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

急性髓系白血病（AML）是一种遗传异质性恶性肿瘤，其特征是髓系祖细胞分化阻滞和不受控制的扩增。由于AML的总生存率低，发病率高，因此对针对疾病病理的AML治疗的需求正在增加。由于AML的低突变负担，很可能是表观遗传因素介导了白血病的发生，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很大的希望。在AML发病机制中起重要作用的关键复合体是SWI/SNF染色质重塑复合体，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复合体是一个多亚基复合体，由10-15个不同的亚基组成，具有独特的功能，有助于维持染色质结构和调节启动子和增强子区域。这些复合物通过修饰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，相应药物的低疗效可能会阻碍临床转化。最近，一种BRG1/BRM中枢atp酶亚基的小分子SWI/SNF抑制剂（BRM014）被开发出来，在体外和体内通过分化和诱导细胞凋亡显著减少AML发病机制。然而，与许多其他药物一样，在AML细胞系（如Fujioka和THP1细胞）中发现了对这种小分子抑制剂的耐药性。这些耐药机制可能会取消临床转译的可行性，或降低这种潜在相关抗癌药物的疗效。因此，本项目旨在了解抗BRG1/BRM atp酶抑制的表观遗传基础。通过利用CRISPR筛选和表观遗传学技术，我们的目标是描述基因调控网络和tf，这些基因调控网络和tf可以作为未来AML联合治疗的靶点。最初，这个项目的目的是确定基因和细胞途径，可能使细胞抵抗抑制剂。这包括细胞毒性试验，以鉴定对BRM014具有不同敏感性的AML细胞系。一种无偏倚的靶向CRISPR筛选方法将用于鉴定与BRG1/BRM抑制剂活性协同的基因和细胞途径，并鉴定可能使细胞对这些抑制剂产生抗性的途径。重要基因将进行进一步分析，包括通过基于crispr - cas9的敲除验证和评估差异BRM014反应。候选基因将通过ChIP-seq和RIME进行分析，以确定在BRM014耐药背景下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，以描述本研究临床翻译的可行性。我们将通过ATAC-seq和乙酰化ChIP-seq分析增强子对BRM014耐药的调控，这将突出在耐药和敏感细胞中，致病性AML染色质景观和3D染色质结构如何响应BRM014而发生变化。进一步的验证将使用dTAG降解器和RNA-seq进行，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点。这可能允许未来开发针对SWI/SNF的双重抑制剂和驱动BRM014耐药的合成致死性相互作用伙伴，以改善患者预后。

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.