Sir Peter MacCallum Department of Oncology - Theses
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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.
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ItemNovel combination therapies targeting Pol I and Pol II transcription to treat AML( 2017)Our laboratory has demonstrated that a small molecule (CX-5461, Senhwa Biosciences; currently in phase I clinical trial for haematological malignancies) that inhibits ribosomal RNA gene (rDNA) transcription by RNA Polymerase I (Pol I), is highly efficacious in various cancer types in preclinical studies. Although CX-5461 has entered the clinic and its therapeutic effects in preclinical study are promising, it is still unclear: 1) what mechanisms confer sensitivity or resistance to Pol I therapy, 2) which patients will benefit the most from CX-5461 and 3) what combination therapies will improve the response rates and survival. The goal of my thesis is to begin to address these questions, by focusing on an aggressive acute myeloid leukaemia (AML), a refractory disease associated with low survival rates and high risk of relapse. We have demonstrated that in AML, CX-5461 acts in part to elicit a nucleolar specific DNA damage response (DDR) via ATM and ATR signaling, resulting in the arrest of leukemia cells in S and G2/M phases (Hein et al, 2017). Importantly, pharmacologic inhibitors targeting DDR were shown to sensitise cancer cells to CX-5461 treatment (Quin et al, 2017). It has been demonstrated that mechanistic interactions underlying the DDR rely on context-specific chromatin structure, including chromatin’s structural rearrangement by histone chaperones and the bromodomain and extra-terminal (BET) proteins. Due to the specialised transcriptional context of rDNA, we hypothesise that targeting epigenetic and transcriptional regulators (eg., bromodomain inhibitors) in combination with CX-5461 may render cancer cells highly sensitive to ATM/ATR activation and result in enhanced anti-tumour activity. Here we used IBET-151 (GSK) to block BET protein binding to acetylated chromatin marks in combination with CX-5461 in a disease model of AML. We observed strong synergy between CX-5461 and IBET-151 across a human AML cell line panel. Importantly, co-treatment with CX-5461 and IBET-151 significantly improved survival over single agents in a preclinical model of aggressive MLL-AF9; NrasG12D as well as AML1-ETO9a; NrasG12D -driven AML. Treatment of AML cell lines with IBET-151 or the knockdown of its target, BRD4, resulted in increased rDNA accessibility to micrococcal nuclease (MNase). We propose that inhibition of BRD4 by IBET-151 increases the ‘open’ state frequency of rDNA chromatin, leading to an enhanced CX-5461-mediated DDR. This model is supported by synergistic checkpoint activation, including a heightened γH2AX response following combined CX-5461 and IBET-151 treatment. Together, our studies highlight the potential therapeutic value of targeting multiple rDNA transcriptional mechanisms for treating patients with AML.