Sir Peter MacCallum Department of Oncology - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemTherapeutically targeting RNA Polymerase II transcription in leukaemia( 2021)RNA Polymerase II (RNAPII) transcription is a discontinuous and unidirectional process, control of which is necessary for normal gene expression and cellular functioning. Regulation occurs at discrete checkpoints throughout transcription and is in part mediated by Cyclin Dependent Kinases (CDKs). This includes CDK7, where it phosphorylates RNAPII among other transcriptional substrates to enable transcriptional initiation and coordination of co-transcriptional processes, such as splicing. However, the genome-wide role of CDK7 in nascent RNA synthesis and splicing, in addition to whether its function is negatively regulated, has not been robustly established. The loss of RNAPII transcription control invariably leads to disease, including cancer. Mutations in cancer can affect several proteins involved in regulating RNAPII and engenders a reliance on sustained functioning of the core RNAPII machinery for continued malignant cell survival and proliferation. This dependence is termed ‘transcription addiction’ and is the basis for targeting RNAPII core components for therapeutic benefit. Small molecule perturbation of transcription in these cancers results in selective gene expression changes attributed to the target gene’s specific chromatin context, which includes the occupancy of oncogenic transcription factors (TFs) and association with genomic elements such as super enhancers (SEs). Whether selective transcriptional changes are also influenced by RNA stability however, is largely unexplored. In this thesis, we demonstrated that RNA decay rates largely determined gene expression changes to clinically relevant transcriptional and epigenetic compounds. Transcripts that were the most down-regulated on the total RNA level had both short half-lives and high production rates and often encoded TFs, including the proto-oncogene c- MYC. Moreover, genetically engineering the three prime untranslated region (3’UTR) of c-MYC to prolong its RNA half-life rendered c-MYC transcripts resistant to transcriptional targeting on the total RNA level. Therefore, these finding highlight an under-appreciated role of RNA stability in gene expression changes to therapeutic RNAPII perturbation in cancer. Among the most promising therapeutic transcriptional targets is CDK7, and inhibitors targeting this protein are currently clinical trials and have been expedited for Food and Drug Administration (FDA) approval. Proteomic and transcriptomic characterization of the CDK7 inhibitor, YKL-5-124, in the THP-1 Acute Myeloid Leukaemia (AML) cell line revealed that it perturbed the phosphorylation of several proteins involved in cell cycling, transcription and co-transcriptional processes, namely splicing. This was paralleled with a global decrease in de novo RNA synthesis, most prominently towards the 5’ ends of genes, and total RNA levels. In addition, CDK7 inhibition increased alternative isoform expression, most strikingly related to exon skipping and intron retention. On a phenotypic level, these changes were also associated with a decrease in THP-1 cell proliferation. Taken together, these findings improve our understanding of the significance of CDK7 kinase activity in RNAPII transcription and splicing. To identify factors that functionally antagonize CDK7 and may confer resistance to sustained CDK7 inhibition, CRISPR-Cas9 knockout positive enrichment screening in THP-1 cells was performed using YKL-5-124 treatment as a selective pressure. The screen showed that knockout of histone acetyltransferase (HAT) and core structural components of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex mediated resistance to CDK7 inhibition. Further investigation into the resistance mechanism revealed that cells knocked out for the SAGA HAT subunit, TADA2B, were less sensitive to nascent RNA down-regulation with YKL-5-124. Moreover, this was concomitant with maintained H2BK120 mono-ubiquitin levels across gene bodies genome-wide. Therefore, these data highlight SAGA complex components as novel negative regulators of CDK7 and how cancers might overcome the effect CDK7 inhibitors in the clinic. Overall, the results presented herein improve our understanding of CDK7 in RNAPII transcription and the importance of RNA decay in defining the anti-cancer effects of therapeutic perturbation of the core RNAPII machinery.
-
ItemCyclin E1 as a therapeutic target in high grade serous ovarian cancer( 2017)The central theme of this thesis is developing therapeutic strategies to selectively target CCNE1 amplified high grade serous ovarian cancer (HGSC). Patients with CCNE1 amplified HGSC represent a key unmet clinical need given that they are associated with primary treatment resistance and poor clinical outcome. Novel therapeutic strategies are urgently required in order to provide these patients with additional treatment options. Using short interfering RNA and short hairpin RNA, I demonstrated selective sensitivity of CCNE1 amplified HGSC to CDK2 gene suppression. However, I did not demonstrate similar amplicon dependent sensitivity to dinaciclib, a potent small molecule inhibitor of multiple CDKs. In order to identify drug combinations that would synergise with dinaciclib, I performed a high throughput compound screen in CCNE1 amplified HGSC cell lines. I identified a combination of dinaciclib and MK-2206, an AKT inhibitor, that was selectively synergistic in in vitro and in vivo models of CCNE1 amplified HGSC. CCNE1 and AKT2 were noted to be co-amplified in primary HGSC samples, and a number of genes in the AKT pathway were found to be required in CCNE1 amplified HGSC cell lines. Furthermore, over-expression of cyclin E1 and AKT isoforms resulted in uncontrolled growth characteristics in TP53-mutant fallopian tube secretory cells, the proposed cell of origin for HGSC. Taken together, these findings suggest that co-operative interaction between CCNE1 and the AKT pathway in HGSC may be exploited therapeutically. I also explored the potential mechanisms of resistance to CDK inhibitors by generating cell lines resistant to dinaciclib. Dinaciclib in combination with multiple BH3-mimetic compounds was noted to be synergistic in CDK-inhibitor resistant cell lines. Upregulation of multiple anti-apoptotic genes was observed in resistant cell lines compared to parental sensitive cell lines, suggesting that this is a potential mechanism of resistance to CDK inhibitors. Targeting homologous recombination (HR) may also be a therapeutic option in CCNE1 amplified HGSC. Proteasome inhibitors such as bortezomib have been shown to be indirect inhibitors of HR, and I showed that CCNE1 amplified cell lines were highly sensitive to bortezomib and MLN9708, a second generation proteasome inhibitor. Potential synergistic combinations with bortezomib were identified in a high throughput compound screen, including a number of HDAC inhibitors, suggesting a possible class effect.