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.

项目摘要 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BRCA 1、BRCA 2和其他同源定向修复（HDR）基因。与其他目标一样， 在治疗中，对PARPi的耐药性经常出现，强调了阐明PARPi如何引起 BRCA突变体中的细胞死亡，而不是正常细胞。单个PARPi可能通过不同的机制起作用， 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 对于更好地预测应答者/无应答者以及患者对PARPi应答的持久性至关重要。 从历史上看，关于BRCA 2突变如何影响基因组完整性的详细机制研究一直是一个复杂的问题。 受到操纵和纯化这种大蛋白质的巨大挑战的阻碍。最近我们 克服这些挑战，使我们能够利用体外生物化学测定和细胞生物学的组合， 用于确定单个致病性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、RAD 51核蛋白丝形成和RAD 51介导的DNA 搁浅入侵除了应用这些技术来询问明确的生化功能外， 由于致病性BRCA 2突变的影响，我们将评估对PARPi的敏感性， 或弱捕获活性（例如分别为Talazoparib、Olaparib和Veliparib）。最后，我们将调查 在PARPi耐药肿瘤患者中发现的“回复”突变重建了功能， 独立识别PARPi灵敏度所需的功能属性。我们的长期目标是揭开 PARPi治疗的分子后果，需要BRCA 2、RAD 51和其他HDR处理 proteins.我们的中心假设是，通过阐明BRCA 2和RAD 51如何在机制上克服 PARPi介导的毒性，我们将提供必要的框架，以了解PARPi抗性如何 在患者中发展。我们的假设是基于令人信服的初步数据，说明了特定的功能 BRCA 2和RAD 51对PARPi的反应。因此，我们的理论基础是揭示 PARPi介导的毒性，将垂直推进围绕PARPi的HDR响应的知识， 最终改善BRCA患者的临床管理。在目标1中，我们将利用患者来源的BRCA 2 逆转等位基因在我们的同基因人类细胞模型，以询问什么特定的功能（S）已经 “重新激活”以促进对PARPi的抗性。在目标2中，我们将确定BRCA 2和RAD 51如何催化 使用纯化的蛋白质和相关的模型DNA底物去除或绕过PARPi捕获的损伤 （反向叉、缺口）。我们的方法是创新的，因为我们独特的 技能集和基于细胞和生物化学功能测定的稳健开发，以剖析HDR机制 BRCA 2和RAD 51。我们目前工作的目标是应用我们的HDR专业知识来解决一个 PARPi领域长期存在的谜团：揭示HDR熟练细胞如何有效地存活治疗。的 预期结果对HDR缺陷肿瘤的临床管理具有积极影响， 治疗抗性和复发是成功治疗患者的关键障碍。