Exploring resistance mechanisms to SWI/SNF inhibition in acute myeloid leukaemia

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Acute myeloid leukaemia (AML) is a genetically heterogeneous malignancy characterized by a differentiation block and uncontrolled expansion of myeloid progenitor cells. Due to its poor overall survival and high rate of incidence, the demand for AML therapies that target disease pathology are increasing. Due to the low mutational burden of AML, it is likely that epigenetic factors are mediating leukaemogenesis, and epigenetic drugs targeting synthetic lethalities have shown great promise in the treatment of this cancer. A key complex with significant roles in AML pathogenesis is the SWI/SNF chromatin remodeling complex, the mutations of which are paradoxically protective in AML, while having opposing effects in other cancers. The SWI/SNF complex is a multisubunit complex consisting of 10-15 distinct subunits with unique functions contributing to the maintenance of chromatin architecture and the regulation of promoter and enhancer regions. These complexes bind enhancer regions through modified histone tails, and recruit crucial transcription factors that induce specific gene regulatory networks. Many subunits of these complexes have been trialed in potential cancer therapeutics, however, the low efficacy of corresponding drugs may impede clinical translation. Recently, a small molecule SWI/SNF inhibitor of the BRG1/BRM central ATPase subunits has been developed (BRM014), that has shown significant promise in reducing AML pathogenesis significantly in vitro and in vivo through differentiation and apoptosis induction. However, like with many other drugs, resistance to this small molecule inhibitor has been noted in AML cell lines, such as Fujioka and THP1 cells. These resistance mechanisms are likely to abolish the viability of clinical translation, or reduce the efficacy of this potentially relevant cancer drug. Therefore, this project aims to understand the epigenetic basis of resistance against BRG1/BRM ATPase inhibition. Through the utilization of CRISPR screening and epigenetic techniques, we aim to delineate gene regulatory networks and TFs that can be targeted by future co-therapies in AML. Initially, this project aims to identify genes and cellular pathways that may render cells resistant to inhibitors. This involves cytotoxicity assays to identify AML cell lines with differential sensitivity to BRM014. An unbiased, targeted CRISPR screening approach will be used to identify genes and cellular pathways that synergize with the activity of BRG1/BRM inhibitors and identify pathways that may render cells resistant to these inhibitors. Significant genes will be subjected to further analysis, including validation through CRISPR-Cas9-based knockout and assessment of differential BRM014 responses. Candidate genes will be analysed through ChIP-seq and RIME, to identify genomic binding sites and genes directly regulated by SWI/SNF in the context of BRM014 resistance. Candidate genes will also be validated in patient samples to delineate the feasibility of clinical translation of this research. Enhancer regulation in BRM014 resistance will be analysed through ATAC-seq and acetylation ChIP-seq, which will highlight how the pathogenic AML chromatin landscape and 3D chromatin architecture changes in response to BRM014 in resistant and sensitive cells. Additional validation will be performed with a dTAG degrader and RNA-seq, to identify how SWI/SNF chromatin binding dynamics changes in differing treatment conditions over time. These experiments aim to deepen our understanding of fundamental SWI/SNF complex functioning in cancer, and how genomic binding is altered in the context of drug resistance. In this project, we are anticipating the discovery of gene targets that interact with SWI/SNF complexes with a synthetically lethal manner. This may allow the future development of dual inhibitors targeting SWI/SNF and synthetically lethal interactive partners that drive BRM014 resistance, in order to improve patient outcomes.