NIHRDataInsights/HRCSData huggingface dataset:
Title: Elucidating the molecular and epigenetic basis of cancer initiation
Funder: Medical Research Council

Abstract: In Objective 1 of this proposed research we will use state-of-the-art technologies to induce epigenetic modifications (or epimutation) and ask what functional consequence they have on cell function. These powerful tools are currently being developed and involve the CRISPR/Cas9 system with slight modifications whereby the Cas9 is inactive and fused to DNA methyltransferases or 5-methylcytosine TET oxidases. We are implementing this system in collaboration with the Jurkowski lab (Stuttgart University) to understand epigenetic processes that occur during cancer initiation. Selection of the genomic regions targeted for epimutations is based on the knowledge accumulated so far in cancer epigenetics research. Numerous promoters are hypermethylated in cancer but key is the identification of combinations of genes that have the potential to be drivers in the process of cancer initiation when no genetic mutation is required. The cell types to test these epimutations on were chosen based on the hypothesis that cancer stem cells can arise from adult stem cell compartments or through dedifferentiation/reprogramming of progenitors or differentiated cells. This is compatible with the knowledge that cancer stem cells harbour stem cell-like properties. Therefore we will start by using mesenchymal stem cells (MSC), neural stem cells (NS), mammary epithelial and myoepithelial cells. Cells harbouring the induced epimutations will be analysed for tumourigenic properties in vitro and injected in immune-compromised mice in order to measure tumour growth in vivo. Subsequently these tumours will be utilised to unravel the molecular analysis of cellular transformation. In Objective 2, we will use the embryonic stem cell model system where transition between two states (primed to ground) is accompanied by widespread DNA demethylation. The methodology involves use of shRNA libraries (whole genome and sub-libraries) to find proteins involved in the DNA demethylation pathway.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Using tumour, peripheral blood and sentinel nodal transcriptomics to understand the interaction between melanomas and the host
Funder: Medical Research Council

Abstract: We will address the objectives described using data from 2 studies 1. We will recruit 300 newly diagnosed melanoma patients to the Ulceration and Immunity Study (164 to date), in which patients are bled at presentation and 4 intervals in the first year. a)The participants allow sampling of the stored paraffin embedded primary tumours and sentinel nodes, and the generation of transcriptomic data from those tissues using the Affymetrix HTA. b) Vitamin D levels are measured at each time point and patients supplemented if indicated by Leeds clinical protocols. c) Peripheral blood white cell RNA is fixed at the bedside using Leukolock filters for gene expression analysis. d) The transcriptomes of melanoma primaries, blood and nodes will be analysed agnostically using pathway analysis (Metacore). Using bioinformatics we will derive data sub-sets which are indicative of the presence of different components of the immune system (the immunome), as pioneered by INSERM U872, Laboratory of Integrative Cancer Immunology, in order to investigate the interaction between tumour and immune cells. e) Previously we have generated primary tumour transcriptomes from 222 melanoma primaries from the Leeds Melanoma Cohort in which application of the bioinformatic approach developed by the INSERM group resulted in the identification of clusters which were strong independent predictors of survival. We will test these clusters in the new samples from the Ulceration and Immunity Study and determine the associations between these tumour sub-groups and the transcriptomic derived "immunomes', in order to understand whether the 'clusters" have survival significance because of the immunological processes evidenced in blood and nodes. 2. We will recruit a second set of patients commencing systemic therapy for stage 4 disease and seek consent to use blood, tumour and nodal samples in which the signatures derived from the Ulceration and Immunity Study can be tested as predictive biomarkers


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Defining Mechanisms of Hormone Therapy Breast Cancer Risk Reduction and Biomarkers of Response
Funder: Medical Research Council

Abstract: Aim: To elucidate how anti-oestrogens modulate MD in order to establish biomarkers to predict which women will be protected by these treatments, and to ultimately identify potentially targetable, alternative pathways that could be modified to reduce risk. Objectives: I) To use primary human in-vitro and ex-vivo model systems to establish genes and pathways modulated by anti-oestrogenic agents. II) To establish the functional significance of pathways identified in (i) using co-culture systems. iii) To investigate the clinical utility of key functional markers in predicting response to anti-oestrogens in a prospectively recruited patient cohort. Methodology: Fibroblasts from patient samples and pieces of breast tissue maintained in culture will be used as model systems. Cultures will be treated with Tamoxifen (+/- estradiol) Aromatase Inhibitor (Letrozole) or vehicle control. Proliferation and fibrogenic signalling will be analysed using MTS assays, Ki67 analysis, western blotting, RT-PCR, ELISA and immunohistochemistry. RNA-Seq will be used to assess genome-wide changes. Targets will be validated by qRT-PCR or western blotting. The impact of pathways identified on epithelial cell proliferation and invasion will be assessed in 2D and 3D co-cultures, using siRNA knock-down, antibody inhibition or gene over-expression approaches. Clinical utility will be assessed in patients offered preventive tamoxifen through Family History clinics, and those receiving adjuvant AIs through the breast clinic. Biopsy and/or blood samples will be taken at baseline and 1 year and markers measured (by ELISA, IHC, RT-PCR), and compared to changes in MD. Scientific and Medical Opportunities: These novel model systems are highly physiologically relevant, applicable to many other areas of research. Medically, this project aims to deliver precision medicine to the clinic.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: 'T cell versus T cell': A Study of the Cellular Immune Response in Cutaneous T cell Lymphoma (CTCL)
Funder: Medical Research Council

Abstract: The clinical progression of cutaneous T cell lymphomas (CTCL) is heterogeneous but the disorder remains largely incurable. One factor that may determine outcome is the nature of the tumour-specific immune response. Several observations indicate that a CTCL-specific immune response can develop but is likely to be suppressed by several mechanisms of tumour evasion. Antibodies such as anti-PD1 or PDL1, which reverse T cell suppression by inhibitory co-stimulatory molecules, are effective in solid tumours and may be introduced in the management of CTCL. However CTCL represent a novel situation in that the tumour cells, being of haemopoietic origin, also express PD ligands. In this fellowship I will undertake a detailed analysis of the reactive T cell infiltrate in patients with CTCL. The aims of the fellowship are to:- - characterise the number, phenotype and functional properties of T cells within the tumour infiltrate of CTCL - define the pattern of expression of co-stimulatory molecules such as PD1 and Tim3 on tumour infiltrating lymphocytes - determine the antigenic specificity of the T cell infiltrate with particular focus on cancer-testis antigens (CTAg) and primary tumour cells - correlate the functional activity of the reactive T cell infiltrate with clinical outcome. Patients will be recruited from the supra-regional CTCL service and blood and skin biopsies taken at different stages of disease. Malignant and reactive T cells will be isolated and examined by multi-parameter flow cytometry, including a complete analysis of co-stimulatory molecule expression. HLA-peptide tetramers will determine the presence of T cells specific for CTAg, and co-cultures used to investigate recognition of tumour cells by the tumour infiltrate. These results will lead to a substantial increase in understanding mechanisms of immune evasion by CTCL and can be used to guide the introduction of a stratified approach to immune-stimulatory antibody therapy.


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Harnessing TNF-mediated cell death in cancer
Funder: Medical Research Council

Abstract: Deregulated cell death and inflammation can drive chronic remodelling and tissue repair, which can contribute to cancer and treatment failure. The principle idea addressed by this application is that cell survival and adaptation mechanisms of tumours are supported, at least in part, by cancer-related inflammation, and that this can be successfully targeted by switching the TNF response from pro-inflammation to activation of cell death, causing tumours to permanently regress. Despite clear evidence that TNF can signal cell survival and death, the mechanisms that can switch between the distinct biological outcomes remain elusive. This is important as its resolution, and putative therapeutic intervention, would allow the diversion of cancer-related inflammation into activation of cell death. Our starting point for this proposal is substantial new data on the regulation of TNF-signalling and cell death. We identified that the Ub-receptor function of cIAP1 critically controls TNF signalling, selectively regulating the pro-death effects of TNF, without inhibiting the NFkB pathway. Our preliminary data indicate that a point mutation in the Ub-binding domain of cIAP1, which abrogates its ability to bind to M1- and K63-linked Ub chains, switches the TNF response to cell death, and completely blocks tumorigenesis. Mechanistically, we find that the Ub-binding domain of cIAP1 regulates intracellular trafficking of TNF-R1, and the consequences of RIPK1 ubiquitylation. Our data are consistent with a scenario whereby Ub-binding of cIAP1 participates with ESCRT-0 in capturing and sorting ubiquitylated TNF-R1 and RIPK1 for recycling. Here, we propose to use mouse models, imaging, and biochemical approaches to decipher how the Ub-receptor function of cIAP1 regulates TNF-induced cell death, and whether switching the TNF response from pro-inflammation to cell death is beneficial or harmful in cancer.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Structural basis of human TRIAP1/PRELI function in mitochondrial lipid transport and apoptosis
Funder: Medical Research Council

Abstract: Mitochondria are dynamic organelles involved in a variety of cellular processes, such as energy. Mitochondria can also orchestrate cell death (apoptosis) via release of cytochrome c and activation of the caspase signalling cascade. Cancer cells often display misregulation in apoptosis, contributing to the generation of chemotherapy-resistant cells. Thus, mitochondria are at the centre of a plethora of cellular processes both under normal physiological circumstances and in disease. Phospholipid composition plays an important role in mitochondrial homeostasis. Changes in lipid content and structure have been associated with mitochondrial dysfunction and a variety of diseases including ischemia, hypothyroidism, aging, and heart failure. The family proteins (Ups1-3 in yeast and PRELIs in humans) together with a novel CX9C chaperone (Mdm35 in yeast and TRIAP1 in humans) regulate the accumulation of phospholipids within mitochondrial membranes. This pathway has been shown to be important for modulating apoptotic through two independent mechanisms. One indirectly through resultant increased cardiolipin accumulation and sequestration of cytochrome C, which is required for apoptosome formation. The other is through a direct interaction with Hsp70 and APAF1 thereby preventing cytochrome C from assembling in the apoptosome. These mechanisms stall apoptosis and gives cell to the opportunity to repair DNA damage This proposal aims to characterize the structure and interactions of human TRIAP1/PRELI using NMR and X-ray crystallography approaches. The structural and dynamic properties of TRIAP1 with PRELI proteins and Hsp70 will be probed by site-directed mutagenesis and functional assay. The outcomes of this research project will provide new insight into the biological roles for the TRIAP1/Mdm35 family of proteins, which have been implicated in mitochondrial lipid homeostasis and cytotoxic drug-resistance through the evasion of apoptosis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Immunotherapy of Epithelial Ovarian Cancer using Autologous Gamma Delta T-cells
Funder: Medical Research Council

Abstract: Epithelial ovarian cancer (EOC) represents the most lethal gynaecological malignancy, with particularly poor outcome seen in patients with high-grade serous tumours (HGSOC). Our goal is to develop a gd T-cell-based immunotherapy to address this need. gd T-cells contribute to cancer immunosurveillance, owing to their ability to recognise markers of cell stress. Vg9Vd2 cells are the primary circulating gd T-cell subset in man and undergo HLA-independent activation by phosphoantigen (PAg) intermediates of mevalonate metabolism. PAgs are increased in EOC cells, owing in part to p53 mutation. Amino-bisphosphonate drugs (NBP) further enhance PAg accumulation, sensitising EOC cells to gd T-cell-mediated killing. We have shown that Vg9Vd2 T-cells can be expanded from EOC patients and that they destroy autologous and immortalised EOC tumour cells and cause regression of derived xenografts. Building on this, we have recently developed a novel GMP-compliant system to expand these cells (method 2), which substantially improves both their yield and intrinsic tumouricidal activity. In this DPFS project, we will evaluate anti-tumour activity of method 2-expanded Vg9Vd2 T-cells from patients against EOC. First, we will use high-throughput in-vitro luciferase assays to determine their anti-tumour activity (alone and in combination with licensed NBPs) against a panel of HGSOC and other EOC cells. Next, we will assess their in-vivo therapeutic activity using a panel of HGSOC xenograft models. Owing to transcoelomic metastasis, all therapeutic agents will be administered to the peritoneal cavity (an established route of drug delivery in EOC). Iterative optimisation will then be undertaken to determine the optimum schedule of repeated cell/NBP administration and need for cytokine support. Finally, we will develop a maximally closed and clinically compliant manufacturing process for large-scale method 2 production of these cells, paving the way for first in man testing in EOC patients.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: CANCER AND NEOPLASMS
Title: Development of the UroMark assay. A non-invasive test for the detection of bladder cancer in urinary sediment cells
Funder: Medical Research Council

Abstract: Cystoscopic investigation for bladder cancer in patients with haematuria and in patients requiring surveillance for recurrent disease incurs a cost to UK healthcare estimated at £55.39 million per year ranking bladder as one of the most expensive cancers to manage. We have developed a highly multiplexed targeted sequencing assay (UroMark) to detect cancer specific epigenetic alterations in urinary sediment, along with a home collection kit allowing the collection of urine through the postal system. The UroMark assay utilises a micro-droplet PCR platform (RainDance Technology) which allows the analysis of a panel of 150 epigenetically altered loci which accurately discriminate between tumour and normal urothelium. Proof of concept testing has shown the potential utility of this assay for the detection of bladder cancer in urinary sediment cells. The aim this project is to validate the UroMark assay through two non-IMP clinical trials. DETECT I, will determine if the assay can rule out bladder cancer in patients with haematuria and DETECT II will validate the diagnostic accuracy of the test in a cohort trial enriched for low grade cancer. Our aim is to show sufficient diagnostic precision to replace cystoscopy in the evaluation of patients with haematuria. The incorporation of a highly sensitive and specific assay will revolutionise bladder cancer pathways, have a profound impact on the requirement for cystoscopy and on patient well being and, reduce the healthcare costs associated with investigation of haematuria and surveillance for disease recurrence.


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: structures and mechanism of BRCA2 in meiotic recombination
Funder: Medical Research Council

Abstract: In reproducing organisms, two sets of genomes need to be exchanged and integrated. Double stranded breaks are required for genetic exchange to occur as they serve as recombination sites. Interestingly, a DSB is also one of the most severe types of damage to our DNA in non-sexually reproducing (mitotic) cells as failure to repair it can cause cell death while mis-repair can cause mutations in DNA that can lead to the development of cancer. Even more remarkable is that mitotic and meiotic DSBs use similar mechanisms for repair. In mitotic cells, homologous recombination utilizes the sister chromatid as templates for repair while meiotic recombination utilizes the homologous chromosome. In homologous recombination, RAD51 recombinase forms filaments on resected ssDNA and this nucleoprotein filament is used for homology search. In meiosis, DMC1, a RAD51 homologue, is the main recombinase although RAD51 is also required. The similarity between the two processes is further exemplified by the requirement of BRCA2. BRCA2 is one of the breast cancer susceptibility proteins, and mutations in BRCA2 gene increase the risk of breast, ovarian and other cancers due to the reduced ability to repair DNA damages. In mice, defects in BRCA2 cause male infertility. However, it is unclear if this is true in human. It is thus intriguing and important to understand what exact roles BRCA2 plays in meiotic recombination and how it fulfills its roles. Furthermore, it is fascinating as why both RAD51 and DMC1 are required in this process and what the exact role of RAD51 has in this process. In this proposal, We plan to use electron microscopy single particle technique to study the 3-dimensional structures of BRCA2 and DMC1 and possibly BRCA2, RAD51 and DMC1 and use biochemical and biophysical approaches to study the influence of BRCA2 on nucleoprotein filament formation to reveal the mechanistic action of BRCA2 and RAD51 in meiotic recombination.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: A BMI1/CHD7 signature in medulloblastoma with poor prognosis.
Funder: Medical Research Council

Abstract: Medulloblastomas are the most common malignant brain tumours of childhood, accounting for a quarter of all cancer-related childhood deaths. Genome-wide expression, copy number analysis and whole genome sequencing have significantly advanced our understanding of the molecular pathogenesis of these tumours, identifying four distinct subgroups affecting prognosis and predicting response to therapy. Despite improvements in the clinical treatment of these tumours, the outlook for aggressive subgroups remains bleak, while survivors often suffer from pronounced neurocognitive delays resulting from the surgical resection of the tumour and subsequent chemotherapy/radiotherapy. We have identified a subset of medulloblastomas that exhibit a novel BMI1highCHD7low molecular signature associated with reduced overall survival. Our initial analyses of primary medulloblastoma cells of this particular subgroup and CHD7-deficient primary mouse granule neuron progenitors have led to the identification of three potential mechanisms of BMI1-CHD7 convergence. We will use these cells and novel mouse models to test the biological relevance of these molecular mechanisms in medulloblastoma development. In addition, we will employ state-of-the-art, unbiased, genome-wide approaches (RNA-seq, ChIP-seq and ATAC-seq) to identify novel tumour-promoting pathways in medulloblastomas displaying this signature. The significance of these genes and pathways in tumour formation and growth will be assessed by over-expression and knock-down experiments in human medulloblastoma cells and xenografts. Comparative analysis of the candidate genes identified in the experimental models with the human datasets followed by functional validation of the most promising candidates in human primary medulloblastoma cells will lay the basis for future high- throughput drug screening to identify new single-agents or combinatorial therapeutics with anti-tumour properties.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Functional Priming of Human Cord Blood Derived Haematopoietic Stem and Progenitor Cells
Funder: Medical Research Council

Abstract: Stem cells are important in normal development, for tissue homeostasis and for regenerative medicine. Understanding the regulation of cell fate in stem and progenitor compartments would lend insight into stem cell physiology and its pathology in diseases such as cancer. Despite the extensive analysis of Haematopoiesis as a paradigm for somatic stem cells, blood stem cells cannot routinely be expanded ex vivo. This limits study of their cell fate control mechanisms but also constrains the use of cord blood units in clinical transplant settings. We have shown that Nov/ CCN3 may enhance the functional capacity of CB stem cells and we propose to evaluate its impact on a number of engraftment parameters in a xenograft model to establish and optimise it potential as a therapeutic. We will also dissect its mechanism of action with a view to establishing genes and pathway that may further enhance stem cell potential but also to understand how Nov effects what we hypothesise to be a more global change in cellular state akin, at least conceptually, to the Naive to Primed transition reported for pluripotent stem cells.


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