Sir Peter MacCallum Department of Oncology - Theses
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ItemEvading the storm: BET inhibitor resistance and the leukaemia stem cell( 2017)Bromodomain and Extra Terminal protein (BET) inhibitors are first-in-class, epigenetic targeted therapies that deliver a new therapeutic paradigm by directly targeting protein-protein interactions at chromatin. Early clinical trials have shown significant promise, particularly in acute myeloid leukaemia (AML), suggesting that these compounds are likely to form an important component of future anti-cancer regimes. However, therapeutic resistance is an inevitable consequence of most cancer therapies, therefore the evaluation of resistance mechanisms is of utmost importance in order to optimise the clinical utility of this novel class of drugs. This work utilises primary murine hematopoietic stem and progenitor cells (HSPC) immortalised with MLL-AF9 to generate several clonal cell lines demonstrating robust resistance, in vitro and in vivo to the prototypical BET inhibitor, I-BET151. Resistant clones harbour cross-resistance to the chemically distinct BET inhibitor JQ1, as well as resistance to genetic knockdown of BET proteins. Moreover, resistance is stably maintained across subsequent cell generations in the absence of ongoing selective pressure. Immunophenotypic immaturity is identified in resistant clones and, through functional limiting dilution assays, resistance is definitively demonstrated to emerge from leukaemia stem cells (LSCs). This finding is further confirmed using an independently generated in vivo model of resistance and in patient derived xenograft (PDX) models of human leukaemia. The underlying mechanism of resistance is identified through examination of the transcriptome and chromatin interface utilising high throughput sequencing techniques. Consistent with the adoption of alternative transcriptional pathways, expression of key target oncogenes such as Myc remain unaltered in resistant clones despite the global loss of chromatin-bound Brd4. Alternatively, increased Wnt/β-catenin signalling in human and mouse leukaemia cells is demonstrated to account, in part, for resistance to BET inhibitors and functions to maintain expression of malignant oncogenes. Negative regulation of this pathway restores sensitivity to I-BET151 in vitro and in vivo and highlights a potential rational combination therapy strategy to circumvent or prevent the development of BET inhibitor resistance. The emergence of BET inhibitor resistance from a LSC population prompted further exploration of rational combination therapies with sound mechanistic basis. LSD1 inhibitors have demonstrated pre-clinical promise in the treatment of AML underpinned by the induction of differentiation of leukaemia cells and the loss of LSC capacity. Using the derived model BET inhibitor resistance, LSD1 inhibitors are demonstrated to function synergistically with BET inhibitors and restore sensitivity to BET inhibitor resistant clones. Induction of differentiation is demonstrated in immunophenotypic and transcriptome assays and suggest a further potential combination therapy approach to enhance the clinical utility of both drugs. Collectively, these findings give insight into the basic biology of AML, demonstrate the role of epigenetically mediated intratumoural heterogeneity and transcriptional plasticity in the evasion of targeted therapies and provide a base from which further investigation of the LSC can occur to identify vulnerabilities which may be exploited for therapeutic gain.