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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ItemThe role of BCL2 family proteins in apoptosis regulation during angiogenesis( 2016)Blood vessels are multicellular tubes, lined with endothelial cells (ECs), that form a hierarchical network essential for the distribution of blood, oxygen, nutrients, hormones and immune cells around the body and removal of metabolic waste products from tissues. Angiogenesis, the growth of new vessels from pre-existing ones, is essential to match the size of the blood vessel network to the metabolic demands of growing tissues. During this process, an over-production of vessels results in the formation of a dense vessel plexus that is inefficient for blood flow. From this dense network, excess vessels undergo a process of selective regression termed ‘pruning’ to produce a mature, hierarchical vessel network. EC apoptosis occurs as part of the angiogenic remodelling processes, but its contribution to angiogenic vessel remodelling, be it vessel pruning or some other purpose, has remained unclear. In this thesis I directly investigated the role of EC apoptosis during angiogenesis by analysing mice in which ECs were unable to execute the apoptotic program regulated by BCL2 family proteins. I found that while EC apoptosis improved the efficiency of selective vessel pruning, it was ultimately dispensable for this process. Instead, blood vessels formed in the absence of EC apoptosis contained excessive numbers of ECs resulting in increased diameter of mature capillaries. Having established that the BCL2 family was essential for promoting EC death during angiogenesis, I investigated whether pro-survival members of the family were required for the survival of ECs during angiogenesis. Using the neonatal retina as a model for angiogenesis, I found that while BCL2 was not required for EC survival during angiogenic vessel growth (Chapter 4), MCL1 was required in a dose-dependent manner (Chapter 5). In contrast to normal angiogenesis, BCL2 and MCL1 were both independently required for the growth of abnormal vascular lesions in a murine model of pathological retina angiogenesis (Chapter 6). These studies have conclusively determined the role for EC apoptosis during angiogenic growth and remodelling and provide evidence that targeting distinct BCL2 family pro-survival proteins may be a useful therapeutic approach for targeting pathological angiogenesis.
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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.