Defining the Roles of BRCA2 and RAD51 in PARPi Response

定义 BRCA2 和 RAD51 在 PARPi 反应中的作用

• 批准号: 10640159
• 负责人: Ryan Brown Jensen
• 金额: $ 37.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-07 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640159
• 关键词: Address; Agreement; Alleles; BRCA1 gene; BRCA2 Mutation; BRCA2 gene; Binding; Biochemical; Biological; Biological Assay; Bypass; Cancer Patient; Cell Death; Cell model; Cells; Cellular Assay; Clinical; Clinical Management; Complex; Coupled; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Structure; DNA replication fork; Data; Development; Drug Design; EMSA; Event; Excision; Exhibits; FDA approved; Fiber; Filament; Gene Mutation; Germ-Line Mutation; Goals; Health; Human; In Vitro; Individual; Invaded; Knowledge; Laboratories; Lesion; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Mission; Modeling; Molecular; Mutation; Nucleoproteins; Pathogenicity; Patients; Play; Poly(ADP-ribose) Polymerase Inhibitor; Positioning Attribute; Process; Proteins; Public Health; Reaction; Relapse; Research; Resistance; Role; Single-Stranded DNA; System; TRAP Complex; Techniques; Testing; Therapeutic; Toxic effect; United States National Institutes of Health; Work; brca gene; cancer cell; cancer therapy; combat; cytotoxicity; drug sensitivity; gene repair; genome integrity; improved; inhibitor therapy; innovation; insight; mutant; novel; novel strategies; novel therapeutics; nuclease; patient response; protein purification; reconstitution; recruit; relapse patients; repair function; repaired; resistance mechanism; response; single molecule; single-molecule FRET; skills; superresolution microscopy; targeted treatment; therapy resistant; tool; treatment strategy; tumor

PROJECT SUMMARY PARP inhibitors (PARPi) hold tremendous therapeutic potential because of their selectivity for cells lacking functional BRCA1, BRCA2, and other homology-directed repair (HDR) genes. However, as with other targeted therapies, resistance to PARPi frequently arises, underscoring the unmet need to elucidate how PARPi cause cell death in BRCA mutant but not normal cells. Individual PARPi may act through distinct mechanisms, either by “trapping” PARP-DNA complexes, or by inhibiting repair of single-stranded (ssDNA) nicks that are subsequently converted to double-stranded breaks (DSBs). Moreover, patients may exhibit differential drug sensitivity depending on the specific causative BRCA gene mutation. Defining this fundamental landscape will be critical to better predict responders/non-responders as well as the durability of patient response to PARPi. Historically, a detailed, mechanistic study of how mutations in BRCA2 influence genome integrity has been hampered by the immense challenge of manipulating and purifying this large protein. Recently, we have overcome these challenges, allowing us to leverage a combination of in vitro biochemical assays and cellular assays to pinpoint how individual pathogenic or targeted mutations influence specific functionalities including: DNA binding, replication fork protection, RAD51 nucleoprotein filament formation, and RAD51-mediated DNA strand invasion. In addition to applying these techniques to interrogate the explicit biochemical function(s) compromised by pathogenic BRCA2 mutations, we will assess sensitivity to PARPi with strong, intermediate, or weak trapping activity (e.g. Talazoparib, Olaparib, and Veliparib, respectively). Lastly, we will investigate the function(s) reconstituted by “reversion” mutations identified in patients with PARPi-resistant tumors, which may independently identify functional attributes necessary for PARPi sensitivity. Our long-term goal is to unveil the molecular consequences of PARPi treatment that necessitate processing by BRCA2, RAD51, and other HDR proteins. Our central hypothesis is that by elucidating how BRCA2 and RAD51 mechanistically overcome PARPi-mediated toxicity, we will provide the necessary framework to understand how PARPi resistance can develop in patients. Our hypothesis is based on compelling preliminary data illustrating the specific functions of BRCA2 and RAD51 in response to PARPi. Thus, our rationale, to reveal the mechanism(s) that underlie PARPi-mediated toxicity, will vertically advance knowledge surrounding the HDR response to PARPi, and ultimately, improve clinical management of BRCA patients. In aim 1, we will utilize patient derived BRCA2 reversion alleles in our isogenic human cell models to interrogate what specific function(s) have been “reactivated” to promote resistance to PARPi. In aim 2, we will determine how BRCA2 and RAD51 catalyze the removal or bypass of PARPi trapped lesions using purified proteins and relevant model DNA substrates (reversed forks, gaps) in reconstituted biochemical assays. Our approach is innovative because of our unique skill set and development of robust cell-based and biochemical functional assays to dissect HDR mechanisms focused on BRCA2 and RAD51. Our objective in the current work will be to apply our HDR expertise to solve a long-standing mystery in the PARPi field: to reveal how HDR proficient cells effectively survive treatment. The results are anticipated to have a positive impact on the clinical management of HDR deficient tumors as therapeutic resistance and relapse are critical barriers to the successful treatment of patients.

项目概要 PARP 抑制剂 (PARPi) 具有巨大的治疗潜力，因为它们对缺乏 PARP 的细胞具有选择性。 功能性 BRCA1、BRCA2 和其他同源定向修复 (HDR) 基因。然而，与其他目标一样 随着治疗的进展，对 PARPi 的耐药性经常出现，这凸显了阐明 PARPi 如何引起的需求尚未得到满足 BRCA 突变体细胞死亡，但正常细胞不死亡。各个 PARPi 可能通过不同的机制起作用，或者 通过“捕获”PARP-DNA 复合物，或通过抑制单链 (ssDNA) 切口的修复 随后转化为双链断裂（DSB）。此外，患者可能会表现出不同的药物 敏感性取决于特定的致病 BRCA 基因突变。定义这一基本景观将 对于更好地预测有反应者/无反应者以及患者对 PARPi 反应的持久性至关重要。 从历史上看，人们已经对 BRCA2 突变如何影响基因组完整性进行了详细的机制研究。 由于操纵和纯化这种大蛋白质的巨大挑战而受到阻碍。最近，我们有 克服这些挑战，使我们能够利用体外生化分析和细胞分析的组合 查明单个致病突变或靶向突变如何影响特定功能的检测，包括： DNA 结合、复制叉保护、RAD51 核蛋白丝形成和 RAD51 介导的 DNA 股入侵。除了应用这些技术来询问明确的生化功能之外 受到致病性 BRCA2 突变的影响，我们将评估对 PARPi 的敏感性，包括强、中、 或弱捕获活性（例如分别为 Talazoparib、Olaparib 和 Veliparib）。最后，我们将调查 通过在 PARPi 耐药肿瘤患者中发现的“回复”突变重建功能，这可能 独立识别 PARPi 敏感性所需的功能属性。我们的长期目标是推出 PARPi 治疗的分子后果需要 BRCA2、RAD51 和其他 HDR 进行处理 蛋白质。我们的中心假设是，通过阐明 BRCA2 和 RAD51 如何机械地克服 PARPi 介导的毒性，我们将提供必要的框架来了解 PARPi 耐药性如何 在患者身上发展。我们的假设基于说明具体功能的令人信服的初步数据 BRCA2 和 RAD51 对 PARPi 的反应。因此，我们的理由是揭示背后的机制 PARPi 介导的毒性，将垂直推进有关 PARPi 的 HDR 反应的知识，以及 最终，改善 BRCA 患者的临床管理。在目标 1 中，我们将利用患者衍生的 BRCA2 我们的等基因人类细胞模型中的逆转等位基因，以询问哪些特定功能 “重新激活”以促进对 PARPi 的抵抗。在目标 2 中，我们将确定 BRCA2 和 RAD51 如何催化 使用纯化蛋白和相关模型 DNA 底物去除或绕过 PARPi 捕获的病变 （反向叉子、间隙）在重组生化测定中。我们的方法是创新的，因为我们独特的 技能组合和开发强大的基于细胞和生化功能分析来剖析 HDR 机制 重点关注 BRCA2 和 RAD51。我们当前工作的目标是应用我们的 HDR 专业知识来解决 PARPi 领域长期存在的谜团：揭示 HDR 熟练细胞如何有效地在治疗中存活下来。这 预计结果将对 HDR 缺陷肿瘤的临床治疗产生积极影响 治疗耐药和复发是患者成功治疗的关键障碍。