Defining synthetic lethal relationships with loss of the homologous recombination factor Rad52

定义同源重组因子 Rad52 丢失的合成致死关系

基本信息

批准号：
10678580
负责人：
Beth Anne Osia
金额：
$ 6.95万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10678580
关键词：
Address Anaphase Award BRCA deficient BRCA2 gene Biological Assay CRISPR/Cas technology Cancer Patient Cell Cycle Regulation Cell Nucleus Cells Chromosome Fragile Sites Cisplatin Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complex Coupled DNA DNA Damage DNA Repair DNA Repair Disorder DNA Repair Pathway DNA biosynthesis Data Dependence Dot Immunoblotting Ensure Event Exposure to FANCD2 protein Fellowship Gamma-H2AX Genes Genetic Genetic Transcription Genome Genome Stability Genomic Instability Genotoxic Stress Goals Human Hybrids Interphase Ionizing radiation Knock-out Knowledge Label Maintenance Malignant Neoplasms Measures Mediating Mission Mitosis Mitotic Pathway interactions Patient-Focused Outcomes Phosphorylation Play Process Proteins RAD52 gene RNA RNA metabolism Radiation exposure Resolution Role Source Stress Structure Synthetic Genes Testing Therapeutic Ultrafine United States National Institutes of Health Work cancer therapy effective therapy fitness genome-wide genotoxicity homologous recombination hydroxyurea improved inhibitor insight interest knock-down malignant breast neoplasm novel novel therapeutic intervention paralogous gene prevent recruit replication stress screening small molecule inhibitor synthetic lethal interaction targeted treatment telomere tumor

项目摘要

SUMMARY. The long-term goal of this project is to define factors and pathways that are synthetic lethal with loss of the human Rad52 protein. Rad52 plays essential roles in several homology-driven DNA repair pathways, including single strand annealing, transcription-coupled homologous recombination, and mitotic DNA synthesis (MiDAS). Although Rad52 is not essential, Rad52 loss with disruption of either the breast cancer 1 (BRCA1) or breast cancer 2 (BRCA2) genes is synthetic lethal. Thus, Rad52 is an intriguing potential target for treatment of BRCA-deficient cancers. However, the full breadth of pathways and factors that create a state of Rad52- dependence when compromised are not understood, and the long-term goal of this proposal is to address this gap in knowledge. In preliminary data, I present my CRISPR knock-out screen in Rad52 Knock-out (Rad52KO) cells vs. wild-type (Rad52WT) to identify factors that are synthetic lethal with Rad52 (defined here as loss of fitness). I then present secondary screening that identified three top hits causing increased persistent DNA damage and loss of viability in the Rad52KO vs Rad52WT: ERCC6L/PICH, DHX9 and GLE1. From these data, my overall hypothesis is that a key regulator of Mitosis (PICH) and RNA metabolism factors (DHX9 and GLE1) are synthetic lethal with Rad52 due to dependence on Rad52 to resolve replication stress from diverse sources. Aim 1: To define the synthetic lethal relationship between Rad52 and PICH. Rad52 protects genome stability through roles in MiDAS and suppression of replication stress. PICH mediates resolution of anaphase ultrafine bridges (UFBs), a separate pathway to mitigate replication stress. Aim 1a: I posit that these two pathways are partially redundant in preventing accumulation of genotoxic damage tied to replication stress. Namely, I posit that PICH- UFBs will be elevated in Rad52KO cells, and conversely that depletion of PICH will cause elevated Rad52 recruitment to replication stress in mitotic cells, as well as MiDAS. Aim 1b: I also posit that replication stress that persists until mitosis is the source of persistent DNA damage in cells lacking Rad52 and PICH. I will test this by assaying phosphorylated RPA2 (pRPA), γH2AX, and FANCD2 localization in mitotic cells. Aim 2: To define the synthetic lethal relationship between Rad52 and RNA metabolism factors DHX9 and GLE1. DHX9 and GLE1 have been shown to suppress RNA-DNA hybrids (R-loops). Thus, I hypothesize that R-loop-related replication stress underlies synthetic lethality between these genes and Rad52. I will assay whether depletion of these genes increases R-loops, causes elevated levels of mitotic replication stress (i.e. Rad52 accumulation into foci, MiDAS, PICH-UFBs, and the measures of replication stress described in Aim 1b). In summary, these studies will provide insight into how loss of these genes create a dependence on Rad52-mediated mitigation of replication stress in mitosis (i.e., MiDAS), enhance our understanding of genome maintenance mechanisms, with a long- term goal of identifing tumor-specific vulnerabilities for Rad52 inhibitors.

摘要：该项目的长期目标是确定合成致死的因素和途径。人类 Rad52 蛋白的缺失在多种同源驱动的 DNA 修复途径中发挥着重要作用，包括单链退火、转录偶联同源重组和有丝分裂 DNA 合成 (MiDAS) 虽然 Rad52 不是必需的，但 Rad52 丢失会破坏乳腺癌 1 (BRCA1) 或乳腺癌 2 (BRCA2) 基因是合成致死基因，因此，Rad52 是治疗乳腺癌的一个有趣的潜在靶点。然而，BRCA 缺陷型癌症产生 Rad52- 状态的途径和因素多种多样。妥协时的依赖尚未被理解，该提案的长期目标是解决这个问题在初步数据中，我展示了 Rad52 敲除 (Rad52KO) 中的 CRISPR 敲除筛选。细胞与野生型 (Rad52WT) 的比较，以确定 Rad52 合成致死的因子（此处定义为丢失然后我提出了二次筛选，确定了导致持久性 DNA 增加的三个热门命中。 Rad52KO 与 Rad52WT 的损坏和活力丧失：ERCC6L/PICH、DHX9 和 GLE1。总体假设是有丝分裂 (PICH) 和 RNA 代谢因子（DHX9 和 GLE1）的关键调节因子是由于依赖 Rad52 来解决来自不同来源的复制压力，因此使用 Rad52 进行合成致死。图1：定义Rad52和PICH之间的合成致死关系，Rad52通过保护基因组稳定性。 PICH 在 MiDAS 中的作用和抑制复制应激介导后期超细桥的解决。（UFB），减轻复制压力的单独途径：我认为这两条途径是部分的。在防止与复制应激相关的基因毒性损伤积累方面是多余的，即，我认为PICH- Rad52KO 细胞中的 UFB 会升高，相反，PICH 的耗尽会导致 Rad52 升高有丝分裂细胞中复制应激的募集以及 MiDAS：我还认为复制应激。持续存在，直到有丝分裂成为缺乏 Rad52 和 PICH 的细胞中持续 DNA 损伤的根源，我将通过以下方法对此进行测试。测定磷酸化 RPA2 (pRPA)、γH2AX 和 FANCD2 在有丝分裂细胞中的定位。 Rad52 与 RNA 代谢因子 DHX9 和 GLE1 之间的合成致死关系。已被证明可以抑制 RNA-DNA 杂合体（R 环），因此，我领导了 R 环相关的复制。应激是这些基因和 Rad52 之间合成致死性的基础，我将分析这些基因是否被耗尽。基因增加 R 环，导致有丝分裂复制应激水平升高（即 Rad52 积累到病灶中， MiDAS、PICH-UFB 和目标 1b) 中描述的复制压力测量方法总之，这些研究将。深入了解这些基因的丢失如何产生对 Rad52 介导的复制缓解的依赖有丝分裂中的应激（即 MiDAS），增强我们对基因组维持机制的理解，具有长期的确定 Rad52 抑制剂的肿瘤特异性漏洞的长期目标。