Medical Biology - Theses
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ItemControl of the Intrinsic Pathway of Apoptosis( 2019)Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.
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ItemAdvancing BH3 mimetics to treat cancers( 2019)The evasion of apoptosis, one of the hallmarks of cancer, is observed in many cancers. This can also impair the efficacy of many conventional chemotherapies. The BCL2 protein family is the central regulator of the intrinsic apoptotic pathway and plays a vital role during tumor development. In particular, the levels of the pro survival family members are often elevated in some cancers. Venetoclax, a BH3 mimetic inhibitor that mimics the BH3-only proteins, natural inhibitors of the pro-survival BCL2 proteins, has proven to be effective for treating hematological cancers by selectively targeting BCL2. This has translated into regulatory approvals of venetoclax for treating a subset of chronic lymphocytic leukemia and acute myeloid leukemia. In addition to targeting BCL2, potent and specific BH3 mimetic inhibitors of its relatives, BCLxL and MCL1, are now also available. However, their full clinical utility is poorly defined. This thesis focuses on advancing the utility of the BH3 mimetic compounds as anti-cancer agents. Previous studies have suggested roles for BCLxL and MCL1 in many solid cancers (e.g. colon, breast, lung). In particular, colorectal cancers have elevated levels of the pro survival protein, one usually associated with chemo resistance. Furthermore, colorectal cancer patients with advanced disease or those who carry poor prognostic markers do not respond well to the current stand of care therapies such as surgery and adjuvant chemo/radiotherapy. Given the pressing need to find better treatments for these patients, we first utilized a panel of validated BH3 mimetics to assess the feasibility of using them for treating colorectal cancer. By using cancer cell lines and patient-derived organoids, we identified and validated BCLxL and MCL1 as the most important survival factors for colorectal cancer. We then validated them as potential targets by pharmacological inhibition in a mouse model in vivo. Moreover, we found that even those tumors that harbor poor prognostic factors respond as avidly as those do not, further highlighting the potential of this approach for treating patients with colorectal cancer. Even though the targeting BCLxL might be a possible approach to kill cancers that depend on it, the clinical use of BCLxL selective inhibitors is limited due to the toxicity of BCLxL inhibition on platelets. I have screened for novel regulators of BCLxL using the CRISPR/Cas9 technology, which might offer potential approaches to target BCLxL safely. The final goal of this thesis is to identify biomarkers that predict response to BH3 mimetics, given that there are few reliable tools to stratify patients that might respond well to these novel anti-cancer agents. By using large scale transcriptomic datasets from publicly available RNA sequencing studies, I was able to identify a few candidate genes and achieve reasonable prediction performance.
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ItemMechanisms of angiogenesis in development and disease( 2019)The expansion of blood vessel networks by angiogenesis is critical for matching blood supply to the metabolic demands of growing tissues. In adult tissues, blood vessels and the endothelial cells (ECs) that make them generally remain in a quiescent state. However, the reactivation of angiogenesis is a hallmark of a number of diseases including cancer and vision-threatening eye diseases. In the case of eye disease, angiogenesis results in abnormal vascular growth that damages the retina. This abnormal angiogenesis often occurs in response to retinal hypoxia that can arise as a result of excessive retinal vessel regression. Understanding the mechanisms by which blood vessels both grow and regress is therefore important for understanding how vascular diseases arise in the eye. In this thesis, I have used in vivo mouse models of normal and pathological angiogenesis in the retina to examine the molecular and cellular mechanisms that control blood vessel growth and regression. In the first part of my thesis, I investigated the role of the histone acetyltransferase HBO1 in retinal blood vessel growth. Through its histone acetyltransferase activity, HBO1 promotes transcriptionally permissive chromatin and is necessary for cells to change their gene expression patterns, particularly during development. I found that HBO1 was required for the expression of genes and pathways that are upregulated by ECs undergoing angiogenesis. ECs lacking HBO1 failed to undergo directed migration during angiogenesis, resulting in reduced blood vessel production both in the normal retina and in a model of pathological retinal vessel growth. In the second part of this thesis, I examined the role of EC apoptosis in disease-causing blood vessel regression using a model of ischaemic retinopathy. In this model, vessel regression associated with EC apoptosis results in retinal ischaemia, causing a hypoxic response that drives abnormal vessel growth similar to that seen in human eye diseases. I found that blocking EC apoptosis did not prevent vessel regression or the onset of retinal ischaemia. Nonetheless, it completely prevented the loss of ECs from ischaemic areas of the retina. These preserved ECs were capable of rapidly reassembling into a functional vessel network that reduced hypoxia and the subsequent pathological vascular response. Vessel reassembly was not impeded by neutralising VEGFA, suggesting that it is not dependent on high levels of VEGFA produced by the ischaemic retina. Overall, my studies provide new insight into the mechanisms of blood vessel growth and regression and may potentially open new therapeutic avenues for preventing abnormal blood vessel growth in retinal vascular disease.
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ItemDefining programs of cell death that can be harnessed to impact on outcomes of chronic viral infection( 2019)Pathogens causing chronic infections have successfully evolved mechanisms to subvert host immunity. Excessive and inappropriate inflammation together with attrition of repeatedly overstimulated high affinity T cells leads to abrogated immunity and persistence of pathogens such as HIV and HBV. T cell exhaustion has been touted as a prelude to T cell deletion during these infections, however, studies indicate that high affinity T cell clones are deleted at the onset of infection. The T cells that remain have lower affinity for pathogen epitopes and hence their response is weaker and more easily antagonised by inhibitory networks, including T-regulatory (Treg) cells. The killing of immune effector cells during chronic overwhelming infections is juxtaposed to the pathogen’s attempts to promote survival of infected target cells. Keeping infected cells alive is imperative for the maintenance of a microbial replicative niche. In this body of work, I dissected the role of host cell molecules and how they contribute to the death and survival of immune and infected cells. Necroptosis did not contribute to the loss of highly functional virus-specific CD8+ T cells during the course of infection. In contrast, when I interfered with death receptor signalling there was a modest rescue of functional CD8+ T cells. This gain in immune function, however, did not translate to improved viral control. The same mechanism I used to promote the survival of T cells made infected target cells refractory to death receptor mediated killing and therefore, offset any gain in immune function. Whilst examining the role of necroptosis in chronic infection, I made the discovery that the necroptotic inducer molecule, RIPK3, has additional non-necroptotic roles. Ripk3-/- mice cleared LCMV with enhanced kinetics compared to wild-type mice and mice that lacked the necroptotic executioner MLKL. I found that in the absence of RIPK3, chronically infected mice had impaired IFNβ responses. Excessive and prolonged IFNβ production is known to impair immunity. This may partially explain why mice lacking RIPK3 had enhanced numbers of granzyme B expressing T cells and controlled infection better than WT animals. The host-viral dynamics that favour displacement of highly functional cells with poorly activated cells makes the immune system highly vulnerable to inhibition through the activity of Treg cells. I next investigated the role of Treg cells in immune dysfunction during chronic infections and I was particularly interested in the cell death and cell survival pathways that contributed to the turnover and accumulation of these cells. I utilised mice with a Treg-specific deletion of Casp8. These mice had twice as many Treg cells as wild-type mice at steady state. Surprisingly, when these mice were infected with chronic LCMV, only 25% of the animals survived to 145 days post infection. Moribund animals succumbed to overt T cell activation and autoimmunity due to a precipitous drop in Treg cell numbers. Survivors, intriguingly, eliminated LCMV in most organs consistent with a massive gain in immune function. The death of the Treg cells was due to necroptosis. When I ablated the necroptotic pathway, through the deletion of Mlkl, I completely prevented the loss of Treg cells and the fatal immune pathology in Treg conditional caspase-8 deficient mice. I found that differential expression of RIPK3 and MLKL in Treg cells made them highly susceptible to necroptosis during chronic infection compared to Tconv cells. This was also the case for human Tregs and I was able to preferentially kill these cells, over Tconv cells, in vitro by driving necroptosis with a clinical stage caspase-8 antagonist called emricasan. Necroptosis is a lytic form of cell death that promotes inflammation and it has been implicated in chronic liver disease. I initially investigated if necroptosis in the liver contributed to the control of chronic LCMV, HBV or the malaria parasite Plasmodium berghei. Ablation of necroptosis had no impact on liver-pathogen dynamics and no impact on general liver function and architecture. In many cell types caspase-8 inhibits death receptor induced necroptosis. So, I reasoned that this molecule must be inhibiting induction of necroptosis in the liver of infected animals. I examined this by infecting mice that had a conditional loss of caspase-8 within hepatocytes. Despite abundant, infection driven, death ligands I observed no necroptosis in the liver. Even drug induced ablation of NF-ĸb survival signalling, downstream of TNF, failed to promote liver necroptosis in the aforementioned scenarios. The liver’s inability to undergo necroptosis was confirmed in mice with a human chimeric liver. I showed this refractoriness was due to liver repression of RIPK3 in humans and mice. The work conducted in this thesis provides important insights into the cell death pathways that are engaged in diverse cell types during chronic viral infections and I provide evidence that antagonising them therapeutically may lead to better clinical outcomes.
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ItemMolecular mechanisms of thymic tolerance induction and recovery from involution( 2019)Thymic epithelial cells (TECs) are vital for the formation of the thymic microenvironment and the differentiation of T cells. This process involves a series of interactions between TECs and thymocytes that govern T cell commitment, progenitor T cell proliferation and differentiation and TCR specificity-based selection of immature T cells to finally allow the release of mature T cells capable of mounting an immune response against foreign antigens (e.g. from pathogens), whilst remaining tolerant of self. Although the role of TECs in mediating these processes is well established, major gaps in our understanding of the molecular mechanisms involved in TEC development, maintenance and function remain. In this thesis we seek to address this knowledge gap and define: (a) the molecular mechanisms underlying TEC survival and death during injury; and (b) the molecular mechanisms that control the expression of peripheral tissue self-antigens (PTAs) in TEC which is required to mediate self-tolerance. Atrophy of the thymus following irradiation or high-dose chemotherapy impairs immune recovery in patients, which is a significant cause of morbidity and mortality. Despite the importance of TECs for immunity, there is little understanding of the mechanisms that control TEC survival and death in the context of treatments that damage the immune system. By using different genetically modified mouse models targeting the intrinsic pathway of apoptosis in both TECs and thymocytes, we found that: (a) TECs die via the intrinsic apoptotic pathway after irradiation; (b) the pro-survival proteins BCL-2 and BCL-XL are crucial for TEC regeneration; and (c) blocking thymocyte death can partially rescue TECs following irradiation. These data provide new insights into the molecular control of TEC survival and regeneration that could be used to inform new treatments that maintain or restore thymic function in immunosuppressed patients. A unique property of TECs is their capacity to express thousands of PTAs to mediate immune tolerance of non-lymphoid self-antigens. The autoimmune regulator, AIRE, is required for the transcription of the majority of these PTAs, and defects in AIRE’s function lead to autoimmune disease in mice and humans. The precise molecular mechanisms by which AIRE orchestrates such broad, tolerogenic transcription of PTAs in TECs remain only partially understood. We here show that the acetyltransferase, KAT7, which mediates the majority of histone 3 lysine 14 acetylation (H3K14ac), is essential for AIRE-mediated expression of PTAs, the establishment of a normal thymic microenvironment and self-tolerance. This study reveals an important role for histone acetylation in AIRE’s function in TEC and immunological tolerance. In conclusion, these studies highlight essential molecular mechanisms underlying TEC survival, regeneration and function that are crucial for immune function and tolerance. This newly gained knowledge will further aid the design of treatment strategies for immune recovery after cytoablative and cancer treatment as well as autoimmune diseases.
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ItemThe role of HECTD1 and MCL-1 in the regulation of normal and malignant haematopoiesis( 2019)Cellular processes important for haematopoiesis are frequently perturbed in malignant cells. Accordingly, healthy immature progenitor cells and malignant cancer cells often share certain properties, e.g. rapid proliferation and self-renewal. Deciphering these developmental pathways can provide information about the critical drivers involved in neoplastic transformation and sustained cancer cell growth. Results in this thesis addresses the role of two proteins, HECTD1 and MCL-1, in haematopoiesis and haematological malignancies. HECTD1 is an E3 ubiquitin ligase required for mouse embryonic development, as homozygous loss of Hectd1 leads to embryonic lethality. HECTD1 is widely expressed in diverse tissues, including haematopoietic cells. However, its role in adult tissues in vivo has not been described. Therefore, we generated mice in which HECTD1 deletion was restricted to the haematopoietic system of adult mice. Analysis of these mice at steady state revealed small perturbations in certain T cell subsets. However, competitive reconstitution experiments revealed that HECTD1 deletion affects the haematopoietic stem and progenitor cell (HSPC) populations. Serial transplantation assays showed that loss of HECTD1 results in a defect in the self-renewal properties of mouse HSPCs. Interestingly, RNA sequencing of Hectd1-/- HSPCs revealed that HECTD1-deficiency led to increased expression of interferon regulated genes, suggesting that HECTD1 plays a critical role in the maintenance of HSPC populations by negatively regulating the interferon signalling pathway. Additionally, I employed the MLL AF9 mouse model of acute myeloid leukaemia and showed that HECTD1-deficiency significantly delayed the latency of tumour development in vivo compared to control mice. MCL-1 is a pro-survival regulator of the intrinsic apoptosis pathway. MCL-1 expression is integral to the survival of many different blood cell types, and to the development and sustained growth of many haematological malignancies. Recently a highly specific MCL-1 inhibitor, S63845, showing 6-fold higher affinity to human MCL-1 compared to mouse MCL-1 was described. To accurately test the efficacy and tolerability of S63845 in preclinical models of disease, we developed a humanised Mcl 1 (huMcl-1) mouse strain in which the genomic region of the murine Mcl-1 locus was replaced with the coding regions for human MCL-1. These mice are phenotypically indistinguishable from wild-type mice, and the intrinsic apoptotic pathway remains intact in their cells. However, as anticipated, huMcl-1 mice were more sensitive to S63845 than wild-type mice. To test whether malignant cells from the humanised MCL-1 mice also show higher sensitivity to S63845, we generated Eµ-Myc lymphomas on a huMcl-1 background. Lymphoma cell lines derived from huMcl-1;Eµ-Myc mice were ~6 times more sensitive to S63845 in vitro compared to Eµ-Myc lymphoma cells expressing mouse MCL-1. Transplantation of huMcl-1;Eµ-Myc lymphoma cells into huMcl-1 mice and treatment with S63845 resulted in tumour-free survival in >60% of mice. Furthermore, combining low doses of S63845 with sub-optimal doses of cyclophosphamide led to almost complete tumour regression. These results show that our huMcl-1 mouse model represents a valuable preclinical tool to test MCL-1 inhibitors, either alone or in combination with other anti-cancer agents, for a broad range of cancers, allowing accurate prediction of efficacy against tumour cells and on target toxicity to normal tissues.