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

• 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
• 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

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。层生癌治疗剂，即诱导染色体DNA双链断裂的药物 （DSB），例如放射疗法，仍然是癌症治疗的基石。 DSB维修的一个主要方面是 两个DSB末端的突触介导了修复，因此DSB端突触是潜在的治疗靶标。 实际上，对于DSB末端突触，DNA依赖性蛋白激酶（DNA-PKC）的一个因素是一个重要的因素，是 几种抑制剂。这包括M3814/peposertib（EMD Sereno），正在临床试验中进行测试。 但是，至少有两个其他因素XLF和53BP1也与DNA端突触有关 维修。这表明这些因素之间可能有一些冗余，可能会影响对的反应 DNA-PKC的抑制剂。在这项研究建议中，我将调查最终突触因素在结束上的作用 （EJ）修复和DNA末端切除的调节。目标1：定义最终突触因子在EJ中的作用。目的 1A（完成）：没有插入或删除的EJ（无indel eJ）是代表高保真性的维修结果 EJ，需要参与规范非同源末端加入（C-NHEJ）的因素，包括Ku70和 XRCC4。我试图定义两个末端突触因子（DNA-PKC和XLF）对无indel的相对影响 EJ。我发现DNA-PKC和XLF促进了indel eJ，但是XLF的损失导致更大的减少 与DNA-PKC相比，无indel EJ。重要的是，我发现破坏DNA-PKC（敲除或治疗 使用抑制剂）当与弱化相互作用接口的XLF突变结合时，会导致协同损失 没有Indel EJ。因此，我发表的研究的结论是，DNA-PKC的作用被放大以促进 当XLF削弱时，没有Indel EJ。 AIM 1B（F99阶段）。在这些发现的基础上，我将调查另一个 末端突触因子，53bp1。具体而言，我将解决53BP1具有部分冗余作用的假设 与DNA-PKC和XLF一起促进染色体EJ。在进行这项研究时，我将扩大我的技能 几种方法，包括学习使用不同的计算方法评估EJ结果 完成的研究。 AIM 2（K00相）。在研究建议的博士后阶段，我将评估 突触因子在切割DNA中的作用结束与DNA-PKC结合以启动最终切除。结束切除 是同源指导修复的第一步，由MRN/CTIP核酸酶复合物介导。最近， 显示MRN/CTIP裂解DNA结束与DNA-PKC结合，这可能是终端的关键启动步骤 切除。我建议检验以下假设，即末端突触的破坏会导致这种开始的增加 最终切除的步骤，我将使用几种技术来扩展我的研究技能。共， 这些研究将填补我们对最终突触因素在调节调节中的作用的主要空白 DNA DSB修复机制。这样的研究对于发展最终突触是癌症的靶标意义重大 治疗，包括确定适用DNA-PKC抑制剂的理想情况（例如M3814/peposertib） 在癌症治疗期间。