Sir Peter MacCallum Department of Oncology - Theses
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ItemA Genome-wide RNAi screen identifies combinatorial efficacy of CX-5461 with homologous recombination deficiency and Topoisomerase I inhibition in ovarian cancer( 2019)High-grade serous ovarian cancer (HGSC) is common, with poor prognosis. Limited therapeutic options are available, and the development of new therapies is of high priority. The RNA Polymerase I (Pol I) transcription inhibitor CX-5461 has shown efficacy in both chemotherapy-sensitive and -resistant ovarian cancer through its ability to activate the DNA damage checkpoint. Here, we combine a genome-wide RNAi screening approach with a focussed drug screen to identify potential targets whose inhibition can enhance the efficacy of CX-5461. We demonstrate that CX-5461 combined with knockdown of homologous recombination DNA repair genes shows cooperative cell proliferation inhibition in several HGSC cell lines. We also demonstrate combinatorial efficacy between CX-5461 and topoisomerase 1 (TOP1) depletion or the TOP1 poison Topotecan. The combination induces cell death, cell cycle arrest and senescence even after drug withdrawal. The mechanism of their cooperativity relies on a cell cycle-independent, nucleolar DNA damage response (DDR) associated with topological stress at the ribosomal DNA and is independent of the ability to inhibit PoI I transcription or induce global replication stress. Despite dose-limiting toxicities hampering the broad use of Topotecan in the clinic, combined treatment with CX-5461 and low-dose Topotecan exhibits striking therapeutic efficacy in vivo, thus providing evidence for a novel strategy to treat HGSC.
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ItemNovel combination therapies with the RNA Polymerase I-mediated transcription inhibitor CX-5461 improve efficacy in the treatment of multiple myeloma( 2019)Multiple myeloma (MM) is a malignant plasma cell disorder that is incurable with currently available therapy. The disease is genetically heterogeneous, with many recurrently mutated genes only seen in small numbers of patients and multiple clones present in each patient. This has limited potential approaches for designing widely applicable genetically targeted therapies. rDNA transcription is consistently dysregulated in cancer, mediated through both oncogenic and tumour-suppressive pathways. RNA polymerase I (Pol I) transcriptional hyperactivity is observed in many cancers, with this dysregulation shown to provoke a survival checkpoint in haematological tumour cells. With the hypothesis that the therapeutic targeting of Pol I transcription may prove an effective strategy across a variety of malignant settings, our laboratory co-developed CX-5461; a highly selective small molecule Pol I-mediated transcription inhibitor, now in phase 1 clinical trials in relapsed / refractory malignancies. We have previously demonstrated that single-agent treatment with CX-5461 provides a significant survival benefit in murine models of B-cell lymphoma and acute myeloid leukaemia. However, despite this improvement, drug resistance and relapse eventually occur, indicating combination drug therapy is essential for long term disease control and implementation in the clinic. This thesis examines combination drug strategies in MM, centred on the therapeutic inhibition of Pol I transcription of ribosomal genes, with the aim of accelerating the clinical use of CX-5461 for MM. A boutique, high-throughput screen in human myeloma cell lines (HMCLs) of CX-5461 in combination with drugs having known clinical or promising preclinical efficacy in MM revealed that CX-5461 increases anti-proliferative effects when combined with a range of other agents, encompassing various targets. The histone deacetylase inhibitor panobinostat and the proteasome inhibitor (PI) carfilzomib demonstrated the most impressive synergy in vitro, both representing drug classes that are actively used to treat patients with MM. In vivo testing demonstrated that the combination of CX-5461 with panobinostat increases survival compared with the single agents in both the Vκ*MYC murine model of MM and in C57BL-KaLwRij mice transplanted with 5T33 myeloma cells. Prolonged combination dosing in the Vκ*MYC model did not cause haematological toxicity beyond that seen with single agents. Investigating the molecular synergistic response to CX-5461 in combination with panobinostat indicated multiple potential mechanisms of synergy, including down-regulation of MYC and enhancement of the DDR elicited by CX-5461 alone. To extend the translation of CX-5461 and its combination with panobinostat into the clinic for MM, where resistance to front-line PI treatment frequently develops, we investigated the synergistic relationship of CX-5461 with each of these drug classes. In addition to the screen finding that CX-5461 synergised with each of panobinostat and carfilzomib, we showed the triplet was synergistic in vitro beyond the individual combinations. Moreover, modelling clinical PI resistance, we generated a cell line that is resistant to the front-line PI bortezomib, and demonstrated that CX-5461 retains its impressive efficacy in this setting, both in vitro and in vivo, using the 5T33-C57BL6/KaLwRij model. Taken together, the results described in this thesis will advance subsequent clinical trials utilising both CX-5461 and its combination with panobinostat in the treatment of relapsed multiple myeloma.
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ItemRole of rDNA chromatin in sensitizing OVCA to RNA polymerase I transcription inhibitors( 2019)The ribosomal RNA (rRNA) genes (rDNA) are arrayed in multiple tandem repeats on the 5 acrocentric chromosomes, and are transcribed by RNA Polymerase I (Pol I) which gives rise to the 47S pre-rRNA, the precursor of 18S, 5.8S and 28S rRNAs. rDNA transcription accounts for around 35-60% of all cellular transcription and is a highly energy consuming process as well as a key determinant of cellular growth and proliferation rates. The highly repetitive and actively transcribed nature of rDNA gives rise to a high recombinogenic potential. Further, structural dynamics and variation in the rDNA loci have been reported in over 50% of solid human cancers (Stults et al. 2009). Although the rate of rDNA transcription is likely to be rate limiting in ribosome biogenesis, only a subset of rRNA genes are transcribed from “active” rDNA at any given time. rDNA chromatin exists in active or silent forms. Active rDNA chromatin is “open” and bound by the upstream binding transcription factor (UBF), which is essential for establishing and maintaining active rDNA states (Sanij et al. 2008). We have reported that rDNA silencing increases during terminal differentiation of granulocytes due to decreased UBF levels (Sanij et al. 2015). Conversely, our studies utilizing MYC-driven B-lymphoma mouse model, demonstrated reactivation of silent rDNA as MYC-driven B-cells progress towards malignancy. We have also demonstrated that inhibition of rDNA transcription by the novel Pol I transcription inhibitor CX-5461 can selectively kill MYC-driven B-lymphoma cells in vivo, while sparing wild-type B-cells (Bywater et al. 2012). Although, CX-5461 activates p53-dependent and p53-independent cellular stress response pathways leading to apoptosis, senescence and cell cycle arrest, the mechanisms underlying tumour cell sensitivity to CX-5461 remain unclear. We have undertaken a systematic approach across a panel of ovarian cancer (OVCA) cell lines to examine their sensitivity to CX-5461. We have demonstrated that OVCA cell lines display similar sensitivities to Pol I transcription inhibition, however, they exhibit differential cellular proliferative growth responses, associated with an immediate or a delayed cell cycle delay and arrest. Furthermore, our studies demonstrate that increased ratio of active to inactive active rDNA chromatin and not rDNA transcription rate per se determines the growth inhibitory sensitivity to CX-5461 treatment of OVCA cells. We utilized RNAi based knock-down of UBF to modulate rDNA chromatin as well as ZFN and CRISPR-Cas9 genome editing knock-out (KO) of rDNA repeats approaches to investigate whether rDNA chromatin states and/or rDNA copy number play a role in determining sensitivity to CX-5461. We demonstrated the important role of rDNA in ribosome biogenesis and genome wide instability. Specifically, reducing rDNA copy number can modulate rDNA chromatin states which possibly mediate global genome instability. We propose that rDNA chromatin states could potentially serve as a biomarker to identify OVCA patients that may benefit from CX-5461 treatment.