NIHRDataInsights/HRCSData huggingface dataset:
Title: Defining the role of PARPs in the DNA repair and genome stability
Funder: Medical Research Council

Abstract: This work will increase our understanding of how Poly(ADP-ribose)-polymerases (PARPs) maintain genome integrity through replication repair and characterise pathways that can compensate for loss of these processes. We have exploited genome editing to generate cell lines defective in the principle DNA damage responsive PARPs alone, or in combination. Using this unique set of reagents in combination with siRNA depletion of DNA repair factors, we have identified PARP1/PARP2 regulate HR-dependent and -independent mechanisms for repair of stalled/damaged replication forks. Moreover, through a genome-wide CRISPR/Cas9 screen designed to identify genes that are synthetic lethal with parp1/parp2 gene disruption, we discovered a novel gene that is required for cells to tolerate replication stress. Building on this work, we will define the mechanistic basis of these pathways. Using genome editing technology, in combination with cell based assays to monitor replication dynamics, DNA damage response activation, cell cycle progression and genome stability, we will define how PARP1/PARP2 regulate HR-dependent and independent DNA repair. Using cutting edge mass spectrometry, we will map ADP-ribosylation sites targeted during replication stress and through mutational analysis define the mechanistic basis of how these modifications regulate genome stability. Additionally, we will assess the role of a novel DDR gene that can compensate for loss of PARP1/PARP2-dependent replication repair mechanisms, identifying the pathway it regulates and the molecular basis of its function. Together these experiments will provide important information regarding how PARP1/PARP2 regulate replication-associated repair and the pathways that can compensate for loss of these processes. This will inform strategies to improve the efficacy of PARP inhibitors in the clinic and identify new synthetic lethal interactions to broaden the use of these agents to treat other tumour types.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Overcoming Ibrutinib and Venetoclax resistance in Chronic Lymphocytic Leukaemia.
Funder: Medical Research Council

Abstract: Inhibitors of BTK and BCL2 have revolutionised the treatment of CLL but they are non-curative, expensive and some patients develop resistance. Resistance has been linked with the clonal expansion of CLL cells exhibiting BTK and BCL2 mutations. However, these mutations are usually only evident in a minor subset of the tumour and in a small percentage of resistant patients. Therefore, other explanations are required for the majority of cases who only partially respond or become refractory to treatment. We, and others, have shown that ibrutinib, both alone and with venetoclax, primes CLL cells from some patients to signalling through the TLR9 pathway inducing drug-resistance. We have developed a TLR9-response migration assay with the capacity to prospectively identify these patients. The aims of this application are: (1) To test our TLR9-response assay's ability to predict for clinical outcome and MRD status in 200 treatment naïve samples from the UK-CLL FLAIR trial. If successful, this would be a simple predictive tool to identify ibrutinib/venetoclax resistant patients. (2) To use samples from these resistant patients to identify novel druggable targets that block TLR9-mediated resistance. Initially, we will focus on NF-kB as BTK, BCL2 and TLR9 converge on this pathway and preliminary data from our TLR9-resonse assay defines CLL groups with different NF-kB signalling. However, we will also consider unexpected routes to resistance using systems biology approaches. (3) Establish the best drug targets using mathematical simulations before confirming their potential in 'wet lab' experiments. We have an on-going collaboration a CRUK-funded drug discovery team who will provide us with novel NF-kB signalling pathway inhibitors to test some of these model-predicted targets. Therefore, this project should predict patient responses to ibrutinib/venetoclax and identify a novel therapy for drug-resistant patients. Insights gained will be applicable to other B-cell malignancies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Elucidating the Rad51-independent pathway of recombination-dependent replication
Funder: Medical Research Council

Abstract: The successful completion of DNA replication is threatened by obstacles, such as DNA-protein complexes and DNA lesions, which block replication fork progression. Upon stalling, the replication fork can "collapse" rendering it unable to continue DNA synthesis due to disassembly of the replication machinery. In such instances, the completion of replication relies either on an opposing fork converging with the collapsed fork or on restoration of DNA synthesis by recombination dependent replication (RDR). Two RDR pathways have been identified in eukaryotes, which can be distinguished by their differing reliance on the recombinase Rad51. Rad51-dependent RDR has been a focus of research for over two decades, whereas, Rad51-independent RDR (RiRDR) has received relatively little attention and remains poorly understood. Interest in RiRDR was recently reawakened by discoveries of its importance in humans, where it appears to be especially key for cancer cells to cope with replication stress and survive without telomerase by driving alternative lengthening of telomeres. These findings highlight the need to discover how RiRDR works. To achieve this goal, we will identify and characterize its key component parts using a unique pipeline of genetic and biochemical approaches that we have established in the model eukaryote Schizosaccharomyces pombe. This includes using site-specific replication fork barriers to trigger fork collapse, enabling detailed in vivo analysis of RiRDR using genetic reporters, native two dimensional gel electrophoresis, chromatin immunoprecipitation and live cell imaging. Feeding into our pipeline will be candidate factors and factors that emerge from a novel forward genetic screen. Through our unique combination of approaches, we will gain unprecedented insight into the molecular mechanism of RiRDR and, in so doing, provide the paradigm for its modus operandi.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Identifying and manipulating molecular mechanisms controlling cancer stem cell metastatic potential in a human oral cancer model.
Funder: Medical Research Council

Abstract: We hypothesise that there is variation within the epithelial cancer stem cell population in a primary tumour - only some of these cancer stem cells possess the plasticity required to initiate the metastatic cascade - that is, to undergo EMT into a mesenchymal phenotype that in turn retains the ability to undergo MET at a secondary site. We propose to test this hypothesis in a human HPV-negative oral cancer model treated with the well-characterised EMT-inducing growth factor TGFbeta. We will use single cell CITE-seq (single cell RNAseq with barcoded antibodies to cell surface markers used in cancer stem cell phenotyping) combined with advanced downstream bioinformatic techniques to identify cell clusters and infer the transitions between these clusters. In this way we will identify epithelial cancer stem cell sub-populations that are able to undergo EMT into a mesenchymal phenotype that in turn retains the ability to undergo MET. Bulk RNAseq on clonal sub-lines with differing plasticity and FACS sorted sub-populations using known markers will support this analysis. We will use this information to identify candidate molecular pathways controlling these cancer stem cell sub-populations. We will then inactivate these pathways using CROP-seq, a CRISPR loss-of-function screening methodology that combines with single cell RNAseq to enable determination of molecular targets whose inactivation can induce loss of key cancer stem cell sub-populations and thus prevent initiation of the metastatic cascade. In selecting candidate targets for the screen, we will focus on druggable nodes within key pathways. We will then test whether target inactivation can prevent metastasis in new engineered metastasis models that we have developed in our lab. Finally, we will test the association of targets with metastasis through immunofluorescent antibody staining of human pathological specimens stratified on metastatic status and bioinformatic analysis of TCGA data.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Identifying novel drugs to treat neurofibromatosis tumours using a unique combinatorial screening strategy
Funder: Medical Research Council

Abstract: Neurofibromatosis type 1 (NF1) is a common genetic disease associated with tumour formation along nerves. The tumours cause a variety of symptoms and can progress into malignant cancers that often result in death. Previous studies have used genetic and compound screens to find candidate drugs for the treatment of NF1 tumours. However, few drugs have emerged and none have yet progressed to clinical use. Our research is focussed on developing novel approaches to genetic screens that greatly improve the chances of identifying effective drug candidates. Using an earlier version of our new methods, existing drugs have already been identified that can be repurposed to treat tuberous sclerosis complex and Vhl-linked kidney cancer, illustrating the power of our screening approach. The aim of this proposal is to apply our new screening methods to find existing drugs that can be used alone or in combinations to effectively kill NF1 tumour cells without affecting healthy cells. We have identified 46 candidate drug targets using genetic screens in Drosophila cells. Furthermore, we have validated two candidates in human cells and a mouse model. In this project, we will first filter the results of the Drosophila screen by testing the candidates in a panel of human NF1 model cell lines. This cross-species filter will identify candidates that kill NF1 mutant cells in diverse genetic contexts, therefore increasing the chance of successful translation to clinical use. In addition, we will apply a new method called high-throughput Variable Dose Analysis (htVDA) to rapidly and accurately assess pairwise combinations of genetic and pharmacological treatments. This will identify pairs of existing drugs that can be repurposed for combinatorial treatment of NF1 tumours. This combination of novel approaches will allow us to identify new candidate treatments for NF1 that have a high chance of translation to clinical use.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Spatio-temporal dependency of radiotherapy induced cardiac toxicity in a novel preclinical model
Funder: Medical Research Council

Abstract: Radiation induced cardiac toxicity (RICT) is a major cause of morbidity and mortality in patients following radiotherapy for thoracic malignancies. The risk of developing RICT after treatment increases linearly with mean absorbed dose to the heart by >7% per Gy with an apparent low dose threshold. Consequently, no clear dose volume constraints can be defined & current radiotherapy planning recommendations advise dose to the heart be kept as low as possible. Current clinical heart dose constraints remain poorly defined and are largely predicted on the heart being a uniformly radiosensitive organ. Recent clinical observations have shown a dose sensitive region in the base of the heart that is associated with poor survival outcomes in lung cancer. We have reverse translated these observations in a mouse model showing significant cardiac dysfunction following base irradiation of the heart. In this project we will use this model to investigate the impact of spatio-temporal radiotherapy delivery parameters on RICT. Our overall aim to optimise treatment planning by integrating regional cardiac dose constraints based on radiosensitive sub-volumes, cardio-pulmonary interplay and effects at ultra-high dose rates (FLASH-RT).


Research activity codes: 5.5 RADIOTHERAPY AND OTHER NON-INVASIVE THERAPIES
Health categories: CANCER AND NEOPLASMS
Title: Investigating the role of vascular endothelial-cell senescence driving resistance to DNA-damaging therapies and metastasis formation in lung cancer
Funder: Medical Research Council

Abstract: We hypothesise that targeting EC-FAK in combination with DNA-damaging therapy controls cancer metastases through senolytic effects. Here we will: 1. Investigate the role of EC-FAK in DNA-damaging therapy induced senescence and metastasis formation in vivo. Syngeneic lung cancer mouse cell lines transfected with a mNIS (sodium iodide symporter) will be injected into the left lung of immunocompetent EC-specific FAK KO and FAK-kinase-dead mice. Mice will receive chemotherapy prior to primary tumour radio-ablation, and monitored for metastasis development by SPECT/CT imaging. Mice will also be crossed with a p16-FDR line, to identify the functional consequence of ablating senescent cells in the regulation of chemoresistance and metastasis. Histologic analysis will determine cellular and molecular changes in primary cancers and metastases. 2. Identify the cellular and molecular mechanisms underlying EC-FAK regulation of metastasis after DNA-damaging therapy. ECs will be isolated and sorted by FACs from treatment naiive vs chemo-treated EC-FAK-knockout or EC-FAK kinase dead mouse pre-metastatic lungs for transcriptomic analysis. End-point metastatic lung foci will be analysed for gene expression. Bioinformatic analyses will compare these data with human lung cancer databases. The molecular mechanisms leading to senescence will be identified using immunofluorescence, western blotting, RIME and Chipseq. 3. Functional validation of the DNA-damage-induced EC-FAK driven mechanisms identified in (2) relevant to metastases formation. Genetic and pharmacological inhibition of the candidates identified above will be tested in physiological 3D microfluidic lung chip models with, and without, chemotherapy. Effects on lung cancer cell seeding and colonisation will be determined. It will also allow discovery and testing of novel alternative strategies, to FAK-inhibition, that target EC-senescence in combination with DNA-damaging therapies in pre-metastatic niche formation


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: The Role of Physical Membrane Properties in Tumour Cell Resistance to Perforin
Funder: Medical Research Council

Abstract: Immunotherapies represent this century's most revolutionary progress in the treatment of cancers. However, in spite of some spectacular success, many patients show incomplete or poor response to treatment, and in many instances, tumours become resistant to immune killing for reasons that are still poorly understood. For the critical effector phase, all these therapies rely on cytotoxic T lymphocytes and, occasionally, natural killer cells, and their killing of target cells mediated by an essential pore forming protein called perforin. We have recently discovered that cytotoxic T lymphocytes are protected against their own perforin by lipid order and charge in their plasma membranes, thus allowing them to kill multiple targets in succession without succumbing to self-harm. Based on that observation, we have hypothesised that cancer cells may use similar lipid-based, physical mechanisms to evade immune killing by resistance to perforin. If so, we anticipate that such mechanisms also play a role in defining tumour cell resistance - and ultimately patient response - to cancer immunotherapies, including CAR T-cell therapy. With this project, we aim to test that hypothesis, based on a combination of biophysical and immunobiological expertise, in addition to access to well annotated patient samples.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Oncogenic signalling through transcriptional repression pathways in oesophageal adenocarcinoma.
Funder: Medical Research Council

Abstract: One of the key emerging molecular events which occur in the transition to oesophageal adenocarcinoma (OAC) is mutational activation of RTK pathway signalling pathway components. In particular ERBB2 locus amplifications are commonly found (>20% cases) but it is currently unclear what the molecular consequences of such amplifications are. One major pathway activated by ERBB2 signalling is the ERK MAP kinase pathway and it is known that a major impact of this pathway is on the transcriptional events in the nucleus. Our recent data implicate a number of ERK-regulated transcriptional repressor proteins as potentially important in OAC cells. However, we know very little about how these proteins operate in cancer and nothing about their function in OAC. Indeed, compared to transcriptional activation, much less is known about transcriptional repression in cancer and how oncogenic signalling pathways influence this activity. The overall aim of this study is to examine the role that transcriptional repression plays in deregulated oncogenic RTK pathway signalling in OAC. We will focus on the RTK-regulated CIC and ERF repressor proteins to gain mechanistic depth and, more generally, we will examine the role of silencer elements and 3D organisation of repressive regulatory hubs in OAC. We will first define the RTK-regulated transcriptional repression circuits in OAC and subsequently identify repressive silencer elements in the OAC genome. We will then derive a mechanistic understanding of the RTK pathway-driven changes to the repressive environment, by focussing on the 3D chromatin environment and the potential role of liquid-liquid phase separation. We will provide clinical relevance by relating our findings to molecular events found in OAC patients and by identifying new potential therapeutic target proteins and pathways lying downstream from oncogenic RTK signalling.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Treating Multiple Myeloma and Diffuse Large B Cell Lymphoma by Targeting the NF-kB Pathway with the First-in-Class GADD45b/MKK7 Inhibitor, DTP3
Funder: Medical Research Council

Abstract: Multiple myeloma (MM) and diffuse large B cell lymphoma (DLBCL) are distinct B cell cancers. Despite several new therapies, MM remains incurable. Similarly, over 40% of DLBCL patients do not respond to therapy or relapse and most of them die from their disease, with worst outcomes in cases of activated B-cell (ABC)-DLBCL. NF-kB is a promising therapeutic target in MM/ABC-DLBCL. Yet, despite a 30-year global effort, no specific NF-kB inhibitor has been clinically approved, due to the toxicities of systemic NF-kB blockade. Several MM/lymphoma agents, e.g. proteasome inhibitors, block NF-kB, but also many other pathways, lack cancer-cell specificity and do not exert their clinical effect via NF-kB. Thus, an attractive alternative to blocking NF-kB would be to target the essential, cancer-specific downstream effectors of the NF-kB oncogenic function. We identified the complex formed by the product of the NF-kB target gene, GADD45B, and the JNK kinase, MKK7, as an actionable target in MM/DLBCL. GADD45b is stably upregulated by NF-kB and promotes MM/DLBCL cell survival by blocking MKK7/JNK-driven apoptosis. Further, we developed the GADD45b/MKK7 inhibitor, DTP3, which kills MM/ABC-DLBCL cells via JNK-mediated apoptosis ex vivo and in mice and is not toxic to normal cells (G0901436). Due to this cancer-selective specificity, DTP3 has similar efficacy to the standard bortezomib in MM cells and xenograft models, but a >100-fold higher ex vivo therapeutic index and far wider safety margin in vivo, ablating MM/DLBCL xenografts without adverse effects. GLP studies established the on-target pharmacology, tolerability and good PK/ADME profile of DTP3 (MR/L005069/1). Early trial data demonstrated no adverse events, with initial efficacy and cancer-specific PD signals in relapsed MM patients, warranting further clinical evaluation. We now aim to conduct a follow-on trial of DTP3 to deliver PoC for a safe and effective NF-kB-targeting therapy and address the unmet needs in MM/DLBCL.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: MICA: PANC-AID: Engineering a novel dynamic pancreatic cancer organoid model (MICA)
Funder: Medical Research Council

Abstract: Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease with 91% of patents dying within five years from diagnosis. The disappointing statistics on pancreatic cancer treatment outputs are partly due to the complex tumour microenvironment (TME) which induces resistance to treatment. Therefore, ensuring accurate mimicry of the TME in pancreatic cancer studies and treatment screening is of high importance. The aim of our project is to develop a high fidelity in vitro pancreatic cancer model, which will enable patient & disease specific treatment optimisation. For the accomplishment of our aim we will use as a basis our static developed 3D pancreatic cancer multicellular model (consisting pancreatic tumour cells, stellate cells and endothelial cells) which is based on a highly porous polyurethane scaffolding system and further advance it with tuneable features of the TME, i.e., different internal structure/porosity, various extracellular matrix protein compositions, different cell rations to tune fibrosis which is a hallmark of PDAC and interstitial flow (to mimic different tissue vascular levels and shear stress). Our preliminary work and existing publications on this direction shows that all cell types are viable in the polymer scaffold for a month in culture, which is extremely encouraging especially for endothelial cells which cannot remain live for so long in a 3D spheroid type organisation. Also, our model captures the fibrotic reaction around the pancreatic cancer cells. Furthermore, the versatility of the synthetic scaffold allows to recapitulate and test multiple critical aspects of the TME including extracellular matrix, stiffness, tissue porosity, diffusion of oxygen, nutrients, metabolites and distribution of the vascularisation. The ability to reproducibly represent & control different biophysical, biochemical and biomechanical features of tumor microenvironments in vitro from presents a clear advantage, compared to current in vitro approaches.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS