Mapping the BRCA2 replication gap suppression domain to uncover themolecular mechanism of chemotherapy response

摘要 遗传性乳腺和卵巢癌易感基因BRCA1或BRCA2的丢失 癌症(HBOC)的特征是通过同源重组(HR)修复DNA缺陷，而在 复制分叉保护(称为分叉保护(FP))。据认为，HR和FP缺乏会产生 癌细胞的脆弱性，因为它们不能修复或防止DNA双链断裂(DSB) 因此，细胞对顺铂和聚腺苷二磷酸核糖等DNA损伤剂很敏感。 聚合酶(PARP)抑制剂(PARPI)。我们最近的发现提供了一个反模型，在这个模型中，这些疗法 诱导单链DNA(SsDNA)缺口，使BRCA缺陷细胞因GAP缺陷而敏化 抑制(GS)。然而，几个BRCA突变细胞模型支持中介反应中的差距 阻力模型至少维持两种功能。因此，不能确定哪个函数是 耐药性，留下了限制临床洞察力的知识缺口。开发有效的治疗方法需要 确定HR、FP和/或GS是否是响应的基本中介。这项研究的目标是 系统地中断并保留BRCA2中的每个职能(人力资源、人力资源、一般事务)，以确定哪些职能对 治疗耐药，阐明耐药的统一机制，并提供对抑制途径的洞察 抗拒告知治疗选择。为此，我们的目标是确定GS的分子机制 通过在BRCA2中定位GS结构域(S)(特定目标1)。在BRCA2缺陷细胞中补充了 野生型VS一系列BRCA突变体，其删除或选择性地靶向特征良好的结构域(即， HR或FP)、蛋白质相互作用区或DNA结合部位，我们将分析常规DNA中的GAP诱导 纤维和免疫荧光分析。如果还没有很好地描述，我们将评估HR的突变 精通标准化验和DNA纤维化验中新生链降解的FP。我们 将使用CRISPR/CAS9在已确定的GS结构域(S)中进行额外的突变，以进一步鉴定 关键残基对GS的调节作用。我们还将测试这些突变表达细胞的PARPI敏感性，以便 评估HR、FP或GS与响应之间的联系。我们的目标也是确定细胞凋亡是否是细胞丢失的原因。 BRCA2缺陷细胞在基因毒素作用下的存活能力(特定目标2)。将使用以下方法测量细胞凋亡 顺铂或PARPI治疗后BRCA2突变的标准检测。此外，我们还将治疗细胞 并确定是否抑制了对PARPI或顺铂的敏感性。我们会核实时间的 以及诱导DSB的剂量与凋亡的比较，并评估抑制凋亡是否会降低DSB 队形。这项拟议研究的基本原理是BRCA2缺乏症最有效的治疗方法是 形成缝隙的疗法，缝隙的形成将是肿瘤反应的生物标记物，并最大限度地提高治疗效果 应对措施方面，应针对限制缺口形成的途径。通过实验获得的洞察力 该提议将对癌症产生影响，并为治疗干预提供新的机会。

Abstract Loss of the breast cancer susceptibility (BRCA1 or BRCA2) genes in hereditary breast and ovarian cancer (HBOC) is characterized by defects DNA repair by homologous recombination (HR) and in the protection of replication forks (known as fork protection (FP)). It is thought that HR and FP deficiencies produce points of vulnerability in cancer cells because they cannot fix or prevent DNA double stranded breaks (DSBs) and therefore cells are sensitive to DNA damaging agents such as to cisplatin and Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). Our recent findings provide a counter model in which these therapies induce single stranded DNA (ssDNA) gaps that sensitize BRCA deficient cells due to a defect in gap suppression (GS). Several BRCA mutant cell models support gaps in mediating response, however, each model of resistance maintains at least two functions. Thus, it is not certain which function underlies the resistance, leaving a knowledge gap that limits clinical insight. The development of effective therapies requires identifying whether HR, FP, and/or GS is the fundamental mediator of response. This goal of this study is to systematically disrupt and retain each function (HR, FP, GS) within BRCA2 to define what function is critical for therapy resistance, elucidate a unified mechanism of resistance, and provide insight into inhibiting pathways of resistance to inform therapeutic choices. To do this we aim to determine the molecular mechanism of GS through mapping the GS domain(s) in BRCA2 (Specific Aim 1). In BRCA2 deficient cells complemented with wild-type vs a series of BRCA mutants that either delete or selectively target well-characterized domains (i.e., HR or FP), protein interacting regions, or DNA binding sites, we will analyze gap induction in our routine DNA fiber and immunofluorescence assays. If not already well characterized, we will assess mutants for HR proficiency in standard assays and FP via examination of nascent strand degradation in DNA fiber assays. We will use CRISPR/CAS9 to make additional mutants in the identified GS domain(s) to further characterize the critical residues mediating GS. We will also test PARPi sensitivity of these mutant expressing cells in order to assess the link of HR, FP, or GS to response. We also aim to determine if apoptosis underlies loss of cell viability in BRCA2 deficient cells following genotoxins (Specific Aim 2). Apoptosis will be measured using standard assays in BRCA2 mutants following treatment with cisplatin or PARPi. In addition, we will treat cells with apoptosis inhibitors and determine if sensitivity to PARPi or cisplatin is suppressed. We will verify the time and dose in which DSBs are induced compared to apoptosis and assess if inhibition of apoptosis reduces DSB formation. The rationale for the proposed research is that BRCA2 deficiency will be most effectively treated by therapies that form gaps, gap formation will be a biomarker of tumor response, and to maximize therapy response, pathways limiting gap formation should be targeted. The insight gained from the experiments proposed will have implications for cancer and provide new opportunities for therapeutic intervention.