Medical Biology - Theses
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ItemDeveloping Preventative and Therapeutic Strategies Against HTLV-1 Using a Novel Humanised Mouse Model( 2022)Satisfactory preventative or therapeutic drugs are lacking for human T cell leukaemia virus 1 (HTLV-1), a disease which lags several decades behind its distantly related cousin HIV in this regard. Consequentially, 5-10 percent of the approximately 10 million people infected with HTLV-1 will progress to serious complications. These include fatalities resulting from a rapidly progressive blood cancer, adult T cell leukaemia/lymphoma, and inflammation of the spinal cord, HTLV-1-assocaited myelitis. Other clinically recognised disease associations include inflammatory diseases involving the eyes, lungs, and skin. In Central Australia, a region in which the adult prevalence of HTLV-1c approaches 40% in some remote Aboriginal communities, HTLV-1 subtype c (HTLV-1c) infection is strongly associated with chronic pulmonary disease and death due to bronchiectasis and is a significant health burden in these communities. Although HTLV-1 subtype a (HTLV-1a) is also associated with pulmonary disease, the extraordinarily high burden of pulmonary disease in Central Australia suggests that there may differences between HTLV-1a and HTLV-1c, which are the two most divergent subtypes. Novel preventative and therapeutic interventions are required to tackle HTLV-1. I investigated the efficacy of antiretroviral and pro-apoptotic BH3 mimetic compounds as preventative and therapeutic agents in a novel humanised mouse model of HTLV-1c infection, the first of its kind. I characterised infection in this model and compared disease to the globally prevalent HTLV-1a. Disease was indistinguishable in mice suggesting that HTLV-1 subtype does not drive distinct disease associations, including HTLV-1 associated pulmonary disease. Tenofovir, a reverse iv transcriptase inhibitor, significantly reduced HTLV-1 transmission in vivo at clinically relevant doses and attenuated de novo viral spread and disease progression during early infection in combination with dolutegravir, an integrase inhibitor. HTLV-1 infection was associated with dysregulation of the intrinsic apoptotic pathway at the transcriptional level, and pharmacological inhibition of MCL-1, but not BCL-2, BCL-xL or BCL-w, killed HTLV-1-infected cells ex vivo and mitigated disease progression in vivo in combination with tenofovir and dolutegravir. Collectively, these data provide evidence that combination antiretroviral and MCL1 antagonism may represent an effective, clinically relevant, curative strategy against HTLV-1. Routine clinical use of these compounds will facilitate their rapid translation to HTLV-1 treatment.
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ItemIdentifying Novel Strategies to Enhance the Anti-cancer Activity of Venetoclax by Manipulating NOXA Expression( 2021)Apoptosis is a form of programmed cell death. The intrinsic pathway of apoptosis is governed by the BCL2 family proteins. Targeting BCL2 proteins by small molecules that mimicking the BH3-only proteins to induce apoptosis has proven to be a successful strategy for cancer therapy. Venetoclax, a specific inhibitor of BCL2, has exhibited remarkable efficacy in treating cancers that rely on BCL2 for survival. However, the activity of venetoclax is often limited in other cancers whose survival relies on MCL1, another BCL2 family member. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials. However, the clinical development of these agents has been hampered by toxicity, especially cardiac toxicity. Potentially, another strategy to target MCL1 is by modulating NOXA, a BH3-only protein that selectively binds to MCL1 and mediates its degradation. I hypothesised that increased NOXA expression would prime cancer cells to venetoclax killing and that this would reduce their co-dependence on MCL1. In order to identify new targets to modulate NOXA expression, I generated and validated cell lines that report on NOXA transcription and then carried out CRISPR-Cas9 genetic screens in those NOXA reporter cell lines. In CRISPR-Cas9 loss-of-function screens focused on epigenetic regulators, I found several genes whose mutation or loss modulated NOXA expression, including CTBP1, CHTOP, ZMYM3, SPEN, HSPA1A, KEAP1, FOXA1, HDAC3 and SAP30. Some of these factors have been targeted for cancer therapies, for example KEAP1 and HDAC3, while the others have not yet been recognized for their therapeutic possibilities. Subject to their validation, my results have identified interesting novel mechanisms of NOXA regulation, thus providing the rationale basis for the development of new anti-cancer agents. In CRISPR-Cas9 tiling screens that focused on the NOXA promoter region, five cis-regulatory elements were identified that contributed to regulation of NOXA expression. Among them, a hypermethylated element on the NOXA promoter was found to be important for repressing NOXA expression across diverse cell lines derived from blood cancers. Disrupting this region led to NOXA induction. Potentially, the findings could provide a rational basis of combining hypomethylating agents with venetoclax in a range of haematological malignancies. In summary, several potential NOXA regulating proteins and DNA elements were discovered by the CRISPR-Cas9 screening approaches. Once validated, these findings should provide new insights into NOXA regulation.
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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.