Investigating PolQ-mediated alternative NHEJ in breast cancer

染色体重排是癌细胞的标志，并通过以下途径构成主要途径 哪些影响肿瘤启动和进展的基因发生突变。这种反常现象可能 由双链断裂(DSB)修复中的缺陷引起。DNA有三条主要途径 哺乳动物细胞的修复--研究广泛的同源重组和非同源重组 末端连接(NHEJ)途径，以及特征不佳但极易出错的Alt-NHEJ (替代-NHEJ)途径。已注意到与ALT-NHEJ一致的基因重排 在自发性和与治疗相关的肿瘤中都是如此。在本提案中，我们将重点介绍聚合酶theta (POLθ)，一种低保真的酶，我们最近发现它是一个关键因素，通过以下途径介导dsb修复 Alt-NHEJ。鉴于HR缺陷肿瘤对通过ALT-NHEJ修复DSB上瘾 途径，我们假设POLθ的诱变活性有助于建立基因组图谱 这有利于肿瘤的侵袭性行为。此外，我们预测在肿瘤中删除POLθ 乳腺癌易感性(BRCA)基因的突变将使细胞对DNA损伤敏感- 诱导治疗药物，包括放射治疗、顺铂和PARP抑制剂。在 第二个目的，我们将调查自动柜员机和PARP 1对POLθ被招募来破坏站点的影响 以及聚合酶如何调节损伤位置以促进错误修复。此外，我们还将 寻求基于蛋白质组学的方法来突出参与的所有分子参与者 Alt-NHEJ。最终，充分理解ALT-NHEJ的机制基础将提供更好的 了解恶性肿瘤过程中基因组不稳定性的来源并指导更多 针对越来越多的HR突变肿瘤患者的有效治疗策略。

Chromosomal rearrangements are a hallmark of cancer cells and constitute a major pathway by which genes that affect tumor initiation and progression become mutated. Such aberrancies can result from defects in double-stranded break (DSB) repair. There are three major pathways of DNA repair in mammalian cells – the well-studied homology recombination (HR) and non-homologous end joining (NHEJ) pathways, and the poorly characterized, yet highly error-prone alt-NHEJ (alternative-NHEJ) pathway. Genetic rearrangements consistent with alt-NHEJ have been noted both in spontaneous and therapy-related tumors. In this proposal we will focus on polymerase theta (Polθ), a low-fidelity enzyme that we recently identified as a key factor that mediates DSB repair by alt-NHEJ. Given that HR-defective tumors are “addicted” to repairing DSBs via the alt-NHEJ pathway, we hypothesize that the mutagenic activity of Polθ help establish a genomic landscape that is conducive for aggressive tumor behavior. Additionally, we predict that deleting Polθ in tumors with mutations in the breast cancer susceptibility (BRCA) genes will sensitize cells to DNA damage- inducing therapeutic agents, including radiation therapy, cisplatin and PARP inhibitors. In the second aim, we will investigate the impact of ATM and PARP1 on Polθ is recruited to break sites and how the polymerase modulates damage sites to promote erroneous repair. In addition, we will pursue a proteomic-based approach to highlight the full spectrum of molecular players involved in alt-NHEJ. Ultimately, a full understanding of the mechanistic basis of alt-NHEJ will provide a better understanding of the source of genomic instability during the course of malignancy and guide more effective treatment strategies for the increasing number of patients with HR mutated tumors.