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 遗传性乳腺癌和卵巢癌易感性基因（BRCA1 或 BRCA2）缺失 癌症 (HBOC) 的特点是通过同源重组 (HR) 进行 DNA 修复缺陷，并且 复制分叉保护（称为分叉保护 (FP)）。人们认为 HR 和 FP 的缺陷会导致 癌细胞的脆弱点，因为它们无法修复或防止 DNA 双链断裂 (DSB) 因此细胞对 DNA 损伤剂敏感，例如顺铂和聚（ADP-核糖） 聚合酶（PARP）抑制剂（PARPi）。我们最近的发现提供了一个反模型，其中这些疗法 诱导单链 DNA (ssDNA) 间隙，由于间隙缺陷而使 BRCA 缺陷细胞敏感 抑制（GS）。几种 BRCA 突变细胞模型支持介导反应中的差距，然而，每种模型 电阻模型至少保持两个功能。因此，尚不确定哪个功能是该功能的基础 阻力，留下限制临床洞察力的知识差距。开发有效的治疗方法需要 确定 HR、FP 和/或 GS 是否是反应的基本调节因子。本研究的目标是 系统地破坏并保留 BRCA2 中的每个功能（HR、FP、GS），以确定哪些功能对于 BRCA2 至关重要 治疗耐药性，阐明统一的耐药机制，并提供对抑制途径的见解 抵制告知治疗选择。为此，我们的目标是确定 GS 的分子机制 通过映射 BRCA2 中的 GS 结构域（具体目标 1）。在 BRCA2 缺陷细胞中补充 野生型与一系列 BRCA 突变体，这些突变体要么删除或选择性地靶向明确表征的结构域（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。如果尚未充分表征，我们将评估突变体的 HR 通过检查 DNA 纤维测定中的新生链降解，熟练掌握标准测定和 FP。我们 将使用 CRISPR/CAS9 在已识别的 GS 结构域中制造额外的突变体，以进一步表征 介导 GS 的关键残基。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​​ 与响应的联系。我们还旨在确定细胞凋亡是否是细胞损失的基础 基因毒素后 BRCA2 缺陷细胞的活力（具体目标 2）。将使用以下方法测量细胞凋亡 用顺铂或 PARPi 治疗后 BRCA2 突变体的标准测定。此外，我们还将处理细胞 使用细胞凋亡抑制剂并确定对 PARPi 或顺铂的敏感性是否受到抑制。我们会核实时间 以及与细胞凋亡相比诱导 DSB 的剂量，并评估细胞凋亡的抑制是否会减少 DSB 形成。拟议研究的基本原理是，BRCA2 缺陷症的最有效治疗方法是 形成间隙的疗法，间隙的形成将成为肿瘤反应的生物标志物，并最大限度地提高治疗效果 响应，限制差距形成的途径应该是有针对性的。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。