NIHRDataInsights/HRCSData huggingface dataset:
Title: How do developmentally-regulated changes in human lymphoid development initiate and propagate treatment resistant childhood leukaemia?
Funder: Wellcome Trust

Abstract: Background:Human lymphopoiesis is a dynamic life-long process that starts in utero, however developmental pathways defining fetal lymphopoiesis are largely unexplored. Detailed characterisation of human fetal lymphoid development is important because most childhood acute lymphoblastic leukaemias (ALL) originate in utero. Hypothesis:Developmentally-regulated characteristics of lymphopoiesis provide the functional and molecular context for initiation and evolution of childhood ALL. Objectives: 1) Define site- and stage-specific characteristics of human lymphoid developmental pathways in fetal and postnatal life. Immunophenotypic, functional, transcriptomic, epigenetic characterisation of human fetal and postnatal stem/progenitors at population/single cell level. 2) Determine molecular/functional characteristics of fetal lymphoid progenitors that initiate, maintain and propagate infant/childhood ALL. In vitro and in vivo models of infant/childhood ALL using human fetal/ postnatal progenitors and primary leukaemias, will be used to investigate cellular context/key dependencies required for initiation and progression. 3) Elucidate ontogeny-related differences in the haematopoietic microenvironment in normal/ leukaemic lymphopoiesis. Molecular, proteomic and imaging mass cytometry approaches will be used to study developmental stage-specific microenvironmental niches 4. a) Integrate research findings into national and international clinical trials for treatment of infant/ childhood ALL. b) Use knowledge gained from Aims1-3 to develop adapted and effective protocols for treatment of infant ALL in low and middle-income countries.

Lay abstract: In humans blood cells are continuously replenished by a process called haematopoiesis which starts before birth. There is growing evidence that many childhood leukaemias start to develop before birth. Those that develop very early (before the first birthday), are called infant leukaemia and are usually associated with rearrangement of a gene called MLL. Although it is now possible to cure 90% of children with the commonest type of leukaemia, sadly only 50% of infants are cured. Much better understanding of the biology of treatment resistant leukaemia is required. I would like to investigate the cells that undergo transformation in infant and childhood leukaemia and how they differ from adults. I will develop a model of infant leukaemia by inserting the abnormal MLL gene into normal cells. We would then be able to use this model to understand the pathways that drive this aggressive disease and test new drugs for treatment.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: CRUK/16/018 PIVOTALboost: A phase III trial of prostate alone vs pelvic lymph node IMRT with or without prostate boost for intermediate and high risk localised prostate cancer
Funder: Cancer Research UK

Abstract: Background: Prostate cancer accounts for 25% of new cancer diagnoses in the UK, and is the most common cancer diagnosis in men. Patients with intermediate and high risk localised prostate cancer are recommended to have either radical prostatectomy or radical radiotherapy combined with hormone therapy. 20-30% of those in higher risk groups are likely to recur following radiotherapy. Intensity modulated radiotherapy(IMRT) and image guidance techniques provide scope for intensifying prostate radiotherapy treatment whilst minimising any associated increase in radiotherapy related side effects. It is, however, currently uncertain whether promising results from planning and cohort studies of these approaches would translate into improved outcomes, without substantial increase in toxicity, in the context of a randomised controlled trial. Aims: In patients with high or intermediate risk localised prostate cancer, to determine whether the addition of pelvic node IMRT and/or prostate boost to standard prostate IMRT, improve failure-free survival (FFS) compared to standard prostate IMRT alone. Secondary aimes include assessment of time to recurrence, metastatic relapse free survival, acute and late toxicity, quality of life and health economics with each technique. Methods: PIVOTALboost is a randomised controlled parallel 4-arm phase III multicentre trial in men with localised high and intermediate risk prostate cancer. Consenting patients will be randomised to receive either: A) Prostate IMRT (control) B) Prostate and pelvic IMRT C) Prostate IMRT and prostate boost D) Prostate and pelvic IMRT and prostate boost All radiotherapy will be delivered using image guidance. Prostate boost will be delivered to the whole gland using HDRB, or to focal disease using focal HDRB or focal IMRT. A comprehensive radiotherapy quality assurance programme will be implemented to support adoption of new techniques at centres. Patients will be followed up for disease outcome, acute and late toxicity and quality of life; health economic details will also be captured. The primary endpoint is FFS at 5 years, defined as biochemical failure, recommencement of hormone therapy, local/pelvic recurrence, distant metastases or prostate cancer death. Assuming a 5-year FFS of 80% in control arm A, the study will recruit 1952 patients to detect a 7% difference between A vs B (85% power) and A vs C or D (80% power) – overall significance 5%, dropout rate 3%. Use of Results: Results will provide potentially practice changing evidence for the treatment of high and intermediate risk localised prostate cancer, seeking to harmonise the current variability in clinical practice.


Research activity codes: 6.5 RADIOTHERAPY AND OTHER NON-INVASIVE THERAPIES
Health categories: CANCER AND NEOPLASMS
Title: Harnessing cell death mechanisms to overcome treatment resistance and improve tumour immunity
Funder: Cancer Research UK

Abstract: Background: Following prolonged mitotic arrest, cells typically die in mitosis or exit without dividing and survive. What dictates the balance between these two fates, and how cells sense mitosis arrest, and in response trigger cell death remains unclear. Mitosis poisons, such as Taxol and vincristine, arrest cells in mitosis, however both the mechanisms that lead to cell death and to acquired therapy resistance are poorly understood. We now have identified the molecular link that connects cell death to mitosis progression. We have uncovered an unanticipated function of the ripoptosome (RIPK1/caspase-8 complex) in cell division, actively promoting faithful chromosome alignment during mitosis, and eliminating those cells that exhibit defects in mitosis progression. Aims: The aim of this proposal is to uncover the link between the cell death machinery and mitosis, and harness it to drive long-lasting anti-cancer immunity. The idea addressed by this application is that cell survival and chromosomal instability of tumours are supported by avoidance of ripoptosome engagement, and that this can be targeted by re-activating the ripoptosome to drive immunogenic cell death or, alternatively, instigate unsustainable amount of chromosomal instability through ripoptosome inactivation, causing tumours to permanently regress. Aim 1: Determining how cell death components regulate physiological mitosis Aim 2: Evaluating the therapeutic benefit of modulating the activity of the mitotic-ripoptosome to trigger long-lasting anti-cancer immunity Methods: We will use biochemical, imaging and in vivo approaches to establish the physiological and pathological role of components of the cell death machinery (ripoptosome) in regulating mitosis, and investigate how ripoptosome components safeguard faithful chromosome segregation (Aim 1). We will also determine how the ripoptosome is activated and elucidate the signals that ultimately shut it down at the end of mitosis. In Aim 2, we will exploit our knowledge of ripoptosome regulation and harness ripoptosome function to sensitise cancer cells to anti-mitotic agents and improve treatment response. We aim to induce a type of tumour cell death that is immunogenic and in combination with immune checkpoint blockade can lead to long-lasting anti-tumour immunity. How the results will be used: Results from this programme will have significant impact on the development of novel treatment combinations aimed at sensitising cancers to anti-mitotic agents and improving cancer immunotherapy. If successful, future clinical testing will be warranted. It will also allow us to stratify patients into responders and non-responders based on the patient’s ability to trigger ripoptosome formation and ripoptosome-dependent cell death.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: An integrated analysis of oral squamous cell carcinoma development and progression
Funder: Cancer Research UK

Abstract: BACKGROUND Oral squamous cell carcinoma (OSCC) arises in the multilayered epithelia lining the mouth and lips. Despite advances in treatment the survival rate remains stubbornly low. It is a cancer of unmet need, associated with social deprivation. AIMS OSCC is highly heterogeneous at both the genetic and cellular levels. I hypothesise that the course of the disease depends on: which epithelial cells acquire mutations; the nature and timing of the mutations; and the effects of those mutations on the local tissue stroma and immune cell infiltrate. We will test this hypothesis using 4-nitroquinoline-1-oxide (4NQO) induced mouse oral carcinogenesis, human OSCC cell lines and an archive of human OSCC and leukoplakias. We will answer three questions: 1. How do specific genetic alterations and the cancer cell of origin influence the biology of individual OSCC? We will monitor tumour development by clonal labelling of the oral epithelium and analyse the impact of carcinogen on stem cell subpopulations, stromal cells and leukocyte subsets. We will perform whole exome sequencing and correlate the characteristics of individual tumours with the underlying genetic alterations. 2. What is the impact of known genetic alterations in OSCC? We will use 4NQO induced mouse oral carcinogenesis to directly test the significance of homozygous and heterozygous NOTCH1, FAT1 and CASP8 gene inactivation. We will determine whether downregulation of KRT76 promotes tumour development by disturbing local and systemic immune responses. 3. Is the biology of individual human tumours indicative of genetic and cellular heterogeneity? Integrating genomic data with specific features of individual human OSCC is challenging because there are so many potential parameters to evaluate. We will overcome this problem by prioritising markers based on their significance in mouse oral carcinogenesis. By analysing archival human leukoplakias and OSCC we will determine whether specific combinations of mutations have a characteristic fingerprint in terms of stromal, immune and epithelial status, as predicted from our mouse models. We will identify underlying mechanisms and test potential therapeutic interventions using our panel of human OSCC lines. METHODS The primary methods are whole exome sequencing, gene expression profiling, 4NQO-carcinogenesis, targeted gene deletions in mice, in vivo fluorescent clonal labelling, histological analysis, and orthotopic transplantation of human OSCC cell lines. HOW THE RESULTS WILL BE USED This project will provide a road map for pursuing new strategies to improve OSCC prognostication and intervention, based on the impact of genetic alterations on the biology of individual tumours.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: The neomorphic role of KBTBD4 in medulloblastoma
Funder: Cancer Research UK

Abstract: BACKGROUND Medulloblastoma is the most common malignant brain tumour in children. Recent genomic data revealed that a subset of aggressive subtypes (group3/4) harbour recurrent indel mutations in KBTBD4, a previously uncharacterised E3 ubiquitin ligase. While mutations in E3 ligases typically disrupt function, we discovered that mutant KBTBD4 gains neomorphic activity by changing substrate specificity. We found that mutations in KBTBD4 promote the recruitment and ubiquitylation of neo-substrates due to structural changes in the substrate-binding domain. The neosubstrates have important roles in controlling chromatin remodelling and have been shown to regulate cerebellar proliferation in mice. Thus, we hypothesise that the KBTBD4-dependent degradation of neo-substrates reprogrammes neuronal differentiation towards uncontrolled proliferation and tumorigenesis. Our discovery identifies a novel pathogenetic mechanism of aggressive medulloblastoma through epigenetic dysregulation which could apply to the majority of aggressive group 3/4 medulloblastomas. Finally, this study will provide new opportunities for safer targeted treatments in children. AIMS We will investigate the oncogenic mechanism of KBTBD4 through three aims. First, we will complete the identification of the neosubstrates recruited by mutant KBTBD4 and establish the type and effect of ubiquitylation. Second, we will elucidate the consequences of neosubstrates ubiquitylation on chromatin remodelling, transcriptional control and tumorigenesis both in vitro and in vivo. Finally, we will determine the structural basis for neomorphic KBTBD4 activity and screen for small molecule inhibitors of mutant KBTBD4 that have potential to selectively kill tumour cells. METHODS We will employ state-of-the art mass spectrometry for proteomic analyses of KBTBD4-specific interactors and define substrates through well-established in vitro and in vivo ubiquitylation assays. We will use CRISPR to establish parallel models of medulloblastoma (cell lines, neuroepithelial stem cells and Patient Derived Xenografts) to define the altered transcriptional programme of neuronal differentiation in tumours and during brain development by RNA sequencing. Additionally, we will employ crystallographic, biophysical and compound screening techniques to establish how indel mutations in KBTBD4 may alter the substrate-binding pocket to selectively engage neo-substrates and small molecules. HOW THE RESULTS OF THIS RESEARCH WILL BE USED Our research will define a new mechanism of tumorigenesis through E3 ligases that parallels the neomorphic activity of mutant IDH1/2 in cancer. Our findings will similarly impact on the fundamental understanding of how epigenetic reprogramming drives tumour development. Finally, our study will support future work towards safer treatments for medulloblastoma by defining a new druggable target for tumour-specific therapies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Dissecting the Roles of the Thrombopoietin Receptor in Myeloproliferative Neoplasms: Towards the Development of New Therapeutic Agents
Funder: Cancer Research UK

Abstract: Background The myeloproliferative neoplasms (MPNs) are clonal haematological malignancies characterised by significant increases in one or more myeloid cell lineages, with over 3,200 new cases diagnosed every year in the UK. JAK2V617F is the most common mutation found in patients with Philadelphia chromosome negative (Ph-) MPNs, identified in 98% of polycythaemia vera (PV) patients and approximately 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Val617 is located in the JAK homology (JH) 2 domain of the JAK2 protein, which is critical in the regulation of the kinase (JH1) domain. Additionally, mutations in the calcium sensing protein calreticulin, have been identified in ~35% of JAK2V617F-negative ET and PMF patients. Recently, we have shown that reducing expression of MPL, the receptor for the haematopoietic cytokine thrombopoietin (TPO), completely prevented JAK2V617F-positive MPN development in vivo. Furthermore, we now have preliminary data suggesting a novel alternative model for JAK2V617F activation, in which the mutated protein drives dimerization of MPL monomers in the absence of TPO. In this proposal, we aim to fully elucidate the mechanisms of JAK2V617F-mediated MPL dimerization and develop new agents designed to antagonise MPL activity in order to reduce MPN pathology. Aims 1. Fully characterise the mechanisms of JAK2V617F and mutant CALR-mediated MPL dimerization. 2. Develop transmembrane-interacting small molecules and MPL-specific monoclonal antibodies and diabody peptides to inhibit MPL dimerization, and determine their effects in MPN models. Methods To address these aims, we have assembled an interdisciplinary team with experience in cell biology, protein biophysics, single molecule fluorescence, synthetic chemistry, small molecule design and clinical haematology. Total internal fluorescence microscopy (TIRF) will be used to determine the effect of mutant CALR and MPL antagonists on receptor dimerization in dual-tagged MPL-expressing cell lines. To gain key mechanistic insights into JAK2V617F-driven MPL dimerization, we will apply a number of biophysical analyses, including SEC-MALLS and AUC and gain structural insights using heteronuclear NMR spectroscopy. Specifically-designed small molecules, monoclonal antibodies and diabodies will be tested initially using an MPL-expressing JAK2 FRET reporter cell line (irf-Bla Ba/F3-MPL) before in vivo analysis using humanized NSG-SGM3 mice. How the results of this research will be used We expect that the findings from this research will provide innovative mechanistic insights into MPN development and initiate the development of novel agents to treat MPNs, with the potential to expand to other myeloid malignancies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Targeting senescence in lung cancer
Funder: Cancer Research UK

Abstract: Background Lung cancer is the most common cause of cancer-related death worldwide. In order to improve poor survival rates, novel approaches and adjuncts to systemic therapies are required. Cellular senescence is characterised by a stable cell cycle arrest that occurs in response to damage and oncogenic stress and involves intense secretion of paracrine factors (termed SASP). I reported in Cell (2013) that senescence plays an active role in orchestrating tissue remodelling. Cellular senescence is a defining feature of premalignant lung tumours and occurs in response to therapy-induced genotoxic stress in cancer, including in response to platinum-based chemotherapies used routinely in lung cancer treatment. Importantly, when senescent cells accumulate they can drive cancer progression, either by releasing complex proinflammatory/protumorigenic cocktails or by re-entering the cell cycle with aggressive clonogenic growth potentials. Therefore, treatment modalities that eliminate senescent cells may be critical for tumour eradication, although this remains a formidable clinical challenge. We have recently reported in EMBO_Mol_Med (2018) how a versatile drug delivery nanocarrier can target senescent cells in cancer models of chemotherapy-induced senescence, demonstrating promise for such a therapeutic approach. Objectives We will test the central hypotheses that therapy-induced senescence can promote lung cancer progression and treatment resistance, and that this can be therapeutically targeted by clearing senescent cells. Our specific aims are: Aim#1: To characterise the extent, role and impact of therapy-induced senescence in NSCLC, and identify targetable biomarkers of senescence. Aim#2: To design, develop and validate novel senotherapies, including therapeutic nanoparticles and second-generation senolytics. Senotherapies will be tested in combination with standard (senescence-inducing) therapies, aiming to eradicate NSCLC cells in murine models. Aim#3: To obtain proof of principle of the therapeutic activity of senotherapies by using human lung tumour organoids and PDX models, in order to identify the most promising candidate(s) for early phase clinical testing. Methods This proposal requires high-throughput analyses (RNAseq and proteomics), cell culturing and NSCLC mouse models. Human lung cancer tissue samples will be obtained in collaboration with Royal Papworth and Cambridge University Hospitals. How the results will be used This is a high-return translational project, based on the development of novel senotherapies, with commercialisation potential that may result in the development of combination therapies to improve lung cancer clinical outcomes. Our ultimate goal at the end of the project is to move our most promising innovative senotherapies into early phase clinical trials to establish their safety and clinical efficacy.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Molecular Mechanisms Safeguarding Genome Stability
Funder: Cancer Research UK

Abstract: Background: Accurate chromosome segregation is critical for the maintenance of a stable genome. It is facilitated by coordinated error correction and checkpoint mechanisms that ensure chromosome mis-segregation during cell division is a relatively rare event. Dissecting these mechanisms is important for cancer research for two reasons: a) the vast majority of human solid tumours (90%) are aneuploid, and we need to understand the molecular reasons for this phenotype to elucidate the aetiology of cancer; b) some of the mechanisms that aid successful chromosome segregation become indispensable in aneuploid tumour cells and could therefore be therapeutic targets. Of particular interest is the spindle assembly checkpoint which halts cell cycle progression until all chromosomes are securely tethered to microtubules. The rational design of treatments that exploit dependencies specific to aneuploid cells relies on the thorough understanding of the mechanisms governing the different stages of chromosome segregation in human cells. Aims: Our first aim is to dissect how the processes of microtubule-kinetochore attachment and error correction are coordinated with spindle assembly checkpoint signalling by the kinases MPS1, PLK1, Aurora B and the phosphatase PP2A-B56. Secondly, we will study how CDK1-cyclin B1 kinase and the opposing phosphatases PP1 and PP2A-B55 regulate kinetochore function at the metaphase-to-anaphase transition. Lastly, we will seek to identify ploidy-dependent vulnerabilities in both the processes of mitotic error correction and spindle checkpoint signalling. Methods: For our investigation we will compare human transformed aneuploid, tetraploid and diploid cells with non-transformed euploid cells. We will use a combination of fixed and live cell microscopy and biochemical analysis to investigate the different steps of chromosome segregation in these different genetic backgrounds. Our research will be aided by CRISPR/Cas9 mediated genome engineering allowing us to fluorescently label the endogenous copy of molecules of interest. This will facilitate live cell microscopy and biochemical studies of the endogenous proteins. How the results of this research will be used: This research programme will elucidate how faithful chromosome segregation is achieved in healthy cells and which regulatory mechanisms are critical to safeguard the success of this process. By comparing the response of aneuploid and euploid cells to disruptions of these control mechanisms we will be able to identify synthetic lethalities between different control pathways as well as vulnerabilities specific to aneuploid tumour cells. This research will provide important insights to the development of future therapies.


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