NIHRDataInsights/HRCSData huggingface dataset:
Title: Computational Analysis of Transcription and Alternative Splicing Events in Squamous Cell Cancer.
Funder: Medical Research Council

Abstract: This project focuses on comprehensively measuring alternative RNA splicing in squamous cell carcinoma (SCC). I will compare SCCs from two different anatomical sites, which arise through distinct aetio-pathological processes: (i) Skin SCC, which is primarily due to ultraviolet radiation exposure (ii) Cervical SCC, caused by high-risk human papillomavirus (HRHPV) infection. From 40 tumour and 20 site, age and gender matched samples from healthy (no cancer) individuals) I will generate high-throughput strand-specific RNA-sequencing data and build transcriptome assemblies. I will compare RNA-seq tools (eg. TopHat/Bowtie/Cufflinks, BWA, Scripture, Kallisto, Sailfish, Salmon, Trinity) and assess their accuracy using RASE-qPCR to determine the optimum toolchain for RNA-seq splicing analysis. I will quantify isoform expression in each tissue sample and quantify alternative splicing events (eg. SUPPA, MISO, MATS). I will investigate the expression of different splicing factors and their targets, as well as differential isoform expression of important cancer genes. Tools including DESeq, DEXSeq, Sleuth, edgeR, voom will be compared and optimised. Expression will be correlated to clinico-pathological features (age, gender, tumour stage and grade) for each cancer type using a support vector machine classifier and will be tested on independent sample sets. Biologically relevant candidates will be validated experimentally by qRT-PCR and gene over-expression/knockout. I will identify targets bound by cancer-specific splicing factors using cross-linking immunoprecipitation high-throughput sequencing. This work will improve our understanding of the role of alternative splicing in SCCs, the mechanisms of splicing deregulation and the functional consequences of altered splicing for SCC-specific regulatory networks. It will also indicate candidate diagnostic and/or prognostic biomarkers and potential targets for novel therapies in SCC


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Characterising cancer-associated fibroblast heterogeneity in lung cancer: relating molecular phenotype to function
Funder: Medical Research Council

Abstract: Lung cancer is the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) represents around 80% of cases. Cancer-associated fibroblasts (CAFs) are the principal stromal cell type in many solid tumours, including NSCLC. These cells are known to drive tumour progression and correlate with poor prognosis. CAFs are conventionally defined by a myofibroblast phenotype; however, the role and impact of CAFs in NSCLC is yet to be adequately characterised. Recent research (including our own preliminary data) has highlighted that CAFs are a heterogeneous population, with functionally distinct subtypes. This project will define the different CAF subtypes found in NSCLC, identify those correlating with poor survival, and investigate their functional role in tumour progression. This will be achieved by single cell RNA-Sequencing (scRNA-Seq) of patient-matched samples from NSCLC and non-involved lung. Transcriptionally distinct fibroblast clusters will be identified through bioinformatic analysis. These groups will be correlated with clinical parameters (e.g. survival) using the publicly available cancer genome atlas dataset. Significant correlations will be validated using digital pathology analysis of an existing NSCLC patient cohort (n=600). Finally, the function of CAF subtypes correlating with disease progression will be analysed in silico using pathway analysis and in vitro using 3D model systems. This project will elucidate the basic biological mechanisms underpinning the role of fibroblasts in lung tissue homeostasis, and how these mechanisms become dysregulated during malignant progression. Identifying pro-malignant CAF subtypes may lead to new diagnostic and therapeutic targets. Furthermore, this work will generate single-cell gene expression profiles from multiple cell types within the NSCLC tumour microenvironment, which will be of significant value to the scientific community.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Combination immunotherapy for solid tumours with oncolytic virus & cognate CAR T-cells
Funder: Medical Research Council

Abstract: Cancer is a leading cause of death globally. Despite advances in immunotherapy, the vast majority of patients with advanced cancer remain incurable. Adoptive cell therapy using chimeric antigen receptor (CAR)-expressing T-cells has considerable promise, but solid tumours have proved resistant to this approach. There are a number of purported reasons for this including a) a lack of homogenously & selectively expressed tumour-associated cell surface antigens, b) a paucity of "dispensable antigens" expressed by solid tumours (unlike CD19+ malignant B-cells where CAR T-cell therapy has proved particularly effective), (c) poor trafficking of CAR T-cells into the tumour microenvironment (TME) and d) impaired CAR T-cell effector function within the TME. This proposed research uses in vitro and in vivo experiments (using an immunodeficient mouse xenograft model) to investigate the efficacy and safety of a novel therapeutic strategy combining oncolytic virotherapy in a synergistic fashion with CAR T-cell adoptive cell transfer for the treatment of advanced solid tumours. The proposal is further refined by engineering the oncolytic virus to alter the TME thus rendering it more permissive to immune attack. The CAR T-cells may also be further edited to facilitate their efficacy and longevity. Thus, I aim to demonstrate proof of concept for this novel & synergistic immunotherapeutic combination strategy.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Investigating the trigger for fibroblast senescence in colorectal cancer: a novel role for extracellular vesicles
Funder: Medical Research Council

Abstract: Background: Fibroblast senescence is a poor prognostic factor in several solid cancers but the trigger for this fundamental process is unknown. E-cadherin-expressing colorectal cancer (CRC) cells produce extracellular vesicles (EVs), attenuating ERK activity in fibroblasts. This corresponds with a proliferation defect, G1 arrest and a higher proportion of senescent cells. EVs from these CRC cells are rich in miR-143, which represses KRAS. Aims and Objectives: This project aims to identify a pathway which leads to fibroblast senescence in CRC through the following objectives: 1. Determine whether miR-143 regulates fibroblast senescence via Ras-inhibition. 2. Ascertain whether extracellular vesicles (EVs) from CRC cells deliver a functionally relevant amount of miR-143 to fibroblasts. 3. Identify changes in the secretory profile of miR-143-modulated fibroblasts and how this alters behaviour of CRC cells. 4. Demonstrate findings in vivo using an orthotopic colorectal cancer mouse model. Methodology: MiR-143 will be overexpressed and inhibited in fibroblast lines. Proliferation, cell cycle profile and senescence will be assessed. Similarly, E-cadherin-expressing CRC cells will have miR-143 levels manipulated, to alter the miR-143 content of their EVs. EVs will be delivered to fibroblasts and senescence assessed. MiR-143-modulated fibroblasts will have their conditioned media profiled for cytokines. CRC cells will be exposed to conditioned media and proliferation, migration, invasion and chemoresistance measured. Finally, an inducible miR-143 fibroblast line will be co-injected with CRC cells intra-caecally in nude mice. Animals will be followed up by in vivo imaging to assess effects of miR-143, including response to chemotherapy. Scientific and Medical Opportunities: Understanding the molecular drivers of fibroblast senescence in colorectal cancer is critical to identifying novel prognostic biomarkers and actionable therapeutic targets.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Epigenomic Mechanisms of Action of Novel Mutant Isocitrate Dehydrogenase Inhibitors in Acute Myeloid Leukaemia
Funder: Medical Research Council

Abstract: Aims and objectives 1. Investigate how abnormal epigenetic gene regulatory mechanisms result in differentiation block and leukaemic transformation in IDHm AML. 2. Investigate the molecular mechanisms of action of mutant IDH inhibitors in inducing differentiation of AML, focusing on effects on transcription factors involved in normal granulocyte-monocyte (GM) differentiation. Methodology 1. Compare gene expression, DNA methylation and chromatin accessibility, performed in parallel (multi-genomics) in normal bone marrow versus AML. Perform unbiased genome-wide bioinformatic analysis, and analysis focused on differentially expressed transcription factors required for normal GM differentiation (GM-TFs) but suppressed in AML. 2. Test the hypothesis that failure to express GM-TFs results in leukaemic transformation using CRISPR knockdown of candidate GM-TFs in IDH1-mutant preleukaemic mouse model. 3. Perform multi-genomic sequencing on mIDHi-treated AML LSC in vitro to investigate how mIDHi upregulates GM-TFs. Identify GM-TFs upregulated with mIDHi in responsive AML samples but which remain repressed in non-responsive samples. Test if re-expression restores response in resistant AML samples. 4. Correlate GM-TFs required for response (from point 3) with gene expression in IDHm AML patients who are resistant to mIDHi treatment in vivo. These may be important targets for novel therapies. Applications and benefits This work will improve our understanding of the molecular mechanisms of differentiation block and its contribution to pathogenesis of AML. It will help explain why differentiation therapies may not cure patients. The data has the potential to identify new genes/ pathways that should be targeted by novel therapies to improve clinical outcomes. It will provide the rationale for design of future clinical trials of combination therapy. Multi-genomic characterisation of patients may be useful as a prognostic tool, and to guide therapeutic decisions.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Structural and Mechanistic Studies of the SWI/SNF Family of Chromatin Remodelling Complexes
Funder: Medical Research Council

Abstract: Chromatin remodelling complexes (CRCs) overcome the barrier presented by the packaging of DNA into nucleosomal and supra-nucleosomal structures and allow access to the factors responsible for transcription, DNA repair, replication and recombination. Genome sequencing of cancer cells has identified the 2MDa human SWI/SNF (BAF) remodelling complex as the most frequently mutated chromatin regulator in human cancer - mutations affecting BAF complex subunits were observed in 20% of all tumors. Although the BAF complex is an important global tumour suppressor, little is known about its architecture and the molecular mechanisms underpinning its recruitment to specific genomic loci and chromatin remodelling activities. My research team will strive to achieve a detailed molecular understanding of the structure, mechanism and promoter recruitment of the eukaryotic SWI/SNF family of chromatin remodelling complexes. Structural work will initially target global architectural details using a proven integrative structural biology pipeline comprising cryo-EM, cross-linking mass spectrometry (CXMS) and computational modelling. High-resolution studies of the complete BAF complex will utilize cutting-edge cryo-EM instrumentation with complementary X-ray crystallography studies targeting sub-complex assemblies. To dissect the molecular determinants of BAF complex recruitment to target gene promoters, we will reconstitute a yeast SWI/SNF-nucleosome-activator protein complex from purified components in vitro. This system will enable us to systematically probe the numerous interactions between CRC, nucleosome and transcriptional activator protein. The structural basis of these CRC-nucleosome interactions will be determined by solving the cryo-EM structure of a BAF-nucleosome complex. Subsequent mechanistic studies will explore the nucleoprotein perturbations accompanying remodelling by trapping "stalled" remodelling intermediates harbouring engineered site-specific DNA-histone crosslinks.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Targeting PPP1R15 in malignancy
Funder: Medical Research Council

Abstract: Deregulated protein synthesis is a feature of cancer and can lead to endoplasmic reticulum (ER) stress. A rectifying response to ER stress involves the phosphorylation of eIF2a by PERK, which inhibits new protein synthesis while triggering a gene expression programme through enhanced translation of ATF4. A target of ATF4 is PPP1R15A, which dephosphorylates eIF2a to restore normal translation. We showed that PPP1R15A contributes to cell death during unremitting ER stress by promoting accumulation of misfolded protein. Phosphorylation of eIF2a has a permissive effect on tumour growth and so PERK inhibition has emerged as a novel anticancer strategy. We observed the loss of PPP1R15A by some cancers leading us to hypothesise that restoration of PPP1R15A activity could serve to oppose PERK and potentiate the anticancer effects of PERK inhibitors. Aim 1. Elucidate the physiological regulation of PPP1R15A: We demonstrated that PPP1R15A is regulated by the binding of G-actin. The abundance of G-actin is determined by cytoskeletal dynamics that are themselves regulated by growth and mobility cues. We will will determine how these cues areguate eIF2a phosphatase activity in health, so that we might target these pathways to augment PPP1R15A activity in disease. Aim 2. Elucidate the regulation of PPP1R15A stability: PPP1R15A has a half-life of <4 hours. We will identify the degradation machinery responsible for this, since inhibiting PPP1R15A degradation could also promote eIF2a phosphatase activity. Aim 3. Elucidate the role of PPP1R15 membrane binding: Binding to the ER, lipid droplets and mitochondria is a prominent but poorly understood feature of the PPP1R15 proteins. We will determine the effect such binding on the function of the organelles and on the activity and stability of PPP1R15. By understanding the regulation, stability and membrane association of PPP1R15A we will be able to develop strategies to control its activity in diseases including cancer.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: RAB11 RECYCLING IN OVARIAN CANCER METASTASIS
Funder: Medical Research Council

Abstract: High grade serous ovarian cancer (HGSOC) is the most common and lethal form of ovarian cancer, and the 5 year survival rate is less than 40%. This statistic has changed very little in the decades since the current surgical and chemotherapy treatments were introduced, and few targeted therapies have been identified. HGSOC preferentially metastasises to the omentum within the peritoneal cavity, and this step is thought to contribute to further dissemination, resistance to therapy and ultimately patient death. We will focus on how HGSOC cells invade into and proliferate within fibronectin-rich islands of stroma within this tissue. We will use surgically resected patient tissues to generate cell lines and explants to understand how HGSOC cells interact with their matrix environment at the metastatic niche. The Rab11 family controls endocytic trafficking, and has 3 members: Rab11a, Rab11b and Rab25. Whilst their role in endocytic recycling is clear, and several effector pathways have been described, how their GTPase cycle is controlled in mammalian cells is not clear. Rab11s control the trafficking of cargoes including the major fibronectin receptor, alpha-5 beta-1 integrins, and Rab25 expression is associated with poor outcome in HGSOC. We aim to understand which Rab11s contribute to HGSOC dissemination, and identify the machinery that controls Rab11-dependent trafficking in HGSOC cells. We will also investigate which cargoes control HGSOC cell invasion, proliferation and resistance to therapy, using cell biology techniques in autologous models of HGSOC in the omentum. Rab11a and Rab11b are expressed ubiquitously, and hence may not be appropriate targets for cancer therapy. However targeting the proteins that promote cargo delivery, or the cargos themselves, could prove effective. We will identify the molecular machinery that controls delivery of specific cargoes to promote HGSOC progression, and thus provide line of sight to therapeutic targets to tackle this disease


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Investigating the role of the actin-myosin regulatory protein MRCK in promoting radiation induced infiltration by glioblastoma cells.
Funder: Medical Research Council

Abstract: Glioblastoma (GBM) is an aggressive and incurable primary brain tumour that causes severe neurological, cognitive and psychological symptoms. Symptoms are caused and/or exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain, disrupting normal function. Tumour infiltration also renders complete surgical resection unachievable, thus contributing to the high recurrence rates that are observed despite aggressive treatment with radiotherapy and chemotherapy. Targeting the mechanisms that drive invasion has potential to alleviate symptoms and extend survival in GBM patients. Resent research has indicated that, while radiotherapy extends life expectancy of patients, it can provoke a more infiltrative phenotype in those GBM cells that survive treatment. This may increase the likelihood of recurrence at or beyond the tumour margin, and adversely affect outcomes after recurrence. We have identified myotonic dystrophy kinase-related CDC42- binding kinase (MRCK) as a key mediator of this response, and have shown that a novel MRCK inhibitor can prevent it, both in vitro and in vivo. We now propose to characterise the signalling cascades up- and downstream of MRCK activation to increase our understanding of the mechanisms by which radiotherapy promotes invasion and thus identify new therapeutic targets and predictive biomarkers. Key to the success of this project will be access to our repertoire of specialised in vitro and in vivo techniques and models that are based on our panel of well characterised primary GBM lines. Specific assets include an ex vivo brain slice model of invasion and an intracranial window model that allows intravital imaging of GBM cells within the brains of live mice. Using these techniques we will characterise the effects of radiotherapy on GBM cell invasion in the most relevant preclinical environment possible, and use our findings to develop and refine new treatments for this cancer of unmet need.


Research activity codes: 5.5 RADIOTHERAPY AND OTHER NON-INVASIVE THERAPIES
Health categories: CANCER AND NEOPLASMS
Title: Elucidation of cellular and molecular mechanisms of lymphoma induction and evolution to identify therapeutic targets
Funder: Medical Research Council

Abstract: Malignant lymphomas are the 5th most common cancers in the world, accounting for ~5% of all tumours. They may be derived from the B-Cell or T-cell lineage and are generally mature in nature. The prognosis of lymphomas greatly varies and is dependent on the stage and histology of the disease. Although up to 60% of young patients (<60y) with aggressive Diffuse Large B-Cell Lymphoma (DLBCL) are cured by combination immune-chemotherapy, older patients have a fairly dismal outlook. However, low-grade indolent disease such as Follicular lymphoma (FL are commonly incurable. Worryingly, the incidence of malignant lymphomas continues to rise, and at 3-4% increase per year appears far in excess of what would be expected simply from the ageing of the population. The identification of critical mediators of lymphomagenesis and novel therapies that target them is therefore a priority for this increasingly common malignancy and unmet medical need. To address these questions, we will use unique mouse models that we have developed engineering in the ability to generate and record further progression events through insertional mutagenesis, specifically within B-cells. This system will allow us to robustly answer the question of the contribution of the initial cell-type to outcome and phenotype of lymphoma. Using the presence of a unique premalignant phase within the model, we will also characterize alterations of transcription, epigenetic state and signaling that occur during lymphoma evolution, through a combination of single cell and bulk RNA-Seq, ChIP-Seq and ATATC-Seq, promoter based capture HiC and intracellular flow cytometry. Finally we will test a priori hypotheses about the requirement for specific DNA repair pathways and residual CREBBP/P300 function in these Crebbp-/- lymphomas, as well as performing a genomewide CRISPR/Cas9 dropout screen to identify further genotype-specific vulnerabilities, a subset of which will be further tested in our in vivo model.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: IMPC- Understanding somatic MSI+ colorectal cancers using conditional coding and non-coding mutations in Mlh1.
Funder: Medical Research Council

Abstract: Microsatellite-unstable (MSI+) tumours are commonly found in the colon where they comprise ~15% of all cancers. MSI+ cancers can occur as a result of the familial Lynch syndrome which is caused by germline mutations in components of the mismatch repair pathway such as MSH2 and MLH1. MSI can also arise in sporadic cancers where, in the majority of cases, MLH1 is silenced by promoter methylation. Colorectal tumours arising in Lynch syndrome patients usually develop via the classical adenoma to carcinoma pathway. In contrast, sporadic MSI+ cancers develop from sessile serrated adenomas. This pathway is characterised by the presence of BrafV600E mutations and has a lower frequency of APC mutations than the classical pathway. Sporadic MSI+ cancers with BrafV600E mutations and promoter silencing of MLH1 form a significant subgroup of colorectal cancer with a good prognosis but a differential response to common chemotherapies. A greater understanding of this group of cancers is essential to develop subtype specific or targeted treatments. Germline Mlh1 mouse knockouts with and without Apc mutations have been studied and proved representative of Lynch syndrome but to date, no model of somatic Mlh1 coding mutation combined with BrafV600E mutation exists. In this proposal we aim to generate and characterise such a model using the IMPC Mlh1tm1c strain and compare it with an existing Mlh1 promoter mutation created in our laboratory. We will use these systems to investigate the initiation and progression of sporadic MSI+ tumours. Our long term goal will be to use either or both models to test existing and novel therapies, including immunological and epigenetic based approaches, in this distinct class of colorectal cancer.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS