The role of DPF2 stability in SMARCB1-deficient cancers

项目摘要 横纹肌样瘤是儿科肿瘤学中最具侵袭性和致命性的癌症之一， 存活率约为20- 25%。SMARCB 1的缺失是在超过90%的人中发现的主要复发性遗传变异。 例最近的进展表明，SMARCB 1的丢失与许多其他癌症有关， SMARCB 1缺陷型癌症SMARCB 1是BAF染色质重塑复合物的重要组成部分， 通过将核小体定位在基因调控区来控制基因转录的复合物。针对性 BAF复合物在增强子上的定位主要归因于SMARCB 1，但没有结构域被 SMARCB 1上识别的，能够识别和结合特定的组蛋白标记。SMARCB 1只能 与核小体非特异性结合。这就提出了一个关键问题：BAF复合体如何识别 调节区域如增强子和结合。最近的研究表明，SMARCB 1的缺失会耗尽一个细胞， 邻近BAF亚基，DPF 2，其能够识别并结合与增强子相关的组蛋白标记。再 SMARCB 1在SMARCB 1缺陷细胞系中的表达导致DPF 2的稳健积累。组蛋白 阅读器蛋白DPF 2先前已涉及BAF复合物向调节性细胞的募集。 地区通过SMARCB 1的深度突变扫描，我们已经确定了四个错义突变， SMARCB 1似乎模拟致癌功能丧失无义突变。预测这些残基 与DPF 2直接相互作用，这表明SMARCB 1至少部分介导其肿瘤抑制作用 通过与DPF 2的相互作用。此外，我们已经确定了一种新的肿瘤抑制作用的DPF 2在肿瘤细胞中， 在两种SMARCB 1缺陷型癌症细胞系中的组成型过表达的背景下。为此我们 假设在SMARCB 1缺陷型癌症中需要DPF 2的耗尽。在目标1中，我将检验假设 DPF 2与BAF复合物的结合减少， 评估每个提出的SMARCB 1突变体的表型、结构和调节作用。在目标2中，我将 验证DPF 2的稳定化可能是恢复转录的有希望的治疗策略的假设 尽管失去了SMARCB 1。在这一目标，我将表征的表型，结构和监管 在SMARCB 1缺陷细胞系中DPF 2过表达的后果，并确定这是否通过 BAF依赖性或BAF独立性机制。这些目标将提供基本的洞察力 SMARCB 1发挥其肿瘤抑制功能的机制，以及扩大我们的研究范围。 了解SMARCB 1缺陷型癌症中的DPF 2作为潜在的治疗靶点。

PROJECT SUMMARY Rhabdoid tumors are one of the most aggressive and lethal cancers in pediatric oncology with overall 5-year survival rates of ~20-25%. Loss of SMARCB1 is the primary recurrent genetic alteration found in over 90% of cases. Recent advances have implicated SMARCB1 loss in a number of other cancers broadly referred to as SMARCB1-deficient cancers. SMARCB1 is a critical component of the BAF chromatin remodeling complex, a complex which controls gene transcription by positioning nucleosomes at gene regulatory regions. Targeted localization of the BAF complex to enhancers has largely been attributed to SMARCB1, yet no domain has been identified on SMARCB1 that is able to recognize and bind specific histone marks. Instead SMARCB1 is only able to nonspecifically bind to nucleosomes. This raises a key question: how does the BAF complex recognize regulatory regions such as enhancers and bind. Recent work has shown that deletion of SMARCB1 depletes an adjacent BAF subunit, DPF2, which is able to recognize and bind histone marks associated with enhancers. Re- expression of SMARCB1 in a SMARCB1-deficient cell line leads to a robust accumulation of DPF2. The histone reader protein, DPF2, has previously been implicated in the recruitment of the BAF complex to regulatory regions. Through deep mutational scanning of SMARCB1, we have identified four missense mutations in SMARCB1 that seem to mimic an oncogenic loss of function nonsense mutation. These residues are predicted to directly interact with DPF2, which suggests SMARCB1 mediates its tumor suppressor role at least partially through its interaction with DPF2. Moreover, we have identified a novel tumor suppressor role of DPF2 in the context of constitutive overexpression in two SMARCB1-deficient cancers cell lines. For this reason, we hypothesize that depletion of DPF2 is required in SMARCB1-deficient cancers. In Aim 1, I will test the hypothesis that reduced DPF2 association with the BAF complex is driving this observed loss of function phenotype by assessing the phenotypic, structural, and regulatory effects of each proposed SMARCB1 mutant. In Aim 2, I will test the hypothesis that stabilization of DPF2 may be a promising therapeutic strategy to restore transcription normalcy despite the loss of SMARCB1. In this aim I will characterize the phenotypic, structural, and regulatory consequences of DPF2 overexpression in SMARCB1-deficent cell lines and determine if this is occurring through either a BAF-dependent or BAF-independent mechanism. Together these aims will provide fundamental insight into the mechanisms through which SMARCB1 exerts its tumor suppressor function, as well as expand our understanding of DPF2 in SMARCB1-deficient cancers as a potential therapeutic target.