Elucidating SAMHD1 in DNA Double-Strand Break Repair

项目总结 人类肿瘤细胞，包括乳腺癌，表现出异常的DNA双链断裂(DSB)修复途径， 它们驱动了基因组的不稳定性和癌症的发生，最重要的是，它们可能成为新的分子靶点。 可用于逆转治疗耐药表型。然而，管理的确切机制 双链断裂的修复以及它们的失调，包括癌症中的上游信号，如何促进肿瘤细胞 抵抗力仍不清楚。DSB是由包括电离辐射在内的多种癌症治疗方法引起的。 (IR)、PARP抑制剂和化疗。因此，这项提案的首要目标是确定 肿瘤细胞对DSB反应的关键过程，如SAMHD1所指示的，以及这些修复是如何进行的 可以利用这些途径来改善乳腺癌的治疗。SAMHD1因其dNTP而被识别 三磷酸水解酶活性，限制HIV-1和其他病毒感染以及与 艾卡迪·古蒂埃雷斯综合征(AGS)，一种自身免疫性疾病。SAMHD1在27%的乳房中也过表达 癌症。我们首次确定了SAMHD1在促进同源基因末端切除步骤中的新作用 重组(HR)修复独立于其dNTPase活性，自那以后已被独立验证。 我们的初步数据表明，SAMHD1的高表达与乳腺癌患者的低生存率有关 接受IR佐剂治疗的患者，表明SAMHD1的过度表达有助于IR抵抗。vbl.使用 蛋白质组学方法，我们发现SAMHD1与许多DNA损伤反应(DDR)蛋白相互作用， 包括CtIP和SIRT1。我们的数据表明SAMHD1招募CtIP到DSB以促进DNA末端切除 和HR不依赖其dNTPase活性，此外，SIRT1在DSB修复中指导SAMHD1功能 通过脱乙酰化。最后，我们开发了一种新的治疗策略，通过靶向SAMHD1治疗 VPX是一种慢病毒辅助蛋白，包装在病毒样颗粒(VLP)中，可使乳房敏感 癌细胞对DSB诱导剂的作用。因此，我们假设除了它在dNTP代谢中的作用外， SAMHD1对SIRT1介导的脱乙酰化作出反应以维持基因组完整性并控制乳腺癌 通过CtIP非催化引导DNA末端切除和HR产生耐药，这可能是 被用来改善乳腺癌的控制。使用创新的遗传、细胞生物学、生化、结构和 在活体方法中，我们提出了以下具体目标：1)确定SAMHD1在指导CtIP中的作用 在DSB修复中；2)通过SIRT1确定SAMHD1在DSB修复中的调节；3)将SAMHD1建立为 有可能成为癌症治疗的新分子靶点。这项工作的完成将定义SAMHD1非 催化指导CtIP在DSB修复中的功能，以维持基因组完整性和指导乳腺癌治疗 抗性，将SIRT1乙酰组与SAMHD1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SAMHD1作为改善乳房的新方法 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.