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

急性髓性白血病（AML）是一种遗传异质性恶性肿瘤，其特征是髓系祖细胞的分化阻滞和不受控制的扩增。由于其较差的总体生存率和高发病率，对靶向疾病病理学的AML疗法的需求正在增加。由于AML的低突变负荷，表观遗传因素很可能介导白血病发生，靶向合成致死性的表观遗传药物在治疗这种癌症方面显示出巨大的前景。在AML发病机制中具有重要作用的关键复合物是SWI/SNF染色质重塑复合物，其突变在AML中具有矛盾的保护作用，而在其他癌症中具有相反的作用。SWI/SNF复合物是由10-15个不同亚基组成的多亚基复合物，其具有独特的功能，有助于维持染色质结构以及调节启动子和增强子区域。这些复合物通过修饰的组蛋白尾部结合增强子区域，并招募诱导特定基因调控网络的关键转录因子。这些复合物的许多亚基已经在潜在的癌症治疗中进行了试验，然而，相应药物的低功效可能会阻碍临床转化。最近，已经开发了BRG 1/BRM中央ATP酶亚基的小分子SWI/SNF抑制剂（BRM 014），其在通过分化和凋亡诱导在体外和体内显著降低AML发病机制方面显示出显著的前景。然而，与许多其他药物一样，AML细胞系（如Fujioka和THP 1细胞）对这种小分子抑制剂具有耐药性。这些耐药机制可能会破坏临床转化的可行性，或降低这种潜在相关癌症药物的疗效。因此，本项目旨在了解对BRG 1/BRM ATP酶抑制的抗性的表观遗传基础。通过利用CRISPR筛选和表观遗传学技术，我们的目标是描绘基因调控网络和TF，这些基因调控网络和TF可以成为AML未来联合治疗的靶点。最初，该项目旨在确定可能使细胞对抑制剂产生抗性的基因和细胞途径。这涉及细胞毒性测定以鉴定对BRM 014具有不同敏感性的AML细胞系。一种无偏见的、有针对性的CRISPR筛选方法将用于鉴定与BRG 1/BRM抑制剂的活性协同作用的基因和细胞途径，并鉴定可能使细胞对这些抑制剂产生耐药性的途径。重要基因将接受进一步分析，包括通过基于CRISPR-Cas9的敲除进行验证和评估差异BRM 014应答。将通过ChIP-seq和RIME分析候选基因，以确定在BRM 014抗性背景下由SWI/SNF直接调控的基因组结合位点和基因。候选基因也将在患者样本中进行验证，以描述本研究临床转化的可行性。BRM 014耐药性中的增强子调控将通过ATAC-seq和乙酰化ChIP-seq进行分析，这将突出显示耐药和敏感细胞中致病性AML染色质景观和3D染色质结构如何响应BRM 014而发生变化。将使用dTAG降解剂和RNA-seq进行额外验证，以确定SWI/SNF染色质结合动力学在不同处理条件下如何随时间变化。这些实验旨在加深我们对SWI/SNF复合物在癌症中的基本功能的理解，以及在耐药性的背景下基因组结合如何改变。在这个项目中，我们期待发现与SWI/SNF复合物以合成致死方式相互作用的基因靶标。这可能允许未来开发针对SWI/SNF和驱动BRM 014抗性的合成致死相互作用伴侣的双重抑制剂，以改善患者结局。

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.