Synergistic DNA repair genes and sensitivity to radiation therapy in prostate cancer

项目概要/摘要 在美国，每年有超过33，000名患者死于前列腺癌（PCa），估计有190，000名新的前列腺癌患者。 病例确诊。这些患者中有10-20%被诊断为转移性PCa（mPCa）。mPCa和 转移性去势抵抗性前列腺癌（mCRPC）获得对已确立治疗的抗性， 对患者生活质量产生深远影响的进步。mCRPC仍具有致死性。 我们的小组是第一个描述新的DNA修复基因FAM 35 A，其状态可能会极大地影响肿瘤 在大部分PCa患者中对放射治疗敏感。这个R15项目将确定一个新的 PCa中FAM 35 A相关治疗抗性/致敏的机制。我们的核心假设是：（1） FAM 35 A调节切除抑制和2）FAM 35 A和DNA聚合酶θ（POLQ）拮抗 同源重组（HR）途径。我们将通过以下两个具体目标进行调查： 目的1：探讨FAM 35 A介导的肿瘤切除抑制机制。虽然我们的FAM 35 A复合体 数据包括BLM作为结合伴侣，但仍不清楚FAM 35 A如何阻断切除酶的活性 （BLM-DNA 2和BLM-EXO 1）和/或阻止BLM与DNA 2或EXO 1的相互作用。我们将测试我们的 FAM 35 A通过结合DNA和/或通过结合BLM来阻止核酸酶活性。我们 实验方法将结合生物化学和细胞生物学方法来确定FAM 35 A的切除 抑制活性首先，我们将a）确认两种FAM 35 A同种型的体外DNA结合活性，和B） 确定FAM 35 A中的BLM结合位点。使用FAM 35 A敲除细胞系补充突变体 FAM 35 A，然后我们将使用克隆形成试验评估DNA损伤敏感性;监测DNA损伤 使用共聚焦显微镜的反应;和使用DNA纤维测定测量切除。目标2：阐明 FAM 35 A促进POLQ KO PCa中DNA损伤反应的机制。我的工作 假设是FAM 35 A/POLQ缺失导致c-NHEJ和alt-NHEJ途径的完全功能障碍， 导致心率过度激活1)为了测试FAM 35 A缺陷如何影响DNA损伤剂， 在没有POLQ的情况下，我们将使用三种FAM 35 A缺失的PCa细胞系进行克隆形成测定， POLQ状态2)为了确定用DNA处理的FAM 35 A耗尽的POLQ KO PCa细胞中的DNA修复活性， 我们将使用共聚焦显微镜和免疫印迹来监测DNA损伤反应 DNA损伤诱导剂我们将进行DSB诱导的HR试验以测量HR活性。 结果有望阐明DNA修复功能障碍在PCa耐药/致敏中的作用， 有助于发现放射治疗和预后靶点。主要的积极影响将是 阐明FAM 35 A和Polq之间的DNA修复途径的作用。该项目将使 对从事卫生和基础科学事业的本科生和研究生进行研究培训。

PROJECT SUMMARY/ABSTRACT In the U.S, over 33,000 patients die yearly from prostate cancer (PCa), and an estimated 190,000 new cases are diagnosed. 10-20% of these patients are diagnosed with metastatic PCa (mPCa). mPCa and metastatic castration-resistant prostate cancer (mCRPC) acquire resistance to established treatments and progress with profound effects on patient quality of life. mCRPC remains fatal. Our group was the first to describe the new DNA repair gene FAM35A whose status may greatly affect tumor sensitivity to radiation treatment in a large proportion of PCa patients. This R15 project will determine a new mechanism of FAM35A-related treatment resistance/sensitization in PCa. Our central hypothesis is that 1) FAM35A regulates resection inhibition and 2) FAM35A and DNA polymerase theta (POLQ) antagonize the homologous recombination (HR) pathway. We will investigate through the following two specific aims: AIM 1: Determine the mechanism of FAM35A-mediated resection inhibition. Although our FAM35A-complex data includes BLM as a binding partner, it remains unclear how FAM35A blocks activity of resection enzymes (BLM-DNA2 and BLM-EXO1) and/or prevents the interaction of BLM with DNA2 or EXO1. We will test our working hypothesis that FAM35A prevents nuclease activity by binding to DNA and/or by binding to BLM. Our experimental approach will incorporate biochemical and cell biology methods to determine FAM35A’s resection inhibition activity. First, we will a) confirm in vitro DNA-binding activity of the two FAM35A isoforms, and b) determine the BLM binding site in FAM35A. Using FAM35A knockout cell lines complemented with mutant FAM35A, we will then assess DNA damage sensitivity using clonogenic assays; monitor the DNA damage response using confocal microscopy; and measure resection using DNA fiber assays. AIM 2: Elucidate the mechanism by which FAM35A contributes to the DNA damage response in POLQ KO PCa. My working hypothesis is that depletion of FAM35A/POLQ causes complete dysfunction of both c- and alt- NHEJ pathways, resulting in HR hyperactivation. 1) To test how FAM35A deficiency affects DNA damaging agents with and without POLQ, we will perform clonogenic assays using three FAM35A-depleted PCa cell lines of varying POLQ status. 2) To determine DNA repair activity in FAM35A-depleted POLQ KO PCa cells treated with DNA damaging agents, we will use confocal microscopy and immunoblotting to monitor the DNA damage response with DNA damage inducing agents. We will perform DSB-induced HR assays to measure HR activity. Results are expected to clarify the role of DNA repair dysfunction in resistance/sensitization in PCa and facilitate discovery of therapeutic and prognostic targets for radiation. The primary positive impact will be clarification of roles of DNA repair pathway between FAM35A and Polq. This AREA project will enable the research training of undergraduate and graduate students pursuing careers in health and basic sciences.