Sir Peter MacCallum Department of Oncology - Theses
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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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ItemInvestigating acquired resistance to Pol I transcription inhibitors for the treatment of haematologic malignancies( 2018)Previous work from our group and others has demonstrated that CX-5461 (Senhwa Biosciences), a first-in-class small molecule inhibitor of RNA Polymerase I transcription of the ribosomal RNA genes, is effective at treating a range of different cancers both in vitro and in vivo, and is currently in clinical trials for haematologic and solid tumours. However, despite initial tumour clearance in response to CX-5461 treatment in preclinical murine models of cancer, mice eventually relapse with tumours that are resistant to further CX-5461 treatment. This thesis investigates the mechanisms via which the tumours can develop resistance to CX-5461 treatment and extrapolates this research to better understand: 1) how CX-5461 functions as an anti-tumour agent; 2) which pathways are required to mediate resistance to CX-5461; and 3) how resistance can be overcome with combination therapy. Using DNA exome sequencing, we found that Top2α is frequently mutated in tumours that have acquired resistance to CX-5461 treatment in vivo. Functional characterization of a Top2α mutant cell line demonstrated that Top2α expression and activity were reduced in these cells. Indeed, we found that knockdown of Top2α was sufficient to cause resistance to CX-5461. This implies that Top2α could provide a novel biomarker for CX-5461 response in clinical trials. Further investigation of the CX-5461 resistance mechanism uncovered that CX-5461 also acts as a Top2 inhibitor in addition to its ability to inhibit rDNA transcription. However, unlike common chemotherapeutic Top2 inhibitors which kill cells by causing genome-wide DNA damage thereby initiating a DNA damage response, CX-5461 treatment causes comparatively fewer DNA breaks enriched at the ribosomal DNA promoter loci. Thus, CX-5461 is able to kill tumour cells via the DNA damage response in the absence of extensive DNA damage thereby potentially limiting the cytotoxicity of drug treatment. Together, the work presented in this thesis identifies novel mechanisms of action and resistance to CX-5461. We propose that CX-5461 and other second-generation inhibitors of RNA Polymerase I and Top2α may provide a viable, less genotoxic alternative to classic Top2 inhibitors.