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.