NIHRDataInsights/HRCSData huggingface dataset:
Title: Molecular genetic characterisation of the pathways by which MEK kinase controls invariant Natural Killer T cell apoptosis and proliferation.
Funder: Cancer Research UK

Abstract: MEK Kinase 1 (MEKK1) is a key signalling molecule for the regulation of lymphocyte biology, playing vital roles in B and T cells and the thymus-dependent (TD) immune response. I have utilised state-of-the-art gene targeting to generate a kinase-deficient MEKK1 mouse model (Map3k1deltaKD) to dissect the critical roles of MEKK1 in lymphocyte biology, and I have analysed Map3k1deltaKD mice and identified an important new role for MEKK1 kinase signalling in the control of invariant Natural Killer T (iNKT) cell, key mediators of the cellular immunity towards tumours. Indeed, alpha-GalCer and analogues as therapeutics to promote iNKT effector function is a promising new therapeutic approach to cancers. Importantly, Map3k1deltaKD iNKT cells have elevated apoptosis and hypoproliferate in response to immunisation of these mice by glycolipid antigen, and this results in a significant deficit in the iNKT cell clonal burst. My research team will further characterise the extent of the MEKK1-dependent defect in iNKT cell biology, and extend the analysis beyond MEKK1 itself to the analyses of the downstream signalling pathways MEKK1 regulates, and the mechanisms by which MEKK1 controls iNKT cell apoptosis and proliferation. My research team will test whether E3 Ubiquitin ligase Itch, a key downstream effector of MEKK1 signalling in T cells, is an important downstream signalling mediator for effector iNKT cell responses to glycolipid antigens by analysis of Itch-deficient mice. I will analyse the downstream MAPK kinase signalling pathways by which MEKK1 controls the iNKT cell clonal burst using MAPK signalling pathway antibodies and MAPK pathway-specific small molecule inhibitors. I will analyse whether MEKK1 influences iNKT cell activation induced cell death (AICD) by crossing Map3k1deltaKD with Fas- or Bim-deficient mice, key regulators of AICD, and then my research team will determine the extent of apoptosis in the double mutant mice following glycolipid antigen immunisation. I will test whether MEKK1 influences iNKT cell killing by Granzyme B cell death pathways by creation and testing of a Map3k1deltaKD and Granzyme B-deficient double mutant mouse model and analysis of the levels of apoptosis present in its iNKT cells in response to glycolipid antigens. I will determine whether MEKK1-mediated proliferative expansion in iNKT cells involves Jun transcription factors (c-Jun, JunB, and JunD) and induction of Cyclins by analysis of their expression in Map3k1deltaKD mice following glycolipid antigen immunisation. I will test whether Cyclin D family members are critical to the normal iNKT cell clonal burst by analysing Cyclin D-deficient mice.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Interaction between the essential replication protein TopBP1 and human papillomavirus 16 E1: a novel anti-viral target
Funder: Cancer Research UK

Abstract: Background Human papillomaviruses (HPV) are causative agents in cervical cancer; in 2007 approximately 310,000 women died from this disease world wide and in the developing world this was the number one cancer killer of women. Although a prophylactic HPV 16/18 (the two most common types associated with cervical cancer) vaccine works, anti-virals are still required as women currently infected remain at disease risk, many developing nations lack resource and more importantly infrastructure to deliver the vaccine, and the vaccine targets only a subset of HPV types; other high risk HPV may become dominant. In addition, an anti-viral offers an alternative way to manage HPV infection; PCR positive patients (only a small fraction of which develop disease) could be given anti-virals to eliminate infection and subsequent disease risk. Viral replication has been a focus for anti-viral drug development. HPV replicate their dsDNA genome using E1 and E2 in concert with host proteins. E2 targets the viral origin, physically recruits E1 which forms a di-hexamer replication helicase. Drugs targeting the E1-E2 interaction have not been clinically developed as different HPV E1-E2 pairs have subtle interaction differences meaning drugs don’t work across HPV type. Therefore the focus has shifted to viral-host protein interactions. We have demonstrated that TopBP1, a protein required for mammalian genome replication initiation, interacts with both HPV16 E1 and E2. A non-TopBP1 interacting E2 has compromised DNA replication. Aims A non-TopBP1 interacting mutant of E1 will be generated. Replication properties of this mutant will be monitored to confirm the essential nature of the E1-TopBP1 interaction for viral replication. A viral life cycle model will confirm the essential nature of this interaction for viral infection. Outline E1-TopBP1 interaction occurs in vivo and in vitro. NMR structural data will reveal contact residues between the two proteins; E1 contact points will be mutated in an HA-tagged E1 mammalian expression vector and mutants tested for TopBP1 interaction in vivo. This will generate a non-TopBP1 interacting E1 mutant. The ability of the mutant to induce DNA replication in vivo will be monitored and to confirm the essential nature of the E1-TopBP1 interaction, the mutant will be introduced into the entire viral genome and life cycle studies carried out in collaboration with Prof. Lou Laimins, Northwestern University. The outcomes of this proposal will provide a novel anti-viral target, the E1-TopBP1 interaction, and a model system for studying TopBP1 DNA replication properties.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: How Sld3 integrates kinase signalling to control DNA replication initiation
Funder: Cancer Research UK

Abstract: Background The control of DNA replication initiation is achieved by the action of multiple protein kinases. My previous work identified two factors, Sld2 and Sld3 as the minimal Cyclin-dependent Kinase (CDK) targets required for replication initiation in budding yeast. In addition I have shown that the checkpoint kinase, Rad53, inhibits replication initiation by phosphorylation of Sld3 and another essential factor Dbf4. My research has therefore established that the phosphorylation of the Sld3 protein is a critical regulatory junction either to promote or to inhibit replication initiation. Despite this, how Sld3 executes its essential functions in budding yeast, or indeed in any eukaryote, is very poorly understood. Relevance to cancer Failure to replicate the genome exactly once leads to genomic instability, which is a hallmark of the earliest stages of tumour formation. Furthermore CDK, checkpoint kinases and the factors that initiate replication are deregulated in many cancers. In addition inhibitors of DNA replication are used in most chemotherapeutic regimens. Therefore investigating how replication initiation is regulated is vital for understanding the causes and furthering the treatment of cancer. Specific aims of the research This project will establish the molecular mechanism of how phosphorylation of Sld3 acts as a switch to control replication initiation. This work will provide insights into the essential functions of Sld3 in vivo and will clarify current dogma of how CDK regulates replication initiation. In addition, a mechanistic understanding of the regulation of Sld3 from budding yeast will serve as a platform to translate studies to the recently identified Sld3 homologues from higher eukaryotes. Outline plan of research We will fully characterise, using biochemical assays and X-ray crystallography, the multilayered interaction between Sld3 and another essential replication factor, Dpb11. This will allow the determination of the specificity between Dpb11 and Sld3 and an analysis of whether this has been conserved in human Dpb11/TopBP1. Finally we will test biochemically the role of the checkpoint kinase Rad53 in inhibition of the interactions mediated by Sld3, both with Dpb11 and another essential factor, Cdc45. Together this will provide a thorough analysis of the regulation of replication initiation through Sld3 phosphorylation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Whole body functional and anatomical MRI: Accuracy in staging and treatment response monitoring in Adolescent Hodgkin's Lymphoma compared to conventional multimodality imaging
Funder: Cancer Research UK

Abstract: Background: Treatment regimens for adolescent Hodgkin's lymphoma are critically dependent on accurate staging and early treatment response triage using cross-sectional imaging. Staging is based on the distribution of nodal and extranodal disease, the former assessed with standard computerized tomography (CT) using nodal size thresholds. Deficiencies of simple size criteria are particularly relevant in the younger population where benign nodal enlargement is common. Lymphomatous tissue exhibits increased glucose metabolism compared to normal tissue, which can be detected using 18F-2-deoxy-2-fuoro-D- glucose Positron Emission Tomography (18FDG PET) providing additional "functional" information about the tumour. CT and PET form the basis of imaging required for the ongoing Euronet PHL trials. Unfortunately CT and PET impart a significant diagnostic radiation dose and for many patients diagnostic imaging generates all radiation exposure. Even small doses of radiation can increase an individual's risk of malignancy and this risk is increased in younger patients. Magnetic Resonance Imaging (MRI) does not use ionising radiation and is therefore a safe alternative. Basic whole body anatomical staging equivalent to CT is possible using MRI but like CT, standard MRI lacks the functional information afforded by PET. However diffusion weighted imaging (a functional MRI technique) provides an estimate of cellular density (expressed as the apparent diffusion coefficient- ADC) which is increased in lymphoma and related to tumour metabolic activity. Data suggests a close a relationship between ADC and 18FDG uptake both before and after treatment Aims and objectives In childhood and adolescent Hodgkin's lymphoma, to prospectively compare diagnostic concordance of whole body functional MRI with the current conventional multi-modality reference of CT, PET-CT and ultrasound in initial disease staging and treatment response triage. Methods: 55 patients aged up to 18 years with confirmed Hodgkin's lymphoma entered into the Euronet PHL Trials will undergo whole body anatomical and functional (diffusion weight imaging) MRI in addition to standard investigations (PET/CT, Chest CT, abdominal ultrasound) at initial disease staging and during treatment response assessment following 2 cycles of chemotherapy. MRI staging will be compared to a multi-disciplinary team derived standard of reference based on full clinical data and all current conventional multi modality imaging investigations. Concordance will be defined on a per nodal station and per patient basis at initial staging and treatment response triage. How the results of the research will be used Prospective validation of whole body functional MRI is an important prerequisite to both local implementation and national multicentre trial investigation


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: How does the loss of EXT gene function induce tumorigenesis?
Funder: Cancer Research UK

Abstract: Background Multiple Osteochondromas (MO) is an autosomal dominant disease that is characterised by the appearance of benign skeletal tumours in children. It is caused by mutations in two genes, EXT1 and EXT2, which encode enzymes that are required for heparan sulphate (HS) synthesis. In addition, patients with MO have a greater than 2% chance of developing chondrosarcoma. We have identified mutations in zebrafish ext2 and have shown that homozygous mutant fish display a cartilage tumour phenotype. We also determined that the absence of ext2 gene function is sufficient to induce early tumour formation. Further analysis led us to propose a new model that the initiation of tumorigenesis is the result of a loss of chondrocyte polarity. Our work as well as work in chick suggests that the planar cell polarity (PCP) pathway establishes chondrocyte polarity in wildtype animals and when disrupted may be involved in the formation cartilage tumours. Aims and Methods Here we aim to elucidate the early steps involved in EXT-dependent cartilage tumour formation and to test whether the PCP pathway plays a role in this process. To do this we will generate transgenic animals that will allow us to turn off HS synthesis in single cells and follow progression of a single chondrocyte into a tumour. We will monitor cell behaviour during the first hours of tumorigenesis and gain a rare glimpse into the cellular mechanics of this process. In addition, we will make lines that block the PCP pathway in individual chondrocytes to determine whether this is sufficient to induce tumour formation. We will make transgenic lines that allow us to visualize PCP pathway activity in single chondrocytes and characterise these in wild type and ext2-/- larvae. These experiments will firmly establish whether PCP signalling plays a role in tumour formation and elucidate how EXT genes initiate tumorigenesis. Outcomes One long-term question is how do EXT induced osteochondromas provide an environment that promotes the development of chondrosarcoma. We propose that chondrosarcoma formation involves initial disruption of PCP signalling with subsequent disruption of other pathways. Once we have generated a robust system for producing cells lacking HS, we will be able to begin to address what other changes are needed to initiate cancer formation. This strain will also provide a platform to screen for drugs which block tumorigenesis. The finding that loss of PCP signalling initiates tumour formation will provide molecular targets for pharmacological intervention.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Structural dissection of intracellular Hedgehog signalling
Funder: Cancer Research UK

Abstract: BACKGROUND AND RELEVANCE TO CANCER: The Hedgehog (Hh) signalling pathway is fundamental for embryonic development and adult stem cell regulation. In humans, activation of the Hh pathway leads to the development of cancers, including basal cell carcinomas, medulloblastomas, gliomas, leukemias, lymphomas and melanomas as well as breast, colon, lung, prostate and pancreas cancers. Hh signalling controls tumour growth by promoting proliferation and cell survival and is required for cancer stem cell self-renewal. In vivo evidence suggests that blocking excessive Hh signalling could provide unique mechanism-based anti-tumour therapies. Modulation of the cell surface receptor Smoothened (Smo), the key protein in Hh signal transduction through the plasma membrane, is a current strategy to target the Hh pathway. Clinical trials demonstrate that GDC-0449, a Smo antagonist, induces rapid tumour regression in meduloblastoma and metastatic basal cell carcinoma. Mutations in downstream components of the Hh pathway (e.g. Suppressor of Fused (Sufu)) also promote tumourigenesis, providing additional potential therapeutic targets. AIMS &amp;amp; OBJECTIVES: A detailed understanding of how the Hh signal is transduced from membrane to nucleus is still lacking. The principal objective of this proposal is to illuminate this process by characterising the molecular interactions between vertebrate intracellular Hh pathway components. What are the molecular mechanisms underlying Hh signal transduction? What are the functional implications of disrupting the observed protein-protein interactions? Specific aims are: - To map protein-protein interaction domains of Sufu, Fused (Fu), Kif7/Kif27 and the intracellular region of Smo. - To detail the molecular architecture of single domains and primary and, where applicable, higher order complexes of Smo, Sufu, Fu and Kif7/Kif27 by X-ray crystallographic analysis. - To characterize these interactions using a combination of structure-guided site directed mutagenesis, biophysical and cell-based methods. - To validate the functional significance of these interactions in vivo using the zebrafish embryo as an assay system. EXPERIMENTAL APPROACHES: The work proposed represents a collaboration between structural and developmental biology labs. Building on pilot expression studies, we will use X-ray crystallography to obtain molecular snapshots of these fundamental processes and combine these atomic details with biochemical and live cell-based studies. Our findings will be placed in an in vivo context in zebrafish embryos. EXPECTED OUTCOMES AND IMPACT ON THE FIELD: Our multi-disciplinary approach will provide a deeper understanding of the Hh pathway providing fundamental biological insights. Importantly, our results and reagents can potentially feed into the development of novel therapeutics targeting Hh signalling.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Mechanism of telomerase activation and inhibition at fission yeast telomeres
Funder: Cancer Research UK

Abstract: Telomeres play a dual role in cancer biology as they act as a barrier to malignant transformation by contributing to genome integrity, and their maintenance is a prerequisite for indefinite cellular proliferation, a hallmark of tumour cells. Telomere maintenance is carried out by the telomerase enzyme, and therefore the characterization of the mechanism of telomerase regulation at chromosome ends has direct implications for an understanding of the carcinogenic process._x000D_ _x000D_ Telomerase acts at individual telomeres in a manner that is regulated by the protein factors bound to telomeric DNA repeats. Whereas telomeres made or longer repeat arrays are non-permissive for telomerase action, shorter telomeric arrays are a preferential substrate. The number of telomere-bound regulators is the critical signal for telomerase inhibition. Recent evidence in budding yeast suggests that telomere shortening promotes increased telomerase recruitment dependent on the Tel1 kinase. However, genetic data in fission yeast, and important differences in the protein composition in the telomeric complex in this organism, which more closely resembles the human complex, raise the question of whether similar mechanisms of telomerase regulation apply to fission yeast and to humans compared with budding yeast._x000D_ _x000D_ We aim to obtain a detailed picture of the protein composition of the fission yeast telomeric complex as a function of cell cycle stage, by using chromatin immunoprecipitation (ChIP). Because telomerase is active in S phase this will identify which changes in the composition of the complex might play a role in telomerase action. We will conduct these studies in strains bearing telomeres of different lengths, to study the dependence of telomerase action on telomere length. This will allow the identification of the molecular events that are associated with telomerase activation, including whether the telomere association of telomerase itself is regulated. In addition, we will focus on the components of the telomeric complex which have a role in either the stimulation or the inhibition of telomerase, or both, thus addressing whether changes in the composition of this complex occur upon telomere shortening, which might be causative of telomerase activation/recruitment. Finally, and in parallel, we will attempt to generate mutations abolishing demonstrated interactions between components of the fission yeast telomeric complex, to assess the role of structural transitions within the complex in regulating telomerase action.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Role of poly(ADP-ribosyl)ation of a transcriptional factor, CTCF, in epigenetic regulation of the p14ARF gene in breast cancer cells
Funder: Cancer Research UK

Abstract: The human p14ARF (mouse p19ARF) is a product of a tumour suppressor gene encoded by the INK4/ARF locus. Deregulation of the p14ARF gene has been associated with breast cancer development, but the molecular events underlying these processes are not well understood. Our recent studies demonstrated that p14ARF gene is regulated by a multifunctional 11 Zn-finger transcription factor, CTCF, and a post-translational modification, poly(ADP-ribosyl)ation (PARylation) of CTCF. CTCF normally activates p14ARF and this activation relies on the presence of both, CTCF and the PARylation enzyme-1, PARP-1, at the CTCF binding site (CTS) identified within the p14ARF promoter. The aim of the proposed investigation is to test two hypotheses: 1. There is a causal link between CTCF and PARP-1 association at the p14ARF CTS, defective CTCF (and possibly PARP-1) PARylation and p14ARF deregulation. 2. CTCF and PARP-1 act as a “rheostat” regulating changes in epigenetic configuration at the p14ARF CTS. To achieve this Aim, we will study the following four Objectives: Objective 1 Analysis of the expression of p14ARF mRNA and the status of CTCF and PARP-1 in the experimental cell models. Objective 2 Analysis of the role of CTCF and PARP-1 in the establishment of the epigenetic states at the p14ARF CTS. Objective 3 Analysis of p14ARF mRNA levels and their correlation with epigenetic states at the p14ARF CTS in normal and tumour breast tissues. Objective 4 Is the p14ARF CTS a “master” p14ARF regulatory element? Analysis of the additional CTSs within the INK/ARF locus and their contribution to the regulation of p14ARF. We will use model cell lines with different levels of p14ARF mRNA expression to assess the role of CTCF and PARP-1 in the regulation of the epigenetic states at the CTS within the p14ARF promoter and also at other CTSs in the INK/ARF locus. Our observations will then be further validated using breast tumour tissues. We will employ a wide range of methods including RT-QPCR, QPCR, ChIP, re-ChIP, bisulfite sequencing, pyrosequencing, Western, gel retardation, methylation interference and various cell/tissue culture techniques. The results obtained will be used to better understand the molecular mechanisms of p14ARF regulation by CTCF and PARP-1. Thus, elucidation of the role of epigenetic alterations in p14ARF can provide an insight into the nature of predisposition to and early stages of breast tumourigenesis. These results may also be valuable for design of anti-cancer treatment because epigenetic changes can potentially be reversed with the right therapies.


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