NIHRDataInsights/HRCSData huggingface dataset:
Title: Dissecting the role of extra centrosomes-induced exosome secretion in PDAC microenvironment
Funder: Medical Research Council

Abstract: Centrosome abnormalities, in particular centrosome amplification, are common features of human tumours. Work from our lab showed for the first time that cancer cells containing supernumerary centrosomes promote non-cell autonomous invasion of surrounding cells (Arnandis et al., 2018). This is mediated by an oxidative stress response induced by extra centrosomes that culminates with secretion of pro-invasive factors. These findings underscore a novel role for these abnormalities in cancer and open exciting questions regarding how they influence tumours and microenvironment. Our unpublished data demonstrates that centrosome amplification is sufficient to induce secretion of small extracellular vesicles, such as exosomes. Moreover, these exosomes can activate pancreatic stellate cells (PSCs). Both exosome secretion and PSC activation are a result of oxidative stress, suggesting that our findings could have a broader relevance in cancer as many oncogenic events induce an oxidative stress response. This is particularly important in pancreatic cancer, where PSC activation-induced fibrosis constitutes a major barrier to drug delivery and efficacy. Here, we propose to use a combination of approaches, such as microscopy, RNA-seq, proteomics and mouse models to: 1) determine the role of extra centrosomes and oxidative stress in exosome biogenesis and secretion; 2) Identify the exosomal factors responsible for PSC activation and the signalling pathways they regulate; 3) characterise fibrosis in vitro and in vivo and assess the role of exosome-induced PSC activation in pancreatic cancer progression and fibrosis. We will also test if preventing PSC activation impacts response to Gemcitabine in vivo. Our work will be the first demonstration for a role for centrosome amplification in tumour microenvironment, will elucidate mechanisms leading to exosome release in cancer and can lead to the development of therapeutic strategies that target fibrosis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Characterisation of the role of NG2-glia and microglia in hypothalamo-pituitary axis coupling.
Funder: Medical Research Council

Abstract: The 5-year survival rate for paediatric cancers has increased to 80%. While extremely positive, this also means that sequelae of anti-cancer treatments become more prominent, such as pituitary hormone deficiencies found in up to 50% of survivors treated by brain irradiation. These are associated with significant morbidity, require monitoring, and replacement or substitution therapies. We do not know why these deficits develop, however cranial irradiation in rats is also associated with pituitary deficits so rodents are a good model to study their aetiology. Obesity can also develop following cranial radiotherapy. In mice, irradiation of the median eminence (ME), the structure at the base of the third ventricle linking hypothalamus and pituitary, drives this effect. More precisely, destruction of the proliferative ME NG2-glia leads to degeneration of Leptin Receptor positive dendrites that it normally supports, therefore loss of hypothalamic leptin sensing and consequently weight gain. We have moreover found that ME NG2 glia are affected in Sox3Y/- mutant mice which show hypopituitarism, as do patients carrying SOX3 mutations. Therefore, we hypothesize that destruction of ME NG2-glia by radiation therapy results in development of hypopituitarism in cancer survivors. To test our hypothesis, we will develop a novel model based on a brain sparing Diphteria Toxin to exclusively eliminate ME NG2-glia, as these are located outside the blood brain barrier. Moreover, microglia will be examined because aspirin can rescue Sox3Y/- hypopituitarism, suggesting an inflammatory contribution to the phenotype, which may also following irradiation. To investigate this aspect, we will eliminate the microglia pharmacologically in these contexts. Single cell analysis will be used to further characterize Sox3Y/- ME phenotypes. Our goal is to understand how hypopituitarism develops following radiation therapies to ultimately provide preventive measures or cures for cancer survivors.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: CANCER AND NEOPLASMS
Title: CD21 directed CAR-T cell immunotherapy for relapsed/refractory T-cell acute lymphoblastic leukaemia (T-ALL)
Funder: Medical Research Council

Abstract: Chimeric Antigen Receptors (CARs) are synthetic signalling molecules used to redirect T-cells to kill tumours expressing a target antigen. CAR T-cells against CD19 are transforming the treatment of children with relapsed/refractory B-ALL. In recent clinical studies of paediatric B-ALL, remission rates close to 100% have been reported, with ~60% of patients achieving sustained responses. However, some 15% of paediatric and 20-25% of adult ALL cases are of T-lineage (T-ALL) and are therefore not suitable for treatment with anti-CD19 therapy. T-ALL remains a challenging clinical problem with an approximately 20% relapse rate. It is a devastating disease for those who fail standard intensive chemotherapy with only approximately 20% achieving remission with salvage chemotherapy resulting in extremely poor long-term survival rates for those cases. With the intensity of both frontline and relapse therapies, treatment-related mortality is a significant cause of treatment failure. Therefore, further intensification of chemotherapy is not an option resulting in the need for targeted therapies with minimal toxicity. No novel therapies have been successfully developed for T-ALL in recent years so there is a high unmet need for new treatments. Given the success of anti-CD19 CARs in B-ALL, development of comparable strategies for T-ALL is highly desirable. However, this is challenging for multiple reasons. Targeting CD19, a pan B-cell antigen, results in B-cell aplasia which an be alleviated by infusion of pooled immunoglobulins. In contrast, targeting a pan-T-cell antigen results in unacceptable immunosuppression which cannot be mitigated, requiring subsequent salvage bone marrow transplantation. A T-ALL surface antigen with expression largely confined to the malignant T-ALL compartment, with acceptable off-tumour expression has long been sought. We believe we have identified such an antigen and would like to develop this further.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Adding Male Single Dose HPV Vaccination to Female HPV Vaccination in Tanzania (Add-Vacc)
Funder: Medical Research Council

Abstract: The Director-General of the World Health Organization (WHO) has announced a target to eliminate cervical cancer as a public health problem. Sub-Saharan Africa has the highest cervical cancer incidence and mortality rates in the world. However, few African countries have HPV vaccination programmes to prevent cervical cancer because of the costs involved. Prophylactic HPV vaccination has been routinely offered to girls prior to sexual debut in 85 countries globally and vaccines have become available to the poorest countries through Gavi support. However, recent data on vaccine coverage in target age groups in some countries has been lower than expected. The recently announced WHO call to eliminate cervical cancer will require novel approaches to reduce HPV acquisition and its sequelae. As well as screening for cervical cancer lesions in older females, other strategies being considered include gender-neutral vaccination that can potentially provide individual protection for boys and can also maximise protection of unvaccinated girls and boys against HPV acquisition. Tanzania introduced two-dose HPV vaccination with the 4-valent vaccine, Gardasil, into its national immunisation programme in 2018. Our trial, Add-Vacc, is a cluster-randomised trial of single dose HPV vaccination given to a multi-year cohort of 14-18-year-old boys alongside routine female HPV vaccination, with a nested observational cohort of vaccinated boys for immunogenicity and safety. Impact evaluation of male vaccination, alongside routine female vaccination, on population HPV prevalence will be measured in a cross-sectional survey of males and females aged 16-22 years three years after the intervention. We will also explore the acceptability of gender-neutral vaccination and the cost effectiveness of adding in male vaccination to a female HPV vaccination programme.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Evaluation of the CD4+ T cell tumour microenvironment of low grade B cell lymphoproliferative disorders
Funder: Medical Research Council

Abstract: This proposal will examine the role that CD4+ T cells have in promoting tumour progression in B-lymphoproliferative disorders. In these diseases an inflammatory pro-tumoural tumour microenvironment (TME) exists, with resultant division and growth of lymphoma cells. Assessment of numbers, co-occurrence, location and spatial relationships of cells and cell clusters within the TME of these diseases has to date been restricted by generally small biopsy material available from routine clinical samples and the limitations of conventional immunohistochemistry. Advanced microscopy-based techniques include imaging mass cytometry and multi-colour immunofluorescent confocal microscopy. These two techniques are complementary: the former provides the ability to determine the anatomical inter-relationship of deeply phenotype tissue at lower resolution, the latter providing high resolution but limited parameter information. This project will use these two techniques in conjunction to explore the hypothesis that CD4+ T cells are drivers of B-lymphoproliferative disorders. Key research themes and questions to answer include: 1. How to optimise an advanced microscopy-based immune mapping pipeline to deep phenotype CD4+ T cells and their interrelationship with lymphoma and non-neoplastic cells, 2. Correlation of these findings with biological and clinical features and, 3. How to apply these analyses to inform related pre-clinical and clinical research and as a clinical bioassay. The pipeline development will necessitate appropriate primary patient sample selection followed by optimisation of panel design for CD4+ subset identification within the TME, image generation using both MCIF and IMC, and development of a bespoke analysis platform using established computer algorithms to perform cell segregation, neighbour analysis and pixel by pixel analyses. The output from the pipeline will give a tool that can be used for pre-clinical and clinical downstream applications.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Application of single cell genomics for precision medicine in myelodysplastic syndromes.
Funder: Medical Research Council

Abstract: As a clinician specialising in MDS and leading a diagnostics laboratory, I have seen how vital it is to obtain the correct diagnosis for patients. I have been in the field of haematology for over 10 years and this has been most striking to me in MDS. This diagnostic difficulty is largely due to cellular heterogeneity, which is masked using our current laboratory tests. Single cell genomic testing will overcome diagnostic difficulties resulting from cellular heterogeneity. Aim: To establish the feasibility and initial utility of introducing single cell genomics into a clinical laboratory, to improve precision diagnostics in MDS. Methods: 1.) Optimisation exercise introducing single cell genomic analysis into the diagnostic evaluation of MDS. I will set up a prospective study and recruit patients referred with suspected MDS, collecting genetic and clinical data. 2.) To correlate single cell genomics data with routine diagnostic modalities in a cohort of MDS patients. Routine laboratory investigations will be performed in parallel with a single cell genomics platform (CITE-seq), which will enable immunophenotyping and RNA-seq of individual bone marrow cells. We aim to benchmark this technology against current standards of diagnosis. The single cell work will be performed by the single cell facility at the WIMM, which is establishing a good clinical laboratory practice accredited laboratory. These data will determine whether CITE-seq correlates with and might ultimately replace current diagnostic modalities for MDS. We will refine these pipelines, to be transferred into a clinical setting.This grant will then enable a future study, powered to investigate the impact on long term patient outcomes. Outcomes: My goal is to address the unmet need for the accurate diagnosis and classification of MDS. Addressing this issue is essential for improved clinical decision making and personalised medicine. Our pipeline will also be of great value beyond MDS.


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: Colour-coded surgery in Neuroblastoma: developing a dual PET/Near-Infrared Fluorescence Imaging probe to visualise tumour from diagnosis to resection
Funder: Medical Research Council

Abstract: Neuroblastoma (NB) is the second most common solid cancer in children, and it affects around 100 new children per year in the UK. One of the main challenges in the treatment of neuroblastoma is to be able to remove the residual tumour following multiple medical treatments. Radical excision of neuroblastoma is challenging because it widely infiltrates into the deepest parts of the abdominal and/or chest cavities. Colour-coded surgery is one of the most promising and needed surgical innovations that could significantly increase the survival in solid tumours. This project aims to develop a dual-labelled PET/NIRF anti-GD2 mAbs probe specific for neuroblastoma. This novel probe will allow for the first time to track active neuroblastoma cells from diagnosis to follow-up, including the real-time visualisation during surgery and at histology. We will run 18 months of laboratory-based experiments (objectives 1, 2 and 3) to prove it is feasible to double label anti-GD2 monoclonal antibodies with an effective near-infrared fluorescence (NIRF) probe and a PET tracer (e.g. Zirconium-89). We will test this novel probe in vitro and in vivo in a series of experiments based on established animal models of neuroblastoma. If successful, we will then design and run a phase 1/2 clinical trials in humans to test the validity of the dual PET-GD2-mAbs-NIRF probe manufactured in a GMP-compliant way (objective 4). The main benefit of this research will be for the children affected by neuroblastoma as the expected results will be essential to achieve the first personalized surgical therapy in the field. Surgical outcomes will improve as surgeons will be able to see better the tumour during the operation. This will improve the chances to resect the entire tumour, and will also reduce the risk of damaging vital tissues often adherent to cancer. Patients will hopefully achieve better long-term survival rate with less surgical complications and less local recurrence of the disease.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.4 SURGERY
Health categories: CANCER AND NEOPLASMS
Title: Molecular mechanisms of eukaryotic ribosome assembly
Funder: Medical Research Council

Abstract: Understanding how the acquisition of a genetic mutation by a single cell drives a cancer by outcompeting its normal healthy cell counterparts is a key question in cancer biology. The autosomal recessive disorder Shwachman-Diamond syndrome (SDS) is an excellent model with which to address this question as affected individuals have an increased propensity to develop acute myeloid leukaemia (AML) and the initiating genetic lesions (germline mutations in the SBDS, EFL1 or DNAJC21 genes) are known. MRC-supported work in my lab defined SDS as a ribosomopathy that is caused by defective maturation of the large ribosomal subunit. However, the basic mechanisms of ribosome assembly and how defects in this process promote cancer remain poorly understood. These are important questions, as haploinsufficiency of ribosomal protein genes is now recognised as a common vulnerability in human cancers and haematological malignancies. However, there are two major barriers to progress in the field. The first is the lack of high-resolution structures of native human pre-60S ribosome assembly intermediates, which are highly dynamic and often transient. The second barrier is the inherent difficulty in tracking haematopoietic stem cell clones in vivo in disease states such as human ribosomopathies. To overcome these obstacles, we will systematically determine the structures of key native pre-60S ribosomal subunit maturation intermediates using the latest advances in single-particle cryo-electron microscopy (cryo-EM). Furthermore, we will use somatic mutations as an endogenous stem cell barcoding tool to determine how ribosomal stress promotes the development of malignant clones in vivo that outcompete their normal counterparts to drive leukaemia. The feasibility and timeliness of this programme is supported by our published work and strong preliminary data that demonstrate our expertise in applying latest advances in cryo-EM to visualise 60S ribosomal subunit maturation states.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Determining the role of the prolyl cis-trans isomerase Cyclophilin A in DNA Repair at stalled DNA replication forks.
Funder: Medical Research Council

Abstract: Cyclophilins, a family of peptidyl-prolyl cis-trans isomerases (PPI), are assumed to mainly play undefined roles at the spliceosome and as chaperones for protein folding. In some specific instances more precise roles influencing cell signalling through receptor binding have been demonstrated. We have generated the first detailed and as yet unpublished interactome of the PPI Cyclophilin A (CyPhA) by BioID-biotin ligase proximity tag-coupled mass spectrometry. Ingenuity analysis highlight putative CyPhA interactors with roles in DNA repair and replication, transcription and G2-M transit. Therefore, our BioID analysis and consequent molecular-cellular preliminary data represent a window onto entirely new biology for CyPhA. The focus of this proposal is to uncover the role of CyPhA in DNA repair (DNA-R), specifically at stalled and erroneously processed DNA replication forks. Following CyPhA-BioID, we demonstrate CyPhA interaction with specific DNA-R proteins through co-immunoprecipitation and show these interactions to be dependent upon the PPI activity of CyPhA. Furthermore, we demonstrate a novel direct interaction between CyPhA and a target protein itself associated with well-known DNA-R proteins. This project involves three integrated aims involving low risk standard biochemical approaches (recombinant protein expression, interaction analysis, deletion mapping, mutagenesis) and cell biology (DNA Damage Response signalling, viability and sensitivity analysis), including cell line manipulation (siRNA, shRNA, CRISPR/Cas9 engineering) and ex vivo model tumour cell systems (3D spheroid and mammosphere co-cultures). DNA fibre combing is an important technique required to assess hyper-resection of nascent DNA at stalled DNA replication forks. We have successfully established and employed this technology. We have also generated shCyPhA-U2OS cells as well as CRISPR/Cas9 CyPhA knockout U2OS (KO), and these reconstituted with isomerase-dead (ID) CyPhA.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: RESETTING AND SCULPTING THE NOTCH RESPONSE
Funder: Medical Research Council

Abstract: The highly conserved Notch signalling pathway functions in diverse developmental and homeostatic processes. Likewise, disease implications from aberrant Notch programmes differ depending on the tissue: Notch activity promotes tumourigenesis in many contexts, including T-cell acute lymphoblastic leukaemia and breast cancers, whereas in others it is protective. Discovering how the appropriate responses to Notch are configured is therefore vital for deciphering when combinations of genetic abnormalities are likely to be oncogenic, and for informing strategies for targeted therapies. Our goal is to use a combination of live-imaging, genetic, biochemical and genomic approaches in Drosophila and in human cells to discover the fundamental mechanisms that reset and sculpt transcriptional responses to Notch. These mechanisms are essential to bring about different outcomes and to avoid inappropriate gene expression programmes being turned on. We have two major aims: (1) to discover what mechanisms ensure that enhancers are correctly remodelled during cell state transitions so that appropriate responses to Notch signals occur and emergence of cancerous cells is avoided; and (2) to learn how Notch-regulated enhancers select and communicate with promoters to produce RNA isoforms with the right regulatory features for the physiological context. Our results will uncover general principles that explain how the diverse effects of Notch activation arise in tissue development They will also provide important insights for distinguishing the likelihood that disrupted Notch signalling will be oncogenic, by helping to decode contexts where other mutations will affect key components or regulatory mechanisms and will provide new paradigms to aid design of therapeutic strategies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: A new paradigm in virus-mediated immunotherapy for liver cancer
Funder: Medical Research Council

Abstract: Oncolytic viruses (OV) are increasingly recognise to achieve anti-cancer efficacy through both cancer-specific replication resulting in cellular lysis, as well as via their pro-inflammatory nature comprising diverse pathogen-associated molecular patterns (PAMP). The balance between how these aspects of OV function result in therapy likely varies according to the virus in question, as well as the tumour against which they are directed. We have established a new paradigm for OV therapy, whereby uv-inactivated human Orthoreovirus (uv-Reo) achieves superior therapy compared with live virus in preclinical models of hepatocellular carcinoma (HCC). Furthermore, uv-Reo is synergistic with Sorafenib in preclinical models, whereas this is again not the case for live virus. Combined with additional preliminary data, we hypothesise that uv-Reo comprises a unique PAMP, the response to which is enhanced by the immune-specific effects of Sorafenib to achieve cancer-limiting immunity. We aim to combine transcriptomic, phenotypic and functional analysis of both human and preclinical murine hepatic/immune cells to define the hallmarks of the response to uv-Reo + Sorafenib. This will be undertaken using ex vivo stimulation of fresh human liver tissue combined with refined orthotopic HCC preclinical models capable of incorporating hepatic fibrosis. Processivity will be enabled through use of the Nanostring transcriptomic and Vectra high throughput IHC systems. Next, we will attempt to validate therapy by blockade of immune-associated kinases, chemokines, or other immune regulators within in vivo models and human liver tissue, with targets determined by preceding transcriptomic, phenotypic and functional analyses. Finally, we will determine retrospectively whether elements of the immunological signatures defined by analysis of successful therapy can be detected in longer-lived HCC patients, or if instead uv-Reo + Sorafenib generates a novel anti-HCC response.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS