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The role of DPF2 stability in SMARCB1-deficient cancers

DPF2 稳定性在 SMARCB1 缺陷型癌症中的作用

基本信息

批准号：
10751414
负责人：
GARRETT COOPER
金额：
$ 4.77万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751414
关键词：
ATAC-seq Acute Myelocytic Leukemia Adenocarcinoma Affect Affinity Apoptosis Automobile Driving Binding Biological Assay Cancer cell line Cell Culture Techniques Cell Line Cell Proliferation Chemotherapy and/or radiation Chromatin Chromatin Remodeling Factor Co-Immunoprecipitations Combined Modality Therapy Complex Data Enhancers Epigenetic Process Flow Cytometry Future Gene Expression Regulation Genes Genetic Transcription Glycerol Goals Histones Human Immunoprecipitation In Vitro Knowledge Lead Malignant Childhood Neoplasm Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Mentors Missense Mutation Modeling Molecular Mutation Nonsense Mutation Normalcy Nucleic Acid Regulatory Sequences Nucleosomes Oncogenic Operative Surgical Procedures Patient-Focused Outcomes Patients Pediatric Oncology Phenotype Play Positioning Attribute Prognosis Proteasome Inhibition Proteins Reader Recurrence Regulator Genes Rhabdoid Tumor Role SMARCB1 gene Scientist Sedimentation process Stains Survival Rate Testing Therapeutic Toxic effect Training Transcriptional Regulation Tumor Suppressor Proteins Urea Western Blotting Work beta-Galactosidase genome-wide improved improved outcome in vivo insight loss of function mutant mutation screening new therapeutic target novel overexpression posttranscriptional recruit senescence success targeted treatment therapeutic target transcriptome sequencing treatment and outcome tumor

项目摘要

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.

项目概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，总体 5 年寿命存活率约为 20-25%。 SMARCB1 缺失是 90% 以上的人中发现的主要复发性基因改变案例。最近的进展表明 SMARCB1 缺失与许多其他癌症（广泛称为 SMARCB1 缺陷型癌症。 SMARCB1 是 BAF 染色质重塑复合物的关键组成部分，通过将核小体定位在基因调控区域来控制基因转录的复合物。有针对性 BAF 复合物对增强子的定位很大程度上归因于 SMARCB1，但尚未确定任何结构域在 SMARCB1 上鉴定出能够识别和结合特定组蛋白标记。相反 SMARCB1 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例如增强子和结合。最近的工作表明，SMARCB1 的删除会耗尽相邻的 BAF 亚基 DPF2 能够识别并结合与增强子相关的组蛋白标记。关于- SMARCB1 在 SMARCB1 缺陷细胞系中的表达会导致 DPF2 的大量积累。组蛋白阅读器蛋白 DPF2 之前曾被认为参与了 BAF 复合物向监管机构的招募地区。通过对 SMARCB1 的深度突变扫描，我们发现了 4 个错义突变 SMARCB1 似乎模仿了致癌功能丧失的无义突变。这些残基被预测直接与 DPF2 相互作用，这表明 SMARCB1 至少部分介导其肿瘤抑制作用通过与 DPF2 的相互作用。此外，我们还发现了 DPF2 在两种 SMARCB1 缺陷癌细胞系中组成型过度表达的背景。为此，我们假设 SMARCB1 缺陷的癌症需要消耗 DPF2。在目标 1 中，我将检验假设 DPF2 与 BAF 复合物的关联性降低正在通过以下方式驱动这种观察到的功能丧失表型评估每个提出的 SMARCB1 突变体的表型、结构和调控作用。在目标 2 中，我将检验 DPF2 的稳定性可能是恢复转录的一种有前途的治疗策略的假设尽管 SMARCB1 丢失，但仍恢复正常。为了这个目标，我将描述表型、结构和调控 SMARCB1 缺陷细胞系中 DPF2 过度表达的后果，并确定这是否是通过以下方式发生的依赖于 BAF 或独立于 BAF 的机制。这些目标共同将提供基本的见解深入研究 SMARCB1 发挥肿瘤抑制功能的机制，并扩大我们的研究范围了解 SMARCB1 缺陷癌症中的 DPF2 作为潜在的治疗靶点。