Elucidating SAMHD1 in DNA Double-Strand Break Repair

项目摘要 包括乳腺癌在内的人类肿瘤细胞表现出异常的DNA双链断裂（DSB）修复途径， 驱动基因组不稳定性和致癌作用，最重要的是， 可用于逆转耐药表型。然而，管理这些问题的确切机制 DSB的修复以及它们的失调，包括癌症中的上游信号传导， 抵抗力仍然不清楚。DSB是由许多类型的癌症治疗引起的，包括电离辐射 (IR)、PARP抑制剂和化疗。因此，本提案的总体目标是确定 肿瘤细胞对DSB反应的关键过程，如SAMHD 1所指导的，以及这些修复如何 可以利用这些途径来改善乳腺癌治疗。SAMHD 1因其dNTP而被识别 三磷酸水解酶活性，其限制HIV-1和其他病毒感染以及与 古铁雷斯综合征（AGS），一种自身免疫性疾病。SAMHD 1也在27%的乳腺癌中过表达。 癌的我们是第一个确定SAMHD 1在促进同源性TNM末端切除步骤中的新作用的。 在一些实施方案中，该化合物具有不依赖于其dNTR活性的重组（HR）修复，这已经被独立地验证。 我们的初步数据表明，SAMHD 1高表达与乳腺癌的生存率低有关。 用辅助IR治疗的患者，表明SAMHD 1过表达有助于IR抵抗。使用 通过蛋白质组学方法，我们发现SAMHD 1与许多DNA损伤反应（DDR）蛋白相互作用， 包括CtIP和SIRT 1。我们的数据表明，SAMHD 1招募CtIP到DSB，以促进DNA末端切除 和HR独立于其dNTR活性，此外，SIRT 1指导SAMHD 1在DSB修复中的功能 通过脱乙酰化。最后，我们开发了一种新的治疗策略， Vpx是一种慢病毒辅助蛋白，包装在病毒样颗粒（VLP）中， 癌细胞对DSB诱导剂的反应。因此，我们假设除了在dNTP代谢中的作用外， SAMHD 1响应SIRT 1介导的去乙酰化以维持基因组完整性并控制乳腺癌 通过非催化引导DNA末端切除和通过CtIP的HR的治疗抗性，其可以是 用于改善乳腺癌控制。利用创新的遗传学、细胞生物学、生物化学、结构学和 在体内方法中，我们提出了以下具体目标：1）确定SAMHD 1在指导CtIP中的作用 2）通过SIRT 1确定SAMHD 1在DSB修复中的调节作用; 3）将SAMHD 1确定为DSB修复中的一个重要调节因子。 潜在的癌症治疗新分子靶点。这项工作的完成将确定SAMHD 1非 在DSB修复中催化指导CtIP功能，以维持基因组完整性并管理乳腺癌治疗 抗性，将SIRT 1乙酰组与SAMHD 1调节的DSB修复连接起来，并建立概念验证 使用含有Vpx的VLP靶向SAMHD 1作为改善乳腺癌的新治疗方法， 癌症控制

PROJECT SUMMARY Human tumor cells, including breast cancers, exhibit aberrant DNA double-strand break (DSB) repair pathways, which drive genomic instability and carcinogenesis, and most importantly, may serve as novel molecular targets that can be used to reverse treatment resistance phenotypes. However, the precise mechanisms governing the repair of DSBs and how their dysregulation, including upstream signaling in cancers, contributes to tumor cell resistance remains unclear. DSBs are induced by many types of cancer treatments, including ionizing radiation (IR), PARP inhibitors, and chemotherapy. As such, the overarching goal of this proposal is to determine the critical processes by which tumor cells respond to DSBs, as directed by SAMHD1, and how these repair pathways can be exploited to improve breast cancer treatment. SAMHD1 is recognized for its dNTP triphosphohydrolase activity, which restricts HIV-1 and other viral infections and for mutations associated with Aicardi Goutières syndrome (AGS), an autoimmune disorder. SAMHD1 is also overexpressed in 27% of breast cancers. We were the first to identify a novel role for SAMHD1 in promoting the end resection step of homologous recombination (HR) repair independent of its dNTPase activity, which has since been independently validated. Our preliminary data indicate that high SAMHD1 expression is associated with poor survival in breast cancer patients treated with adjuvant IR, suggesting that SAMHD1 overexpression contributes to IR resistance. Using a proteomic approach, we found that SAMHD1 interacts with a number of DNA damage response (DDR) proteins, including CtIP and SIRT1. Our data suggest that SAMHD1 recruits CtIP to DSBs to facilitate DNA end resection and HR independent of its dNTPase activity, and moreover, that SIRT1 directs SAMHD1 function in DSB repair through deacetylation. Finally, we developed a novel therapeutic strategy whereby targeting SAMHD1 for degradation with Vpx, a lentivirus accessory protein, packaged in virus-like particles (VLPs), sensitizes breast cancer cells to DSB-inducing agents. Thus, we hypothesize that in addition to its role in dNTP metabolism, SAMHD1 responds to SIRT1-mediated deacetylation to maintain genome integrity and govern breast cancer treatment resistance by non-catalytically directing DNA end resection and HR through CtIP, which may be exploited to improve breast cancer control. Using innovative genetic, cell biological, biochemical, structural, and in vivo approaches, we propose the following specific aims: 1) Determine the role of SAMHD1 in directing CtIP in DSB repair; 2) Determine the regulation of SAMHD1 in DSB repair by SIRT1; 3) Establish SAMHD1 as a potentially new molecular target for cancer therapy. Completion of this work will define how SAMHD1 non- catalytically directs CtIP function in DSB repair to maintain genome integrity and govern breast cancer treatment resistance, connect the SIRT1 acetylome with SAMHD1-regulated DSB repair, and establish proof of concept for the use of VLPs containing Vpx to target SAMHD1 as a novel therapeutic approach for improving breast cancer control.