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

• 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
• 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

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子和途径， 人Rad 52蛋白的丢失。Rad 52在几种同源驱动的DNA修复途径中起重要作用， 包括单链退火、转录偶联同源重组和有丝分裂DNA合成 （MiDAS）。尽管Rad 52不是必需的，但Rad 52的丢失伴随乳腺癌1（BRCA 1）或乳腺癌细胞的破坏。 乳腺癌2（BRCA 2）基因是合成致死的。因此，Rad 52是一个有趣的潜在治疗靶点， BRCA缺陷型癌症然而，创造Rad 52状态的全部途径和因素- 当妥协时的依赖性不被理解，本提案的长期目标是解决这一问题 知识的差距。在初步数据中，我在Rad 52 Knock-out（Rad 52 KO）中展示了我的CRISPR敲除筛选 细胞与野生型（Rad 52 WT）比较，以鉴定与Rad 52合成致死的因子（在此定义为 健身）。然后，我提出了二次筛选，确定了三个最大的命中造成增加持久的DNA Rad 52 KO与Rad 52 WT：ERCC 6L/PICH、DHX 9和GLE 1中的损伤和活力丧失。根据这些数据，我 总体假设是有丝分裂的关键调节因子（PICH）和RNA代谢因子（DHX 9和GLE 1）是 由于依赖于Rad 52来解决来自不同来源的复制应激，因此Rad 52具有合成致死性。目的 1.确定Rad 52与PICH的综合致死关系。Rad 52通过以下途径保护基因组稳定性 在MiDAS和抑制复制应激中的作用。PICH介导后期超细桥的拆分 （UFB），一个单独的途径，以减轻复制压力。目标1a：我认为这两条途径部分是 在防止与复制应激相关的遗传毒性损伤积累方面是多余的。所以，我认为，PICH- 在Rad 52 KO细胞中UFB将升高，相反，PICH的消耗将导致Rad 52升高 募集到有丝分裂细胞中的复制应激，以及MiDAS。目标1b：我还重申，复制强调， 持续存在，直到有丝分裂是缺乏Rad 52和PICH的细胞中持续DNA损伤的来源。我将通过 测定磷酸化RPA 2（pRPA）、γ H2 AX和FANCD 2在有丝分裂细胞中的定位。目标2：定义 Rad 52与RNA代谢因子DHX 9和GLE 1的合成致死关系DHX 9和GLE 1 已显示抑制RNA-DNA杂合体（R环）。因此，我假设R环相关的复制 胁迫是这些基因和Rad 52之间的合成致死性的基础。我将分析这些物质的消耗 基因增加R环，导致有丝分裂复制应激水平升高（即Rad 52聚集到病灶中， MiDAS、PICH-UFB和目的1b）中描述的复制应激指标。总之，这些研究将 深入了解这些基因的缺失如何依赖于Rad 52介导的复制缓解 有丝分裂中的应力（即，MiDAS），增强我们对基因组维持机制的理解， 确定Rad 52抑制剂的肿瘤特异性弱点的长期目标。