NIHRDataInsights/HRCSData huggingface dataset:
Title: Gene network engineering: quantifying emergent gene expression in cancer and antibiotic resistance
Funder: Wellcome Trust

Abstract: RESEARCH QUESTION ABSTRACT ONLY Gene network engineering: quantifying emergent gene expression in antibiotic and cancer drug resistance. My aim is to understand and build gene networks that behave predictably, consistently and robustly, in the context of solving medically-relevant problems. The research is carried out at various levels of complexity, from bespoke engineering of regulatory proteins and small networks, through analysing cellular networks, and ultimately carrying out gene therapy in whole organisms (PNAS 109:E 3136-45, 2012). In this 5-year proposal, I will tackle the problem of emergence in network behaviours, or gene expression according to need , particularly in the context of antibiotic resistance genes in bacteria and multi-drug resistance genes in cancer cells. A main concept at the centre of this proposal is that natural selection occurs not only on genes (on genetic variation), but also at the level of gene expression (on heritable phenotypic variation). For example, if a gene product is requ ired for increased cell division in cancer, low expression will slow down division, giving time for expression levels to build up stochastically, reinforcing the presence of high-expressing cells. Feedbacks between gene expression levels and division rates therefore drive expression higher than expected from hard-wired network regulation alone. Fit epigenetic states with upregulated antibiotic resistance genes or certain oncogenes are thus actively selected, in the absence of molecular signallin g or genetic variation. As I outlined in a brief commentary (Nature 488: 40-41, 2012; final 2 paragraphs) there are several possibilities for these feedbacks (involving cell division, secretion, compartmentalisation, inactivation, degradation) and a systematic, quantitative analysis is currently lacking. By combining wetlab experiments and computational modelling, we will develop a new theoretical basis for understanding emergent gene expression. Key questions 1. Can we identify or build mo del examples of each class of emergent feedback in bacteria and mammalian cells? 2. Can we establish quantitative rules describing emergence? i.e. perturb regulation with tunable repression of an emergent drug-resistance gene (and controls) to compare expected-versus-observed expression levels, in combination with computer models. 3. Can we apply our synthetic zinc fingers and TALENs to regulate or override emergence? Key words Systems biology, Synthetic biology, zinc finger engineerin g


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS;INFECTION
Title: The relative importance of human and animal sources of vancomycin-resistant Enterococcus faecium in immunocompromised patients in hospital.
Funder: Wellcome Trust

Abstract: Antibiotic resistance is recognised by the WHO as one of the three greatest threats to human health and its spread is challenging the ways we practice medicine. Enterococcus faecium (Efm), a bacterium carried harmlessly in the gut of humans and animals, has emerged as a leading cause of infections in severely immunocompromised patients in hospitals. It has a propensity to accumulate and disseminate antibiotic resistance determinants, in particular to vancomycin, one of the workhorse antibiotics in hospital. This makes its treatment more challenging and can lead to hospital outbreaks that are difficult to control. This study proposes to use a cutting-edge technique, whole-genome sequencing, to elucidate the origins of vancomycin-resistant Efm (VREfm) in patients admitted for the treatment of haematological cancers at Addenbrooke's Hospital. In addition, I will sample slurry from livestock farms and wastewater from sewers to determine the frequency of VREfm in farms and the community, respectively. I will use computer software to compare the evolution of individual bacterial isolates and their resistance genes, establish links between patients on the same ward, and links between the 3 different reservoirs. By understanding the epidemiology of Efm, effective prevention strategies for healthcare-associated Efm infection (including VREfm) could be developed.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: CANCER AND NEOPLASMS;INFECTION
Title: Gene Regulation and Chromatin
Funder: Medical Research Council

Abstract: Development of multicellular organisms requires that cells divide and proliferate to generate the organs and structures of the body, Cells must also activate and repress specific gene expression programmes at different stages of differentiation. Disruption of this regulation is a major cause of disease including cancer, autoimmune disease and metabolic disorders. The overall aim of the group is to study the mechanisms that regulate gene expression during development with a particular focus on how cell cycle regulators integrate with external signals and intracellular factors in the control of transcriptional initiation and elongation. We have identified several important regulatory pathways that are regulated by the Aurora B kinase. Aurora B is an epigenetic regulator that phosphorylates histone H3 serine 10 and serine 28 in mitosis and during development, and also phosphorylates multiple proteins that are involved in regulating different stages of the cell cycle. We are using mouse models to investigate roles for Aurora B in regulating multiple cellular functions. These include regulating protein ubiquitination during embryonic stem cell differentiation, the functioning of the polycomb proteins in positive and negative regulation of gene expression, and the activity of transcription factors and co-factors that control B lymphocyte activation. We use the study of cell cycle kinase functions in gene regulation as a paradigm for understanding how regulation of the cell cycle has been co-opted during evolution to integrate cell proliferation with developmental processes and to control developmental transitions. We are also investigating how dysregulation of these processes contributes to tumourigenesis and cancer progression.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: Leucocyte and Pathogen Trafficking through Lymphatics in Inflammation and Immunity
Funder: Medical Research Council

Abstract: Goals To investigate mechanisms of lymphatic trafficking in inflammation, immunity and cancer. In addition to maintaining fluid balance and tissue homeostasis the lymphatics also provide a conduit for trafficking of antigen presenting cells to lymph nodes for activation of nave T cells, and in so doing they allow a route for tumour metastasis. Recent work has focused on understanding the key molecular mechanisms regulating entry of leukocytes and tumour cells to lymphatics and their subsequent trafficking to lymph nodes. We have discovered a separate new integrin and chemokine dependent pathway for translymphatic migration of dendritic cells (DC) that functions in inflammation (1) and demonstrated its importance for dermal antigen delivery during T cell responses - identifying a new potential target for intervention (2). We have focused on two lectin-like receptors of the Link superfamily, LYVE-1 and CD44. LYVE-1 is the major HA receptor of lymphatic endothelial cells and was originally cloned and characterized in my laboratory (3). We have shown that in addition to mediation of lymphatic trafficking of DC, LYVE-1 also plays a role in lymphatic invasion in tumour metastasis and in the resolution of inflammation in a model of acute lung injury. We have identified tumour lymphatic endotherlial cell markers (TLEM) and have shown these to be important in nodal metastasis in a range of murine and human cancers (4). The crystal structure and activation mechanism of CD44, an inflammatory leukocyte HA receptor closely related to LYVE-1 and which plays a pivotal role in transmigration of activated leukocytes across inflamed vascular endothelium have been determined (5). Future research plans Future work will concentrate on the definition of the lymphatic entry points and molecular mechanisms for transmigration of different leukocyte populations. The use of microscopic imaging and structure/function analyses will allow us to determine how receptors such as LYVE-1 mediate cell trafficking in animal models of inflammation. Moreover, we will pursue the 3D structure of the LYVE-1 ectodomain and elucidate the mechanisms regulating it's interaction with hyaluronan, search for additional LYVE-1 ligands mediating leukocyte trafficking, define downstream signalling pathways mediating transendothelial migration and investigate function blocking antibodies of potential therapeutic value for inflammation and cancer. References: (1) Johnson et al. 2006 J Exp Med 203: 2763; (2) Teoh et al. 2009 J Immunol 182: 2425; (3) Gale et al 2007 Mol Cell Biol 27: 595; (4) Clasper et al. 2008 Cancer Res 68:7293; (5) Banerji et al. 2007 Nature Struct Mol Biol 14:234


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: A multi-disciplinary approach to understanding the immunological basis and potential prevention of graft versus host disease.
Funder: Medical Research Council

Abstract: Our programme seeks to understand the regulation of immune function in the early post-transplant period in order to separate the Graft versus leukaemia response (GVL) and Graft versus Host disease (GVHD). To achieve this, our Programme is based on 3 main areas 1) immune reconstitution during the period of very early immune reconstitution, (2) Regulation of migration of antigen-specific T cells to target tissues during GVHD and (3) development of chemokine blockade as a mechanism to prevent GVHD. We have shown that functional donor lymphocytes are detectable within the first two weeks after transplant and that kinetics of recovery predict clinical outcome. Here we seek to extend studies of VEIR to specifically monitor development of the alloimmune response utilising flow FISH for detection of donor T cells, multimer technology and High throughput sequencing. These techniques will allow us to identify both GVL and GVHD responses, and to assess the ability of these developing cells to migrate towards, and kill patient tissue. The patient microenvironment will be key to T cell homing during immune reconstitution and therefore we will monitor changes in chemotactic cytokines during VEIR. A major part of this section of work will be to study the influence of the local tissue microenvironment on the development of T-cell responses incorporating a novel skin blister model, tissue explants and fluorescent microscopy. As GVHD is a tissue specific disease, this will be key to understanding disease pathogenesis. We have shown that chemokines guide T cells towards the tissues in GVHD. Here we will combine our studies of VEIR with our migration work to predict which patients will develop disease, and in which tissues. Once established we will develop pre clinical assays to block T cell migration to classical GVHD sites, using both neutralizing antibodies and siRNA. Development of these represents an opportunity to prevent GVHD without abrogating GVL.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: Dissecting the impact of L-selectin on T lymphocyte dependent tumour immunity
Funder: Medical Research Council

Abstract: T lymphocyte infiltration of tumours correlates with improved outcome in patients. However, tumours actively suppress T cell recruitment by inhibiting the expression of homing associated cell adhesion molecules and chemokines on blood vessels in draining lymph nodes and within the tumour. The overall effect is to limit priming of T cells to tumour derived antigens in lymph nodes draining the tumour. Even if T cells are primed, recruitment into the tumour will be restricted because tumour blood vessels fail to support T lymphocyte homing. Strategies that boost the localisation of effector T cells capable of tumour destruction within the tumour may improve the efficacy of immunotherapies to control tumour growth. Several studies link the expression of the leucocyte specific homing molecule L-selectin/CD62L and tumour growth but the underlying mechanisms are unknown. Since L-selectin is an essential lymph node homing molecule, the role of L-selectin in regulating tumour immunity may be to facilitate T cell priming and differentiation in response to tumour derived antigens in draining lymph nodes. However, studies using L-selectin knockout mice have shown that reduced control of tumour growth in these mice is not simply due to lack of cytotoxic T lymphocyte (CTL) generation but correlated with a markedly reduced recruitment of leucocyes into the tumour. This highlights an important role for L-selectin in regulating leucocyte recruitment into tumours. We have found that the rapid growth of primary tumours in L-selectin deficient mice is reduced by maintaining expression of L-selectin on T lymphocytes. These data demonstrate that T cell expressed L-selectin controls tumour immunity and reveal a novel way of promoting tumour immunity by altering expression of homing molecules on T cells. The aim of this proposal is to study the properties of these L-selectin enhanced T lymphocytes and to determine whether they could be used to treat tumours.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: Integrating innovative technologies for genotyping and phenotyping in stratified medicine
Funder: Medical Research Council

Abstract: Stratified medicine is applied to group patients based on specific disease phenotype, mechanism of onset or progression and response to treatment; these mechanisms are specific to individual patients and means a personalised approach to management throughout a patient journey is required to maximise patient survival and minimise healthcare costs. To develop novel stratified medicine approaches, we will integrate multiple clinical and molecular datasets for diagnosis and management of disease, with the aim of delivering a system of monitoring and treatment which is tailored towards the individual patient. These type of data integration must (i) inform research into disease causation and development, (ii) contribute to patient management and (iii) provide insights towards novel therapeutics. We are proposing to build upon the unique integrated clinical and research setting within Birmingham by creating a West Midlands Stratified Medicine Innovation & Translation Facility through major infrastructure investment, aligned to an ambitious research programme which will enhance our mechanistic knowledge as well as diagnostic and clinical capabilities. Research will particularly focus on immune-mediated inflammatory diseases and blood cancers, which are disciplines of great local strength. This will not only build on local investments such as the £24m Institute for Translational Medicine (co-funded by government, along with our NHS partners University Hospitals Birmingham) and the new £4.1m Centre for Computational Biology, but also concurrent national investments in new technology such as CyTOF cell sorting and the recent MRC-NIHR National Phenome Centre, all of whom we are collaborating with to enable a step-change in national capabilities in stratified medicine research.


Research activity codes: 4.5 RESOURCES AND INFRASTRUCTURE (DETECTION)
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: The ACKR2-CCR2 axis in development and disease.
Funder: Medical Research Council

Abstract: Our in vivo approaches depend on a number of mouse strains all but one of which we currently have. We propose to generate a novel mouse strain in which ACKR2 expression is driven by tetracycline in a cell-specific manner. These mice will be important for a number of aspects of the proposed study and will be produced by Taconic who have extensive expertise in this area. Finally, our plans to identify therapeutically relevant antagonists of ACKR2 function will benefit from access to a number of chemical libraries. We have an agreement with MRC-T for access to their compound library. In addition, through an agreement between the University of Glasgow and AstraZeneca (AZ), we have access to AZ chemical libraries and, importantly, to a library sub-selected for molecules able to interact with chemokine receptors. The existing agreement between the University and AZ allows us to screen these libraries with no commitment, other than first right of refusal, to AZ.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.2 CELLULAR AND GENE THERAPIES
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: Single Cell-Level Functional Proteomics and Genomics exemplified in Cancer and Immunology
Funder: Medical Research Council

Abstract: We propose to create a state-of-the-art facility for single cell functional analysis, combining the latest developments in cell sorting with microfluidics-based cellomics, optical proteomics and single cell expression and genomics analysis. The expertise and methodologies brought together by the consortium create an opportunity to share technological benefits in an overlapping set of systems. The approach is innovative in being highly enabling for research across multiple clinical arenas; we will be operative in relation to multiple source materials (tissue, blood, culture) and levels of abundance. We will exploit the power of mass cytometry (CyTOF already in place) to maximize pre-sort capability, with the bonus of higher purity and data focusing. The modular design presents a challenge in terms of ensuring that platforms can be linked; but offers the considerable advantage of not being locked into a single commercial technology platform that runs the risk of scientific and budgetary constraint. The strategy thus increases our research possibilities whilst reducing the overall risk. The system will be developed in collaboration with industry, and exemplified via cohesive research programmes in immune-mediated and infectious disease, regenerative medicine and cancer. These provide distinct challenges but have significant cross-talk, making them ideal for exploring the full potential of the facility. For example, cancer growth and dissemination are characterised by immune evasion and tolerance to self/neo-antigens, whereas autoimmunity is distinguished by failure of self-antigenic tolerance, and the transfer of heterologous and autologous cells in regenerative medicine can engender responses that oppose tolerance induction. Through the synergistic development of new technology platforms that analyse complex cellular processes in cancer, inflammation, autoimmunity and alloimmunity on one site, we aim to resolve complex disease processes at the single cell level.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: Vascular endothelium as the amplifying machinery of oncogene-induced senescence surveillance
Funder: Medical Research Council

Abstract: Oncogene-induced senescence (OIS) is a tumour suppressor mechanism. Critical to this function, senescent hepatocytes, non-autonomously drive their own immune-mediated destruction through the inflammatory senescence-associated secretory phenotype (SASP). Liver sinusoidal endothelial cells (LSEC) are the gatekeepers for immunocyte recruitment into the liver and are known to utilise unique recruitment mechanisms. Our preliminary data suggest that OIS cells could regulate NF-kB activity and inflammatory cytokine expression in LSEC, suggesting a SASP amplification loop. Currently we do not understand: 1) how senescent cells recruit immunocytes and whether this involves the endothelium; 2) how senescent hepatocytes affect LSEC behaviour and immunocyte recruitment; 3) which immune cells are critical for immune-mediated clearance; 4) can we target LSEC biology to promote the immune-mediated clearance of pre-cancer? Our research questions are: 1) Do LSECs underpin SASP amplification in the liver, immune surveillance of senescence and prevent tumorigenesis? 2) Are these effects dependent on LSEC NF-kB activity? We plan to: 1) completely describe the LSEC and immunocyte response to in vivo OIS-hepatocytes through transcriptional profiling of senescent hepatocytes and LSEC and deep immunophenotyping of the liver, utilising mass cytometry; 2) model senescence-endothelial cell interactions in vitro to describe and manipulate key senescence-inducible pathways in LSEC, underpinning secretion and immunocyte recruitment; 3) Develop an inducible endothelial-specific NF-kB-knockout mouse in combination with RAS-senescence of hepatocytes to study the dependence of immunocyte recruitment and tumour suppression on LSEC NF-kB activity; we will validate our findings in healthy and diseased human liver tissue. This will allow us to understand the basis of immunity to the earliest forms of cancer, why it fails and how to promote immune-mediated clearance in cancer-prone human tissues.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM
Title: The effect of the bone marrow microenvironment upon the function of anti-leukaemia T cells.
Funder: Medical Research Council

Abstract: Although adoptive transfer of T cells engineered to express chimeric antigen receptors can successfully induce remissions in patients with acute lymphoblastic leukaemia (ALL), many patients fail to respond or respond only transiently. Recent work from the host laboratory has shown that T cell persistence and function in the bone marrow are dependent upon access to the homeostatic cytokine, IL-15, through its trans-presentation on the surface of IL-15 receptor alpha-expressing cells. In this proposal, I am seeking: (1) to determine which cells in the bone marrow microenvironment are essential for CAR T cell function, and (2) to define how prior treatment or leukaemia growth affects the T cell microenvironment in the bone marrow. To identify putative IL-15 receptor alpha+ cell population(s) that are required for CAR T cell function and survival, I will use a combination of flow cytometric, imaging and genetic approaches. Using established models of ALL, I will seek to understand how leukaemia growth and/or treatment affects the bone marrow microenvironment, specifically those cells or elements that support CAR T cell function. Knowledge regarding how the bone marrow microenvironment supports CAR T cell function and persistence could be used to improve anti-leukaemia T cell therapies, either through manipulating the bone marrow microenvironment or through designing CAR T cells that can substitute for microenvironmental factors damaged as a result of leukaemia growth or treatment.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM