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.

项目概要 同源重组 (HR) 是一种修复 DNA 双链断裂 (DSB) 的途径，经常被 HR 缺陷的癌症容易出现基因组不稳定，并且严重依赖于其他癌症。 DNA 修复机制包括 DNA 损伤传感器 PARP，以及 PARP 抑制剂。 因此，被证明是消除 HR 缺陷癌症的有效疗法。然而，一些 HR 缺陷肿瘤确实如此。 对 PARP 抑制剂没有反应，大多数肿瘤最终会复发并对药物产生耐药性。 因此，迫切需要治疗 HR 缺陷型癌症并克服 PARP 的治疗策略 在潜在的策略中，越来越多的注意力集中在替代末端连接（Alt-）上。 EJ)，一种备用 DSB 修复途径，在正常细胞中是可有可无的，但在 HR 处于低水平时对细胞生存至关重要。 虽然越来越多的证据表明抑制 Alt-EJ 可能是一种强有力的策略。 尽管克服了对 PARP 抑制剂耐药的问题，但 Alt-EJ 通路的特征仍然很差，限制了 可以开发的潜在治疗靶点数量。 为了解决这一知识差距，我的实验室设计了一种新颖的方法来识别 Alt-EJ 因素，使用 使用这种方法以及基于修复产生或的 Alt-EJ 检测进行全基因组 CRISPR/Cas9 筛选。 Alt-EJ 介导的端粒融合，我们已确定核酸酶 APE2 是 Alt-EJ 中的关键蛋白。 令人信服的发现提出了令人兴奋的可能性，即 APE2 抑制可用于专门针对 HR- 缺陷的癌细胞，从而预防或克服对 PARP 抑制剂的耐药性。 然而，需要首先解决 APE2 在 Alt-EJ 中的确切功能及其在 HR 缺陷细胞中的作用 仍然难以捉摸。 首先，我们将在分子水平上表征 APE2 在 Alt-EJ 中的功能和作用机制。 我们将确定：(1) APE2 在 Alt-EJ 中的作用，(2) 涉及的域和生化活性，以及​​ (3) DSB 的招募机制的目的是显着改善我们的基本面。 了解 Alt-EJ 修复途径并获得有关 APE2 的足够知识，以实现未来 抑制剂的开发。 其次，我们将揭示 APE2 抑制对 HR 缺陷癌细胞的治疗潜力 (1) 我们。 将通过 HR 识别驱动其合成致死性的 APE2 修复功能，并且 (2) 我们将确定 APE2 抑制与 PARP 抑制剂协同作用并预防或克服对 PARP 耐药性的潜力 抑制剂。 我们的新发现是 APE2 在选择性末端连接中发挥着关键作用，以及创新工具 我们为研究 APE2 在 Alt-EJ 中的作用而创建的项目使我的实验室处于一个独特的位置，可以成功地开展 本研究提出。