Defining the Roles of BRCA2 and RAD51 in PARPi Response

项目总结 PARP抑制剂(PARPI)具有巨大的治疗潜力，因为它们对缺乏细胞的细胞具有选择性 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， 治疗中，对PARPI的耐药性频繁出现，突显了阐明PARPI如何引起的未得到满足的需要 BRCA突变型细胞死亡，而正常细胞不死亡。单个PARPI可以通过不同的机制起作用，或者 通过“捕获”PARP-DNA复合体，或通过抑制单链(SsDNA)缺口的修复 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基因BRCA突变。定义这一基本格局将 对于更好地预测应答者/无应答者以及患者对PARPI的反应的持久性至关重要。 从历史上看，一项关于BRCA2突变如何影响基因组完整性的详细机械研究 受到操纵和提纯这种大蛋白质的巨大挑战的阻碍。最近，我们有 克服这些挑战，使我们能够利用体外生化分析和细胞 确定单个致病基因或靶基因突变如何影响特定功能的分析方法包括： DNA结合、复制分叉保护、RAD51核蛋白细丝形成和RAD51介导的DNA 海岸线入侵。除了应用这些技术来讯问显性的生化功能(S) 受致病BRCA2突变的影响，我们将评估PARPI对强、中、 或较弱的诱捕活性(例如，分别为他唑巴利布、奥拉帕利布和维利帕利)。最后，我们将调查 由PARPI耐药肿瘤患者中发现的“逆转”突变重建的功能(S)，这可能是 独立确定PARPI敏感度所需的功能属性。我们的长期目标是揭开 需要BRCA2、RAD51和其他HDR处理的PARPI处理的分子后果 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理解PARPI抗性如何 在病人身上发展。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 BRCA2和RAD51对PARPI的响应。因此，我们的理论基础，揭示了(S)背后的机制 PARPI介导的毒性，将垂直推进围绕HDR对PARPI的反应的知识，以及 最终提高BRCA患者的临床管理水平。在目标1中，我们将使用患者来源的BRCA2 在我们的等基因人类细胞模型中逆转等位基因，以询问特定的功能(S) “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 使用纯化的蛋白质和相关的模型DNA底物去除或旁路PARPI困住的病变 (倒叉、缺口)在重组生化分析中。我们的方法是创新的，因为我们独特的 用于分析HDR机制的强大的细胞和生化功能分析的技能集和开发 专注于BRCA2和RAD51。我们当前工作的目标是应用我们的人类发展报告专业知识来解决 PARPI领域长期存在的谜团：揭示HDR熟练细胞如何有效地存活下来。这个 这些结果有望对HDR缺陷肿瘤的临床治疗产生积极影响，因为 治疗耐药和复发是患者成功治疗的关键障碍。

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.