NIHRDataInsights/HRCSData huggingface dataset:
Title: How does cohesin regulate transcription in mammalian cells and how does this go awry in tumour cells?
Funder: Cancer Research UK

Abstract: Untimely activation of oncogenes or repression of tumour suppressors can lead to cell proliferation, transformation and cancer. Inappropriate communication between otherwise unaltered genes and distal regulatory elements can lead to aberrant gene expression and therefore cancer. Recent Hi-C studies have shown that mammalian chromosomes are compartmentalized into domains within which long range interactions between DNA sequence pairs are more frequent than interactions with sequences outside such compartments. These compartments are referred to as Topologically Associating Domains (TADs). TADs constrain long-range DNA interactions and could therefore limit the possible enhancer promoter interactions. Changes in chromosome structure resulting from deletion or inactivation of a TAD boundary can thus lead to inappropriate (onco)gene expression. There is growing evidence for a strong correlation between TAD disruption and oncogene activation in several cancers. The key question is how are TADs formed. In order to explain how mammalian chromosome organization is brought about by cohesin and condensin I suggested that both the Smc/Kleisin complexes are capable of trapping small chromosome loops and then extrude them in a processive manner (Loop Extrusion). TADs are likely a property that emerges from the continuous extrusion of loops by cohesin complexes in a process that is punctuated by CTCF sites, which thereby define the TAD borders. Loop Extrusion (LE) provides the only coherent explanation for how CTCF prevents distant enhancers from activating the wrong promoter. Somatic mutations of cohesin have been reported in a wide range of human cancers, the cohesin SA2 subunit is one of only 12 genes that were significantly mutated in four or more cancer types. Importantly, these cohesin mutations are not necessarily accompanied by defects in sister chromatid cohesion, but there is a growing evidence for TAD disruption in these cancer cells, possibly due to alteration of LE dynamics. Understanding how TADs are formed is therefore of utmost importance in the fight against cancer. We will use biochemical, molecular and cell biological techniques to address the following important questions concerning the LE process and its biological and pathological ramifications. Can we observe TAD formation in living cells and assess the consequence of cohesin inactivation on this process? Can we observe LE by cohesin in vitro? What powers the cohesin motor? How does CTCF function as a boundary element?


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: MICR Unallocated Research Services
Funder: Cancer Research UK

Abstract: CRUK Manchester Institute core facilities not allocated to specific labs. Coded to overall lab profile


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: Understanding the function of MACROD1 and MACROD2 proteins and their relevance to cancer
Funder: Cancer Research UK

Abstract: Background: ADP-ribosylation is an important and dynamic posttranslational protein modification with crucial roles in a wide variety of cellular processes, including maintenance of genomic integrity, mitochondrial metabolism and cell death which has multiple implications for human health and longevity. The major family of enzymes that synthesise ADP-ribosylation are poly(ADP-ribose) polymerases (PARPs) and many have been extensively studied. On the other hand, much less is known about the cellular enzymes that remove protein ADP-ribosylation. In particular, the two enzymes that can specifically reverse protein mono-ADP-ribosylation, MACROD1 and MACROD2, have been identified only very recently. Consequently much remains to be discovered about their functional importance in vitro and in vivo. In addition, relevant to human disease, MACROD1 and MACROD2 are found amplified in cancer (for example breast cancer) and this over-expression was suggested to be associated with a worse prognosis. Aims: The goal of this project is elucidation of the physiological and pathophysiological functions of MACROD1 and MACROD2. In doing so, we also aim to establish whether MACROD1 and MACROD2 play a role development of cancer (breast and prostate cancer in particular) and anti-cancer drug resistance. Methods: In order to achieve our goal we will use cellular and mouse models supported by biochemical and structural biology approaches. How the results of this research will be used: Overall, our research will contribute to a better understanding of the post translational modification, and address fundamental gaps in our knowledge with regards to the molecular mechanisms that regulate ADP-ribosylation mediated signalling and genome stability. More importantly, these studies will clarify the importance of novel hydrolases (such as MACROD1 and MACROD2) in cancer and may uncover novel biological mechanisms of therapeutic interventions and/or mechanisms of resistance to therapies.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: Defining molecular interactions between tumor suppressors and oncogenes in the Wnt signalling pathway
Funder: Cancer Research UK

Abstract: The Wnt/ß-catenin signalling cascade is an ancient cell communication pathway with key roles in embryonic development of all animals, and in stem cell compartments of adult tissues. Hyperactivation of this pathway drives a wide range of cancers. Notably, most colorectal cancers are initiated by the mutational inactivation of the APC tumour suppressor, a negative regulator of ß-catenin, or by activating ('oncogenic') mutations in ß-catenin itself, a key effector of the pathway. Oncogenic ß-catenin mutations are also widespread in other cancers. Despite its importance in cancer, there are no well-validated small-molecule inhibitors of this pathway, due to the lack of ‘druggable’ targets and an incomplete understanding of the key molecular interactions that govern the signalling flow from the plasma membrane to the nucleus. Defining these interactions therefore remains a high priority. We plan to address major unsolved questions in the membrane-proximal part of the Wnt pathway, focussing on the molecular mechanisms that allow the Wnt signalosome to block the APC-dependent destabilisation of ß-catenin by the Axin degradasome. This signalosome is assembled by Dishevelled whose polymerisation enables it to bind to Axin and to recruit the degradasome to the plasma membrane. This results in the phosphorylation of the cytoplasmic tail of the LRP6 co-receptor. We plan to identify the proteins binding to this tail that enable it to block the degradasome and to define the molecular role of the WTX tumour suppressor in this process. We also plan to investigate how Dishevelled impacts on the ß-catenin-dependent transcription switch by the Wnt enhanceosome via its PDZ domain, and to determine the protein ligands of this domain that divert Wnt signals from ß-catenin to non-canonical effectors. We thus aim to complete a molecular map of the physical interactions between key components of the Wnt/ß-catenin pathway and to understand the molecular switch that diverts Dishevelled signalling from ß-catenin to non-canonical Wnt effectors. Our strategy is to combine biochemical, biophysical and structural approaches with functional assays in human cell lines, to identify new signalling partners in the Wnt/ß-catenin pathway and to define the molecular interfaces that underlie critical switches in Wnt signal transduction. We shall also retain the Drosophila model as a rigorous genetic test system, to check physiological relevance and to obtain novel functional clues. Our results will inform strategies for therapeutic intervention in ß-catenin-dependent cancers, and may open up new avenues and molecular targets for drug development.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: Molecular Mechanisms of Morphogen Signalling at the Cell Surface
Funder: Cancer Research UK

Abstract: BACKGROUND A handful of secreted, gradient-dependent morphogen signalling molecules orchestrate the development of multicellular organisms. Among these, the functionally-related Hedgehog (Hh) and Bone Morphogenetic Protein (BMP) pathways are crucial for embryogenesis and stem cell maintenance, but deregulated signalling is a frequent and important driving force behind various cancers. This programme seeks to unravel the molecular mechanisms underlying Hh and BMP morphogen reception and signal transduction through the membrane, and how they are changed in cancer. AIMS 1. Unravel the molecular architecture of the Hh receptors Patched-1 (PTC1) and Growth Arrest Specific-1 (GAS1) and their binding determinants to Hh morphogens. This will provide a mechanistic understanding of oncogenic mutations in PTC1, and will potentially shed light on how PTC1 inhibits the Hh signal transducer Smoothened (SMO). 2. Discover the molecular mechanisms of Hh signal transduction by the G-protein coupled receptor and oncoprotein Smoothened (SMO). This will provide a structural template for the development of the next generation of SMO inhibitors against Hh-driven cancers, which are known to become resistant against drugs currently used in the clinic. 3. Define the structural determinants of extracellular BMP signal antagonism and agonism by the crucial co-receptors Repulsive Guidance Molecule (RGM) and BAMBI. This will provide insights into structural interfaces, interaction specificities and BMP co-receptor regulation, and can help to develop novel therapeutic strategies and reagents for modulating BMP signalling. 4. Structurally and functionally characterise full-length transmembrane BMP receptor complexes. These analyses will reveal important insights into stochiometries and determinants of protein-protein interactions in BMP signalling complexes and their site of action, and may unravel novel strategies for manipulating both the BMP pro- and anti-tumour effects in cancer. METHODS We will use structural biology methods (X-ray crystallography, small angle X-ray scattering and cryo-electron microscopy) to obtain molecular snapshots of active extracellular and membrane protein complexes, and combine these studies with mutagenesis, biophysical studies with purified proteins, cellular reporter assays and live-cell imaging. HOW THE RESULTS OF THIS RESEARCH WILL BE USED. Our multi-disciplinary analyses will provide a deeper understanding of important and novel mechanisms of Hh and BMP signal transduction, unravelling the role of cancer-causing mutations and basic cancer mechanisms. Importantly, our molecular analyses can lead to the identification of novel strategies and reagents (engineered nanobodies and protein domains, and small molecules) feeding into basic and translational cancer research and facilitating development of novel therapeutics targeting Hh and BMP signalling.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: Understanding Brf2-dependent RNA Polymerase III transcription in cancer: structures, mechanisms and potential for intervention
Funder: Cancer Research UK

Abstract: The occurrence of abnormally high levels of RNA Polymerase (Pol) III transcripts is a hallmark of many forms of cancer. Recent studies have demonstrated a direct causative link between deregulation of RNA Pol III transcription and tumourigenesis. We have recently established Brf2 as a master regulator of the oxidative stress response pathway via a mechanism which involve oxidative modifications of Brf2. Importantly, we derived a mechanistic link between the genetic activation of Brf2 observed particularly in lung and breast carcinoma with evasion of oxidative-stress induced apoptosis, a hallmark of cancer cells (Cell, in press). Following up this initial and exciting discovery, I am planning to use a holistic approach integrating structural and biochemical data with genome-wide analysis in living cells, in order to obtain a comprehensive understanding of the molecular events underlying Brf2-dependent transcription deregulation in cancer cells and to assess the potential of Brf2-dependent RNA Pol III transcription as a novel anticancer target. In particular, I am aiming to: I) Structurally and functionally characterize Brf2 as part of a complete TFIIIB complex bound to natural promoters. II) Unravel the molecular basis of RNA Polymerase III recruitment at Brf2-dependent promoters and its regulation. III) Elucidate oxidative stress-mediated regulation of the Brf2 transcriptional program and its interplay with the Nrf2-Keap1 pathway in normal and cancer cells at a genome-wide level. IV) Mechanistically understand the tumorigenic role of Brf2 in different cancer types using three-dimensional culture systems. V) Establish a cell-based high-throughput assay for screening small molecule inhibitors of Brf2-dependent transcription and a cell-free transcription assay for follow-up studies. Structural biology techniques (Aims I and II: x-ray crystallography) will be integrated with biochemical and biophysical methods (Aims I-II) and functional and genome-wide analysis in 2D and 3D cultured cells (Aim III-IV: inducible shRNA/cDNA expression, spheroid analysis, immuno-staining, ChIP-seq, RNA-seq). Aim V will focus on the establishment of cell-based (RT-qPCR based) and cell-free assays for identification of chemical tools to specifically target Brf2-dependent transcription in order to evaluate its potential as a novel anticancer target. The proposed research will generate outputs that will enhance our understanding of basic cancer biology but, most importantly, has the potential to open up a unique and yet unexplored avenue for the development of therapeutic agents, which target cancer enhanced antioxidant defense by inhibition of Brf2-dependent RNA Pol III. (Checked eGMS - only lung and breast models will be used)


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: The molecular origin of ductular cancers in tubular morphogenesis.
Funder: Cancer Research UK

Abstract: Background: Ductular cancers of the liver and pancreas have a very poor prognosis, with many patients succumbing to disease within a matter of months following diagnosis. These ductular cancers, cholangiocarcinoma in the liver and pancreatic ductular adenocarcinoma in the pancreas, often have their origins in tissues experiencing chronic disease, where immune cells and fibroblasts influence damaged ducts providing them with pro-growth signals. I hypothesise that in ductular cancers, the earliest cancer cells co-opt these pro-growth signals to drive their proliferation and establish a cancer. Aims: 1. Determine what the signals are that promote duct cells to proliferate following injury 2. Understand what factors influence the growth and elongation of a regenerating duct 3. Define whether duct cancer cells use the same mechanisms to grow as repairing ducts and investigate whether these represent new therapeutic vulnerabilities to treat ductular cancer. Methods: I will use a combination of cell lines from cancer tissue and normal ducts to investigate the signals that govern duct growth in vitro. Having defined these signals, I will use sophisticated animal models of duct repair and cancer to see in vivo whether the signals that control duct repair are utilised to initiate ductular cancer. I will use the data generated from this research in two ways: Firstly, by defining the signals and processes that control duct growth following injury I will gain a deeper understanding of how different cell types interact to promote duct growth. Using this knowledge I will then be able to determine whether these signals are important for tumour initiation in biliary cancers – if this is the case, and biliary cancers are dependent on these signals for their initiation, I will develop future collaboration and therapeutic strategies to understand whether inhibiting these pathways can prevent tumour growth or disease progression.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: Haematopoietic stem cells and cancer evolution - from single cells to population dynamics
Funder: Cancer Research UK

Abstract: BACKGROUND The process by which adult stem cell populations are established and maintained and how individual stem cell clones “compete” with each other is a key component to understanding ageing and cancer. The high prevalence of clonal haematopoiesis in otherwise healthy elderly individuals has demonstrated that aberrations in clonal competition can be a key indicator of early cancer development. Using a suite of single cell and clonal approaches, this programme of work will comprehensively map the normal blood forming system, establishing the relatedness of haematopoietic stem cells (HSCs) to each other and their mature cell progeny while also exploring the intrinsic and extrinsic drivers of clonal competition in cancer patients. AIMS The proposal is divided into two aims that study the clonal fitness of HSCs and their progeny. Aim 1 delves into questions that we cannot yet ask in human samples, studying the complete set of HSC clones in mice to establish whether HSCs contribute equally and stably to mature blood cell production. Aim 2 is focused on determining the impact of cellular stress in primary samples from patients with haematological malignancies. Specifically, we will study patients with pre-leukaemic bone marrow failure syndromes to assess HSC clonal dynamics and to identify potential systemic regulators of clonal competition. This aim is sub-divided to study the effect of intrinsic cellular stresses (2A) and extrinsic microenvironmental stresses (2B) on HSC genomic integrity and cancer evolution using bone marrow failure disorders as a model of early stage tumourigenesis. METHODS This programme is built around three core techniques that are all well-established in our lab: 1) whole genome sequencing of HSC clones to identify somatic mutations as a genetic barcode to track HSC population dynamics (Lee-Six et al., Nature 2018); 2) single cell RNA-sequencing combined with single cell transplantation to identify molecular regulators of HSCs (Wilson/Kent/Buttner et al., Cell Stem Cell 2015); and 3) serum cytokine profiling with comprehensive genetic and clinical data to identify new regulators, or potential biomarkers, of disease evolution (Obro/Grinfeld et al., under review). HOW THE RESULTS OF THIS RESEARCH WILL BE USED Overall, this programme aims to deliver a more refined understanding of the intrinsic and extrinsic drivers of HSC clonal expansion and how HSCs compete against one another throughout ageing, stress, and cancer development. This work will also have much broader implications, informing efforts in transfusion and transplantation medicine, and to manipulate clonal dynamics to improve cancer outcomes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE
Title: B-cell receptor signalling in B-cell malignancies: Identifying strategies for optimal therapeutic intervention based on common and distinct responses
Funder: Cancer Research UK

Abstract: Background The purpose of this research is to investigate function and therapeutic targeting of the B-cell receptor (BCR) in chronic lymphocytic leukaemia (CLL) and follicular lymphoma (FL), the two most prevalent lower-grade B-cell malignancies. We will identify mechanisms by which BCR signalling operates from upstream initiating events to downstream effector pathways, and determine how activated pathways and drug responses differ between the tumour types. Aims The programme has three specific aims; 1. To characterise downstream signalling pathways linking MYC and mRNA translation in CLL, and investigate effects of targeted inhibitors in vitro and in vivo. 2. To identify novel features of anergic CLL cells and develop strategies for selective elimination of this latent but potentially dangerous population. 3. To use large-scale cell imaging to compare early signalling events and BCR endocytosis in FL and CLL, and to characterise downstream effects of the FL BCR-binding lectin DC-SIGN on apoptosis, MYC and mRNA translation in FL. Methods Experiments will focus on primary patient-derived material, with extension to established B-cell lines and mouse models for mechanistic studies. Comparison to normal B cells will reveal tumour-specific phenomena. Analysis of mRNA translation will focus on the regulation of translation initiation factors and ribosome components, and on responses to inhibitors of mRNA translation and MYC. In vivo analysis will be performed using the EmuTCL1 mouse model and xenografted human samples in NSG mice. Translationally-regulated mRNAs will be identified using ribosome profiling with parallel miRNA analysis to probe regulatory mechanisms. Analysis of anergy will focus on expression and modification of candidate anergy-associated proteins, including BIM and SHIP1, as well as responses to BH3-mimetics and small molecule SHIP1 activators. We will also perform miRNA and mRNA profiling to identify novel anergy-associated features. This will be performed in close collaboration with Professor Goodnow allowing powerful cross-comparison to datasets from mouse models of anergy prior to functional assessment using transfection studies. We will use high-content, large-scale imaging to analyse signalling pathways and endocytosis, including in response to DC-SIGN in FL. Downstream analysis will focus on regulation or MYC and mRNA translation in FL, and apoptosis, including response to BH3-mimetics. Outcomes The research will provide important new insight into the critical growth control pathways in B-cell malignancies. It is likely to provide new biomarkers to guide treatment decisions and new therapeutic approaches (including new drug therapies and rational combinatorial approaches) for the treatment of these currently incurable diseases.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE