NIHRDataInsights/HRCSData huggingface dataset:
Title: Establishing the mechanism for RUVBL2 essentiality in acute myeloid leukaemia
Funder: Medical Research Council

Abstract: Research in our groups is focussed on understanding the molecular basis of acute leukaemia. We reported previously that the AAA+ ATPase RUVBL2 is essential for acute myeloid leukaemia (AML) survival. In this proposal we present further evidence for the selective requirement of RUVBL2 in AML, but not normal haematopoietic progenitors. We demonstrate that RUVBL2 binds to the transcription factor c-MYB and regulates its activity, ensuring repression of myeloid differentiation-associated c-MYB target genes. c-MYB is known to play an essential role in multiple subtypes of AML. The mechanism underlying the control of c-MYB activity by RUVBL2 in AML is unclear and the subject of the current proposal. We propose to determine how RUVBL2 controls c-MYB activity, by establishing the impact of RUVBL2 on co-repressor and co-activator complexes recruited to c-MYB and to c-MYB target genes, and examine changes to this recruitment and to the target gene chromatin landscape following RUVBL2 silencing. We will generate deletion and point mutations to define which regions and amino acid residues of RUVBL2 are necessary for c-MYB binding and its regulation, and examine the effects of inhibitory RUVBL2 mutants on essential leukaemia-specific transcriptional networks, and on AML survival and progression in experimental models of the disease. Further analysis of our data also demonstrated changes in E2F1 target gene expression in AML cells following RUVBL2 silencing. Following on from a recent study reporting RUVBL2 binding to E2F1 and regulation of its transcriptional activity in hepatocellular carcinoma, we will examine interactions of RUVBL2 with E2F1 in AML cells and the impact of RUVBL2 on target gene binding by E2F1. We envisage that this project will provide crucial information on how RUVBL2 contributes to the establishment of aberrant leukaemia-associated transcription factor networks and will facilitate future drug development initiatives.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: The cell death regulator FLIP: evaluation of novel inhibitors in colorectal cancer
Funder: Medical Research Council

Abstract: FLIP is a nodal regulator of cell death and chemotherapy resistance in colorectal cancer (CRC), and high FLIP expression correlates with poor prognosis. We recently developed the first small molecules that inhibit FLIP. The overall goal of this project is to use these unique FLIP-targeting reagents and complementary genetic approaches to determine the therapeutic potential of this class of agent in CRC. This project is organised into 5 workpackages: 1: Efficacy and mechanism-of-action of novel FLIP inhibitors in molecularly-defined CRC cell lines: the aim here is to identify optimal treatment combinations and target CRC subgroup(s) for FLIP inhibitors and understand the mode(s) of cell death induced using a panel of genetically and molecularly characterised CRC cell line models. 2: FLIP inhibitor efficacy in patient-derived models: in collaboration with Prof Andrea Bertotti (Turin), we will analyse patient-derived models of CRC in vitro and in vivo to confirm the optimal combination partner(s) and clinical context(s) identified in WP1. 3: Impact of FLIP inhibitors on murine Apc/Trp53/Kras mutant CRC: given FLIP's roles in the tumour immune microenvironment, the impact of FLIP inhibitors on tumour drug resistance in immune-competent animals (including assessment of tumour-associated immune cells) will be determined using a novel protocol to induce formation of colorectal tumours in genetically-engineered mouse models (GEMMs). 4: The association of FLIP with CRC is clear; however, the requirement of FLIP for tumourigenesis has not yet been established. Here, we will generate the first GEMMs to address this by generating animals in which we can simultaneously induce CRC and delete FLIP. 5: FLIP is a known inhibitor of anoikis (detachment-induced apoptosis), the suppression of which is required for cancer cells to disseminate from their primary site; here, the requirement of FLIP for CRC metastasis and impact of FLIP inhibitors on metastasis will be assessed.


Research activity codes: 5.5 RADIOTHERAPY AND OTHER NON-INVASIVE THERAPIES
Health categories: CANCER AND NEOPLASMS
Title: Characterising novel recombination pathways at DNA adducts of the environmental mutagen BPDE
Funder: Medical Research Council

Abstract: We will investigate mechanistic toxicology of BPDE in human relevant models. The proposed experiments will primarily be performed in human U2OS osteosarcoma cells. We have U2OS cells for inducible PrimPol depletion and CRISPR-deleted for PARP1 and PARP2, and will generate further U2OS lines for combined depletion/expression of PrimPol for mutagenesis studies. BPDE adducts will be induced using nanomolar concentrations of commercially available (+)-anti-BPDE and adduct levels will be quantified by HPLC. PrimPol exerts primase-, polymerase-, and TLS activity and plasmids expressing separation-of-function variants of PrimPol will be used to distinguish the contributions of these activities to BPDE-induced recombination. PrimPol-dependent ssDNA formation and resection will be assessed by immunostaining for native BrdU, RPA, and phospho-Serine S4/8 RPA, as well as S1 endonuclease-modified fibre assay and SMART fibre assay. Impact on replication fork progression will be measured using standard DNA fibre analyses. We will further decipher the mechanisms of ssDNA gap resection and RAD51 loading at BPDE adducts using chemical inhibitors of resection factors and recombination mediators such as MRE11 and PARP1/2. Other resection proteins and recombination mediators such as BRCA1, BRCA2, RAD52, DNA2, and Exo1 will be siRNA-targeted. Initiation of recombination will be measured by RAD51 foci formation and chromatin recruitment, as well as proximity ligation assay. iPOND may be used as backup if needed. We will investigate potential competition between PrimPol-dependent recombination and translesion synthesis by DNA polymerase kappa. This will involve siRNA depleting Pol kappa and quantify eGFP-Pol kappa foci formation. Impacts of PrimPol-dependent recombination on mutagenesis will be measured by classic mutagenesis assays e.g. HPRT, recombination assays such as sister chromatid exchange and whole genome sequencing followed by bioinformatics analyses.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: CANCER AND NEOPLASMS
Title: Finding therapeutic targets in FLT3-ITD AML using a systems biology approach
Funder: Medical Research Council

Abstract: Acute myeloid leukaemia (AML) is caused by mutations interfering with hematopoietic differentiation and mutations conveying a proliferative advantage. FLT3-ITD mutations are the most common growth-promoting mutations in AML. The prognosis of FLT3-ITD AML is extremely poor and new therapies are urgently needed. For this we need to obtain systems-level information on the targets affected by drugs and mechanistic information on how drugs work. We have extensively profiled FLT3-ITD AML using multi-omics approaches and uncovered a common set of FLT3-ITD AML-specific deregulated genes. We identified the core FLT3-ITD-specific deregulated transcriptional network and pathways. Central to this network was FLT3-ITD signalling to MAPK, AP-1 and RUNX1. In collaboration with John Bushweller in the USA, we showed that small molecule inhibitors of RUNX1 can suppress the FLT3-ITD network. We also established conditions to propagate primary FLT3-ITD cells both in vitro and in vivo. In this proposal we will build on this work to define the role of such genes and pathways with respect to FLT3-ITD AML development and maintenance. (1) We will use an shRNA-based drop-out screen targeting the complete list of FLT3-ITD-AML-specific genes. We will use this to identify genes essential for tumour formation in a FLT3-ITD xenograft model, or for colony formation using an in vitro model. (2) We will validate the role of specific signalling pathways, transcription factors and other genes using patient-derived FLT3-ITD AML cells. We will target these parts of the FLT3-ITD network using (a) shRNAs identified via the shRNA screen, (b) RUNX1 inhibitors, and (c) A dominant negates FOS which acts as a broad inhibitor of AP-1. (3) We will also use drugs to target essential pathways in FLT3-ITD AML. We will test combinatorial therapy of FLT3 inhibitors plus different MAPK inhibitors. (4) We will reveal new therapeutic targets by identifying pathways used by FLT3-ITD AML to develop resistance.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Clonal heterogeneity and therapy resistance in Burkitt lymphoma
Funder: Medical Research Council

Abstract: Burkitt lymphoma (BL) is a rare non-Hodgkin lymphoma with a good chance of cure. However, this requires the use of intensive multi agent chemo-immunotherapy and is associated with significant acute toxicity, prolonged in-patient stays and the need for broad supportive care measures. Furthermore, many older adults are not suitable for such intensive therapy and their outcome is worse. For patients of any age who relapse, cure is very unlikely as disease is commonly resistant to multiple agents. Little is known about the cellular, genetic or transcriptomic factors driving relapse. This fellowship will investigate the heterogeneity of tumours at diagnosis and how clonal, mutational and transcriptomic changes occur under therapeutic pressure and contribute to therapy resistance. To achieve this, I have developed patient-derived xenograft (PDX) models of BL suitable for treatment with multi-agent chemotherapy, analogous to that used initially in humans. Without treatment escalation it is predicted that these models will develop therapy resistance, as occurs in patients. Untreated and chemo-resistant models will be characterised by exome, targeted mutation and RNA sequencing and their clonality assessed by sequencing of the clonally unique V(D)J region. Evolutionary modelling will be conducted in collaboration with the ICOS computing group, Newcastle. Integrated pathway and network analyses will determine the clonal and transcriptomic drivers of relapse. Drivers of relapse, identified in PDX models, will be validated using samples from therapy resistant patients and functionally examined in vitro. Therapeutically tractable targets will be assessed in pre-clinical studies using PDX models of relapsed disease, in collaboration with the CRUK Drug Development group in Newcastle. An improved understanding of factors predisposing to relapse or causing therapy resistance will allow patient stratification and guide the development of future relapse treatment strategies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Investigating the role of the Notch atypical ligand delta-like homologue 1 (DLK1) in adrenocortical carcinoma
Funder: Medical Research Council

Abstract: Adrenocortical carcinoma (ACC) is a rare malignancy with a devastating prognosis. Treatment options are limited. Early surgical resection offers the only chance of cure but frequently disease presents late and in the advanced stage five-year survival is <15%. Histopathological diagnosis of ACC is complex, based on a prediction of malignancy, and there are few widely used biomarkers in clinical practice as yet. We have identified a novel cell population in the adrenal cortex that express the protein delta-like homologue 1 (DLK1). DLK1 expression is low in most healthy tissues; however, expression is reported in numerous malignancies and is associated with a worse prognosis. We have identified that DLK1 is overexpressed in ACC compared to normal adrenals and non-malignant adrenal pathology. In preliminary experiments using the ACC cell line H295R, we have shown that DLK1+ cells have increased colony forming ability, chemoresistance and features of adrenocortical progenitor/stem cells compared to DLK1- cells. Aims: 1) To investigate the role of DLK1 as a novel biomarker in adrenocortical carcinoma 2) To characterise the features of DLK1+ adrenocortical carcinoma cells in vitro and ex vivo Methods: - Correlation of immunohistochemical expression of DLK1 with survival rates and details of clinical and biochemical disease presentation - Assessment of serum DLK1 levels in patients with ACC vs control population of non-malignant adrenal pathology before and after surgical resection - Assessment of characteristics of DLK1+ ACC cells (3 cell lines and primary tumours): spheroid generation, chemo-resistance, colony formation, RNA extraction and quantitative PCR, tumour-initiating ability Impact: This project promises to identify and characterise a new molecular biomarker for diagnosis and prognosis of ACC, to deliver new biology defining a cell population in this malignancy and to identify targets which may be amenable to novel therapeutic intervention.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Interrogating the relationship between tumour microenvironment and its infiltrating immune cells in high-grade serous ovarian cancer
Funder: Medical Research Council

Abstract: Tumour biopsies will be obtained from 30 patients with HGSOvCa at diagnosis and after neoadjuvant chemotherapy at definitive surgical intervention. Following tissue digestion, millions of cells will be dissociated into a single-cell solution. Tumour infiltrating immune (TIL) cells (CD45+) will then be magnetically selected from the tumour microenvironment. The single-cell sequencing technique, Drop-Seq, will analyse both the tumour (CD45-) and TIL (CD45+) populations. Here, 2000 individual cells are suspended in droplets containing barcoded oligonucleotide beads and lysed in situ in preparation for next generation sequencing. Bioinformatic analysis will determine the specific transcriptome of individual cells by analysing up to 10,000 reads per cell. This will profile the heterogeneity of tumour and TIL populations, identifying the most highly expressed transcripts. These findings will be related to clinical outcomes, such as chemo-resistance, disease recurrence and survival. Major findings will be further investigated by analysis of protein expression using multi-colour flow cytometry to detail the pattern of lymphocyte memory phenotype, co-stimulatory status and activation status. Immunohistochemical and immunofluorescence staining will validate our results by detecting highly expressed proteins in the primary tumour tissue. Multiplex immunostaining will acquire additional information on RNA expression: key markers for tumour and immune cells will visualise the proportion and location of differing infiltrating immune cells in relation to certain tumour cell populations. This will enable us to study the relationship between tumour cells and specific immune infiltrates. Finally, immune cells will be isolated from the tumour bed using flow cytometric sorting and co-cultured with primary tumour cells. A range of functional assays, such as cytotoxicity and cytokine analysis, will investigate the capacity of different immune cells to respond to tumour tissue.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: A protein-based method for the generation of allogeneic chimeric antigen receptor T-cells
Funder: Medical Research Council

Abstract: Chimeric Antigen Receptor (CAR) T-cell therapy is growing in application. Currently, CAR T-cell products are generated as individual autologous products for each patient. This has several disadvantages: each patient must undergo leukapheresis; production failure rate is ~10%; product quality is not guaranteed; there is a 3-4 week lag time to treatment; and economies of manufacturing scale cannot be applied. A potential solution is allogeneic CAR T-cells from healthy donors. However, unrelated donor CAR T-cells would cause severe/ fatal graft-versus-host-disease (taGvHD) mediated by the T-cell receptor (TCR) of donor T-cells. To deliver allogeneic CAR T-cells, TCR signalling must be prevented. To date, allogeneic CAR T-cell strategies have used genome-editing to delete the TCR. Indeed, we were the first to describe clinical use of anti-CD19 CAR T-cells generated from allogeneic TALEN-edited cells. While some clinical efficacy has been seen, a complex multi-stage manufacturing process is required with highly stringent depletion of TCR positive T-cells. This difficult manufacture negates much of the advantage in terms of practicality/ cost of goods for allogenic cells. We have since developed an alternative platform for allogeneic CAR T-cell manufacturing without genome-editing. By co-expressing a TCR-specific single-chain variable fragment with a Golgi retention signal along with the CAR (TCR-KDEL), TCR-negative CAR T-cells can be generated with a single retroviral transduction. Further, additional co-expression of a GMP-usable marker gene facilitates facile highly stringent selection of TCR-negative, CAR-positive cells in a single sorting step. With this grant, we propose to test the TCR-KDEL allogeneic CAR-T platform using a next-generation CD19 CAR (CAT19) currently in several studies at UCL. While we do not expect TCR-KDEL to be limited to CD19 CAR, testing with CAT19, testing with CAT19 allows direct comparison with ongoing autologous CAR studies.


Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: CANCER AND NEOPLASMS
Title: Characterisation of novel genetic determinants of Craniopharyngioma tumours
Funder: Medical Research Council

Abstract: Adamantinomatous Craniopharyngiomas (aCPs) lead to severe endocrine dysfunction and other sequelae, due to their effect on local structures such as the hypothalamus, optic and cranial nerves. Treatment is currently limited to surgery and radiotherapy, which are associated with significant morbidity. There is an urgent need to understand the molecular basis leading to formation and progression of aCPs to allow improved and personalised clinical management. My group's preliminary work has identified that 70% of aCPs carry mutations in B-catenin and these tumours have a high frequency of mutations in important negative regulators of the Wnt/B-catenin pathway. The group has identified that negative regulators of the the Wnt/B-catenin pathway are also mutated in the aCP-B-catenin-negative tumours, suggesting these genes could contribute to the development of aCPs independently of B-catenin. Importantly the identified mutational heterogeneity of aCPs tumours could confer variability in tumour phenotype, which may explain the variable clinical outcomes of aCPs. My project aims to understand the role of mutations in negative regulators of WNT signalling in the aetiology of aCPs 1) functional characterisation of the identified mutations 2) transgenic in vivo modelling of the identified mutations 3) clinical phenotype correlation of aCP patients with mutations identified. To achieve these objectives I will use well established lab techniques to determine the role of identified mutations on their protein function. I will use transgenic modelling to genetically ablate, within the pituitary, the two most commonly mutated genes and study the role of these genes in pituitary development and tumour formation. Finally, in collaboration with a tumour bio-informatician and cancer statistician, I will use correlation studies to understand if the variable clinical phenotype in aCP cohort is due to mutational heterogeneity within these tumours.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Breast Cancer: Early diagnosis using materials immortalisation
Funder: Medical Research Council

Abstract: Breast cancer remains a major cause of cancer death, with more than 11,500 deaths per year in the UK, despite advances in detection and treatment. Improvements in both early diagnostic and prognostic methods are required, not only to reduce breast cancer mortality, but also to reduce the large number of unnecessary interventions resulting from overdiagnosis and also address the very poor positive predictive value of screening. A primary marker for malignancy is the particular morphological and distributive nature of microcalcifications observed through mammography. However, little is known definitively about the natural history, chemistry or microstructure of these ectopic precipitates. A common consensus is that both oxalates and apatites form two major groups ('type I' and 'type II' respectively). However, in contrast to the somewhat stolid and phlegmatic oxalates, apatites have extremely flexible molecular structures that are able to accommodate high levels of foreign cations and anions. This enigmatic material then, has the inherent ability to immortalise chemical features of the microenvironment/tissue in which it precipitates, and is thus potentially a rich target for novel biomarkers. This proposal builds on pilot studies that have demonstrated correlations between chemical characteristics of breast calcifications and tumour type. We will adopt a multi-analytical, retrospective examination of tumour subtypes to generate a new understanding of the materials physicochemistry of calcifications. As a result, we expect this will identify important new biomarkers that will ultimately enable in vivo (at the mammography stage), novel diagnostic methods capable of high accuracy identification of breast cancers.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Two-stage genome-wide 5-hydroxymethylcytosine (5hmC) reprogramming during colorectal carcinogenesis and liver metastasis
Funder: Medical Research Council

Abstract: The purpose of this programme is to discover the function of 5-hydroxymethylcytosine (5hmC) during colorectal cancer (CRC) and liver metastasis. 5hmC is produced as an intermediate DNA demethylation product when 5-methylcytosine is oxidised by the TETs (Ten-Eleven-Translocation proteins). In normal tissue 5hmC is a stable epigenetic mark associated with active gene expression. Recently 'Loss of 5hmC' has emerged as a hallmark of many primary cancers, an early detection and potential cancer staging tool. The TETs are exceptionally responsive to metabolic stimuli and this opens up several avenues for therapeutic intervention. However, the therapeutic potential seems to be cancer-specific, since unexpectedly, in some cancers, (e.g. prostate cancer), metastasis and recurrence are associated with increased levels of 5hmC and poor survival. We have preliminary data that suggest that this may also be the case for CRC. In this project we are using a combination of bioinformatics, mouse models and a unique series of CRC model cell lines in an organoid culture system to discover the causal relationship between 5hmC levels, the tumour phenotype and metastatic transformation in CRC. We propose to test two models for 5hmC function. The first model proposes that changes in 5hmC mediate oncogenic splice switching to drive cancer progression. The second model proposes that 5hmC levels regulate the stem-like phenotype of cancer cells. These models are not mutually exclusive. The outcome of this research will inform how 5hmC contributes to the cancer phenotype and whether it is a viable therapeutic target. Although the focus is on the role of 5hmC, an additional outcome of this project will be to further elucidate the catalytic function of TET enzymes in cancer progression.


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