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dc.contributor.authorTyler, Dean Sydney
dc.description.abstractA prominent theme in malignant transformation is abnormal regulation of epigenetic processes that facilitate gene expression profiles underpinning malignant initiation and progression. The plasticity of the epigenome lends itself to therapeutic intervention within these settings. Novel small molecules designed to target members of the BET protein family (BRD4 BRD3, and BRD2) represent effective application of this premise and promising pre-clinical data have formed the basis for early phase clinical trials. However, molecular mechanisms of BET inhibition are yet to be fully characterized and may be driven by a range of cellular mechanisms and transcriptional processes presently undefined. In order to derive the most clinical benefit from these novel therapies, an urgent need exists to develop tools to better understand and clarify the molecular and cellular mechanism as well as pharmacokinetic properties potentiating the pre-clinical efficacy of these small molecule drugs. The development of methodologies to better characterize drug mechanisms by tracking activities of targeted cancer therapies within a cellular context is an exciting and essential area of current research. My thesis aims to improve the assessment of drug mechanisms by developing novel complementary strategies amenable to Click Chemistry that overcomes the need to radically modify small molecule drugs in order to utilize them as molecular probes in vitro and in vivo. Application of Click-proteomics and Click-sequencing provide new mechanistic insights into gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. This study utilizes Click-sequencing to directly assess levels of BET inhibitor contained across genomic transcriptional units. Analysis of Click-sequencing data reveals BET inhibitor responsive genes to correlate with relative levels of drug-chromatin occupancy and proposes alternate binding modes of BRD4 at responsive or unresponsive genes. Furthermore, it is shown by co-administration of BET inhibitors in cellular assays, preferential binding to one domain of BRD4 is exhibited, whilst another domain remains engaged at the genomic interface. Assessment of BET inhibitors using Click Chemistry technique are not restricted to studies at the genomic level, the versatility of the techniques developed also permits these small molecules to be assessed whole-organism wide. In mouse models of acute leukaemia, high resolution microscopy and flow cytometry are employed to highlight an underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. Differential distributions and effects of the drug in normal and malignant cells in vivo are also demonstrated. Such observations are not only pre-clinically relevant to drug development processes but may be used to better understand clinically relevant pharmacokinetic parameters. Together these data provide critical insights into the cellular and molecular environment that governs the efficacy and limitations of these agents. This study also provides an innovative framework for the pre-clinical assessment of other conventional and targeted therapies that may be used to limit uncertainties during pre-clinical drug development and increase the probability of success within the clinical drug development process.en_US
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dc.titleClick chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapiesen_US
dc.typePhD thesisen_US
melbourne.affiliation.departmentSir Peter MacCallum Department of Oncology
melbourne.affiliation.facultyMedicine, Dentistry & Health Sciences
melbourne.thesis.supervisornameDawson, Mark
melbourne.contributor.authorTyler, Dean Sydney
melbourne.accessrights This item is embargoed and will be available on 2021-03-08. This item is currently available to University of Melbourne staff and students only, login required.

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