2020 phd projects and supervisory teams doctoral ......restriction, nature, 2016 4. beale r*, wise...

2020 PhD projects and supervisory teams Doctoral Fellowships for Clinicians

Influenza infection and the autophagy machinery. Supervisory team: Rupert Beale (primary supervisor, Crick) and Wendy Barclay (Imperial College London) Identification and validation of novel therapeutic targets for pancreatic cancer using human PDAC organoids. Supervisory team: Axel Behrens (primary supervisor, Crick) and Debashis Sarker (King’s College London) Resistance to infection and Parkinson’s disease: an exploration of LRRK2, PD risk alleles and macrophage function. Supervisory team: Maximiliano Gutierrez (primary supervisor, Crick) and Huw Morris (UCL) The molecular mechanism underlying Loeys-Dietz syndrome. Supervisory team: Caroline Hill (primary supervisor, Crick), David Abraham (UCL) and Nitha Naqvi (Royal Brompton Hospital) Molecular mechanisms for astrocyte misfunction in ALS. Supervisory team: Nicholas Luscombe (primary supervisor, Crick), Rickie Patani (Crick/UCL) and Jernej Ule (Crick/UCL) Linking neutrophils changes with tumour malignancy: investigating the potential of neutrophil-driven immunotherapy. Supervisory team: Ilaria Malanchi (primary supervisor, Crick), Victoria Sanz Moreno (Barts Cancer Institute/QMUL) and David Propper (Barts Cancer Institute/QMUL) Understanding intra-tumour heterogeneity in the response of ovarian cancer to PARP inhibitors. Supervisory team: Erik Sahai (primary supervisor, Crick) and Iain McNeish (Imperial College London) Profiling the evolutionary history of rare atypical cancer cells. Supervisory team: Peter Van Loo (primary supervisor, Crick) and Nischalan Pillay (UCL) Oncolytic Vaccinia virus therapy of ovarian cancer: finding novel targets for combination therapies. Supervisory team: Michael Way (primary supervisor, Crick) and Iain McNeish (Imperial College London)

2020 Doctoral fellowships for clinicians

Influenza infection and the autophagy machinery A PhD project for the 2020 doctoral clinical fellows programme with Rupert Beale (primary supervisor, Crick) and Wendy Barclay (Imperial College London)

We have discovered that influenza infection triggers an intracellular pathway that is related to autophagy but has a distinct molecular basis. Influenza M2 proton channel activity triggers a collapse in proton gradients, and hence intracellular pH gradients, within the cell. This causes lipidation of the key autophagy protein LC3, but this requires a domain of ATG16L1 (linked by GWAS studies to Crohn’s disease) that is dispensable for autophagy. Provocatively, M2 also encodes a highly conserved LIR motif which binds directly to LC3. M2 proton channel activity also causes inflammasome activation. There is at present no clear idea of how these mechanisms relate to one another or their biological purpose either for the host or the pathogen. This project aims to define the biological purpose of the influenza/autophagy machinery interaction – from the point of view of both host and pathogen. As well as generating M2 mutant influenza viruses and infecting model organisms, the student will explore the biological effects of mutations in genes previously defined in the lab to be important for this interaction. We anticipate there will be important effects on both innate and adaptive immune functions. This project would suit applicants from any medical or surgical speciality with an interest in infection and immunity. The partner institution for this project is Imperial College London.

References: 1. Singanayagam A, Zambon M, Barclay W. Influenza virus with increased pH of HA activation has

improved replication in cell culture but at the cost of infectivity in human airway epithelium., J Virol 2019

2. Fletcher K*, Ulferts R*, Jaquin E, Veith T, Gammoh N, Aresteh JM, Mayer U, Carding SR, Wileman T, Beale R†, Florey O†. The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes. EMBO J, 2018

3. Long JS, Giotis ES, Moncorge O, Frise R, Mistry B, James J, Morisson M, Iqbal M, Vignal A, Skinner MA, Barclay WS, Species difference in ANP32A underlies influenza A virus polymerase host restriction, Nature, 2016

4. Beale R*, Wise H*, Stuart A, Ravenhill BJ, Digard P, Randow F. A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability. Cell Host and Microbe 2014


Identification and validation of novel therapeutic targets for pancreatic cancer using human PDAC organoids A PhD project for the 2020 doctoral clinical fellows programme with Axel Behrens (primary supervisor, Crick) and Debashis Sarker (King’s College London)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging cancers to treat, with unchanged 5 year overall survival of <5% for the last 30 years. Identifying new and targetable pathways in human tumours is an important goal in order to develop more effective therapies. We have made fundamental contributions to the understanding of PDAC biology using mouse models [1-3]. Rspondin-based 3D tumour organoids derived from patient biopsies closely recapitulate several properties of the original tumour [4]. To extend our work towards patient benefit, in close collaboration with King's College Hospital, we have established a human PDAC (hPDAC) tumour organoid biobank. We have established a workflow that allows the isolation of PDAC organoids and wild-type organoids from the same patient (Figure 1 ).

Figure 1 : Workflow to isolate human wild-type ductal and PDAC organoids. To date, we have established more than 20 matched normal ductal cells and PDAC cell pairs from PDAC patients, a number that is constantly increasing. The human PDAC organoid lines are routinely characterised by subcutaneous and orthotopic transplantation into immunodeficient mice. Exome sequencing and RNAseq is performed on both cell types isolated from PDAC patients. While all hPDAC patients enrolled in our biobank carried a mutation in the KRAS gene, the additional mutational landscape is diverse. Thus, we have established a human PDAC biobank that captures some of the genetic diversity of human PDAC. Here, we propose to use human pancreatic tumour organoids to develop and assess the efficacy of novel therapies. We have performed genetic and pharmacological screens and identified specific PDAC vulnerabilities. In one experimental approach, we investigated the role of deubiquitinases (DUBs) in PDAC. DUBs counteract the activity of E3 ubiquitin ligases. It has become clear that DUBs are druggable and several companies have DUB inhibitors in their portfolio. Since different DUBs are involved in multiple cancer-related signalling pathways [5], we hypothesized that specific DUBs may be involved in PDAC. As a first step to identify DUBs required for PDAC growth and maintenance, we used a custom-made lentiviral DUB shRNA library and compared the effect of DUB depletion in human wild-type ductal organoids with human PDAC organoids. Depletion of several DUBs, strongly inhibited the growth of PDAC organoids without having a measurable effect on wild-type pancreatic ductal cells. In this project, we want to further analyse the most interesting DUBs required for human PDAC growth, to validate them as potential therapeutic targets. Several complementary approaches, including biochemical characterisation, CRISPR/Cas-mediated genetic gene engineering of human PDAC organoids, usage of


specific DUB inhibitors (where available) and transplantation studies will be used to elucidate DUB function in PDAC. The partner institution for this project is King’s College London.

References: 1. Ferreira, R. M. M., Sancho, R., Messal, H. A., Nye, E., Spencer-Dene, B., Stone, R. K., . . .

Behrens, A. (2017) Duct- and Acinar-Derived Pancreatic Ductal Adenocarcinomas Show Distinct Tumor Progression and Marker Expression. Cell Rep 21: 966-978. PubMed abstract

2. Messal, H. A., Alt, S., Ferreira, R. M. M., Gribben, C., Wang, V. M., Cotoi, C. G., . . . Behrens, A. (2019) Tissue curvature and apicobasal mechanical tension imbalance instruct cancer morphogenesis. Nature 566: 126-130. PubMed abstract

3. Gruber, R., Panayiotou, R., Nye, E., Spencer-Dene, B., Stamp, G. and Behrens, A. (2016) YAP1 and TAZ Control Pancreatic Cancer Initiation in Mice by Direct Upregulation of JAK-STAT3 Signaling. Gastroenterology 151: 526-539. PubMed abstract

4. Boj, S. F., Hwang, C. I., Baker, L. A., Chio, II, Engle, D. D., Corbo, V., . . . Tuveson, D. A. (2015) Organoid models of human and mouse ductal pancreatic cancer. Cell 160: 324-338. PubMed abstract

5. Ge, Z., Leighton, J. S., Wang, Y., Peng, X., Chen, Z., Chen, H., . . . Liang, H. (2018) Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types. Cell Rep 23: 213-226 e213. PubMed abstract


Resistance to infection and Parkinson’s disease: an exploration of LRRK2, PD risk alleles and macrophage function A PhD project for the 2020 doctoral clinical fellows programme with Maximiliano Gutierrez (primary supervisor, Crick) and Huw Morris (UCL)

LRRK2 is the most important Mendelian gene causing Parkinson’s disease (PD) and the G2019S mutation is particularly common in Ashkenazi Jewish and North African populations, with the G2385R mutation common in Asian populations [1]. The LRRK2 gene has also been implicated in the risk of developing mycobacterial infections and inflammatory bowel disease, raising the possibility that PD risk alleles may be protective in early life and in populations with high risk of infectious disease (ID), and deleterious in later life leading to neurodegeneration [2]. Understanding these relationships will have important implications for understanding global variation in neurodegenerative disease risk, and in predicting beneficial and harmful effects of LRRK2 directed therapy. The Gutierrez lab has recently shown that LRRK2 regulates phagosome maturation [3] and Prof. Morris leads a community based study of familial PD and over 100 UK based LRRK2 PD families have been identified. This project will involve using clinical and genetic data from large scale datasets to identify the relationship between infectious disease and PD (cross disorders analysis systematically exploring the overlap, at a genetic and clinical level, between PD and ID), including patients carrying rare and common LRRK2 risk alleles and using population genetic data related to LRRK2 haplotypes and rare variants to explore the possibility of balancing positive selection for LRRK2-PD risk alleles in specific global populations. The major part of the project will involve studying biological samples from PD patients in the Gutierrez lab to determine the effects of variation at the LRRK2 locus on macrophage function. For that, blood samples from patients with different types of Parkinson’s disease will be collected and blood monocyte-derived macrophages isolated and differentiated under different conditions. Macrophage function such as phagocytosis, lysosomal function, ROS and RNS production as well as the response to infection with several pathogens (e.g. M. tuberculosis) will be analysed to establish possible correlations with clinical manifestations in the LRRK2 PD families. This project will offer training in cell biology and infection as well as bioinformatics/data analysis/genetics, developing evolutionary approaches to studying neurodegeneration and in cellular approaches to studying inflammatory and immune function. The partner institution for this project is UCL.

References: 1. Cookson MR. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson’s disease. Nat Rev

Neurosci. 2010 Dec;11(12):791–7. 2. Wang D, Xu L, Lv L, Su L-Y, Fan Y, Zhang D-F, et al. Association of the LRRK2 genetic

polymorphisms with leprosy in Han Chinese from Southwest China. Genes Immun. 2015 Mar;16(2):112–9.

3. Härtlova A, Herbst S, Peltier J, Rodgers A, Bilkei-Gorzo O, Fearns A, et al. LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages. EMBO J. 2018 Jun 15;37(12). Available from: http://dx.doi.org/10.15252/embj.201798694

4. Herbst S and Gutierrez MG. LRRK2 in Infection: Friend or Foe? ACS Infect Dis. 2019 Jun 14;5(6):809-815. doi: 10.1021/acsinfecdis.9b00051. Epub 2019 Apr 5.


The molecular mechanism underlying Loeys-Dietz syndrome A PhD project for the 2020 doctoral clinical fellows programme with Caroline Hill (primary supervisor, Crick), David Abraham (UCL) and Nitha Naqvi (Royal Brompton Hospital)

Background Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder, related to Marfan syndrome and characterised by vascular tortuosity and aneurysm in association with craniofacial and skeletal manifestations (1). Marfan syndrome itself is caused by mutations in the extracellular matrix protein, Fibrillin 1 (FBN1) (2), which are thought to increase the bioavailablity of TGF-b. Interestingly, six components of the TGF-b signalling pathway have been shown to be mutated in LDS. Loss-of-function mutations have been found in the type I and type II TGF-b receptors and in two TGF-b ligands – TGFB2 and TGFB3, and mutations have also been found in two downstream signal transducers of the TGF-b signalling pathway – SMAD2 and SMAD3 (1). In SMAD3, 61% are missense mutations and 23% are frameshift mutations; in SMAD2 all the mutations are missense mutations. Another Marfan-related syndrome, Sprintzen-Goldberg syndrome (SGS), is caused by mutations in a transcriptional repressor, SKI that acts negatively in the TGF-b pathway (3).

TGF-b signalling is initiated by ligand binding to the type I and type II serine/threonine kinase receptors. The type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates SMAD2 and SMAD3. The phosphorylated SMADs form complexes with SMAD4, which accumulate in the nucleus. There, they are recruited to DNA in conjunction with other transcription factors and both positively and negatively regulate target gene transcription (4). Prior to signalling, some target genes are repressed by SKI and/or a related protein, SKIL. In response to signal, SKI and SKIL are rapidly degraded, allowing the target genes to be activated by phosphorylated SMAD2/3–SMAD4 complexes. Recent work on SGS in the Hill lab (Francis Crick Institute) has shown that the reported SGS mutations in SKI result in a protein that is resistant to TGF-b-induced degradation, which has a dominant negative effect on TGF-b signalling.

The project Given the role of excess TGF-b signalling in Marfan syndrome, it has been difficult to understand how loss-of-function mutations in core components of the signalling pathway result in a syndrome (LDS) with remarkable similarities to Marfan syndrome. Paradoxically, mouse knockins of selected TGFBR1 and TGFBR2 mutations actually show elevated TGF-b signalling in the aortic wall and upregulation of TGF-b1. Exactly how this result relates to the diminished activity of the mutated receptors has not been resolved (5). Moreover, little is known about the functional effects of the LDS SMAD2/3 mutations.

The project aims to use CRISPR/Cas9-mediated genome editing to determine the effects of the LDS SMAD2/3 mutations on TGF-b pathway activity and also on the activity of other TGF-b superfamily pathways. Given that aortic aneurysms are a major feature of LDS, we will focus mainly on generating the mutations in human aortic endothelial cells and vascular smooth muscle cells, cultured separately and as a co-culture. RNA-seq will be used to investigate how the gene expression programmes are altered by these mutations in response to TGF-b superfamily signals, and CRISPR/Cas9-induced knockout will be used to determine the function of key downstream targets. The project will involve strong links with patients at the Royal Brompton Hospital through the collaboration with Dr Nitha Naqvi. The partner institution for this project is UCL.

References: 1. Schepers, D., Tortora, G., Morisaki, H., MacCarrick, G., Lindsay, M., Liang, D., Mehta, S. G.,

Hague, J., Verhagen, J., van de Laar, I., Wessels, M., Detisch, Y., van Haelst, M., Baas, A., Lichtenbelt, K., Braun, K., van der Linde, D., Roos-Hesselink, J., McGillivray, G., Meester, J., Maystadt, I., Coucke, P., El-Khoury, E., Parkash, S., Diness, B., Risom, L., Scurr, I., Hilhorst-Hofstee, Y., Morisaki, T., Richer, J., Desir, J., Kempers, M., Rideout, A. L., Horne, G., Bennett, C., Rahikkala, E., Vandeweyer, G., Alaerts, M., Verstraeten, A., Dietz, H., Van Laer, L., and Loeys, B. (2018) A mutation update on the LDS-associated genes TGFB2/3 and SMAD2/3. Hum Mutat 39, 621-634.

2. Verstraeten, A., Alaerts, M., Van Laer, L., and Loeys, B. (2016) Marfan Syndrome and Related Disorders: 25 Years of Gene Discovery. Hum Mutat 37, 524-531.


3. Doyle, A. J., Doyle, J. J., Bessling, S. L., Maragh, S., Lindsay, M. E., Schepers, D., Gillis, E., Mortier, G., Homfray, T., Sauls, K., Norris, R. A., Huso, N. D., Leahy, D., Mohr, D. W., Caulfield, M. J., Scott, A. F., Destree, A., Hennekam, R. C., Arn, P. H., Curry, C. J., Van Laer, L., McCallion, A. S., Loeys, B. L., and Dietz, H. C. (2012) Mutations in the TGF-b repressor SKI cause Shprintzen-Goldberg syndrome with aortic aneurysm. Nat Genet 44, 1249-1254.

4. Wu, M. Y., and Hill, C. S. (2009) TGF-b superfamily signaling in embryonic development and homeostasis. Dev Cell 16, 329-343.

5. Gallo, E. M., Loch, D. C., Habashi, J. P., Calderon, J. F., Chen, Y., Bedja, D., van Erp, C., Gerber, E. E., Parker, S. J., Sauls, K., Judge, D. P., Cooke, S. K., Lindsay, M. E., Rouf, R., Myers, L., ap Rhys, C. M., Kent, K. C., Norris, R. A., Huso, D. L., and Dietz, H. C. (2014) Angiotensin II-dependent TGF-b signaling contributes to Loeys-Dietz syndrome vascular pathogenesis. J Clin Invest 124, 448-460.


Molecular mechanisms for astrocyte misfunction in ALS A PhD project for the 2020 doctoral clinical fellows programme with Nicholas Luscombe (primary supervisor, Crick), Rickie Patani (Crick/UCL) and Jernej Ule (Crick/UCL)

Neurodegeneration is widely considered to progress through deterioration of specific subclasses of neurons in a cell autonomous manner. However, this ‘neuron-centric’ view is been increasingly challenged, with astrocytes being implicated as key determinants of neurodegeneration. This project will investigate the role of astrocytes in amyotrophic lateral sclerosis (ALS). ALS has a ~1 in 400 lifetime risk: it is rapidly progressive, incurable and invariably fatal. a progressive, fatal and incurable condition. It is characterized by motor neuron (MN) degeneration, but its aetiology remains unknown. For therapeutic development, we need a unified understanding of common primary events leading to ALS pathogenesis.

Studies have generally focused on astrocyte-mediated injury to neurons by searching for toxic factors and alternative mechanisms for astrocyte involvement – such as their neuroprotective capacity - are comparatively understudied. Our labs have pioneered the development and characterisation of patient-derived induced pluripotent stem cells (iPSCs) to study ALS (Hall et al Cell Reports 2017; Tyzack et al Nature Communications 2017; Kelley et al Neuron 2018). In doing so, we have confirmed a role for astrocytes in ALS: i) they show a survival phenotype themselves (Hall et al Cell Reports 2017) and ii) there is a cell intrinsic failure in neuroprotection of neighboring MNs (Tyzack et al Nature Communications 2017). However, the underlying molecular mechanisms remain unresolved. In a breakthrough discovery, we recently identified abnormal RNA processing in the form of erroneous intron retention, as the earliest detectable change in ALS (Luisier et al. Nature Communications 2018). This study focused on events in MNs, but given the clear cellular and molecular phenotypes in (isogenic) ALS astrocytes, it is clearly a research priority to characterize any defects in RNA metabolism that might represent convergent or (cell type-specific) divergent mechanisms.

The core hypothesis of this project is that our established and published astrocyte cellular phenotypes are determined by key defects in RNA metabolism. To investigate this, we will perform deep RNA-sequencing of astrocytes during their lineage restriction from iPSCs so that we can identify temporal defects in RNA metabolism. Crucially we have 13 years of experience in this area and fully validated / published methods. Targeted RNAseq experiments will identify differentially expressed genes / splicing events discriminating deleterious from neuroprotective astrocytes in ALS. We will validate candidate events by modulating identified pathway(s) using genome editing or small molecular pharmacology and test the functional consequence by co-culture with MNs in longitudinal co-culture.

This project harnesses fully established platforms in hiPSC astrocyte and MN specification, in vitro assays (in monoculture and coculture) to generate focused datasets. The successful candidate will receive comprehensive training in iPSC manipulation, molecular, cellular and genomic techniques as well as computational biology and bioinformatics. In summary this work aims to elucidate new molecular mechanisms by which astrocytes contribute to motor neuron death in ALS. This raises the prospect of considering astrocytes as a new cellular target for molecular intervention in ALS. The partner institution for this project is UCL.

References: 1. Luisier R, Tyzack GE, Hall CE, Mitchell JS, Devine H, Taha DM, Malik B, Meyer I, Greensmith L,

Newcombe J, Ule J, Luscombe NM, Patani R. Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS. Nat Commun. 2018; 9:2010.

2. Serio A, Patani R. Concise Review: The Cellular Conspiracy of Amyotrophic Lateral Sclerosis. Stem Cells. 2018; 36:293-303.

3. Chakrabarti AM, Haberman N, Praznik A, Luscombe NM, and Ule J. Data Science Issues in Studying Protein–RNA Interactions with CLIP Technologies. Annu Rev Biomed Data Sci. 2018; 1, 235–261.

4. Hall CE, Yao Z, Choi M, Tyzack GE, Serio A, Luisier R, Harley J, Preza E, Arber C, Crisp SJ, Watson PMD, Kullmann DM, Abramov AY, Wray S, Burley R, Loh SHY, Martins LM, Stevens MM, Luscombe NM, Sibley CR, Lakatos A, Ule J, Gandhi S, Patani R. Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS. Cell Rep. 2017; 19:1739-1749.

5. Tyzack GE, Hall CE, Sibley CR, Cymes T, Forostyak S, Carlino G, Meyer I, Schiavo G, Zhang SC, Gibbons GM, Newcombe J, Patani R, Lakatos A. EphB1 is a neuronal signal that induces a neuroprotective astrocyte state, but fails in ALS. Nat Commun. 2017; 8:1164.


Linking neutrophils changes with tumour malignancy: investigating the potential of neutrophil-driven immunotherapy A PhD project for the 2020 doctoral clinical fellows programme with Ilaria Malanchi (primary supervisor, Crick), Victoria Sanz Moreno (Barts Cancer Institute/QMUL) and David Propper (Barts Cancer Institute/QMUL)

Tumours aggressiveness is linked to intrinsic features of cancer cells which influence the composition of the tumour microenvironment (TME). Sanz Moreno’s lab has recently shown that in melanoma, ROCK activity (a regulator of cell actomyosin contractility) in cancer cells, which is directly linked to its aggressiveness, modulate the pro-tumorigenic activity of tumour associated macrophages (1). Malanchi’s lab has shown that during breast cancer metastasis more contractile cancer cells are directly supported by signals derived by surrounding neutrophils (2). It is well accepted now that neutrophils are mobilized by systemic cancer derived signals and a high circulating neutrophil- to-lymphocyte ratio is a robust biomarker of poor clinical outcome in various cancers (3). There are evidences in mouse and human that aggressive cancers are able to generate different neutrophils already in the bone marrow (4).

In a more recent study, Malanchi’s lab highlighted the broad perturbation found in neutrophils in the lung metastatic environment (niche) of breast cancer cells, in comparison to lung neutrophils not in contact with the niche (5). This indicates that neutrophils, not only respond to cancer systemically, but modify their properties locally by cancer derived signals. Sanz Moreno’s lab also found that highly metastatic melanoma releases high levels of neutrophils stimulating chemokines such as IL8 (1). It is reasonable to hypothesise that specific neutrophil perturbations induced by more or less aggressive tumours will have important impact in cancer behaviour. Moreover, anti-cancer activity of neutrophils has also been reported. This rises the exciting possibility that signals controlling neutrophil behaviour could be used for therapeutic intervention.

We propose to investigate the phenotype, behavior and localization of neutrophils in the TME of tumours with different tumorigenic potential. We will concentrate on the tumour types our labs have more experience with (melanoma and breast cancer). We aim to extend our studies to pancreatic cancer, since both labs have tools and preliminary data to test similar hypothesis. We will combine the use of human samples, human cell lines and established mouse genetic models. Year 1. Sanz Moreno’s lab has generated human tissue microarrays of melanoma (1), breast and pancreatic cancer. We will analyse the immune infiltrate (including neutrophils), stromal components and cancer cell heterogeneous characteristics to gain quantitative, qualitative (phenotype) and spatial (cell-proximity) information about tumour infiltrating neutrophils. Year 2/3. We will perform similar studies in xenograft tumours generated using differently malignant human cell lines of the various tumours types. Using these animal models, we will also investigate the perturbations occurring systemically in the neutrophil compartment in the bone marrow. This design represents a discovery platform that will help to generate hypothesis driven models to mechanistically investigate in preclinical studies. Year 2/3. An important unanswered question is the impact of systemic (long) vs local (fast) responses that can influence neutrophil behaviour in the TME. To address this important question, we will perform adoptive transfer experiments where labelled neutrophils from healthy Actin-GFP mice will be injected in the circulation of mice harbouring different tumours. We will analyse the localization, phenotype and behaviour in the TME of non-pre-conditioned labelled vs pre-conditioned unlabelled neutrophils. We believe that the combination of our expertise in cancer cell biology and cancer inflammation will give us a unique advantage in carrying out this important project. The partner institution for this project is Barts Cancer Institute/QMUL.

References: 1. Georgouli M., Herraiz C., Crosas-Molist E., … Sanz-Moreno V. (2019) Regional Activation of Myosin

II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment. Cell. 7;176(4):757-774

2. Wculek SK, Malanchi I. (2015) Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Nature. 17;528(7582):413-7.


3. Shaul M. E. and Fridlender Z. G. (2019) Tumour-associated neutrophils in patients with cancer. Nat Rev Clin Oncol. doi: 10.1038/s41571-019-0222-4

4. Zhu, Y. P., Padgett, L., Dinh H. Q., ... Hedrick C. C. (2018) Identification of an Early Unipotent Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow. Cell Reports 24, 2329–2341

5. Ombrato L., Nolan E., Kurelac I., … Malanchi I. (2019) Metastatic niche labelling reveals tissue parenchyma stem cell features. Nature in press.


Understanding intra-tumour heterogeneity in the response of ovarian cancer to PARP inhibitors A PhD project for the 2020 doctoral clinical fellows programme with Erik Sahai (primary supervisor, Crick) and Iain McNeish (Imperial College London)

Despite great advances in our understanding of the genetic and molecular basis of solid tumours, our ability to cure them with either conventional or targeted chemotherapies remains sadly limited. One reason for this is the inter-cellular variability in how cancer cells within a tumour respond to therapeutic drugs. This variation enables cancer cells that are either intrinsically less sensitive to the drug or in an environment that provides protection against the drug to continue proliferation, leading ultimately to the evolution of therapy resistant disease (McGranahan and Swanton, 2015). PARP inhibitors are starting to improve the outlook for cancer patients, in particular those with BRCA1 or 2 mutations. However, both inter- and intra-tumour heterogeneity in response are undermining clinical benefit in ovarian cancer (Bowtell et al., 2015). This project will use high resolution imaging methodologies to track inter-cellular heterogeneity in response to PARP inhibitors. Imaging will enable the linkage between drug-target engagement, the initial DNA damage response, and ultimate cell fate to be determined in cultures that recapitulate the complexity of the tumour microenvironment (Hirata et al., 2015; Hirata and Sahai, 2017). Particular attention will be paid to factors that contribute to heterogeneous responses, including inter-cellular heterogeneity in cell cycle phase and the role of stromal cells including fibroblasts, adipocytes, and macrophages. Both patient-derived cultures and murine co-cultures using ID8 cells with engineered mutations in BRCA1 & BRCA2 will be interrogated (Walton et al., 2017). In the later stages of the project, orthogonal single cell techniques such as CyTOF and single cell RNA sequencing will be used to understand more about cancer cells that contribute to residual disease and ultimately relapse. Finally, therapeutic strategies that aim to reduce the inter-cellular variability in response will be tested for their ability to improve responses to PARP inhibitors, with a likely focus on those that interfere with the signals derived from the tumour microenvironment. The candidate should be enthusiastic about understanding the cellular events triggered in response to DNA damaging drugs and how tumours function as a system of communicating cell types. They should be comfortable interacting with researchers from different disciplines and highly motivated. Some prior experience of either molecular biology or imaging methods is desirable, but not considered essential, and a quantitative mind-set would be an asset for interactions with computational biologists building in silico models of cancer evolution. A track record of publications and/or an intercalated degree would be an advantage. Good record keeping and time management will also be important. The partner institution for this project is Imperial College London.

References: 1. Bowtell, D. D., Bohm, S., Ahmed, A. A., Aspuria, P. J., Bast, R. C., Jr., Beral, V., Berek, J. S.,

Birrer, M. J., Blagden, S., Bookman, M. A., et al. (2015). Rethinking ovarian cancer II: reducing mortality from high-grade serous ovarian cancer. Nat Rev Cancer 15, 668-679.

2. Hirata, E., Girotti, M. R., Viros, A., Hooper, S., Spencer-Dene, B., Matsuda, M., Larkin, J., Marais, R., and Sahai, E. (2015). Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin beta1/FAK signaling. Cancer cell 27, 574-588.

3. Hirata, E., and Sahai, E. (2017). Tumor Microenvironment and Differential Responses to Therapy. Cold Spring Harb Perspect Med 7.

4. McGranahan, N., and Swanton, C. (2015). Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer cell 27, 15-26.

5. Walton, J. B., Farquharson, M., Mason, S., Port, J., Kruspig, B., Dowson, S., Stevenson, D., Murphy, D., Matzuk, M., Kim, J., et al. (2017). CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity. Sci Rep 7, 16827.


Profiling the evolutionary history of rare atypical cancer cells A PhD project for the 2020 doctoral clinical fellows programme with Peter Van Loo (primary supervisor, Crick) and Nischalan Pillay (UCL)

Cancers acquire somatic mutations as they evolve, including single-nucleotide variants, insertions and deletions, structural variants and copy number alterations. A tumour’s genome therefore carries an archaeological record of its evolutionary past, and much of this history can be reconstructed from the tumour’s genome sequence (1, 2). In addition, single-cell DNA and RNA sequencing approaches allow tracing the origin of single cancer cells to cancer clones or subclones (3) and inferring their cell states, respectively.

Interestingly, some mutational processes, such as chromothripsis (4), can generate large numbers of changes to the genome in a single event. Such punctuated events can substantially reconfigure the cancer genome, leading to large leaps in tumour evolution. Chromothripsis is observed in multiple cancer types, and is particularly common in several sarcomas, including osteosarcoma and liposarcoma. We hypothesise that due to the extensive damage chromothripsis causes to the genome, most of these events are detrimental to their host cells, and therefore, most chromothripsis events remain unobserved through genome sequencing of bulk tumour tissue. Single-cell sequencing approaches hold the potential to observe and study these unselected chromothripsis events.

Some cancers, particularly connective tissue tumours, harbour scattered morphologically highly atypical cells. Such cells are observed in a number of sarcomas, specifically undifferentiated pleomorphic sarcomas, pleomorphic rhabdomyosarcoma, pleomorphic liposarcoma and pleomorphic leiomyosarcoma, and also in glioblastomas. It is noteworthy that scattered atypical cells with enlarged polymorphic nuclei are also seen in some benign connective tissue tumours, in particular schwannomas, osteoblastomas, and chondromyoid fibroma. The genotypes of these cells and their role in tumour evolution have not been characterised.

Fig. 1: atypical cells across cancer types. A and B: monster cells (200 µm and 150 µm respectively) in undifferentiated sarcomas. C: Mitosis in this same tumour as B, demonstrating multipolarity as well as micronuclei. D: giant cell glioblastoma (source: Oh et al., Brain Pathology).


In this project, we plan to study these rare scattered atypical cells across sarcoma and benign connective tissue tumours, using single-cell laser capture microdissection (5) and single-cell DNA and RNA sequencing. Our aims are three-fold:

(i) Study the evolutionary history of these rare atypical cells and their relationship to their neighbouring tumour cells, across multiple sarcomas, as well as a few benign connective tissue tumours with scattered highy atypical cells such as those seen in ‘degenerate schwannona’, using single-cell DNA sequencing.

(ii) Study their phenotype and cell state using single-cell RNA sequencing. (iii) Evaluate whether these rare atypical cells (or other cancer cells studied through single-

nucleus sequencing), are chromothriptic, and if so, leverage them to study the landscape of chromothripsis outside of the context of positive selection.

This project is suitable for a pathologist in training, with an interest in genomics.

The partner institution for this project is UCL.

References: 1. M. Gerstung et al., The evolutionary history of 2,658 cancers. bioRxiv preprint, doi:

https://www.biorxiv.org/content/10.1101/161562v3 (2018). 2. J. Demeulemeester et al., Tracing the origin of disseminated tumor cells in breast cancer using

single-cell sequencing. Genome Biol 17, 250 (2016). 3. P.J. Stephens et al., Massive genomic rearrangement acquired in a single catastrophic event

during cancer development. Cell 144, 27-40 (2011). 4. A.K. Casasent et al., Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell

Sequencing. Cell 172, 205-217.e12 (2018). 5. C.D. Steele et al., Undifferentiated Sarcomas Develop through Distinct Evolutionary Pathways.

Cancer Cell 35, 441-456.e8 (2019).


Oncolytic Vaccinia virus therapy of ovarian cancer: finding novel targets for combination therapies A PhD project for the 2020 doctoral clinical fellows programme with Michael Way (primary supervisor, Crick) and Iain McNeish (Imperial College London)

The poor outcome of recurrent ovarian cancer to current interventions justifies exploration of new therapeutic approaches. Vaccinia virus is being developed as an oncolytic agent given its proven safety profile as the vaccine to eradicate smallpox. Moreover, it does not transform cells, preferentially accumulates in tumours and has been shown to spread to metastases in vivo. Interestingly, Vaccinia has a strong tropism for ovarian tissue and an intrinsic ability to kill ovarian epithelial cancer cells in mouse models (Zhao, Adams, and Croft 2011,).

Current oncolytic Vaccinia viral backbones used in clinical trials have deletions in the viral Thymidine Kinase (JX-594) or TK and Vaccinia Growth Factor, VGF (JX-929/963) (Kim et al., 2009). These deletions increase the therapeutic index of the virus but do not enhance its intrinsic cytolytic activity. More recent strains are also “armed” to express non-viral proteins that increase anti-tumour activity and/or attract immune cells (Breitbach et al., 2011). Another less explored approach is to design recombinant viruses that increase their intrinsic oncolytic potential when combined with chemotherapeutic agents and other novel inhibitors.

The Way Laboratory is investigating Vaccinia-induced cell death. We have found that two virus-encoded proteins, VGF and F1L, synergise to prevent apoptosis during infection (Postigo et al., 2009). A recombinant virus lacking these proteins causes an increase in cell death compared to wild type virus, without impacting on viral replication or spread and therefore has great clinical potential. In addition, we have performed a genome-wide RNAi screen using our recombinant virus and have identified approximately 100 hits that further accelerate virus-induced death of HeLa cells. The aim of the project is to validate and explore these hits as potential targets for combination therapies.

Initial validation of one hit from the screen, using siRNA and chemical inhibitors, has confirmed it to be synthetic lethal with the recombinant virus. Ongoing work is addressing the molecular basis for this synthetic lethality and examining the impact of the loss of the protein in a murine model of ovarian high-grade serous carcinoma using our recombinant virus and genome-edited ID8 murine ovarian cancer cells (Walton et al., 2016). The project will build on our ongoing analysis and mouse work and assess the potential of other candidates from our original RNAi screen.

In summary, the project constitutes an exciting, novel approach to improve Vaccinia-based oncolytic strategies and seeks to translate these basic findings towards the clinic. The partner institution for this project is Imperial College London.

References: 1. Zhao, Y., Y. F. Adams, and M. Croft. Preferential replication of vaccinia virus in the ovaries is

independent of immune regulation through IL-10 and TGF-beta. Viral Immunology 24: 387-96. 2011 2. Kirn, D. H., and S. H. Thorne. Targeted and armed oncolytic poxviruses: a novel multi-

mechanistic therapeutic class for cancer. Nature Reviews Cancer 9: 64-71. 2009 3. Breitbach, C. J., J. Burke, D. Jonker, J. Stephenson, A. R. Haas, L. Q. Chow, J. Nieva, T. H.

Hwang, A. Moon, R. Patt, A. Pelusio, F. Le Boeuf, J. Burns, L. Evgin, N. De Silva, S. Cvancic, T. Robertson, J. E. Je, Y. S. Lee, K. Parato, J. S. Diallo, A. Fenster, M. Daneshmand, J. C. Bell, and D. H. Kirn. 2011. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 477: 99-102. 2011

4. Postigo, A., M. Martin, M. P. Dodding and M. Way. Vaccinia-induced EGFR-MEK signalling and the anti-apoptotic protein F1L synergize to suppress cell death during infection. Cellular Microbiology. 11: 1208-1218. 2009

5. Walton, J., J. Blagih, D. Ennis, E. Leung, S. Dowson, M. Farquharson, L. A. Tookman, C. Orange, D. Athineos, S. Mason, D. Stevenson, K. Blyth, D. Strathdee, F. R. Balkwill, K. Vousden, M. Lockley, and I. A. McNeish. 2016. CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma. Cancer Research 76: 6118-29. 2016

2020 phd projects and supervisory teams doctoral ......restriction, nature, 2016 4. beale r*, wise...

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