Pathology - Theses
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ItemThe identification and characterisation of novel genes involved in sensitivity to the histone deacetylase inhibitor, vorinostat( 2013)Histone deacetylase inhibitors (HDACi) are a novel class of anti-cancer agents that elicit a range of anti-tumour responses including apoptosis. Vorinostat is an FDA-approved broad-spectrum HDACi, which has achieved remarkable clinical success in some patients, particularly those with Cutaneous T cell lymphoma and Peripheral T cell lymphoma, however it remains unclear why certain patients remain unresponsive. Constitutive STAT activation, overexpression of pro-survival Bcl-2 proteins and loss of HR23B have been identified as potential biomarkers of HDACi resistance, however none have improved the clinical utility of HDACi. Therefore, the study described within aimed to further elucidate vorinostat resistance mechanisms through a functional genomics screen to identify novel genes that when knocked down by RNA interference (RNAi) sensitised cells to vorinostat-induced apoptosis. A synthetic lethal functional screen using a whole-genome RNAi library was used to identify genes that when knocked down co-operated with vorinostat to induce tumour cell apoptosis in otherwise resistant cells. The primary SMARTpool siRNA screen yielded 450 gene hits, of which 106 validated in a secondary ‘deconvolution’ screen using the four individual constituent siRNAs of each SMARTpool. Tertiary screening was conducted to evaluate specificity of these genes to co-operate with vorinostat compared to conventional chemotherapeutics. Thirteen vorinostat-resistance candidate genes were identified, which sensitised specifically to vorinostat in two or more of the four cell lines tested and these were validated in multiple independent assays for apoptosis. For ten of these genes, knockdown was greater than 90% while three appeared to be off-target. Comparative gene expression analysis was undertaken upon knockdown of vorinostat-resistance candidates, however transcriptome analysis did not identify common molecular network(s) through which these genes contributed to vorinostat resistance. Detailed analyses were conducted on two vorinostat-resistance candidates, GLI1 and to a lesser degree, PSMD13. GLI1 is a known oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI small molecule inhibitor, GANT61, phenocopied the effect of GLI1 knockdown. The mechanism by which GLI1 loss of function sensitised tumour cells to vorinostat-induced apoptosis was through interactions with vorinostat to alter gene expression in a manner that favoured apoptosis. Finally, a xenograft model was established to test this combination in vivo. Together, the results presented in this thesis describe the identification and characterisation of ten vorinostat-resistance genes, eight of which had not previously been described as important for HDACi response. These ten vorinostat-resistance candidates may serve as biomarkers for stratification of patients for HDACi treatment. Furthermore, the mechanism of action of one of these candidates, GLI1, was investigated and results herein provide a strong rationale for development of GLI1 inhibitors for clinical use in combination with HDACi therapy.