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

项目总结 同源重组(HR)是一种修复DNA双链断裂(DSB)的途径，是一种常见的 在癌症中发生突变。缺乏HR的癌症很容易发生基因组不稳定，并且严重依赖于其他 DNA修复机制是生存的基础。其中包括DNA损伤传感器PARP，以及PARP抑制剂 因此，这被证明是一种消除HR缺陷癌症的有效方法。然而，一些HR缺乏的肿瘤会 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抗药性。新的 因此迫切需要治疗策略来治疗HR缺乏的癌症和克服PARP 抑制剂抗性。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- Ej)，这是一条备用的DSB修复途径，在正常细胞中是必不可少的，但在HR 妥协了。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来 克服了对PARP抑制剂的耐药性问题，Alt-EJ途径仍然缺乏特征性，限制了 可开发的治疗靶点的潜在数量。 为了解决这一知识差距，我的实验室设计了一种新的方法来识别Alt-EJ因子，使用 全基因组CRISPR/Cas9筛选。使用此方法以及基于修复报告或 在ALT-EJ介导的端粒融合中，我们发现核酸酶APE2是Alt-EJ中的一个关键蛋白。这 令人振奋的发现提出了一种令人兴奋的可能性，即APE2抑制可能被用来专门针对HR- 它可以抑制肿瘤细胞的生长，从而防止或克服对PARP抑制剂的耐药性。几个问题 然而，需要首先解决的是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，我们将从分子水平上研究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)涉及的结构域和生化活性，以及(3) 其向DSB招募的机制。这里的兴趣既是为了显著改善我们的基本面 了解Alt-EJ修复途径并获得足够的APE2知识以支持未来 一种抑制剂的开发。 其次，我们将发现APE2抑制在HR缺陷的癌细胞中的治疗潜力。(1)我们 将确定APE2的修复功能，从而驱动其与HR的合成杀伤力，以及(2)我们将确定 APE2抑制与PARP抑制剂协同作用及预防或克服对PARP的耐药性 抑制剂。 我们的新发现APE2在另一种末端连接中起关键作用，以及创新的工具 我们为研究APE2在Alt-EJ中的S角色而创建的这一点使我的实验室处于一个独特的位置，可以成功地开展 这项拟议的研究。

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.