Deciphering the function of the APE2 nuclease during repair by alternative end-joining and its role in HR-deficient cells

解读 APE2 核酸酶在选择性末端连接修复过程中的功能及其在 HR 缺陷细胞中的作用

• 批准号: 10339167
• 负责人: Nausica C. Arnoult
• 金额: $ 35.16万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339167
• 关键词: APEXL2 Gene; Address; Attention; Back; Base Excision Repairs; Biochemical; Biological Assay; CRISPR screen; Cells; Chemicals; Comprehension; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; Data; Defect; Development; Double Strand Break Repair; Drug resistance; Exonuclease; Future; Genome Stability; Genomic Instability; Human; Immunofluorescence Immunologic; Impairment; In Vitro; Knowledge; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mediating; Methods; Modeling; Molecular; Mutate; Mutation; Nonhomologous DNA End Joining; Normal Cell; Ovarian Carcinoma; Pathway interactions; Peptides; Phosphodiesterase I; Play; Positioning Attribute; Progression-Free Survivals; Proteins; Proteomics; Relapse; Reporter; Research; Resistance; Role; Surgical Flaps; Testing; Therapeutic; Up-Regulation; Zinc Fingers; base; cancer cell; cell growth; chromatin immunoprecipitation; cost; design; endonuclease; experimental study; genetic approach; genome-wide; homologous recombination; improved; inhibitor; innovation; interest; malignant breast neoplasm; new therapeutic target; novel; novel strategies; novel therapeutic intervention; nuclease; phosphoric diester hydrolase; prevent; recruit; repair function; repaired; sensor; telomere; therapeutic evaluation; therapeutic target; tool; tumor

PROJECT SUMMARY Homologous recombination (HR), a pathway that repairs DNA double strand breaks (DSB), is frequently mutated in cancers. HR-deficient cancers are prone to genomic instability and are critically dependent on other DNA repair mechanisms for survival. Among them is the DNA damage sensor PARP, and PARP inhibitors have therefore proved an efficient therapy to eliminate HR-deficient cancers. However, some HR-deficient tumors do not respond to PARP inhibitors, and most tumors eventually relapse and become resistant to the drug. New therapeutic strategies are therefore urgently needed to treat HR-deficient cancers and overcome PARP inhibitor resistance. Among the potential strategies, growing attention has focused on alternative end-joining (Alt- EJ), a back-up DSB repair pathway that is dispensable in normal cells but critical to cellular survival when HR is compromised. While mounting evidence suggests that inhibition of Alt-EJ could be a powerful strategy to overcome the problem of resistance to PARP inhibitors, the Alt-EJ pathway remains poorly characterized, limiting the potential number of therapeutic targets that could be developed. To address this gap in knowledge, my lab has designed a novel approach to identify Alt-EJ factors using genome-wide CRISPR/Cas9 screens. Using this method as well as Alt-EJ assays based on repair reporters or Alt-EJ-mediated fusion of telomeres, we have identified the nuclease APE2 as a critical protein in Alt-EJ. This compelling discovery raises the exciting possibility that APE2 inhibition could be used to specifically target HR- deficient cancer cells, and thereby prevent or overcome resistance to PARP inhibitors. Several questions however need to be addressed first, as the exact function of APE2 in Alt-EJ and its role in HR-deficient cells remain elusive. First, we will characterize, at the molecular level, the function and mechanism of action of APE2 in Alt-EJ. We will determine: (1) the role of APE2 in Alt-EJ, (2) the domains and biochemical activities involved, and (3) the mechanism of its recruitment to DSBs. The interest here is both to significantly improve our fundamental understanding of the Alt-EJ repair pathway and to gain sufficient knowledge on APE2 to enable the future development of an inhibitor. Second, we will uncover the therapeutic potential of APE2 inhibition in HR-deficient cancer cells. (1) We will identify the repair function of APE2 that drives its synthetic lethality with HR, and (2) we will determine the potential of APE2 inhibition to synergize with PARP inhibitors and to prevent or overcome resistance to PARP inhibitors. Our novel discovery that APE2 plays a critical function in alternative end-joining, and the innovative tools that we have created to study APE2’s role in Alt-EJ places my lab in a unique position to successfully carry out this proposed research.

项目摘要 同源重组（HR）是一种修复DNA双链断裂（DSB）的途径，经常是 在癌症中突变。缺乏HR的癌症容易发生基因组不稳定性，并且严重取决于其他 生存的DNA修复机制。其中是DNA损伤传感器PARP，PARP抑制剂具有 因此，证明是一种有效的疗法，可以消除HR缺陷癌症。但是，一些缺乏HR的肿瘤会 不对PARP抑制剂反应，大多数肿瘤最终都会中继并对药物具有抵抗力。新的 因此，迫切需要治疗策略来治疗HR缺乏癌症并克服PARP 抑制剂耐药性。在潜在的策略中，日益增长的关注集中在替代最终结合上（alt- EJ），一种备用的DSB修复途径，在正常细胞中是可分配的，但在HR为HR时对细胞存活至关重要 妥协。虽然越来越多的证据表明，抑制alt-ej可能是一个有力的策略 克服对PARP抑制剂的抗性问题，Alt-EJ途径的特征仍然很差，限制 可以开发的治疗靶标的潜在数量。 为了解决知识中的这一差距，我的实验室设计了一种新颖的方法来识别Alt-Ej因素 全基因组CRISPR/CAS9屏幕。使用此方法以及基于维修记者的Alt-EJ分析或 Alt-Ej介导的端粒融合，我们将核酸酶APE2鉴定为Alt-EJ中的临界蛋白。这 引人入胜的发现增加了令人兴奋的可能性，即APE2抑制可用于专门针对HR- 缺乏癌细胞，从而防止或克服对PARP抑制剂的抗性。几个问题 但是，首先需要解决，作为APE2在Alt-EJ中的确切函数及其在HR缺陷细胞中的作用 保持难以捉摸。 首先，我们将在分子水平上表征APE2在Alt-EJ中的作用和机理。 我们将确定：（1）APE2在alt-ej中的作用，（2）所涉及的域和生化活动，以及（3） 其招募到DSB的机制。这里的兴趣是显着改善我们的基本 了解Alt-EJ维修途径并获得有关APE2的足够知识以使未来 开发抑制剂。 其次，我们将发现APE2抑制在HR缺陷型癌细胞中的治疗潜力。 （1）我们 将确定APE2的维修功能，该功能驱动其使用HR的合成致死性，（2）我们将确定 APE2抑制与PARP抑制剂协同作用并防止或克服PARP的潜力 抑制剂。 我们的小说发现APE2在替代最终连接和创新工具中起着关键作用 我们创建的是为了研究Ape2在Alt-Ej中的作用，使我的实验室处于成功执行的独特位置 这项拟议的研究。