Sir Peter MacCallum Department of Oncology - Theses
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ItemUnderstanding and manipulating epigenetics in cancer( 2019)The appropriate regulation of gene expression programs is essential for normal cellular function. In cancer, mutations derail normal developmental gene expression programs resulting in a malignant epigenetic state. Traditionally, therapies have attempted to kill cancer cells by targeting rapid proliferation or by directly disrupting the mutated protein. More recent therapies, such as BET inhibitors, attempt to directly target the oncogenic epigenetic state. All of these therapeutic approaches are frequently hampered by the emergence of drug resistance. In many cases, resistance is acquired through genetic changes. However, there is increasing evidence that drug resistance can also arise through non-genetic/epigenetic mechanisms. This has major implications for cancer treatment, as the processes that drive genetic and non-genetic resistance are completely different and epigenetic changes, unlike genetic changes, are potentially reversible. Our laboratory previously generated a model of non-genetic resistance to BET inhibitors in acute myeloid leukaemia (AML). Resistance coincided with the acquisition of a less differentiated stem cell like phenotype and was stable upon drug withdrawal. To discover targets that can overcome the resistant epigenetic state, I performed a focused CRISPR-Cas9 screen, which identified the enhancer regulator, LSD1, as key to maintaining the resistance phenotype. Treatment with an LSD1 inhibitor overcame resistance through time-dependent reprogramming of the resistant cells. This epigenetic reprogramming resulted in both differentiation and the formation of new enhancers around critical BET inhibitor target genes. Through functional genomics experiments, I demonstrated that re-sensitization of the resistant cells was driven by the new enhancer formation, rather than differentiation. Mechanistically, the enhancer remodeling is caused by upregulation of Irf8, which together with the pioneer factor, Pu.1, initiates the formation of new enhancers around BET inhibitor target genes, leading to a restored BET inhibitor transcriptional response. Motivated by the poor clinical responses and rapid acquisition of resistance to BET inhibitors in AML, I also sought to develop an assay that could identify new therapeutic targets that may be more effective. Transcription factors (TFs) are ideal targets, however they are difficult to disrupt directly. To circumvent this, I developed an assay that can identify what cofactors (which are often druggable) are required to drive the transcriptional activity of a given TF. This assay works by combining the Gal4 transactivation system with CRISPR-Cas9 screening. Preliminary screens using the transcription factors, VP64 and MYB, were able to identify previously validated cofactors for each TF. VP64 was dependent on MED25 and MYB was dependent on P300. A number of other potential specific cofactors were identified and will be validated in future work. This assay not only provides the potential to identify novel therapeutic targets, but will also provide insight into the poorly characterized interface between TFs and cofactors.
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ItemDefining signalling pathways that control the response of endothelium to cancer therapy( 2018)Targeting non-transformed stromal components of the tumour microenvironment (TME) has become clinically attractive in treating cancer over the last few decades. On this basis, vascular endothelial growth factor A (VEGFA) inhibitors which suppress blood vessel sprouting (angiogenesis) by blocking VEGFA signalling have been developed and integrated into modern cancer treatment regimens. However, tumour response to VEGFA inhibitors is highly complex and variable. In addition to cancer cells, the TME is composed of various stromal cell types that play an important role in modifying the tumour response by, for example, supporting the development of resistance to VEGFA blockade. Moreover, the indiscriminate large-scale application of VEGFA inhibitors (with or without chemotherapeutic agents) in clinical oncology, resulting in overall modest patient benefit and the inevitable occurrence of resistance, has underscored a pressing need for rational use of these expensive agents. To address the challenges in deciphering the role of each TME component and thus the mechanisms of resistance, this Thesis focused on the main cellular target of VEGFA inhibitors — human microvascular blood endothelial cells (ECs). To identify molecular modifiers of the EC response to VEGFA inhibitors (in this Thesis bevacizumab, a humanised anti-VEGFA neutralising monoclonal antibody, was used), a pooled genetic screening platform was developed. This involved a three-dimensional microcarrier-based culture system, CRISPR–Cas9-driven genetic loss-of-function (LOF) and VEGFA-dependent serum-free culture conditions for applying selective pressure. A pooled kinome-wide CRISPR–Cas9-based screen identified 18 candidate genes that upon LOF were significantly enriched or depleted in the bevacizumab versus control treatment arm. Candidate evaluation using small interfering RNA (siRNA) validated ACTR2, BRD2, BRD3, BRD4, TAOK1 and TRRAP LOF as mediators of EC resistance to bevacizumab; TLK1 and TLK2 LOF as sensitisers of ECs to bevacizumab. Further analysis of the most significant validated candidate genes BRD2, BRD3 and BRD4 (encoding members of the bromodomain and extraterminal domain (BET) family of proteins) using the BET bromodomain inhibitors (BETi) JQ1 and I-BET762 reproduced the effect of siRNA-mediated knockdown of BRD2, BRD3, or BRD4 on the EC response to bevacizumab. Markedly, a survival- and/or proliferation-inhibiting effect of BETi was observed regardless of the presence of bevacizumab. However, this inhibitory effect was unexpectedly attenuated when cells were co-treated with bevacizumab under VEGFA-dependent culture conditions. These results collectively indicated an interaction between BETi and bevacizumab. Investigation of the mechanistic basis for such interaction using RNA sequencing suggested a role for epigenetic regulation of chromosomal activity in modifying the EC response to co-treatment with BETi and bevacizumab. With development and application of a minimally biased and systematic screening approach, this Thesis identified and validated novel molecular modifiers of the EC response to bevacizumab. A previously unreported interaction between BET protein activity and VEGFA signalling in the context of bevacizumab treatment in ECs was revealed. Importantly, these observations will prompt further investigation of the role of epigenetic regulation in vascular biology, tumour angiogenesis and response to cancer therapy. These findings could facilitate clinical development of predictive and/or response biomarkers and strategies to overcome therapeutic resistance, ultimately enabling the rational use of VEGFA 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.
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ItemBET bromodomain inhibition as combined apoptotic and immunomodulatory therapy for the treatment of MYC-driven lymphoma( 2017)Bromodomain and Extra-Terminal (BET) proteins are a conserved family of ‘epigenetic readers’ that bind to acetylated lysine residues on histone and non-histone proteins to modulate transcription. BET proteins are enriched at promoter and enhancer regions and recruit the positive transcription elongation factor b (P-TEFb) complex to activate RNA polymerase II. Anti-tumour responses elicited by BET inhibitors have been associated with the suppression of genes required for cellular proliferation and survival, including oncogenic transcription factors. Suppression of the proto-oncogene MYC was initially reported as a key mechanistic property of BET inhibitors, however more recent evidence suggests that additional target genes are mechanistically implicated. In this thesis, the Eμ-Myc model of aggressive B-cell lymphoma was utilized to investigate the full repertoire of genes modulated by JQ1 and their functional significance in mediating therapeutic responses. JQ1 did not suppress the expression of transgenic Myc in this model, allowing the determinants of apoptosis induction to be assessed, independently of changes in Myc expression. This apoptotic response was p53-independent and associated with modulation in the ratio of pro- and antiapoptotic Bcl-2 family members to favor activation of the intrinsic mitochondrial apoptotic pathway. Therapeutic administration of JQ1 to mice bearing Eμ-Myc lymphomas led to robust clinical responses, however, universal treatment failure was observed despite ongoing therapy. Using RNA-Seq, disease progression and secondary JQ1 resistance was found to be associated with RAS pathway activation and Bcl-2 upregulation. In addition, the efficacy of JQ1 was found to be dependent on an intact host immune system, where a 50% reduction in the survival advantage was observed upon transplantation into immune-deficient mice. Using RNA-Seq, the immune checkpoint ligand Cd274 (Pd-l1) was found to be potently suppressed by JQ1. Mechanistically, BET inhibition decreased Brd4 occupancy at the Cd274 promoter, leading to promoter-proximal pausing of RNA polymerase II, and loss of Cd274 mRNA production. Rapid epigenetic remodeling of the Cd274 locus in response to interferon gamma (IFN-γ) stimulation led to recruitment of Irf1, Brd4, RNA polymerase II, as well as increased local histone acetylation. Accordingly, BET inhibition suppressed constitutive and IFN-γ-induced PD-L1 expression in genetically diverse tumour models. Ectopic expression of PD-L1 in Eμ-Myc lymphomas was sufficient to reduce the efficacy of JQ1, demonstrating the significance of PD-L1 suppression to the observed therapeutic responses associated with BET inhibition. Finally, treatment of mice bearing Eμ-Myc lymphomas with JQ1 in combination with a checkpoint inhibitor (anti-PD-1) or immune stimulating antibody (anti-4-1BB/CD137) led to improved therapeutic responses. The results presented herein demonstrate the importance of MYC-independent apoptotic signaling to therapeutic responses associated with BET inhibition, as well as acquired drug resistance. In addition, these results demonstrated the ability of BET inhibitors to directly engage the host immune response during anti-cancer therapy. Finally, BET inhibitors can suppress oncogenic PD-L1 transcription for therapeutic gain, leading to augmented anti-tumour immunity. These studies establish a strong rationale for clinical investigation of BET inhibitors in combination with immune modulating therapies.
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ItemEvading the storm: BET inhibitor resistance and the leukaemia stem cell( 2017)Bromodomain and Extra Terminal protein (BET) inhibitors are first-in-class, epigenetic targeted therapies that deliver a new therapeutic paradigm by directly targeting protein-protein interactions at chromatin. Early clinical trials have shown significant promise, particularly in acute myeloid leukaemia (AML), suggesting that these compounds are likely to form an important component of future anti-cancer regimes. However, therapeutic resistance is an inevitable consequence of most cancer therapies, therefore the evaluation of resistance mechanisms is of utmost importance in order to optimise the clinical utility of this novel class of drugs. This work utilises primary murine hematopoietic stem and progenitor cells (HSPC) immortalised with MLL-AF9 to generate several clonal cell lines demonstrating robust resistance, in vitro and in vivo to the prototypical BET inhibitor, I-BET151. Resistant clones harbour cross-resistance to the chemically distinct BET inhibitor JQ1, as well as resistance to genetic knockdown of BET proteins. Moreover, resistance is stably maintained across subsequent cell generations in the absence of ongoing selective pressure. Immunophenotypic immaturity is identified in resistant clones and, through functional limiting dilution assays, resistance is definitively demonstrated to emerge from leukaemia stem cells (LSCs). This finding is further confirmed using an independently generated in vivo model of resistance and in patient derived xenograft (PDX) models of human leukaemia. The underlying mechanism of resistance is identified through examination of the transcriptome and chromatin interface utilising high throughput sequencing techniques. Consistent with the adoption of alternative transcriptional pathways, expression of key target oncogenes such as Myc remain unaltered in resistant clones despite the global loss of chromatin-bound Brd4. Alternatively, increased Wnt/β-catenin signalling in human and mouse leukaemia cells is demonstrated to account, in part, for resistance to BET inhibitors and functions to maintain expression of malignant oncogenes. Negative regulation of this pathway restores sensitivity to I-BET151 in vitro and in vivo and highlights a potential rational combination therapy strategy to circumvent or prevent the development of BET inhibitor resistance. The emergence of BET inhibitor resistance from a LSC population prompted further exploration of rational combination therapies with sound mechanistic basis. LSD1 inhibitors have demonstrated pre-clinical promise in the treatment of AML underpinned by the induction of differentiation of leukaemia cells and the loss of LSC capacity. Using the derived model BET inhibitor resistance, LSD1 inhibitors are demonstrated to function synergistically with BET inhibitors and restore sensitivity to BET inhibitor resistant clones. Induction of differentiation is demonstrated in immunophenotypic and transcriptome assays and suggest a further potential combination therapy approach to enhance the clinical utility of both drugs. Collectively, these findings give insight into the basic biology of AML, demonstrate the role of epigenetically mediated intratumoural heterogeneity and transcriptional plasticity in the evasion of targeted therapies and provide a base from which further investigation of the LSC can occur to identify vulnerabilities which may be exploited for therapeutic gain.