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

绘制 BRCA2 复制间隙抑制域图谱以揭示化疗反应的分子机制

• 批准号: 10679641
• 负责人: Jenna Whalen
• 金额: $ 6.95万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679641
• 关键词: Acceleration; Address; Apoptosis; Apoptosis Inhibitor; BRCA deficient; BRCA1 gene; BRCA2 gene; Binding Sites; Biological Assay; Biological Models; Breast Cancer gene; CRISPR/Cas technology; Cell Death; Cell Survival; Cell model; Cells; Chemoresistance; Cisplatin; Clinical; Clone Cells; Complement; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA replication fork; Defect; Development; Dose; Fiber; Future; Genes; Goals; Hereditary Breast Carcinoma; Hereditary Breast and Ovarian Cancer Syndrome; Immunofluorescence Immunologic; Induction of Apoptosis; Inhibition of Apoptosis; Knowledge; Lesion; Link; Malignant Neoplasms; Maps; Measures; Mediating; Mediator; Modeling; Molecular; Mutagens; Mutate; Mutation; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Point Mutation; Poly(ADP-ribose) Polymerase Inhibitor; Prediction of Response to Therapy; Predisposition; Proteins; Regulation; Research; Resistance; Resistance development; Role; Sampling; Series; Single-Stranded DNA; Stress; Testing; Therapeutic; Therapeutic Intervention; Time; Tumor Markers; anti-cancer; brca gene; cancer cell; cell killing; chemotherapy; effective therapy; experimental study; homologous recombination; insight; malignant breast neoplasm; mutant; prevent; resistance mechanism; response; restraint; tissue culture; treatment response; tumor

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.

抽象的 遗传性乳房和卵巢中乳腺癌易感性（BRCA1或BRCA2）基因的丧失 癌症（HBOC）的特征是通过同源重组（HR）和在 保护复制叉（称为叉保护（FP））。人们认为人力资源和FP缺陷会产生 癌细胞的脆弱性是因为它们无法修复或预防DNA双链断裂（DSB） 因此，细胞对DNA损伤剂（例如顺铂和聚（ADP-核糖））敏感 聚合酶（PARP）抑制剂（PARPI）。我们最近的发现提供了一个抗衡模型 诱导单链DNA（ssDNA）间隙，由于间隙缺陷而使BRCA缺陷的细胞敏感 抑制（GS）。但是，几种BRCA突变细胞模型支持介导反应中的差距 电阻模型至少保持两个功能。因此，不确定哪个功能是基于 阻力，留下限制临床见解的知识差距。有效疗法的发展需要 确定人力资源，FP和/或GS是否是反应的基本中介。这项研究的目标是 系统地破坏并保留BRCA2中的每个功能（HR，FP，GS），以定义哪种功能至关重要 治疗抗性，阐明了统一的抵抗机制，并提供了抑制途径的见解 阻力为治疗选择提供信息。为此，我们旨在确定GS的分子机制 通过在BRCA2中映射GS域（特定目标1）。在BRCA2缺乏的细胞中 野生型与一系列删除或选择性靶向良好特征域的BRCA突变体（即 HR或FP），蛋白质相互作用区域或DNA结合位点，我们将分析常规DNA中的间隙诱导 纤维和免疫荧光测定。如果还没有很好地表征，我们将评估HR的突变体 通过检查DNA纤维测定中的新生链降解，可以熟练标准测定法和FP。我们 将使用CRISPR/CAS9在已确定的GS域中制作其他突变体，以进一步表征 介导GS的关键残留物。我们还将测试这些表达细胞的PARPI敏感性，以便 评估人力资源，FP或GS与响应的联系。我们还旨在确定凋亡是否构成细胞损失 基因毒素后BRCA2缺乏细胞的生存能力（特定目标2）。凋亡将使用 用顺铂或PARPI治疗后，BRCA2突变体的标准测定。另外，我们将处理细胞 用凋亡抑制剂并确定对PARPI或顺铂的敏感性是否抑制。我们将验证时间 与凋亡相比，诱导DSB的剂量并评估抑制凋亡是否会降低DSB 形成。拟议研究的理由是，BRCA2缺乏症将受到最有效的治疗 形成间隙的疗法，间隙形成将是肿瘤反应的生物标志物，并最大化治疗 响应，应限制差距形成的途径。从实验中获得的见解 提议将对癌症产生影响，并为治疗干预提供新的机会。