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; 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，因为它们对缺乏细胞的选择性 功能性BRCA1，BRCA2和其他同源指导修复（HDR）基因。但是，与其他目标一样 疗法，对PARPI的抵抗力经常出现，强调未满足的需要阐明PARPI的原因 BRCA突变体但不是正常细胞的细胞死亡。单个PARPI可以通过不同的机制作用 通过“捕获” PARP-DNA复合物，或通过抑制单链（ssDNA）划痕的修复 随后转换为双链断裂（DSB）。此外，患者可能存在差异药物 敏感性取决于特定的欢呼BRCA基因突变。定义这个基本景观将 对于更好地预测反应/非反应器以及患者对PARPI反应的持久性至关重要。 从历史上看，BRCA2突变如何影响基因组完整性的详细机械研究已经 受到操纵和净化这种大蛋白的巨大挑战的阻碍。最近，我们有 克服这些挑战，使我们能够利用体外生化测定和细胞的组合 测定以指出个体病原或靶向突变如何影响特定功能，包括： DNA结合，复制叉保护，RAD51核蛋白丝形成和RAD51介导的DNA 链入侵。除了应用这些技术来询问显式生化功能（S） 由于致病性BRCA2突变所妥协，我们将评估对PARPI的敏感性 或弱捕获活性（例如Talazoparib，Olaparib和Veliparib）。最后，我们将调查 在抗parpi耐药性肿瘤患者中鉴定出的“恢复”突变的功能，可能 独立地识别PARPI灵敏度所需的功能属性。我们的长期目标是揭露 PARPI处理的分子后果，BRCA2，RAD51和其他HDR的必要处理 蛋白质。我们的中心假设是，通过阐明BRCA2和RAD51的机械克服 PARPI介导的毒性，我们将提供必要的框架来了解Parpi抗性如何 在患者中发育。我们的假设基于引人入胜的初步数据，说明了特定功能 响应PARPI的BRCA2和RAD51。我们的理由是揭示基于的机制 PARPI介导的毒性，将垂直提高HDR对PARPI响应的知识，并 最终，改善了BRCA患者的临床管理。在AIM 1中，我们将利用患者派生的BRCA2 在我们的等源性人类细胞模型中的恢复等位基因，以询问哪些特定功能是 “重新激活”以促进对PARPI的抗性。在AIM 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底物的PARPI捕获病变的去除或旁路 （反向叉，间隙）在重构的生化测定中。我们的方法是创新的，因为我们的独特 鲁棒基于细胞和生化功能测定的技能集和开发，以剖析HDR机制 专注于BRCA2和RAD51。我们目前的工作目标是应用我们的HDR专业知识来解决 在PARPI领域的长期神秘：揭示HDR熟练的细胞如何有效地生存。这 预计结果将对HDR缺乏肿瘤的临床管理产生积极影响 治疗性耐药性和缓解是成功治疗患者的关键障碍。