Defining the role of chromosomal break end synapsis factors for DNA repair

定义染色体断裂末端突触因子在 DNA 修复中的作用

基本信息

批准号：
10743770
负责人：
Metztli Cisneros
金额：
$ 3.32万
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：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10743770
关键词：
Address Affect Binding Biological Assay Catalytic Domain Cell Line Chromosomal Breaks Chromosome Pairing Clinical Trials Complex DNA DNA Binding DNA Double Strand Break DNA Repair DNA-dependent protein kinase Defect Development Distal Double Effect Double Strand Break Repair Event Excision Frequencies G22P1 gene Ionizing radiation Knock-out Knowledge Learning Link Liquid substance Malignant Neoplasms Measures Mediating Mission Molecular Genetics Mutation Nonhomologous DNA End Joining Outcome Pathway interactions Patient-Focused Outcomes Phase Phosphotransferases Poison Postdoctoral Fellow Publications Publishing Radiation therapy Regulation Research Research Personnel Research Project Grants Research Proposals Role Single-Stranded DNA Site Synapses Techniques Testing Therapeutic Topoisomerase Training Treatment outcome United States National Institutes of Health Work XRCC4 gene cancer cell cancer clinical trial cancer therapy chromatin immunoprecipitation clastogen genetic approach improved inhibitor insertion/deletion mutation insight mutant nuclease p53-binding protein 1 protein kinase inhibitor repaired response skills small molecule inhibitor targeted cancer therapy therapeutic target treatment response

项目摘要

SUMMARY. Clastogenic cancer therapeutics, i.e., agents that induce chromosomal DNA double-strand breaks (DSBs), such as radiotherapy, remain a cornerstone of cancer treatment. A central aspect of DSB repair is synapsis of the two DSB ends to mediate repair, such that DSB end synapsis is a potential therapeutic target. Indeed, one factor important for DSB end synapsis, DNA-dependent protein kinase (DNA-PKcs), is the target of several inhibitors. This includes M3814/Peposertib (EMD Sereno), which is being tested in clinical trials. However, at least two other factors, XLF and 53BP1, have also been implicated in DNA end synapsis during repair. This suggests there may be some redundancy between these factors that could influence response to inhibitors of DNA-PKcs. In this research proposal, I will investigate the role of end synapsis factors on end joining (EJ) repair, and the regulation of DNA end resection. Aim 1: Define the role of end synapsis factors on EJ. Aim 1a (Completed): EJ without insertions or deletions (No Indel EJ) is a repair outcome that represents high-fidelity EJ, which requires factors involved in canonical non-homologous end joining (C-NHEJ), including KU70 and XRCC4. I sought to define the relative influence of two end synapsis factors (DNA-PKcs and XLF) on No Indel EJ. I found that DNA-PKcs and XLF promote No Indel EJ, however loss of XLF caused a much greater decrease in No Indel EJ compared to DNA-PKcs. Importantly, I found that disrupting DNA-PKcs (knockout or treatment with inhibitor) when combined with XLF mutations that weaken interaction interfaces, caused a synergistic loss of No Indel EJ. Thus, the conclusion of my published study is that the role of DNA-PKcs is magnified to promote No Indel EJ when XLF is weakened. Aim 1b (F99 Phase). Building on these findings, I will investigate another end synapsis factor, 53BP1. Specifically, I will address the hypothesis that 53BP1 has a partially redundant role with DNA-PKcs and XLF to promote chromosomal EJ. In performing this research, I will expand my skill set in several ways, including learning to assess EJ outcomes using different computational approaches from the completed research. Aim 2 (K00 Phase). In the postdoctoral phase of the research proposal, I will be assessing the role of synapsis factors on cleaving of DNA ends bound to DNA-PKcs to initiate end resection. End resection is the first step of homology-directed repair, and is mediated by the MRN/CtIP nuclease complex. Recently, MRN/CtIP was shown to cleave DNA ends bound to DNA-PKcs, which may be the key initiating step of end resection. I propose to test the hypothesis that disruption of end synapsis causes an increase in this initiation step of end resection, which I will test with several techniques that will expand my research skill set. Altogether, these studies will fill a major gap in our understanding of the role of end synapsis factors on the regulation of DNA DSB repair mechanisms. Such research is significant for developing end synapsis as a target for cancer therapy, including identifying the ideal circumstances to apply inhibitors of DNA-PKcs (e.g. M3814/Peposertib) during cancer treatment.

概括。断裂基因癌症疗法，即诱导染色体 DNA 双链断裂的药物（DSB），例如放射治疗，仍然是癌症治疗的基石。 DSB 修复的一个核心方面是两个 DSB 末端的突触介导修复，因此 DSB 末端突触是潜在的治疗靶点。事实上，DNA 依赖性蛋白激酶 (DNA-PKcs) 对 DSB 末端突触很重要，它是几种抑制剂。其中包括正在临床试验中进行测试的 M3814/Peposertib (EMD Sereno)。然而，至少有两个其他因素，XLF 和 53BP1，也与 DNA 末端突触有关。维修。这表明这些因素之间可能存在一些冗余，可能会影响对 DNA-PKcs 抑制剂。在本研究计划中，我将研究末端突触因素对末端连接的作用 (EJ)修复，以及DNA末端切除的调节。目标 1：定义末端突触因子对 EJ 的作用。目的 1a（Completed）：没有插入或删除的EJ（No Indel EJ）是代表高保真的修复结果 EJ，需要参与规范非同源末端连接 (C-NHEJ) 的因素，包括 KU70 和 XRCC4。我试图定义两个末端突触因子（DNA-PKcs 和 XLF）对 No Indel 的相对影响 EJ。我发现 DNA-PKcs 和 XLF 促进 No Indel EJ，但是 XLF 的丢失导致了更大的减少与 DNA-PKcs 相比，No Indel EJ。重要的是，我发现破坏 DNA-PKcs（敲除或治疗与抑制剂）当与削弱相互作用界面的XLF突变结合时，导致协同损失无 Indel EJ。因此，我发表的研究的结论是，DNA-PKcs的作用被放大以促进 XLF 减弱时无 Indel EJ。目标 1b（F99 阶段）。基于这些发现，我将调查另一个末端突触因子，53BP1。具体来说，我将提出 53BP1 具有部分冗余作用的假设与 DNA-PKcs 和 XLF 一起促进染色体 EJ。在进行这项研究时，我将扩展我的技能多种方法，包括学习使用不同的计算方法来评估 EJ 结果完成研究。目标 2（K00 阶段）。在研究计划的博士后阶段，我将评估突触因子在切割与 DNA-PKcs 结合的 DNA 末端以启动末端切除中的作用。末端切除是同源定向修复的第一步，由 MRN/CtIP 核酸酶复合物介导。最近， MRN/CtIP 被证明可以切割与 DNA-PKcs 结合的 DNA 末端，这可能是末端的关键起始步骤。切除。我建议测试以下假设：末端突触的破坏会导致这种启动的增加末端切除的步骤，我将使用多种技术进行测试，以扩展我的研究技能。共，这些研究将填补我们对末端突触因子在调节中的作用的理解的重大空白。 DNA DSB 修复机制。此类研究对于开发末端突触作为癌症靶点具有重要意义治疗，包括确定应用 DNA-PKcs 抑制剂的理想环境（例如 M3814/Peposertib）在癌症治疗期间。