xiang, M. Zaccolo, Physiology (Bethesda) 34, 240-249 (2019).
12. Schwach V, et al., Front. Cardiovasc. Med. 7:50 (2020). 13. Rodriguez B Alternatives to Laboratory Animals, Vol. 47(5-6) 221–227 (2019).
14. C. N. Toepfer et al., Sarctrack. Circ Res, (2019). 15. C. N. Toepfer et al., Circulation 141, 828-842 (2020). 16 N. C. Surdo et al., Nat Commun
8, 15031 (2017). 17. Giacomelli e. et al, Cell Stem Cells, 26, 862-879 (2020). 18. Y. Dai et al., Sci Rep 10, 209 (2020). 19.D. Barefield et al., J
Mol Cell Cardiol 79, 234-243 (2015). 19. M. Paci et.al., Heart Rhythm 14, 1704-1712 (2017).

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 Genome-wide screening to identify factors impacting on cellular
survival upon acute depletion of BRCA2 or PALB2 – Prof.
Esashi1,2,3A,3B
Primary Supervisor: Fumiko Esashi
Additional Supervisors: Shibani Nicum, Sarah Blagden
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.
Project Summary
DNA repair enzymes are crucial for maintaining genomic stability, and defects in the DNA damage
response (DDR) is intimately linked to the development of cancer. Defective homology-directed repair
(HDR), arising from, e.g., loss of BRCA1/2 or PALB2, is associated with several types of cancer, including
breast and ovarian cancer. Cancer cells displaying defective HDR are typically highly sensitive to a
number of drugs, including crosslinking agents and poly (ADP-ribose) polymerase (PARP) inhibitors,
but the development of resistance to therapy is common and poses a critical clinical problem. Here,
we propose a state-of-the-art approach to screen for synthetically lethal interactions and genes
conferring resistance to treatment in HDR deficient cells, utilizing genome-wide modulation of
transcription in cell lines in which endogenous BRCA2 or PALB2 can be rapidly depleted. In doing so,
we hope to identify novel diagnostic markers and therapeutic targets.
Individuals with inherited mutations within genes encoding the breast cancer susceptibility 2 (BRCA2)
and the partner and localizer of BRCA2 (PALB2) exhibit increased risk to develop a wide range of
cancers, including breast cancer, ovarian cancer and acute myeloid leukaemia (AML). It has been
widely demonstrated that BRCA2- or PALB-defective cells display increased sensitivity to genotoxic
drugs, such as crosslinking agents (e.g., MMC) and poly (ADP-ribose) polymerase (PARP) inhibitors
(e.g., olaparib), and these treatments have been used to treat affected patients (1). However, it is
becoming increasingly clear that cancers often develop resistance to treatment, leading to a poor
prognosis. There is an urgent need to identify the mechanisms by which these cells develop resistance
to treatment, and to identify new therapeutic strategies. This study will tackle this important question
by a genome-wide screening for factors that modify the survival of BRCA2 or PALB2 defective cells
upon MMC or olaparib treatment. The study is expected to reveal, in an unbiased manner, factors that
increase or reduce the sensitivity of BRCA2- or PALB2-defective cells against existing cancer drugs,
MMC or olaparib. By doing so, we will learn how BRCA2 or PALB2 deficient cells develop drug
resistance and how to best tailor the treatment of these types of cancers.

Research objectives and proposed outcomes

The genetic concept of synthetic lethality, in which the
combination or synthesis of mutations in multiple genes
results in cell death, has attracted increasing attention
with the advent of clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-associated
protein 9 (Cas9)-mediated gene editing, and the
prospect of finding novel therapeutic targets in hard-to-
treat cancers (2). Typically, studies to identify synthetic
lethal interactions (SLIs) have been done by genome-
wide loss-of-function screens, using, for example, RNA
interference (RNAi) or CRISPR knockout (CRISPRko), in
knockout cell lines (where both alleles of the gene of
interest have been rendered defunct). There are,
however, several pitfalls associated with this approach,
including the inability to study the direct effects of
protein depletion before the phenotype becomes

65
obscured by a range of secondary effects and mutations. To overcome these issues, a new approach
to screen for SLIs is in urgent need. In this project, we propose to combine auxin-inducible degron
(AID) technology (3, 4) and CRISPR-mediated modulation of transcription, namely CRISPR
interference/activation (CRISPRi/a) (5, 6, 7). This allows for highly-specific and non-toxic systematic
interrogation of genomes, including studies on the direct effects of protein depletion. Using this
approach, we will screen for SLIs and mechanisms of drug resistance in cells, where endogenous
BRCA2 or PALB2 can be rapidly depleted. In doing so, we also avoid the stochastic nature of
CRISPRko. Our lab has already generated HCT116 cell lines expressing OsTIR1 and endogenously AID-
tagged BRCA2/PALB2, and confirmed that, upon auxin-induced depletion of BRCA2 or PALB2, cells
exhibit increased sensitivity to a crosslinking agent (MMC) and a PARP inhibitor (olaparib) (Figure 1,
and not shown). The next step is to introduce the CRISPRi/a machinery (dCAS9-KRAB/VPR) in these
cells, followed by transduction with sgRNA libraries. HCT116 cell lines harboring OsTIR1, mAID-
BRCA2/PALB2, dCas9-KRAB/VPR, and sgRNA will then be used to screen for SLIs. Factors identified
through this screening will reveal the molecular mechanism by which cell maintain their survival in the
absence of BRCA2 or PALB2. Additionally, acquired results will help identify new diagnostic markers
for cancers and therapeutic strategies to treat cancers. We will initially collaborate with Dr. Joey
Riepsaame, head of genome engineering at the Sir William Dunn School of Pathology, University of
Oxford. The award facilitates additional new collaborations with clinicians, Dr. Shibani Nicum and Prof
Sarah Blagden, so that the outcome can be directly transferred to a clinical setting.

Translational potential of the project: By finding and targeting synthetically lethal interactions, it is
possible to take advantage of weaknesses specific to HDR deficient cancer cells, allowing for more
effective treatments while simultaneously reducing the rate and severity of the adverse effects. Prior
studies have implicated a number of genes as synthetically lethal with BRCA2, PALB2, and PARP1.
However, we believe that by using this non-canonical approach, where direct effects of protein
depletion can be studied, that we will be able to find novel synthetic lethal interactions while also
reducing the false discovery rate. This could, in turn, enable the development of new therapeutic
strategies for hard-to-treat HDR deficient cancers (e.g., breast-, ovarian-, and pancreatic cancer),
including therapies to sensitize cells to PARP inhibition. This non-canonical pipeline could also prove
to be a powerful tool for future synthetic lethal screens.

References: 1. Pommier, Y. et al. (2016). Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms
of action. Science Translational Medicine, 8, 362ps17. 2. Lord, C. J., & Ashworth, A. (2017). PARP inhibitors:
Synthetic lethality in the clinic. Science, 355, 1152–1158. 3. Natsume, T. et al. (2016). Rapid Protein Depletion in
Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Reports, 15, 210–218. 4.
Nishimura, K. et al. (2009). An auxin-based degron system for the rapid depletion of proteins in nonplant cells.
Nature Methods, 6, 917–922. 5. Qi, L. S. et al. (2013). Repurposing CRISPR as an RNA-guided platform for
sequence-specific control of gene expression. Cell, 152, 1173–1183. 6. Gilbert, L. A. et al. (2013). CRISPR-
mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154, 442–451. 7. Gilbert, L. A. et
al. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell, 159, 647–661.

66
 Galectin-3 promotes glioblastoma emergence from the
subventricular zone stem cell niche – Prof. Szele1,2,3A,3B
Primary Supervisor: Francis Szele
Additional Supervisors: Eric O’Neill
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
Galectin-3 (Gal-3) has profound roles in promoting inflammation, is uniquely expressed in the adult
subventricular zone (SVZ) stem cell niche, is commonly expressed by and regulates cancer Together
with Eric O’Neill, we have found that the pro-inflammatory Gal-3 regulates several signalling
molecules including Wnt1,2. Whereas Gal-3 increases Wnt signalling in cancer cells, it decreases it in
the healthy SVZ. We also demonstrated in pilot work that Gal-3 regulates Hippo/Yap/Taz signalling in
human ES cells and apical basal polarity (ABP) of human ESC derived rosette formation. Here we will
test to role of Gal-3 in coordinating Wnt function in ABP. ABP is a key hallmark of SVZ stem cells (apical
primary cilia and basal blood vessel contact) and loss of ABP promotes gliomagenesis. Previously, we
showed that the IDHR132H human cancer-relevant mutation promotes gliomagenesis when specifically
expressed in the murine SVZ3. The work in this studentship will define how these signalling pathways
disrupt ABP in this relevant SVZ gliomagenesis model. The student will use established in vitro and in
vivo approaches and also novel state-of-the art live imaging to visualise the generation and evolution
of SVZ glioblastomas. This work will position us for future funding to use a powerful new drug
screening approach which we developed with the Target Discovery Institute to find small molecule
inhibitors of glioma infiltration4. We will test the novel hypothesis that Gal-3 signalling in the SVZ
induces tumorigenesis via loss of ABP5.

Background. Gliomas contacting the SVZ are aggressive, are likely to spread/reappear and thus have
a worse prognosis than gliomas not contacting the SVZ6,7. Inflammation predisposes cancer
development via cytokines which can activate developmental
pathways shifting the tumour toward a more undifferentiated state
and increasing the number of cancer stem cells, which are
characterised by loss of ABP8. In the brain, the SVZ is a uniquely
inflammatory region that can predispose the niche to cancer
development by the action of the pro-inflammatory regulator Gal-3
on stem cells5. Gal-3 has been linked with cancer aggressiveness,
not only due its pro-inflammatory role, but because it upregulates
several pro-tumorigenic pathways and Gal-3 is correlated with brain
tumor grade and prognosis9,10

Pilot data. In unpublished work, the O’Neill and Szele labs have
shown that Gal-3 expression is increased in the SVZ during IDH1R132H tumourigenesis. Additionally
macrophages were activated in and around the tumour, which is interesting because they secrete Gal-
3 and are chemotactic to it, causing a feed-forward cycle of inflammation11. Using human ES cells we
have also shown that anti-Gal-3 antibodies disrupt rosette formation and that Gal-3 and YAP are
differentially expressed in the apical and basal poles of stem cells in rosettes (Fig. 1). We further
showed that Gal-3 binds to b-catenin1 and hypothesise that this regulates ABP via YAP signalling. In
other work we developed and are using intravital 3-photon (3PM) microscopy capacity in DPAG (with
Adam Packer (DPAG), Martin Booth (Engineering) and Chris Xu (Cornell). With 3PM, we can visualise
the entire SVZ in live animals at cellular resolution (In revision) and his will be used here.

67
Research objectives and proposed outcomes
Workpackage 1. In vitro studies of ABP. Wnt pathways have been implicated in malignancy and
“stem-ness” of gliomas, and are a possible therapeutic target12. Since Gal-3 modulates Wnt signalling
in opposite directions in cancer compared to the healthy postnatal SVZ, malignant transformation of
SVZ cells could involve altered Gal-3 function. The student will modulate Gal-3, Wnt signaling molecule
and Yap/Taz in vitro to uncover their role in ABP of healthy and IDH1R132H mutant SVZ stem cells. They
will learn and employ the murine neurosphere stem cell assay. This in vitro stem cell assay is well
established in the Szele lab and will be used to assess ABP as well as the key features of SVZ lineage
progression: self-renewal, proliferation, fate choices and migration. The student will complement this
with parallel functional studies in human ES cells (Fig. 1). ABP in Rosettes will be employed to dissect
its molecular regulation.
Workpackage 2. In vivo studies of ABP. They will next confirm our in vitro findings with in vivo
approaches well-established in the Szele lab including knockdown, conditional knockout and rescue
experiments of Gal-3 and Wnt signalling partners in the SVZ. We will determine ABP of SVZ stem cells
(type B1) and loss of ABP in IDH mutant mice. We will also ascertain whether altering Gal-3, Wnt and
Yap/Taz signalling in these stem cells affects self-renewing symmetric divisions, increases their
proliferation or emigration into surrounding tissues.
Workpackage 3. In vivo studies of inflammation. We will assess the level of in vivo inflammation in
the mice generated in WP 2 and this will be correlated with the extent of changes seen in ABP and SVZ
stem cell behaviour mapped in WP 2. In other work we will pharmacologically manipulate
inflammation in mice to directly determine its effect on ABP of SVZ stem cells. We will determine
macrophage infiltration into the gliomagenic lesion by carrying out adoptive transfer experiments with
CD68-GFP mice generated in the Greaves Lab, Oxford, monitor the expression of inflammatory
cytokines/chemokines and microglial and macropglial activation.
Workpackage 4. Imaging studies. We will next turn to imaging ABP loss and inflammation in live
animals. Based on the results of the above experiments we will judiciously choose which functional
manipulations to image. The student will learn 3P microscopy in live mice and 2P time-lapse in slices13,
(which is easier and will provide finer temporal and spatial resolution than 3P microscopy). We will
use a bespoke 3PM time-lapse in DPAG to determine how the IDH mutation alters the ABP,
inflammation, cell proliferation and migration. We will also image tumour evolution in the same mice
with the MRI (installed at BSB in South Parks Road).
Translational potential of the project. In parallel work, we will carry out pharmacological targeting of
signalling pathways. We have recently published a unique medium-throughput 3D approach – the
“spheroid migration assay” to measure SVZ cell migration/iinvasion4. In conjunction with the TDI, we
plan to use molecular libraries targeted towards the most relevant pathways revealed in Aims 1 and
2. Thus the translational potential of this project is very good as it will screen for molecular targets
that limit glioma infiltration. Gal-3 conferred resistance to traditional treatment with chemotherapy
and radiotherapy in glioblastoma10, and there are several inhibitors of Gal-3 described, some in clinical
trial for cancer14,15. In addition to screening for molecules that block downstream Gal-3 signaling
partners we will also determine the role of direct Gal-3 inhibitors in SVZ cell migration with the
spheroid migration assay.

References: Al-Dalahmah, Stem Cells, 38:1149, 2020. 2.Al-Dalahmah, Glia, 68:435, 2020. 3.Bardella, Cancer
Cell, 30:578, 2016. 4.Ducker, Stem cell reports, 2020. 5.Bardella, Prog Neurobiol, 170:37, 2018. 6.Sanai, New
England Journal of Medicine, 353:811, 2005. 7.Mistry, J Neurooncol, 131:125, 2017. 8.Arnold, Cancer Growth
Metastasis, 8:1, 2015. 9.Bresalier, Cancer, 80:776, 1997. 10.Wang, Cancer Epidemiology Biomarkers
&amp;amp; Prevention, 28:760, 2019. 11.Li, Cell, 167:973, 2016. 12.He, Journal of Cellular Physiology, 234:2217,
2019. 13.Nam, J Comp Neurol, 505:190, 2007. 14.Wdowiak, International journal of molecular sciences, 19:210,
2018. 15.Stegmayr, Sci Rep, 9:2186, 2019.

68
 Effects of androgen deprivation on multimodality prostate cancer
therapy – Prof. Bryant1,2,3A
Primary Supervisor: Richard Bryant
Additional Supervisors: Ian Mills, Jens Rittsher, Freddie Hamdy
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Radiotherapy (RT) with concomitant androgen deprivation therapy (ADT) is a widely used
standard of care for patients with high risk localised or locally advanced prostate cancer.
Whilst this treatment is often successful, it can have significant long-term side effects, and a
third of patients develop recurrence with limited treatment options. Vascular-targeted
photodynamic therapy (VTP) is a novel minimally invasive surgical technique which can focally
ablate PCa with high precision. VTP can treat low-risk low-volume prostate cancer with
minimal side effects, but it has not been used in combination with other treatments such as
RT or ADT.
The Bryant laboratory is currently investigating the potential for additive/synergistic effects
by combining RT and VTP in pre-clinical immunocompetent murine models of prostate cancer.
Initial results have established that sub-lethal doses of RT can have pro- and anti-tumorigenic
immune responses within the tumour microenvironment (1), and can alter the function and
structure of the tumour microvasculature. These initial experiments have characterised the
necessary schedules and doses of RT and VTP for tumour control, and identified the sub-lethal
treatment conditions for these immunological and vascular changes. Experiments combining
RT and VTP in these pre-clinical models are on-going, testing the hypothesis that these two
treatments may lead to benefit when administered sequentially, such that the dose of RT and
VTP needed for complete tumour control/cure might be reduced. Such an observation would
lead to early phase clinical trials in patients, with the potential to improve clinical practice by
reducing RT-related side effects and increasing the cure rate of prostate cancer. However, to
date these pre-clinical experiments have not incorporated ADT. Given that ADT plus RT is a
standard-of-care for high-risk localised and locally advanced prostate cancer, it is necessary
to investigate whether administration of ADT plus RT modulates any additive/synergistic
effects of VTP. ADT modulates the tumour immune microenvironment (2-4), and in addition
may initially reduce prostate cancer tumour microvascularity and increase hypoxia (5-9) prior
to a later revascularisation and reoxygenation phase. This initial prostate cancer tumour
vessel degeneration post-ADT may be mediated by dysfunction of androgen-sensitive
endothelial cells. Other evidence suggests that ADT results in an improved and highly
functional vascularization of prostate cancer tumours one month post initiation of ADT (10-
14), which may be driven by initial hypoxia, stabilisation of HIF-1α, expression of HIF-1α target
genes including VEGF, and acquisition of alternative mechanisms for androgen-sensitive
endothelial cells to reproduce the vascular network. Therefore, it is possible that concomitant
ADT influences any beneficial effects of combining RT and VTP, i.e. it may be that combining
RT and VT may be best done around one month after commencing ADT, rather than
immediately after ADT. It may also be that ADT prior to VTP may obviate the need for RT to
induce neovascularisation. This hypothesis requires testing in our established pre-clinical
models, as it would have important implications for the design of early phase clinical trials. In
conclusion, this research proposal aims to test the effects of ADT on combined RT and VTP
treatment of pre-clinical prostate cancer models

69
Figure 1. A) In vivo tumour growth delay of flank prostate cancer allografts post-RT; B) DCE-MRI imaging of
flank prostate cancer allografts; C) Immunofluorescence analysis of tumour vasculature.

Research objectives and proposed outcomes:

The proposed Clinical DPhil project is particularly suitable for an academic surgery or
oncology trainee, wishing to gain broad experience in pre-clinical models, multi-modality
treatment, with early translational potential. The project is also suitable for a medical
undergraduate or science graduate. It aims to understand the complex interplay between
ADT, RT and VTP in prostate cancer treatment, such that these effects may be harnessed in
improved anti-cancer treatment. The project also aims to cement a recently forged
collaboration between the Bryant, Mills and Rittscher laboratories in the study of this theme.
Translational potential of the project:
Given that ADT plus RT is a standard of care treatment for localised and locally advanced
prostate cancer, and given that VTP has already been shown to be a safe and efficacious
treatment of low-risk low-volume prostate cancer, combining these treatments with the
correct timing and sequence is eminently feasible, although this needs to be informed by pre-
clinical results. An early phase clinical trial of these treatment combinations could be in
progress within a short timescale (~3 years, though outside of the remit of this DPhil
proposal). Such a trial, if successful, could lead to a phase 3 randomised clinical trial
investigating this combined treatment approach against standard of care ADT and RT. Such a
trial has the potential to change practice by reducing toxicity and improving outcomes for
patients with this common malignancy. It also has translational potential for other cancer
types, including lung, breast, pancreas and colorectal cancer.

References: 1) Philippou. Brit J Cancer 2020. Online ahead of print. 2) Kalina. Cancers 2017;9:13. 3) Wu.
Cancers 2019;11:20. 4) Aragon-Ching. Front Biosci 2007;12:4957. 5) Byrne. BJC 2016;114:659. 6) Lekas.
Urol Res 1997;25:309. 7) Shabsigh. Prostate 1998;36:201. 8) de la Taille. Prostate 1999;40:89. 9) Hayek. J
Urol 1999;162:1527. 10) Røe. Radiation Oncology 2012;7:75. 11) Godoy. Am J Physiol Endocrinol Metab
2011;300:E263. 12) Semenza. Nat Rev Cancer 2003;3:721. 13) Marignol. Cancer Treat Rev 2008;34:313. 14)
Stewart GD. BJUI 2009;105:8.

70
 Characterising the developmental origins in the pathogenesis of
mesenchymal tumours of the central nervous system – Prof. Sauka-
Spengler1
Primary Supervisor: Tatjana Sauka-Spengler
Additional Supervisors: Olaf Ansorge, Sanjeeva Jeyaretna
Eligibility: Track 1 students only are eligible to apply for this project.

Project Summary
Primary mesenchymal tumours of the skull base include aggressive meningioma, chordoma and
chondrosarcoma. Though hypothesised to arise from different cell types, classification subtypes share
histological similarities. All are characterised by slow growth but are locally aggressive and likely to recur
with significant morbidity and mortality. Surgery and radiotherapy are currently the only available
treatments. There is emerging evidence that meningiomas with particular molecular profiles have a spatial
phenotype which strongly correlates with the developmental origin of the meninges. The development of
the cranial bones and meninges includes contributions from the neural crest and cranial mesoderm with
evidence of signalling interdependence for normal development. Common mutations have been identified
across aggressive meningioma, chordoma and chondrosarcoma including mutations affecting hedgehog
and PI3K signalling, and SMARCB1. This project seeks to undertake spatial and temporal transcriptomic
characterisation of the human embryonic skull base and skull base mesenchymal tumours to unlock new
mechanistic insights into the origin of these tumours that may identify new targeted treatments.

Research objective and Proposed Outcomes

The developmental origin of cancer is increasingly recognised; however, study of relevant cellular and
molecular pathways in human tissues is rarely done. This proposal aims to address this, focussing on the
interface of neural crest and cranial mesoderm at the skull base, a site of locally aggressive cancers with
poor outcomes. Our project is novel; no study has directly addressed the comparative biology of these
tumours in relation to human development.

Meningiomas are the most common primary brain tumour, and their incidence and prevalence are
increasing. Patients with atypical (WHO grade II) and malignant (WHO grade III) meningioma suffer from a
high morbidity and mortality, with reported 10-year survival of 63% and 15% respectively. For chordoma,
5-year and 10-year survival is 67.6% and 39.9% respectively, while chondrosarcoma has a recorded 5-year
mortality of 11.5% with a median survival of 24 months. Current treatment of surgery and radiotherapy is
associated with a high risk of morbidity due to effects on peritumoural critical neurovascular structures.
The epigenomic landscape is of increasing importance, particularly given its predictive utility in
meningioma, chordoma and chondrosarcoma classification including response to therapeutics1,2. It is thus
of mechanistic significance and hypothesised that further understanding of gene regulatory networks
underpinning skull base bone and meningeal development could unlock new insights.

The meninges are hypothesised to develop from a combination of neural crest and mesoderm, the pattern
of this development correlates strongly with the mutation profiles of meningiomas. Chromosomal
instability and mutations affecting chromosome 22 (22q) and Hedgehog signalling result in meningiomas
in neural-crest cell derived meninges, while somatic mutations affecting PI3K signalling, TRAF7, KLF4 and
POLR2A result in meningiomas in the mesodermal-derived meninges (Figure 1). The presence of neural
crest cell subpopulations in meningiomas indicates that tumourigenesis may capitalise on existing gene
regulatory networks in development. Chordoma is a malignant tumour thought to arise from the
embryonic remnant of the notochord, while chondrosarcoma develops from chondrocytes within rests of
endochondral cartilage. These are anatomically intimately related to the mesodermally-derived meninges
of the skull base, and the driver landscape of these tumours have identified common use of the PI3K and
Hedgehog signalling pathways3. Furthermore, chordoma and WHO grade II chordoid meningioma have a
striking histological similarity to chordoma and chondrosarcoma. While hyperostosis of bone overlying

71
meningioma and even primary intraosseous meningioma can occur, conversely chondrosarcoma can
develop in the meninges mimicking an atypical meningioma4.

The meninges and underlying cranial base bone both have contributions from the neural crest and
mesoderm, with mouse studies identifying a signalling interdependency for normal development
particularly in ossification. This project thus seeks to characterise the gene regulatory networks
underpinning normal development of the skull base in humans, while conducting analysis on mesenchymal
skull base tumours to identify mechanistic insights.

Figure 1 – Anatomical depiction of meninges with brain and spinal cord removed displaying skull base including tentorium
cerebelli on the right side. A) Distribution of meningioma by mutation gene pathway, B) Meningeal development by tissue
of origin, and C) Overlying bone development by tissue of origin.

This project would be supervised by Professor Sauka-Spengler, an expert on gene regulatory networks in
the neural crest, which we will apply to foetal cranial base bone and meningeal tissue and tumour5-8. Oxford
is well placed with existing national multi-centre collaborations for meningioma, sarcoma, and
chondrosarcoma tissue. These include the Oxford skull base unit (collaboration with Sanjeeva Jeyaretna,
lead consultant for skull base surgery and committee member of the British-Irish Meningioma Society) and
the Oxford Sarcoma Service (collaboration with Jeremy Reynolds, lead consultant for Oxford’s Bone and
Soft Tissue Tumour Service).

Translational Potential
There are currently no targeted treatments for aggressive mesenchymal tumours including aggressive
(recurrent and WHO grade II-III) meningioma, chordoma and chondrosarcoma. Characterisation of the
gene regulatory landscape of these tumours, in the context of discovery work in developmental networks
of embryonic tissues, will yield improved understanding of the mechanisms resulting in cellular
vulnerability to these tumours and potential targets for future treatments.

References: 1. Sahm, F. et al. DNA methylation-based classification and grading system for meningioma: a multicentre,
retrospective analysis. Lancet Oncol 18, 682-694, doi:10.1016/S1470-2045(17)30155-9 (2017). 2. Nicolle, R. et al. Integrated
molecular characterization of chondrosarcoma reveals critical determinants of disease progression. Nat Commun 10, 4622,
doi:10.1038/s41467-019-12525-7 (2019). 3. Presneau, N. et al. Potential therapeutic targets for chordoma:
PI3K/AKT/TSC1/TSC2/mTOR pathway. Br J Cancer 100, 1406-1414, doi:10.1038/sj.bjc.6605019 (2009). 4. Korten, A. G., ter
Berg, H. J., Spincemaille, G. H., van der Laan, R. T. & Van de Wel, A. M. Intracranial chondrosarcoma: review of the literature
and report of 15 cases. J Neurol Neurosurg Psychiatry 65, 88-92, doi:10.1136/jnnp.65.1.88 (1998). 5. Sauka-Spengler, T. &
Bronner-Fraser, M. A gene regulatory network orchestrates neural crest formation. Nature Reviews Molecular Cell Biology
9, 557-568, doi:10.1038/nrm2428 (2008). 6. Hockman, D. et al. A genome-wide assessment of the ancestral neural crest gene
regulatory network. Nat Commun 10, 4689, doi:10.1038/s41467-019-12687-4 (2019). 7. Williams, R. M. et al. Reconstruction
of the Global Neural Crest Gene Regulatory Network In Vivo. Dev Cell 51, 255-276 e257, doi:10.1016/j.devcel.2019.10.003
(2019). 8. Ling, I. T. C. & Sauka-Spengler, T. Early chromatin shaping predetermines multipotent vagal neural crest into neural,
neuronal and mesenchymal lineages. Nat Cell Biol 21, 1504-1517, doi:10.1038/s41556-019-0428-9 (2019).

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 Investigating pathological crosstalk between mature tumour cells
and haematopoietic progenitor cells in chronic myelomonocytic
leukaemia – Prof. Mead1
Primary Supervisor: Adam Mead
Additional Supervisors: Lynn Quek
Eligibility: Track 1 students only are eligible to apply for this project.
Project Summary
Chronic myelomonocytic leukaemia (CMML) is characterised by proliferation of myelomonocytic cells,
bone marrow (BM) failure (anaemia, thrombocytopenia), and high risk of transformation to acute myeloid
leukaemia (AML). It is incurable in most patients (median survival ~30 months), and there are no disease
specific treatments available. The mechanism by which the CMML clone exerts a fitness advantage over
normal haematopoietic cells is poorly understood. It is increasingly recognised that CMML is associated
with chronic inflammation, likely caused by the expansion of pro-inflammatory monocytes due to
mutations such as in TET2, found in ~60% of cases. We propose that aberrant cell-cell crosstalk between
expanded pro-inflammatory myelomonocytic cells in CMML and both wild-type and CMML haematopoietic
stem and progenitor cells (HSPC) is a key driver of clonal expansion, aberrant myelomonocytic
differentiation and suppression of normal HSPC in CMML. To explore this, we will apply high-throughput
single cell RNA sequencing to characterise the cellular landscape in CMML and normal controls. We will
use a novel computational package developed in our laboratory, SCINDY, to identify aberrant cell-cell and
ligand/receptor interactions that might be responsible for driving abnormal HSPC function in CMML. We
will validate our findings using mass spectrometry and iCyTOF to demonstrate changes in protein levels of
these putative interactions, to confirm direct cell-cell interactions and explore alterations in downstream
signalling. We will also validate these interactions functionally using co-culture and differentiation assays
alongside addition of soluble ligands/small molecule inhibitors/CRISPR knockdown of targets to restore
normal HSPC function with the ultimate aim to identify novel druggable targets in CMML.

Background and Translational Importance: CMML is associated with an adverse outcome and lack of
effective treatments. The genetic basis of CMML has been extensively studied of in the last few years
leading to the discovery of a number of recurrent driver mutations within epigenetic modifiers,
spliceosome apparatus and cell signalling pathways (Patel, B. J. et al. Leukemia 31, 2815–2823, 2017).
However, few patients have a mutation which is directly targetable with small molecule inhibitors.
Furthermore, the genetics of CMML does not explain many aspects of the disease phenotype, with many
CMML patients carrying the same mutations but with markedly heterogeneous clinical presentation and
disease course. This supports that additional epigenetic or cell extrinsic factors are important in shaping
CMML pathogenesis. CMML causes a dysregulation of the immune system leading to a chronic state of
inflammation that can be detected through the presence of increased levels of proinflammatory cytokines
and chemokines in patient serum (Niyongere, S. et al. Leukemia 33, 205–216, 2019). It is not known exactly
which cells are responsible for the production of these inflammatory mediators, but monocytes in CMML
patients develop a proinflammatory transcriptome with enrichment for cytokine and chemokine signalling,
making them a likely candidate (Franzini, A. et al. Blood Adv 3, 2949–2961, 2019). Furthermore, the most
frequent driver mutations in CMML are inactivating mutations of TET2, a known regulator of inflammation
(Zhang, Q. et al. Nature 525, 389–393, 2015).
Single cell sequencing and single cell culture have identified early haematopoietic stem and progenitor cells
(Lin-/CD34+/CD38- HSPCs) as being the disease initiating cells and the source of myelomonocytic
differentiation bias (Itzykson et al. Blood 121, 2186–2198, 2013 and Wiseman et al. EBioMedicine 58,
102904, 2020). These cells however do not exist in isolation but are surrounded by a complex architecture
of multiple different haematopoietic and stromal cells as well as extracellular matrix molecules. HSPC
function is profoundly influenced by ligand-receptor interactions with this complex BM microenvironment
in both health and disease, for example, in regulating the ‘stress haematopoiesis’ response to inflammation
(Batsivari et al. Nat Cell Biol 22, 7–17, 2020). Stress haematopoiesis causes progenitor cells to preferentially
differentiate to myelomonocytic cells and suppress red cell production, paralleling key aspects of the
CMML phenotype. Cell-cell interactions have also recently been identified as an important cancer specific

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mechanism promoting leukaemic stem cell quiescence and treatment resistance in chronic myeloid
leukaemia (Rothe et al. Cell Stem Cell 27, 110–124.e9, 2020). We therefore hypothesise that crosstalk
between mature myelomonocytic cells and HSPCs is an important driver of CMML pathogenesis. The
molecular and cellular components of this crosstalk would make excellent targets for treatment of CMML.
Indeed, therapies targeting the microenvironment in MDS such as luspatercept are now entering routine
clinical practice.

Project outline: Work package 1 will focus on identifying aberrant interactions between mature
myelomonocytic cells and HSPCs in TET2 mutant CMML by using the high throughput microfluidic-based
Chromium platform (10x Genomics) to perform single cell sequencing of all BM mononuclear cells. We
have access to 14 well-annotated TET2 mutant CMML patient BM samples via the Oxford based HaemBio
biobank. We will analyse a total of 42000 cells from 9 CMML samples with TET2 mutations and 3 age-
matched controls. We will use hashtag antibodies to process 6 samples simultaneously and improve cost
efficiency. This will produce a comprehensive dataset for further analysis. Using SingCellaR, a
computational pipeline for single cell analysis developed by our collaborator Supat Thongjuea, we will
comprehensively characterise the cellular landscape in CMML alongside age-matched healthy donor
controls, thereby cataloguing all distinct haematopoietic clusters in normal BM and CMML. We will then
apply MONOCLE to define differences in the myeloid and erythroid differentiation trajectories and key
branch points in normal versus CMML. We will then apply SCINDY, a novel single cell interactome analysis
package developed by the Mead lab, to identify putative ligand-receptor interactions that might promote
aberrant myeloid differentiation in CMML at the expense of normal haematopoietic development. SCINDY
also ranks interactions that are most likely to be biologically relevant by searching publicly available
databases. The most promising 30 will be investigated by literature review to choose 10 candidates for
further analysis. The Mead Laboratory is experienced in the application of single cell techniques to study
malignant haematopoiesis (e.g. Giustacchini et al, Nature Medicine 23, 692-702, 2017 and Rodriguez-Meira
et al, Molecular Cell 73,1292-305, 2019).
In Work package 2 we will validate these candidate interactions at the protein level through the use of
mass cytometry (CyTOF) and imaging CyTOF, CyTOF analysis is established as a technique in the Mead
laboratory (Psaila et al, Molecular Cell 78, 477-92, 2020). We will use this to confirm that putative ligands
and their receptors are present at the protein level in greater quantity within expanded cell populations in
CMML samples and explore whether downstream signalling cascades are triggered by targeting phosphate
specific epitopes on downstream molecules. We will also use imaging CyTOF, to visualise cell membrane-
based ligand/receptor interactions. This will be performed on BM trephine samples that will be obtained
through recruitment of CMML patients from Oxford’s large haematology patient population. By using
histological BM samples we will gain architectural information about which cells reside in close proximity
to HSPCs in CMML adding a crucial additional layer of validation to prioritise key cell-cell and receptor-
ligand interactions.
In Work package 3 we will conduct additional functional validation of the candidate interactions and
explore direct link to TET2 dysfunction. We have previously developed a primary cell culture assay for
differentiating HSPCs from CMML patient samples in semi-solid methylcellulose (colony forming assay) and
liquid media to give detailed readouts of HSPC differentiation. We and others have found that early
CD34+/CD38- HSPCs are primed to myelomonocytic differentiation in CMML but this is not found in more
mature C34+/CD38+ HSPCs. We will adapt this to include co-culture of HSPCs with mature myelomonocytic
cells from CMML patients or control samples to investigate whether this alters HSPC function. We will also
use this as a model to test our candidate interactions by adding additional ligand to the media or using
targeted inhibition/ligand-trap (if available) to recreate changes seen on co-culture. We will then perform
CRISPR knockdown of candidate ligand/receptor interactions in CMML patient samples to restore
differentiation to normal. Finally, to validate direct role of TET2, we will perform enhanced reduced
representation (eRR) methylation sequencing (MethylSeq), using TAPS techniques to generate libraries
that discriminate between unmethylated cytosine, 5-methylcytosine and 5-hydroxymethylcytosine. We
will look for differential methylation between TET2m progenitors and normal controls, to see if this may
be the mechanism of altered gene expression. To ascertain the role of TET2 in modulating of gene
expression via chromatin accessibility, we will perform ATAC-seq (as a proxy measure of active or
repressive histone marks). These techniques are established in the Mead/Quek laboratories.

74
 Mathematics of Lymphoma Immunotherapies: Application of
Mechanistic Models to Accelerate and De-Risk Therapeutic
Development for Blood Cancers – Prof. Coles1,2,3A,3B
Primary Supervisor: Mark Coles
Additional Supervisors: Eamonn Gaffney
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
Lymphoma is mostly a disease of older age diagnosed in over 15,000 people in the UK each year and
although many people respond to first line chemo and radiotherapy a significant proportion of
patients fail to respond effectively in the longer term. Although antibody based therapeutics (e.g. anti-
CD20) have had a profound effect on patient treatment, longer term outcomes can be poor. More
recently activating immunotherapies, including CART (Chimeric Antigen Receptor T) cells and bi-
specific antibodies (dual specificity antibody therapies) have been developed for non-Hodgkin’s
lymphoma. However, understanding dosing, efficacy and the management of immune related adverse
events, as well as the potential for therapeutic synergy, is clinically challenging and limiting
development and application of these therapies in the clinic. Additionally, how to effectively translate
from pre-clinical animal models to human disease is unclear. We have previously developed
physiological scaled computational models of human, primate and rodent immune cell dynamics to
understand human CART cell dosing and we are currently expanding this work to understand CART
cell and tumour cell dynamics in lymphoma. In this proposal, we will extend these models to capture
the dynamics of endogenous CTLs in both mouse lymphoma models and human patients to determine
optimal strategies for bi-specific and combination therapies to optimise the therapeutic window for
next generation therapeutics in non-Hodgkin’s lymphomas.

Preliminary Data leading to the project: There is an explosion of antibody (including bi-specifics),
small molecule and cell based therapeutics targeting haematological cancers and solid tumours,
leading to a combinatorial explosion in possible treatments and schedules, to the extent that there
are insufficient patients to adopt classical clinical trial approaches to distinguishing the most
appropriate treatment for the most appropriate patient. Consequently, there is an extensive interest
in, and demand for, novel technologies aimed at improving the exploitation of current and future
clinical data to facilitate evidence-based and rationalised decision-making in immuno-oncology. In
haematological disorders the mechanism of action of next generation activating immune-therapeutics
requires enabling cytotoxic lymphocytes or CART cells to kill target cells. This is dependent on
dynamics and interactions with the target cells and thus the pharmacology of the drug (biologic or
CART) is dependent on the trafficking of immune cells. This process is key for immunosurveillance of
malignant haematopoietic cells: human splenectomy shows a significant increased risk of
haematological disorders including non-Hodgkin’s lymphoma, indicating the key role of lymphocyte
circulation and the spleen in protection against human haematological disorders (Fig1). To model
immune cell dynamics a set of ordinary differential equations (ODEs) were developed for T cell
trafficking in the circulatory and lymphatic systems (Fig2) (Brown et al., bioaRxiv, doi:
https://doi.org/10.1101/759167 2020). Although cellular therapies including CART cells have shown
clinical efficacy in leukaemia and lymphomas, these therapies are delivered in lymphocyte depleted
patients and marked by a 100-1000-fold initial cellular expansion. In contrast, activating
immunotherapies (e.g. CD20-CD3) are delivered in replete patients and thus trafficking rates of
endogenous CTLs to the tumour site are a potential rate-limiting event. Through parameterising the
simulation across different species, utilising physiologically relevant datasets, has permitted
identification of how T cell trafficking is non-allometric across species due to physiological differences
in blood flow. We have validated model predictions using PET/radiological imaging data. To capture
CTL function, we have built models incorporating spatial aspects of killer T cell–target cell interactions,

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thus permitting exploration of tumour microenvironmental changes. Together these technologies
provide underpinning technology development to build models of next generation therapeutic
approaches for DLBCL.

Research Objectives: In this project we will develop a quantitative computational model to explore
next generation treatments for DLBCL focusing on bi-specifics and combination immunotherapies.
Specifically, in the PhD project the student will:
Objective 1: Develop a computational model of CTL trafficking of human and mouse parameterised
using a combination of next generation PET data (human) and Kaede florescent protein (KFP) photo-
labelling and tracking of CTLs (mouse), permitting quantitative understanding of CTL dynamics in
health and lymphoma. KFP labelled cells will be tracked using confocal microscopy and flow cytometry.
Objective 2: Incorporate therapeutic pharmacology and mechanistic biology for bispecific and check-
point inhibitors targeting T cells into the simulation to model temporal DLBCL cancer cell dynamics
during treatment resulting from T cell mediate killing and inhibit CTL anergy.
Objective 3: Develop virtual patient cohorts to understand variability in response and adapt the
computational models to reflect anatomic differences in (NOD/SCID/GAMMA) mouse models to
better understand translation from commonly use murine lymphoma models and human disease.
Expected outcomes: Outcomes from this PhD will be a mechanistic QSP model of DLBCL that permits
exploration of animal model data in the context of human disease and can be used to simulate mono
and combination immunotherapies for DLBCL. These simulations will be used to identify improved
treatments using neural network based genetic evolutionary algorithms (Alden, K et al. EEE/ACM
Trans Comput Biol Bioinform, doi: 10.1109/TCBB.2018.2843339 2020).
Translational potential: Through working with pharmaceutical industry we aim to have direct impact
on accelerating therapeutic approaches, providing an evidence-based approach for combination
therapeutics, helping to optimise clinical trial design and identify right patients for different treatment
combinations.
Role of collaborations: This interdisciplinary project will involve close collaborations with scientists in
Oxford, Cardiff and Birmingham to develop a model of non-Hodgkin’s lymphoma treatment, bringing
together key expertise in oncology, immunology and mathematics.
Relevance to Patient Care: This project will work with primary human clinical trial datasets with the
potential to directly impact on patient care. The ultimate long term goal of this approach will be to
enable personalised delivery of immunotherapies for lymphoma and to accelerate translation from
lab to clinic.

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 Pre-clinical testing of a novel mitochondrial inhibitor (NBS037) in
combination with radiotherapy and immunotherapy – Prof. Higgins
1,2,3A
Primary Supervisor: Geoff Higgins
Additional Supervisors: Karl Morten
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.
Project Summary
The use of radiation in the treatment of cancer has been in existence for nearly a century. Important advances
in radiotherapy (RT) technology have led to more precise physical targeting of the tumour to increase efficacy.
The identification and exploitation of biological determinants that specifically enhance tumour radiosensitivity
is an important clinical objective. Mitochondria are thought to play a critical but not fully understood role in the
response to RT. One important variable in the repair of DNA is the ‘oxygen enhancing effect’. Long lasting
damage is enhanced by the intra-tumoral level of oxygen which reacts with broken DNA strands creating stable
and difficult to repair peroxides structures. Although it is well known that solid tumours typically have a poorly
developed vasculature and a reduced presence of oxygen (hypoxia), overall tumour oxygenation is a balance
between delivery (vasculature) and consumption (oxygen consumption rate, OCR) through mitochondrial
respiration. While much research is ongoing looking to target and normalise the tumour vasculature, targeting
of cancer cell mitochondria is an area that has only recently received greater attention. Thus, one of the more
critical aspects of hypoxia is it renders cancer cells less responsive to cancer therapies resulting in markedly
worse clinical outcomes. Furthermore, hypoxia induced changes in the tumour microenvironment (TME) create
a well described immunosuppressive state. Thus, identification of agents that reduce tumour hypoxia may
enhance radiosensitivity and T cell anticancer function as well as current immune checkpoint blockade
strategies. We recently reported on the ability of a select group of compounds which target both mitochondrial
respiration and tumour hypoxia (1). This work demonstrated that pharmacological inhibition of cellular oxygen
consumption reversed hypoxia and caused a tumour growth delay when combined with RT. The objective of
these proposed investigations is to further explore and understand the mechanistic basis of the cooperation
between mitochondrial functional inhibition and radiotherapy sensitivity in the first part and then to examine
and determine if this same functional inhibition can sensitise to immune checkpoint inhibition.

Figure 1. EF5 stain, a marker of hypoxia

is shown using HCT116 (a) and H1299
(b) spheroids treated with NBS037 for
48hr. Spheroid diameters (c) are similar
compared to controls. (d) EF5 (green)
stained spheroids untreated (top) and
treated (bottom). Nuclei (blue) stained
with Hoechst stain.

Figure 2. EF5 probe hypoxia levels (a)

control vs ETC inhibitor treated
tumours. (b, c) Tumour survival curves.
Treatments: ETC inhibitor (ETCi),
Checkpoint blocking therapy (aPD-L1), T
cell depletion by anti-CD8 antibody
(aCD8).
Project Proposal: We have been working with an experimental compound in preclinical development called
NBS037 (Mitox Therapeutics) whose activity is based on a modified antibiotic which binds and inhibits the
mammalian mitochondrial ribosome. The compound possesses tumour-specific mitochondrial accumulation
through (i) incorporation of a delocalized cation targeting group (2) and (ii) the reported higher mitochondrial
membrane potentials in cancer cells (3, 4). Disruption of mitochondrial ribosomal translation interferes with
electron transport chain (ETC) protein expression, activity, and oxygen consumption. We have preliminary data
(Figure 1) showing hypoxia levels from NBS037 treated colon (fig 1a, 1c-top graph, 1d) and lung (fig 1b, 1c-
bottom) tumour spheroids are reduced in a dose dependent manner (fig 1a, b). Importantly, spheroid size is
mostly unaffected (fig 1c), consistent with hypoxia reversal due to reduced OCR.

77
Objectives and Proposed Outcomes:
Objective 1: We have been supplied with NBS037 through a collaboration with Mitox Therapeutics. NBS037 is
presently under pre-clinical development but has already shown to effectively target ETC protein expression
both in vitro and in vivo as well as OCR in vitro. The studentship will support investigative studies using NBS037
to demonstrate radiosensitisation. Three dimensional (3D) in vitro and in vivo models will be employed. In vitro
tumour spheroids, a 3D model of nutrient-restricted tumour-like conditions will be used in these studies and
cultured across a range of nutrient restricted tumour-like conditions to examine responses to the inhibitor.
Oxygen consumption rates in 2D cultures, hypoxia induction and reversal will be examined. In vivo studies will
assess tumour growth and growth delay. Tumour tissue will be examined and analysed using a hypoxia specific
probe for its development and reversal under treatment. Additional experiments: examine DNA damage and
repair to assess for treatment-induced increases, Colony formation assays in 2D cultures to determine whether
NBS037 potentiates IR independently of hypoxia, compound toxicity/tolerability studies in vivo.
Objective 2: Cancer immunotherapy using checkpoint blocking strategies is an exciting breakthrough in the
treatment arsenal of clinicians but is limited in effectiveness due to an immunosuppressive TME. One of many
studies that highlights these effects, Najjar et al (5) showed that tumour hypoxia inhibited T cell activity.
Furthermore, deregulated tumour cell oxidative metabolism, not glycolytic metabolism was associated with
hypoxia-induced T cell exhaustion and decreased immune activity. Importantly, their analysis of patient samples
identified oxidative phosphorylation (OXPHOS) metabolism as being associated with disease progression during
PD-1 blockade. It has been demonstrated using either oxygen supplementation (6) or a hypoxia activated pro-
drug strategy (7) that responses to anti-PD-1 or anti-CTLA-4 can be enhanced in pre-clinical models. However,
these strategies are poorly translatable to the clinic and a more direct strategy using small molecule inhibitors
is needed. In preliminary studies from our laboratory using a small molecule ETC inhibitor (referred to as ETCi)
we identified reduced hypoxia signal in tumour tissue (figure 2a). Furthermore, this agent was effectively
combined with anti-PD-L1 blocking therapy as a significant growth delay was observed compared to anti-PD-L1
or ETCi treatments alone (figure 2b). The growth delay was most likely a specific effect of T cells as it was lost
with anti-CD8 induced T cell depletion. Furthermore, combination treated mice with complete tumour
regression showed no tumour growth when re-challenged (figure 2c). An effect not observed in treatment naïve
mice or inoculation with a different murine cell line (4T1) strongly suggesting the development of a memory,
long-lasting and specific anti-tumour immune response. The studies proposed herein will extend these
investigations by focusing on the interaction between anti-PD-L1 treatment and ETC inhibition by a
mechanistically different inhibitory agent, NBS037.
In our preliminary studies with the ETCi, we found no direct effect on T cell activation or effector function,
however rotenone and antimycin A, both potent ETC inhibitors, were shown to suppress T cell activation (8). We
will expose stimulated splenocytes to NBS037 to determine if T cell activation is inhibited or potentiated by the
compound. Growth inhibition and OCR levels will be examined in vitro using the well-established syngeneic
murine cancer models CT26 and MC38 models to determine sensitivity to NBS037. Then using the CT26 and
MC38 models in vivo, these investigations will seek to determine if NBS037 synergizes with anti-PD-L1 therapy
to produce a growth delay or regression. Anti-CD8 antibody will be used as a co-treatment in these studies to
deplete cytotoxic T lymphocyte and verify CTL specificity through rescue of a growth delay if it exists. Tumour
tissue will be analysed using the EF5 probe to determine if NBS037 causes a reversal of hypoxia. As treatment
with a mitochondrial inhibitor could potentially alter the TME and affect other immune cell types, we will profile
tumour immune infiltrates using RNAseq, whose analysis will help predict the quantities of specific immune cells.
Flow cytometry analysis will be used to verify the RNAseq analysis of immune infiltrates and to assess the levels
of tumour-reactive T cells using CT26 and MC38 antigen-specific tetramers.

Translational Potential: The successful development of a tumour-specific radiosensitizer would lead to

substantial benefits in cancer therapy due to enhanced therapeutic indices. Demonstrating the efficacy of a new
therapeutic strategy in combination with radiotherapy and immunotherapy will be important first steps prior to
planned clinical studies with NBS037.

References: (1) Ashton, T. M., et al. (2016). Nat Commun 7: 12308. (2) Ross, M. F., et al. (2008). Biochem J 411(3): 633-645. (3) Houston, M.
A., et al. (2011). Int J Cell Biol 2011: 978583. (4) Bonnet, S., et al. (2007). Cancer Cell 11(1): 37-51. (5) Najjar, Y. G., et al. (2019). JCI Insight
4(5). (6) Hatfield, S. M. et al. (2014) J Mol Med (Berl) 92. (7) Jayaprakash, P. et al. (2018) J Clin Invest 128, 5137-5149. (8) Chang, C. H. et al.
(2013) Cell 153, 1239-1251.

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 Mechanisms of therapeutic response and resistance to BCMA-
directed therapy in multiple myeloma- a systems biology approach
– Prof. Oppermann1,2,3A,3B
Primary Supervisor: Udo Oppermann
Additional Supervisors: Karthik Ramasamy, Adam Cribbs , Sarah Gooding , Martin Philpott
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
Multiple myeloma (MM) is a cancer of aberrant immunoglobulin-secreting plasma cells
residing in the bone marrow. Myeloma recurrently relapses during its clinical course, with
inevitable evolution to a fatal multidrug-resistant phenotype. Although MM survival rates
have improved due to the availability of new drug classes including immunomodulatory drugs
and proteasome inhibitors, patients eventually become resistant to these therapies. Although
several novel drug classes are currently investigated, it is only incompletely understood why
effective drug molecules only work in subsets of patients. The next wave of therapeutics is
targeted against BCMA (B cell Maturation antigen). CART cells, bispecific antibodies and
antibody drug conjugates targeting BCMA are in clinical trials.
One such novel therapeutic agent is Belantamab Mafodotin, an antibody-drug conjugate that
targets B-cell maturation antigen (BCMA), an essential myeloma surface protein, approved by
the FDA recently. Clinical data is encouraging however, a high proportion of patients do not
respond, and drug resistance develops in a fraction of patients. The proposed DPhil project
will investigate in depth the mechanisms underlying Belantamab Mafodotin drug resistance
using a systems-based approach to correlate genomic, epigenomic and proteomic data in ex
vivo systems and patient derived myeloma samples (including banked sequential samples
from Belantamab treated patients). This will inform on patient populations that are likely to
benefit from a targeted therapy.
Research Objectives and Outcomes: Multiple myeloma (MM) is the second most common
haematological malignancy and is a clonally heterogeneous cancer of antibody-producing
plasma cells residing in the bone marrow. Therapeutic outcomes depend upon underlying
genomic and mutational alterations besides evolution in the clonal landscape following
exposure to sequential therapy combinations. MM is currently incurable, and it is suggested
that the combination of therapy-driven clonal evolution together with the ability of malignant
plasma cells to adopt a reversible, microenvironment-dependent dormancy are fundamental
reasons why even deep remissions inevitably relapse. It is known that lower minimal residual
disease (MRD) levels following induction and consolidation therapy correlate with longer
progression free and overall survival. In order to advance towards deeper remissions and
potentially towards a cure, suitable approaches to identify optimal patient populations for a
given treatment and strategies for targeting persistent and therapy resistant tumour cells are
required.
B-cell maturation antigen (BCMA, CD269, TNFRSF17) is a member of the TNF-receptor
superfamily and is preferentially expressed in mature B-lymphocytes and malignant plasma
cells. The receptor binds to its ligands BAFF and APRIL, leading to NF-kappaB and MAPK8/JNK
activation and thus may transduce signals for cell survival and proliferation. Belantamab
Mafodotin (GSK2857916) is a novel antibody-drug conjugate (ADC) that targets BCMA, and
upon receptor binding and internalisation displays strong cancer cell responses mediated in

79
part by its chemotherapeutic drug conjugate auristatin, a microtubule-targeting agent. The
responses to single agent Belantamab Mafodotin therapy in relapsed myeloma are very
exciting, showing an exceptional overall response rate of 60% in a Phase 1 study in an
extensively pre-treated myeloma patient population1. This monotherapy is a step change in
available therapeutics for MM. However, responses are durable only for a proportion of
patients: some patients are primary refractory to this agent whilst some patients respond but
eventually relapse. Accordingly, the mechanisms driving both response and relapse require
systematic scientific evaluation.
Objectives and proposed work: in order to investigate the mechanisms underlying
therapeutic response and resistance to BCMA-ADC therapy, a two-tiered approach will be
taken. Our laboratory has established a defined set of human myeloma cell lines which
represent distinct genomic aberrations and reflect the main determinants of risk status found
in myeloma patients. Isogenic wild-type and drug-resistant cell lines are available, including
various proteasome inhibitor (PI; bortezomib, carfilzomib) and immunomodulatory drugs
(IMiD; lenalidomide, pomalidomide), currently the main myeloma therapeutics. These cell
lines have been extensively characterised using a systems approach including transcriptomic
(RNAseq), proteomic (including phosphoproteomic and ubiquitomic, as well as mass
cytometry) and epigenomic (ATACseq, Hi-C, CHIPseq) techniques. Single-cell data are
currently generated to understand the clonal trajectories underlying PI and IMiD resistance.
In this DPhil project, the student will first use these well-characterised tools to investigate the
responses to Belantamab Mafodotin using the suite of techniques described above. Selected
cell lines will then be used to generate BCMA-ADC resistance and will also include
experiments with the chemotherapeutic payload auristatin as single agent. Collectively this
will deliver essential ex vivo information on candidate pathways that define therapeutic
responses to BCMA-directed drugs. By investigating bone marrow samples from patients
treated with Belantamab Mafodotin using the systems approach detailed above, the student
will be in a position to correlate ex vivo data with patient data. Moreover, single-cell readouts
(single cell seq and mass cytometry) used for the patient bone marrow assays will provide
important datasets and information on the interactions between myeloma cells and the bone
marrow immune environment.
Outcome: Data generated in this project will inform on mechanisms of response and
resistance and mode of action of a novel therapeutic in myeloma. The project will provide the
student with state-of-the art systems approaches to investigate drug resistance. It is
anticipated that the successful student will be trained to advance a future career as clinician
scientist or as biomedical researcher in academia or industry.
Translational Potential: This systems approach will deliver new information on how primary
and acquired drug resistance against a novel therapeutic develops by defining the dynamic
changes in the integrated myeloma cancer and bone marrow microenvironment. The
generated large data sets will be instrumental to identify mechanisms underlying the
pathways that determine both primary resistance and which are altered during the
acquisition of drug resistance. This information is essential to (i) select the appropriate patient
population that will best respond to therapy and furthermore (ii) will provide target driven
hypotheses to possibly overcome acquired drug resistance to BCMA targeted therapeutics.
References: 1. Trudel et al Lancet Oncol 2018, 19(12): 1641–1653. doi: 10.1016/S1470-2045(18)30576-X

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 Personalised monitoring intervals for cancer surveillance – Dr
Oke1,2,3A,3B
Primary Supervisor: Jason Oke
Additional Supervisors: Rafael Perera, Brian Nicholson , Richard Hobbs
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary:An increasing proportion of precancerous conditions and less advanced

cancers are being managed conservatively with surveillance rather than immediate treatment
and an increasing number of cancer survivors are being monitored for recurrence. Current
surveillance protocols suggest periodic testing to monitor for disease progression. Whilst the
evidence for which tests to use is of higher quality, the evidence for the timing of surveillance
intervals is often weak with recommendations involving convenient periods of time e.g. every
6 months or annually. Guidelines rarely incorporate patient level risk factors or offer clear
guidance on when surveillance could cease. For example, active surveillance for localised
prostate cancer involves monitoring changes in PSA or PSA velocity every 3 to 4 months for
the first year and every 6 months thereafter (1). We propose the development of a dynamic
method to derive personalised monitoring intervals based on the patient’s current risk.

Research objectives and proposed outcomes

The aim of this project is to develop a statistical monitoring method that could be applied to
a wide range of clinical areas including cancer recurrence. We illustrate how the evidence for
the frequency of monitoring is often underdeveloped with two examples; the management
of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Barrett's
Oesophagus. According to the British Society of Gastroenterology guidelines (2) on the
diagnosis and management of Barrett’s oesophagus monitoring should take into account the
presence of intestinal metaplasia (IM) and length of the Barrett’s segment with patients with
Barrett’s oesophagus shorter than 3 cm, with IM, should receive endoscopic surveillance
every 3–5 years and patients with segments of 3 cm or longer should receive surveillance
every 2–3 years but the quality for the evidence for monitoring is considered low. Similarly,
the British Journal of Haematology guidelines for the investigation of newly detected M-
proteins and the management of MGUS (3) states that there is “no published evidence on
which to base recommendations for the frequency of follow-up and guidance is, of necessity,
pragmatic but should seek to take into account information which is known about risk factors
for progression and patterns of progression”. After a review of the current evidence and the
selection of target applications, the candidate will focus on developing an algorithm that is
capable of effectively stratifying a patient by their risk of progression or recurrence and then
using this information to inform when to re-measure or cease monitoring. Fundamentally,
the more likely the immediacy of risk the more closely we should check the patient’s condition
and conversely, if the risk of an event is low then intervals can be safely be extended and
eventually stopped. Validation would follow model development and as we currently don’t
know the best way to do this, this part of the project would present an opportunity for the
development of novel methodology.

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Academic Value
This DPhil studentship has broad relevance to monitoring of precancer and the follow-up of
cancer survivors. It addresses two of the strategic scientific themes of the Oxford Centre: Big
Data and blood (e.g. MGUS monitoring for progression to myeloma) and digestive cancers
(e.g. Barrett's oesophagus monitoring for oesophageal cancer, CEA monitoring for colorectal
cancer recurrence). As this is a methodological project we have two supervisors with
statistical expertise (Prof Perera and Dr Oke) and two supervisors who are academic GP with
expertise in risk prediction modelling (Dr Nicholson and Prof Hobbs). Prof Hobbs and Prof
Perera has supervised many DPhil students to completion. Dr Oke is currently supervising 3
DPhil students and taken one student to completion.

Collaborations
This project will be based in the Nuffield Department of Primary Care Health Sciences but we
would expect the candidate to further already established collaborations with experts from
oncology and laboratory medicine at Oxford University Hospitals Trust once appropriate
clinical areas have been agreed.

Translational potential of the project

This project has great potential to impact patients and the NHS. More effective monitoring
would reduce opportunities for unnecessary harm from overtesting and to focus health
system resources to scenarios where monitoring is most likely to produce measurable clinical
benefit.

References: 1.National Institute for Health and Care Excellence. NG131: Prostate cancer:
diagnosis and management. BJU Int. 2019/06/18 ed2019. p. 9-26. 2.Fitzgerald R, di Pietro M,
Ragunath K. New British Society of Gastroenterology (BSG) guidelines for the diagnosis and
management of Barrett's oesophagus. Gut. 2006;55(4):442. 3.Bird J, Behrens J, Westin J,
Turesson I, Drayson M, Beetham R, et al. UK Myeloma Forum (UKMF) and Nordic Myeloma
Study Group (NMSG): guidelines for the investigation of newly detected M-proteins and the
mana

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 Presentation, Diagnosis and Outcomes of Hodgkin Lymphoma: the
role of Primary Care – Prof. Bankhead1,2,3A,3B
Primary Supervisor: Clare Bankhead
Additional Supervisors: Graham Collins, David Cutter, Toby Eyre, Richard Hobbs, Brian
Nicholson, Rafael Perera
Eligibility: Track 1, 2, 3A and 3B students are eligible to apply for this project.

Project Summary
Lymphoma is the 5th commonest cancer in the UK and is broken down into 2 distinct types:
Hodgkin (HL) and Non-Hodgkin lymphoma (NHL). Significant challenges exist within primary
care, to recognise and refer a patient with lymphoma. These include:
• The demographic of patients. Classical Hodgkin Lymphoma is the commonest cancer
in the teenage and young adult (TYA) population with a peak incidence in the 15 to
35-year-old age group. Diagnosis in younger patients can take longer, with more GP
consultations before referral to a specialist (Furness et al, 2017).
• Both NHL and HL are far less common that the ‘big 4’ cancer types of lung, breast,
bowel and prostate limiting the experience of primary care physicians in recognising
these conditions
• Lymphoma frequently has a non-specific symptom profile with the lack of a specific
blood test (or other diagnostic test) to facilitate a diagnosis in general practise (Howell
et al, 2019).

The Nuffield Department of Primary Care Health Sciences (NDPCHS) has direct access to
linked primary and secondary care electronic health records data resources, including the
Clinical Practice Research Datalink (CPRD), QResearch, OpenSafely and the Oxford Research
Oxford and Royal College of General Practitioners Clinical Informatics Digital Hub
(ORCHID). These longitudinal data sources include millions of patients. The supervisory team
also have access to linked routinely collected datasets from Public Health England for all
lymphoma patients diagnosed nationally from 1997 to 2017 including cancer registration,
hospital episode statistics, cancer waiting times, radiotherapy (RTDS), systemic anti-cancer
therapy (SACT), diagnostic imaging, cause of death and linked cardiovascular databases via
the Virtual Cardio-Oncology Research Institute (VICORI). These data sources may be
harnessed to address specific research questions to improve the diagnosis of lymphoma and
the management of lymphoma survivors in primary care.
This DPhil project aims to generate evidence to address all or some of the following research
questions:

1. What is the potential role of simple currently available blood tests available in primary
care and the potential role of more specialist blood tests (e.g. serum CCL17 or TARC)
in lymphoma diagnosis?
2. What are the early signs and symptoms of lymphoma, and how can they be identified
and acted upon earlier?
3. How are symptoms of lymphoma, such as non-resolving lymphadenopathy, managed
in primary care?
4. How are routes to diagnosis, and treatments received, associated with clinical
outcomes?

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 5. Does the early diagnosis of lymphoma improve outcomes?
6. What are the long-term health and psychological impacts of lymphoma treatment,
and how can they be managed?

This is likely to involve a combination of the following methods developed with the successful
DPhil candidate:
• Cohort and case-control studies utilising large linked routinely collected health care
datasets
• Evidence synthesis to identify potential biomarkers of use in the diagnosis of
lymphoma, and their effectiveness
• Analysis of early laboratory research and clinical records of emerging diagnostic or
prognostic tests

Research objectives and proposed outcomes

There is scope to develop the research objectives and outcomes within the broad framework
of the proposed project. Essentially the objectives are likely to include: utilising routinely
collected data to explore the utility of blood and other tests, or combinations of symptom
profiles to identify lymphoma with the aim to shorten the diagnostic interval.
The team have extensive contacts and collaborations within the cancer research and clinical
arena, including both primary and secondary care and have excellent access to large routinely
collected healthcare data (CPRD, OpenSafely, ORCHID, QResearch) and registry data (Deaths,
Cancer Registrations and specialist Registers). NDPCHS is a member of the NIHR School for
Primary Care Research which is a national collaboration of the leading Departments of
Primary Care, including close links with Exeter University who also have a focus on cancer
research.

Translational potential of the project.

Harnessing large routine and research datasets to identify signature patterns prior to
diagnosis would be translatable to tangible use in clinical care, in a relatively short time
period. Earlier diagnosis has been identified as of importance to patients and may be
associated with less intensive chemotherapy and radiotherapy with fewer short and life-long
detrimental consequences such as psychological consequences, reduced fertility, increased
risk of second malignancies and chemotherapy induced cardiomyopathy. In addition,
identification of the workload placed on primary care services from patients treated with
lymphoma can lead to proper planning and resourcing of care.

References: Furness, C.L., Smith, L., Morris, E., Brocklehurst, C., Daly, S. & Hough, R.E. (2017)
Cancer Patient Experience in the Teenage Young Adult Population— Key Issues and Trends
Over Time: An Analysis of the United Kingdom National Cancer Patient Experience Surveys
2010–2014. Journal of Adolescent and Young Adult Oncology, 6, 450–458. Howell, D.A., Hart,
R.I., Smith, A.G., Macleod, U., Patmore, R. & Roman, E. (2019) Disease-related factors
affecting timely lymphoma diagnosis: a qualitative study exploring patient experiences.
British Journal of General Practice, 69, e134–e145

84
 The impact of microenvironmental components on cellular
plasticity in colorectal cancer – Prof. Buczacki1,2,3A
Primary Supervisor: Simon Buczacki
Eligibility: Track 1, 2 and 3A students only are eligible to apply for this project.

Project Summary
Colorectal cancer (CRC) is the third commonest solid organ cancer in the world and contributes to
significant morbidity, mortality and related healthcare costs. Considerable advances have been made
in the understanding of CRC within the last decades. Modern management of CRC uses a combination
of surgical resection, chemotherapy or radiotherapy. Despite, progress in these domains, recurrence
of tumour (either locally or at distant locations) is common and contributes to poor outcome. It is
postulated that cancer stem cells (CSCs) underlie this process of recurrence by fuelling regrowth,
mediating resistance to anti-cancer therapies and seeding metastases [1, 2]. These CSCs therefore are
important potential therapeutic targets.

In spite of the well-accepted Fearon-Vogelstein theory of colorectal carcinogenesis [3], emerging data
shows colorectal cancer to be heterogenous, both in terms of mutational background and cellular
identity (stem cell versus differentiated) [4]. While targeting CSCs could in theory reduce disease
recurrence and progression, recent evidence suggests that certain subpopulations of differentiated
cancer cells retain a context dependent ability to revert back to a stem cell like identity that is
independent of mutational background [5, 6, 7]. We hypothesise that different clonal subpopulations
may interact co-operatively or competitively to achieve these properties [8]. Further, other
components of the tumour microenvironment may also be involved in crosstalk to achieve cellular
plasticity. Recently published data strongly implicates the stroma and immune system as involved
although these reports fail to elucidate how different mutational background changes this paracrine
phenomenon [9, 10, 11].

Objectives
This project aims to experimentally understand how tumour cell mutational background and proximity
to stromal or immune populations changes cancer cell behaviour and fate.

Methodology
Tools used in the project will include fluorescence-activated cell sorting (FACS), single cell RNA
sequencing, organoid technology, flow cytometry and CRISPR targeting.

The Buczacki lab already has expertise in applying CRISPR gene editing techniques to primary colon
epithelial cell cultures grown as organoids. Combining these contemporary tools enables genetically
clean CRISPR-targeted clonal organoid cultures. By sequentially mutating common driver mutations
in normal colonic organoids we can generate a robust in vitro tool that can be combined with co-
culture to explore cellular interactions and dissect the involvement of specific mutations. Co-culture
of organoids with immune and stromal components has previously been shown technically possible
and the Buczacki lab already has performed pilot experiments to demonstrate feasibility.

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 Fig 1: Human colon organoids CRISPR
targetted to mutate TP53
This project will initially continue with the
progress in the lab in generating a CRISPR
targeted colon organoid bank containing
clonal samples containing combinations of
the common driver mutations of CRC. Next
co-culture of these organoids with both
stromal cells and components of the
immune system will be performed. Co-
cultures will be screened for phenotypes
using live cell imaging, RT-PCR and flow
cytometry. Co-cultures that show positive phenotypes will then be taken forward for multiplexed
single cell RNAseq. Single cell data will be analysed using well validated bioinformatic pipelines that
enable fate readouts (pseudotime or entropy). Results will be validated using xenograft models in
NSG mice (stroma) or humanised mouse models (immune) in collaboration with the Saeb-Parsy Lab
(University of Cambridge).

The results of the experiments described above should lead to:

1. A characterisation of molecular interactions between tumour and supporting cell populations
during early tumorigenesis
2. Robust identification of markers for cell subpopulations that possess plasticity (the ability to
revert to CSC)
3. Potential biomarkers of plasticity that may form targets for therapeutic interventions

Translational potential of the project.

As a consequence of cellular plasticity, simply targeting CSCs in a bid to reduce disease progression or
recurrence is inadequate, and instead, reserve cells need to be targeted as well. The results of this
project should lead to consistent and accurate identification of these reserve cells thereby paving the
way for novel targeted therapies. Technologies generated during the project synergise with other
ongoing projects within the Buczacki laboratory and could equally be applied to other tumour types
to explore similar interactions.

References: 1. O’Brien, C., Pollett, A., Gallinger, S. et al. A human colon cancer cell capable of initiating tumour growth in
immunodeficient mice. Nature 445, 106–110 (2007). 2. Batlle, E., Clevers, H. Cancer stem cells revisited. Nat Med 23, 1124–
1134 (2017). 3. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 61, (5):759-767. (1990) 4. Buczacki
SJ, Davies RJ. The confounding effects of tumour heterogeneity and cellular plasticity on personalized surgical management
of colorectal cancer. Colorectal Dis 16, (5):329-31 (2014) 5. Kreso A, O'Brien CA, van Galen P, Gan OI, Notta F, Brown AM, Ng
K, Ma J, Wienholds E, Dunant C, Pollett A, Gallinger S, McPherson J, Mullighan CG, Shibata D, Dick JE. Variable clonal
repopulation dynamics influence chemotherapy response in colorectal cancer. Science 339, (6119):543-8. (2013) 6. van Es
JH, Sato T, van de Wetering M, Lyubimova A, Nee AN, Gregorieff A, Sasaki N, Zeinstra L, van den Born M, Korving J, Martens
AC, Barker N, van Oudenaarden A, Clevers H. Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. Nat
Cell Biol 14, (10):1099-104. (2012) 7. Buczacki SJA, Popova S, Biggs E, et al. Itraconazole targets cell cycle heterogeneity in
colorectal cancer. J Exp Med 215, (7):1891-1912. (2018) 8. Cleary AS, Leonard TL, Gestl SA, Gunther EJ. Tumour cell
heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers. Nature 508, (7494):113-7. (2014) 9.
van der Heijden M, Miedema DM, Waclaw B, et al. Spatiotemporal regulation of clonogenicity in colorectal cancer xenografts.
Proc Natl Acad Sci U S A, 116(13):6140-6145. (2019) 10. Lee HO, Hong Y, Etlioglu HE, et al. Lineage-dependent gene expression
programs influence the immune landscape of colorectal cancer. Nat Genet 52, (6):594-603. (2020) 11. Wei C, Yang C, Wang
S, et al. Crosstalk between cancer cells and tumor associated macrophages is required for mesenchymal circulating tumor
cell-mediated colorectal cancer metastasis. Mol Cancer 18, (1):64. (2019)

86
 Investigating the role of inflammation in pancreatic cancer – Dr.
Jiang1,2,3A
Primary Supervisor: Shisong Jiang
Additional Supervisors: Eric O’Neill
Eligibility: Track 1, 2 3A and 3B students are eligible to apply for this project.

Project Summary
Pancreatic cancer is one of the most lethal type of cancers, with a five-year survival rate of less than
5%. It is usually diagnosed at an advanced stage with limited therapeutic options. There are no
efficacious treatments available for patients with advanced pancreatic cancer. This may be due to the
highly immunosuppressive tumour microenvironment conferring resistance to conventional therapy
in pancreatic cancer. There is an unmet need to
understand cellular and molecular mechanisms underlying
the immune microenvironment of pancreatic cancer so
that we can design immunotherapy that eliminates
pancreatic cancer (Ref 1 and 2).
The aetiology of PC is not clear but inflammation
plays an important part. The transcription factor NF-kB, a
driving factor for cytokine release in inflammation, is
critical in pancreatic cancer (Figure 1). Moreover, many
other factors play roles in the process of pancreatic
tumorigenesis. For example, 1) proinflammatory cytokines
such as TNF, IL-6 and IL-1 have been shown to facilitate PC.
Blocking the master proinflammatory cytokine TNF
binding to its receptors in PC has certain in vitro
advantages but clinical trials of TNF inhibitors in pancreatic
cancer have been disappointing (Ref 3 and 4). 2) Genetic
factors such as KRAS mutation may lead to inflammatory
pancreatic pathogenesis. 3) Epigenetic dysregulation such as DNA methylation or histone acetylation
is related to autoimmunity/inflammation (Ref 5) as well as pancreatic cancer (Ref 6). In general,
chronic inflammation (pancreatitis) is one of the major factors that may lead to PC.
From published and unpublished data of lab, we have discovered that among 10-30% of
healthy population (Ref 5), there exists autoimmunity against TNF (e.g. autoantibodies and/or
autoimmune T cells). In normal situation, the autoimmunity causes no pathogenesis but in TNF-related
inflammation, binding of autoantibodies to TNF forms immunocomplex which can stimulate
monocytes/T cells to produce more cytokines (e.g. more TNF). This positive feedback loop of TNF
production prevents resolution of Positive feedback
inflammation. We therefore call the
autoimmunity factors Inflammation
Enhancer (IE). IE has been found by us in
Positive feedback

TNF Agonist anti-TNF Ab

acute inflammatory disease sepsis and
chronic inflammatory disease Inflammation enhancer
rheumatoid arthritis. In both situations,
Epigenetic changes +++

NF-kB
IE deteriorated the diseases (Figure 2,
unpublished data and Ref 7). Since Inflammation
inflammation plays a major role in PC PC/PDAC(?)
development, we are keen to determine
the contribution of the core of NF-kB – Figure 2. positive feedback loop of agonist anti-TNF antibody. TNF stimulates body to
TNF pathway- related inflammation to produce agonist antibodies, these antibodies will enhance TNF activities including activation
of NF-kB leading to more cytokines including TNF secretion. Moreover, epigenetic changes
emergence of initiating PC and/or PDAC of NF-kB may facilitate inflammation and PC/PDAC.
cells.

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Research Objectives and Outcomes
Goal 1 – Establish the correlation between inflammation and PC/PDAC
Goal 2- Identify factors that affect inflammation

The student candidate enrolled in this study will not only gain in-depth understanding of inflammation
and its role in tumourigenesis of PC/PDAC but also will master the following technologies:
1. Measurement of cytokines in the patient’s samples – multiplex or ELISA assays
2. Measurement of autoantibodies against cytokines – ELISA assay
3. Measurement of apoptosis, necrosis and necroptosis – these are highly related to
inflammation so we could investigate these cell death related inflammation in pancreatic
cells. Shisong Lab has developed a good methods to measure
apoptosis/necrosis/necroptosis.
4. The candidate will be trained in the detection and bioinformatic processing of epigenetic
data (e.g. 5'methylcytosine and 5'hydoxymethylcytosine) as well as correlation with PC
subtypes and inflammatory processes in 1. 2. and 3. This will involve processing FFPE tissue,
DNA methylation analysis, illumina EPIC chip processing and bioinformatics using various R
packages including iCluster.

References
1. https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-
type/pancreatic-cancer/survival#ref-0
2. https://cancerres.aacrjournals.org/content/canres/early/2014/03/25/0008-5472.CAN-14-
0155.full.pdf
3. A multicenter, phase II study of infliximab plus gemcitabine in pancreatic cancer
cachexia. Wiedenmann, P. Malfertheiner, H. Friess, P. Ritch, J. Arseneau, G. Mantovani et al. J.
Support Oncol., 6 (2008), pp. 18-25.
4. Disrupting cytokine signaling in pancreatic cancer: a phase I/II study of etanercept in combination with
gemcitabine in patients with advanced disease.
C. Wu, S.A. Fernandez, T. Criswell, T.A. Chidiac, D. Guttridge, M. Villalona-Calero et al.
Pancreas, 42 (2013), pp. 813
5. Surace Anna Elisa Andrea, Hedrich Christian M. The Role of Epigenetics in Autoimmune/Inflammatory
Disease. Frontiers in Immunology. 10 (2019), pp.1525
6. Neureiter, D., Jäger, T., Ocker, M., & Kiesslich, T. (2014). Epigenetics and pancreatic cancer:
pathophysiology and novel treatment aspects. World journal of gastroenterology, 20(24), 7830–7848.
7. Lu W, Chen Q, Ying S, Xia X, Yu Z, Lui Y, Tranter G, Jin B, Song C, Seymour LW, and Jiang S. Evolutionarily
conserved primary TNF sequences relate to its primitive functions in cell death induction. Journal of
Cell Science 2016; 129(1): 108 – 120.
8. Herreros-Villanueva, M., Hijona, E., Cosme, A., & Bujanda, L. (2012). Mouse models of pancreatic
cancer. World journal of gastroenterology, 18(12), 1286–1294.
9. Torres MP, Rachagani S, Souchek JJ, Mallya K, Johansson SL, Batra SK (2013) Novel Pancreatic Cancer
Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic
Adenocarcinoma: Applications in Diagnosis and Therapy. PLoS ONE 8(11): e80580.
10. Jesús Espada, Manel Esteller, Mouse models in epigenetics: insights in development and
disease, Briefings in Functional Genomics, Volume 12, Issue 3, May 2013, Pages 279–287.

88
 The Epitope Abundance-Avidity-Efficacy Axis In Cancer – Prof.
Elliot1,2,3A
Primary Supervisor: Tim Elliot
Additional Supervisors: Mark Middleton, Xin Lu, Tao Dong
Eligibility: Track 1, 2 and 3A students are eligible to apply for this project.
Project Summary
The infiltration of tumours with CD8+ T cells (particularly CD103+ Resident memory CD8+ T cells) correlates
with better prognosis (1) and a positive outcome in checkpoint blockade immunotherapy (2); and
correlates with a tumour gene signature in which the antigen processing and presentation module is
upregulated (3). Furthermore, loss of expression of APM genes frequently correlates with poor outcome
(4-6); and loss of MHC I heterozygosity during tumour evolution is a marker of poor overall survival (7).
Consequently, epitopes targeted by CTL in tumours is currently a subject of fierce interest. Neoantigens,
ie epitopes encoded by tumour-specific mutations (or tumour specific post-translational peptide
modification) are emerging as crucial targets and although there is some correlation between the
mutational burden of a tumour (and therefore the theoretical number of neoepitopes), this is insufficient
to explain differences in tumour infiltration with CD8+ CTL and other markers of effective CD8+ T cell
mediated immune control []. New paradigms are emerging aimed at understanding (and predicting) the
likelihood of specific neoepitopes prompting effective antitumour CTL responses in immunotherapeutic
settings such as checkpoint blockade therapy CBT and therapeutic vaccination. These include peptide
affinity, homology to microbial peptides and probability scores for TcR recognition. Factors relating to the
antigen-processing pathway are also important and include the source and abundance of translated
products that enter the processing pathway, processing enzymes including highly polymorphic ERAP1,
competition between peptides during the selection process, and the action of tapasin, which varies
depending on HLA type of the patient. This project will investigate how these factors control the relative
abundance of different peptide:MHC complexes at the cell surface, and how this determines the hierarchy
of CTL responses in vivo. The ultimate goal is to identify events in the antigen processing pathway that
underpin the generation of CTL that are more likely to improve outcomes following CBT and therapeutic
vaccination.

Background
We have shown that the antigen presentation machinery regulates antigen abundance at steady state on
the surface of cells and that this correlates with immunodominance in a DNA vaccine, viral infection and
tumour setting. In the latter case, we increased tumour immunogenicity by increasing the abundance of
an epitope that is regulated by ERAP1.
There are many examples in the literature that indicate an inverse relationship between antigen dose and
T cell avidity (including peptide-vaccine studies in cancer patients. We have shown that peptide-specific T
cells primed to recombinant virus in mice that lack tapasin – where epitope abundance is significantly
reduced, have a functional avidity two orders of magnitude higher than T cells primed in tapasin-competent
mice where epitope abundance is much higher.
Though the superiority of high avidity T cells in infections and cancer is often asserted, other studies point
to the relevance of low-avidity T cells in controlling chronic virus infections and established tumours.
Indeed, low-avidity T cells might (a) better distinguish between tumours overexpressing self-antigens and
healthy self-tissue, (b) be less sensitive to checkpoint regulation, activation-induced cell death, senescence
and exhaustion, leading to protracted survival of functionally-competent T cells, and (c) be less likely to
induce tumour escape.
In the CT26 tumour model where the immunodominant epitope GSW11 is highly abundant, we observe
the priming of a diverse population of GSW11-specific CD8+ T cells which are suppressed or exhausted in
the tumour microenvironment. These T cells have a range of avidities, yet the low avidity clones are more
readily suppressed by Treg and their expansion (in response to either Treg depletion or immune checkpoint
blockade with anti-PD1) correlates with protection in immunotherapy experiments. Moreover, we have
found that the immunophenotype of this population, in contrast to high avidity CD8+ T cells recognising
the same peptide, resembles that of precursor exhausted T cells seen in chronic viral infection and cancer.

89
Importantly, this population has been shown to be responsive to reinvigoration by anti-PD1 CBT, unlike its
terminally differentiated counterpart.
Taken together with recent models of epitope fitness – based on their homology to microbial peptides, we
have investigated the quality of CTL responses generated in a syngeneic mouse tumour model (CT26)
where some target epitopes are derived from an endogenous retroviral glycoprotein that is not expressed
in neonatal thymus and therefore superficially resembles a neoepitope with a high quality score.
One of these epitopes (GSW11) is particularly interesting because although it has a very low affinity, it is
abundant at the cell surface and is especially sensitive to editing in the antigen processing pathway.
Previously, we have shown that epitope abundance controls immunodominance for several non-cancer
epitopes in two vaccination settings, and we have also shown that the (tumour) cell surface abundance of
GSW11 controls its immunogenicity in the CT26 model. GSW11 is sensitive to tapasin editing, and its
abundance at the cell surface increases when ERAP1 is inhibited leading to its enhanced immunogenicity.
Low affinity CTL recognising this peptide are preferentially suppressed by Treg and can be re-activated
upon Treg depletion where they become therapeutically highly effective (Sugyarto et al 2020,
Immunotherapy Advances (in press). De-suppression of the same CTL also correlates with therapeutic
efficacy in anti-PD1 immunotherapy. We have shown that these CTL have a partially exhausted phenotype
similar to those described in chronic viral infection and PD1-responsive human melanoma (Sugyarto et al,
in preparation). Taken together, these data suggest that an inverse correlation may exist between the
avidity of antitumour CTL and their quality, and furthermore that the generation of low avidity CTL may
correlate with a high abundance of epitope presented at the tumour cell surface – a parameter that is
ultimately under control of the antigen processing pathway.

Proposed Research
3.1 Proof of immunological concepts in mouse models.
The relationship between antigen dose, T cell avidity and function in the CT26 model: We have shown that
it is possible to enhance the immunogenicity of CT26 by increasing the abundance of the GSW11:Dd
complex as a SCT transgene. To test the relationship between p:MHC abundance, immunogenicity, CTL
avidity and response to CBT, we will generate a panel of CT26gp90-/- transfectants expressing graded levels
of the GSW11:Dd SCT. These will be correlated to tumour growth, TIL specificity, avidity of anti-GSW11 T
cells expanded in response to CBT. We have also shown that it is possible to increase the abundance of
GSW11:Dd by manipulating ERAP1 expression, and this leads to better immunogenicity. We will isolate TIL
from regressing CT26ERAP1-/- tumours and enumerate high and low avidity (GSW11-specific) TIL, using
both tetramer run-off experiments and a new technical platform: the Lumicks Movi-Z cell interaction
platform which is capable of measuring the force (in pico Newtons) required to separate T cells from their
targets using an ultrasound forcefield.
The relationship between antigen dose and CD8+ T cell avidity in an anticancer cDNA vaccine setting: Our
observation may be relevant to anti-cancer vaccination, where a significant barrier is the tumour-induced
exhaustion (or inactivation) of vaccine-induced T cells. We have shown that it is possible to manipulate
immunodominance to competing SV40T epitopes delivered as DNA fusion vaccines by altering p:MHC
abundance via peptide affinity. We will evaluate the response (relative abundance of the 4 specificities in
TIL, and their avidity) of tumour-bearing mice to vaccination with constructs delivering different
abundancies of dominant and subdominant epitope. Experiments will be performed initially in B6 mice
transplanted with the prostate cancer SV40T-expressing TRAMP-C1 line, then the TRAMP GMM, which
uniformly and spontaneously develop prostate tumours driven by SV40T. We will evaluate responses
(specificity and avidity) to anti-PD1 CBT in the 40-50% of vaccinated mice in which we observe progressing
tumours by isolating low and high avidity CD8+ anti- SV40T TIL at the point where response is evident, using
tetramers, quantifying T cells, and characterising their immunophenotype.

3.2 Low avidity CTL in human cancer

In HNSCC, we have observed the presence of low avidity oligoclones recognising the well-characterised
HPV-1 E6/7 derived HLA-A*0201 restricted LV9 epitope (low tetramer staining example in Figure below).
The Lumicks z-movi platform offers the possibility of measuring average T-cell avidities for oligoclonal TIL
of unknown specificity, and avidity-sorting them. We are currently validating the platform using the
tetramer-sorted cell populations described above and autologous tumour target cells. As part of this

90
project, we will isolate high and low-avidity T cells recovered from TIL as they become available from
patient biopsies and/or resected tumours. Where possible, we will integrate this analysis with an ongoing
investigation into the correlates of response to combined anti-PD-L1 in combination with chemo-
radiotherapy for gastro-oesophogeal cancer (the LUD2015 005 trial in collaboration with Profs Mark
Middleton (Oncology) and Xin Lu (Ludwig Institute)). Avidity-sorted CD8+ T cells will be processed for bulk
RNAseq to look for different gene signatures, particularly those correlating with precursor vs terminal
exhaustion phenotypes, which have been observed by others in TIL. We will relate these data to
longitudinal clinical data to determine whether there is a correlation between the expansion of low avidity
T cells and tumour regression.

Figure: Proof of concept that high and low avidity anti E6/E7 CD8+ T cells can be identified among TIL of an HNSCC patient

Publications relevant to this project

1. Protective low avidity anti-tumour CD8+ T cells are selectively attenuated by regulatory T cells. Sugiyarto G., Prosser D.,
Dadas O., Elliott T, James E. bioRxiv 481515; doi: https://doi.org/10.1101/481515 (submitted) 2. HPV Epitope Processing
Differences Correlate with ERAP1 Allotype and Extent of CD8+ T-Cell Tumor Infiltration in OPSCC Reeves E, Wood O,
Ottensmeier CH, King EV, Thomas GJ, Elliott T, James E. (2019) Cancer Immunol Res DOI: 10.1158/2326-6066.CIR-18-0498 3.
Induction of protective anti-tumor immunity through attenuation of ERAAP function. James E., Bailey I., Sugiyarto G. and
Elliott T.J. (2013) J Immunol. Jun 1;190(11):5839-46. doi: 10.4049/jimmunol.1300220 4. CD8+ T-cell cross-competition is
governed by peptide-MHC class I stability. Galea I., Stasakova J., Dunscombe M.S., Ottensmeier C.H., Elliott T.J.,
Thirdborough S.M. (2012) Eur J Immunol 42(1):256. 5. Differential suppression of tumour specific CD8+ T cells by Regulatory
T cells. James E., Yeh A., King C., Korangy F., Bailey I., Murray N., Van den Eynde B. and Elliott T.J. (2010) J.Immunol. Nov
1;185(9):5048-55.

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 Cell stress and the premalignant niche in ovarian – Prof.
Blagden1,2,3A
Primary Supervisor: Sarah Blagden
Additional Supervisors: Ahmed Ahmed
Eligibility: Track 1, 2 and 3A students are eligible to apply for this project.

Project Summary
When normal cells are exposed to intrinsic or extrinsic stress they immediately attenuate cap-
mediated protein translation and, if the stress is severe or sustained, undergo programmed
cell death. Perversely, when cancer cells are exposed to stress they proliferate instead of
dying and the stress signal is subverted to drive their malignant behaviour such as
encouraging new vessel formation (angiogenesis), chemotherapy resistance, invasion and
metastasis (Cubillos-Ruiz et al 2017). It is now known that a class of proteins called RNA
binding proteins (RBPs) are crucial for this response. While some RBPs (such as eIF4E) are
canonical and required for protein synthesis in all cells, others are present at low levels in
normal adult cells but are upregulated in diseases associated with cell stress such as cancer
and diabetes whereupon they bind to messenger RNAs (mRNA) encoding stress response
proteins and alter their stability and half-life. In this way, the RBPs post-transcriptionally
regulate gene expression and ensure that, even when the majority of protein translation is
halted, the survival mRNAs are preserved (Chen & Cubillos-Ruiz, 2020) (Backlund et al, 2020)
and the cell survives. Interestingly, levels of RBP are high in cancers, for example the RNA
binding protein LARP1 is highly expressed in ovarian cancer. We have shown that ovarian
cancer cells are “addicted” to LARP1 as it enables then to live in the hypoxic or nutrient-
deprived cancer environment. When LARP1 is depleted, the cancer cells die. We have
identified that LARP1 is packaged in exosomes and enters the circulation of cancer patients
and suspect that it signals to the immune system. Ovarian cancer originates from dysplastic
lesions in the fallopian tube called STICs (serous tubal intraepithelial carcinomas) that are
present for an average of 6.5 years before becoming ovarian cancer. Although STICs are highly
curable with surgical resection, they are “invisible” to blood tests or radiological imaging and
the opportunity to intervene is lost. We have found high levels of LARP1 within STICs but also
changes in the immune cells around the STIC lesions. In this project, we will investigate how
LARP1 regulates the cellular response to ER stress and drive malignant transformation and
how it communicates with the immune cells. We will use murine and human tumour
organoids and a LARP1-/- mouse model to characterise the role played by LARP1 in
tumorigenesis and, specifically, in the formation of ovarian cancer from its origins in the
fallopian tube. The overarching aim of this proposal is to explore the contribution of stress
and the immune response in driving the formation of ovarian cancer from preinvasive STIC
lesions. This has the potential to help identify STIC lesions and reduce the high mortality
associated with ovarian cancer.

Techniques
Mouse work, crossing GEMMs, extracting fallopian tubes, IHC, multiplex imaging, DNA and
RNA extraction and sequencing, proteomic analysis, cell line work, RT-PCR, generating and
maintaining organoids.

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References
1. Hopkins TG et al. The RNA-binding protein LARP1 is a post-transcriptional regulator of
survival and tumorigenesis in ovarian cancer. Nucleic Acids Res. 2016
2. Kim J et al. Cell Origins of High-Grade Serous Ovarian Cancer. Cancers (Basel). 2018 Nov
12;10(11):433
3. Stavraka C, Blagden S. The La-Related Proteins, a Family with Connections to Cancer.
Biomolecules. 2015 Oct 16;5(4):2701-22.

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 The immune landscape of the pancreas during neoplastic
transformation – Prof. O’Neill1,2,3A, 3B
Primary Supervisor: Eric O’Neill
Additional Supervisors: Tim Elliot
Eligibility: Track 1, 2 and 3A/B students are eligible to apply for this project.

Project Summary
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death with a
5-year survival below 5%. Poor survival statistics arise from the lack of early symptoms
resulting in advanced stage at diagnosis together with mostly ineffective chemotherapeutic
regimes. A major difficulty to identifying individuals at-risk of PDAC earlier is that symptoms
are vague, non-specific and indicate a very low progression rate even in conditions known to
pre-dispose to the disease. Therefore, identifying key biological events associated with
progression form pre-neoplastic or benign lesions is key to providing biomarkers that can
properly diagnose early. Pancreatic intraepithelial neoplasia (PanIN) lesions are the most
common precursor and proposed to originate from both smaller pancreatic ducts and via
dedifferentiation of the acinar cells. Inflammation of the pancreas is well described to
promote PanIN formation and progression to PDAC, but as not all PanINs progress to invasive
carcinoma in situ, diversity of the immune landscape in early lesions is likely to be important
in the establishment of disease. Upon progression to PDAC the microenvironment shows
extensive fibrotic stroma, an abundance of T-regulatory (Tregs) and M2-polarised tumour
associated macrophages (TAMs), blocking immune effector functions. Although there is low
immunogenicity, there is variability across in patients with low CD4/CD8 T-cell infiltrates
associated with poorer survival.

The aim of this project is understand the fine balance between the immune system and the
emerging cancer.

This project will characterise the establishing tumour immune-microenvironment (TIME) in

pre neoplastic tissue to identify key events that result in tumour initiation, but also highlight
how evasion mechanisms get embedded and therefore potential intervention strategies for
PDAC.

The KPC model (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) is a well-defined mouse

model of human pancreatic cancer and this rapidly develops spontaneous tumours. The KC
model (KrasG12D/+;Pdx-1-Cre) allow identification of early PanIN lesions, with a subset going
onto form PDAC with a long latency that more closely mirrors the human condition. We aim
to utilise syngeneic orthotopic injection of KPC tumour cells or KC pancreatic ductal organoids
together and KC mice to interrogate the immune landscape of benign vs pre-cancerous
lesions. We also aim to validate findings in ex-vivo tissue slices of resected PDAC vs
comparative healthy tissue, where live tissue responses to interventions can be monitored in
real-time.

Research objectives and proposed outcomes

Our general goals are:
1. To establish the Tumour immunological microenvironment of developing neoplasms in
the pancreas

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2. Identify how the immune suppressive environment becomes established and where
intervention strategies are likely to provide benefit.

The specific aim for Goal 1 - Establish the immune landscape of developing lesions in the
pancreas:
We have developed an Aurora Cytek panel covering a broad panel of immune cell markers to
give an overview of immune cell populations in blood and tissue. Here we aim to use to
determine the tissue resident and emerging immune populations present as a tumour
initiates and evolves in the pancreas of KC mice. A secondary aim is to determine whether
immune profiling give biomarker information for early detection, monitoring and intervention
strategies in per-neoplasm to early disease.

The specific aims for Goal 2 - To establish the chronology of how a developing tumour escapes
immunoediting and an immune suppressive environment becomes established. A secondary
aim is to ascertain key immune signalling events crucial to maintenance of the repressive state
and where intervention may induce greater tumour control. We also employ an othrotopic
model of pancreatic cancer by injection of KPC tumours cells directly into the pancreas,
allowing greater tractability around the onset and monitoring immune landscape.

It is anticipated that the information obtained here will lead to a better understand of early
lesion biology and will advance in detection as well as potential treatments with immune
targeting agents.

Translational potential of the project. Describe the relevance of the project to cancer
The research has very high translational potential: Through this project, we hope to establish
in-depth understanding of the relationship immunity and pancreatic cancer. This project will
initiate collaborations between Prof. Tim Elliott (Cancer Immunology, NDM), Prof. Eric O’Neill
(Pancreatic Cancer, Oncology as well as research medical oncologists (Mark Middleton and
Shivan Shivakumar) and surgeons at the NHS Churchill). As a centre for early diagnosis,
pancreatic cancer and immune-oncology any findings here can be rapidly brought forward
through our clinical partners to interventional trials.

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