Elucidating the role of DNAPKcs in chromosomal break end joining and clastogen resistance

阐明 DNAPKcs 在染色体断裂末端连接和断裂剂抗性中的作用

• 批准号: 10296356
• 负责人: Jeremy Michael Stark
• 金额: $ 40.26万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10296356
• 关键词: Address; Binding; Biological Assay; Biological Markers; Catalytic Domain; Cells; Chromosomal Breaks; Chromosomes; Clinical; Clinical Trials; Code; Combined Modality Therapy; Complex; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Damage; DNA Double Strand Break; DNA biosynthesis; DNA replication fork; DNA-dependent protein kinase; Data; Defect; Double Strand Break Repair; Ensure; Event; Fibrinogen; G22P1 gene; Genetic; Genome; Genome Stability; Genomic Instability; Human; Ionizing radiation; Laboratories; Lasers; Ligase; Magnetic Resonance Imaging; Maintenance; Measures; Mediating; Mission; Modeling; Mutation; Nonhomologous DNA End Joining; Outcome; PRKDC gene; PTEN gene; Pathway interactions; Patient-Focused Outcomes; Phosphotransferases; Play; Radiation therapy; Regulation; Reporting; Resistance; Ribosomal RNA; Role; Series; Signal Transduction; Site; Technology; Testing; Time; Treatment Protocols; Treatment outcome; United States National Institutes of Health; V(D)J Recombination; XRCC4 gene; biomarker development; cancer cell; cancer therapy; clastogen; experimental study; improved; inhibitor/antagonist; innovation; insertion/deletion mutation; insight; kinase inhibitor; mutant; novel; nuclease; radiation resistance; recruit; repaired; replication stress; response; response biomarker; scaffold; synergism; therapeutic target; treatment strategy; tumor

SUMMARY. Kinase inhibitors targeting the catalytic subunit of the DNA-dependent protein kinase (DNAPKcs) are being developed to sensitize tumors to clastogenic (chromosomal-breaking) agents, such as radiotherapy. The DNAPKcs kinase inhibitor M3814 (EMD Serono) is in clinical trials, and other new potent inhibitors have been reported. Elucidating the role of DNAPKcs in the repair of chromosomal breaks is significant because it will enable the identification of the contexts (e.g. tumor genetic background and treatment regimens) in which DNAPKcs kinase inhibitors are most effective, and thereby develop biomarkers to predict tumor response. DNAPKcs has long been recognized as important for promoting radioresistance, but its role in chromosomal break repair has remained elusive. Namely, DNAPKcs associates with factors in the canonical non-homologous end joining (C-NHEJ) pathway for DNA double-strand break (DSB) repair but is not required for all C-NHEJ events (e.g. is largely dispensable for V(D)J recombination signal EJ). Furthermore, until recently, specific assays to detect C-NHEJ of chromosomal DSBs, apart from V(D)J recombination, have not been available, due to partial redundancy with the alternative EJ (ALT-EJ) pathway. To address this gap in technology, our laboratory recently identified a specific hallmark of C-NHEJ. Namely, we reported that several C-NHEJ factors (i.e. XLF, KU70, and XRCC4) are required for EJ between blunt-ended DSBs (induced by the Cas9 nuclease) that are joined without causing insertion/deletion mutations (indels), i.e. for accurate/No Indel EJ. In preliminary data, we show that DNAPKcs is partially required for No Indel EJ, but its role is substantially magnified in cells with XLF reduced-function mutations (hypomorphs), including mutations in an XLF motif important for binding the KU heterodimer and DNA. Building on these findings, we will define the role of DNAPKcs in chromosomal EJ and clastogen resistance. In Aim 1, we hypothesize that XLF and DNAPKcs function synergistically to ensure No Indel EJ, and this synergy is critical for clastogen resistance. We will also examine the interplay between these two factors in DNA replication, and define the key domains of XLF and DNAPKcs important for each of these functions. In Aim 2, we investigate the interplay between DNAPKcs and a set of KU-binding factors, as well as ALT-EJ, on clastogen resistance and genome stability. We test whether DNAPKcs functions synergistically not only with XLF, but also with each of four KU-binding factors (CYREN/MRI, PAXX, APLF, and WRN), for No Indel EJ, clastogen resistance, and robust recruitment of KU to DNA damage. These studies will fill a major gap in our understanding of C-NHEJ mechanisms. Finally, we posit that combined disruption of DNAPKcs and ALT-EJ causes aberrant DSB end processing, leading to large deletions, and/or persistent, unrepaired DSBs, thereby causing reduced clonogenic survival and/or clastogen sensitization. In summary, we will test the overall hypothesis that DNAPKcs promotes accurate EJ (i.e. No Indel EJ), that its role in such EJ is magnified by defects in a set of KU-binding factors, and that it is critical for genome stability in cells deficient in ALT-EJ.

概括。针对 DNA 依赖性蛋白激酶 (DNAPKcs) 催化亚基的激酶抑制剂 正在开发使肿瘤对染色体断裂剂（例如放射疗法）敏感的药物。 DNAPKcs激酶抑制剂M3814（EMD Serono）正处于临床试验阶段，其他新型强效抑制剂也已上市 被举报。阐明 DNAPKcs 在染色体断裂修复中的作用具有重要意义，因为它将 能够识别背景（例如肿瘤遗传背景和治疗方案） DNAPKcs 激酶抑制剂是最有效的，因此可以开发生物标志物来预测肿瘤反应。 DNAPKcs 长期以来一直被认为对于促进放射抗性很重要，但它在染色体中的作用 断裂修复仍然难以实现。即，DNAPKcs 与典型的非同源因子中的因子相关联。 DNA 双链断裂 (DSB) 修复的末端连接 (C-NHEJ) 途径，但并非所有 C-NHEJ 都需要 事件（例如对于 V(D)J 重组信号 EJ 来说很大程度上是可有可无的）。此外，直到最近，具体 除 V(D)J 重组外，还没有检测染色体 DSB 的 C-NHEJ 的方法，因为 与替代 EJ (ALT-EJ) 途径部分冗余。为了解决这一技术差距，我们的实验室 最近发现了 C-NHEJ 的一个特定标志。也就是说，我们报告了几个 C-NHEJ 因素（即 XLF、 平端 DSB（由 Cas9 核酸酶诱导）之间的 EJ 需要 KU70 和 XRCC4），这些 DSB 是 加入时不会引起插入/删除突变 (indels)，即准确/无 Indel EJ。在初步数据中，我们 表明 DNAPKcs 对于 No Indel EJ 是部分必需的，但其作用在具有 XLF 的细胞中显着放大 功能减少突变（亚型），包括对结合 KU 很重要的 XLF 基序中的突变 异二聚体和DNA。基于这些发现，我们将定义 DNAPKcs 在染色体 EJ 和 断裂剂抗性。在目标 1 中，我们假设 XLF 和 DNAPKcs 协同作用以确保不 Indel EJ，这种协同作用对于断裂剂耐药性至关重要。我们还将研究这些之间的相互作用 DNA 复制中的两个因素，并定义了对每个因素都很重要的 XLF 和 DNAPKcs 的关键域 功能。在目标 2 中，我们研究了 DNAPKcs 和一组 KU 结合因子之间的相互作用，以及 ALT-EJ，关于断裂剂抗性和基因组稳定性。我们测试 DNAPKcs 是否具有协同作用 仅适用于 XLF，还适用于四种 KU 结合因子（CYREN/MRI、PAXX、APLF 和 WRN）中的每一种，用于无插入缺失 EJ、断裂剂抗性以及 KU 对 DNA 损伤的强力募集。这些研究将填补我们的一个重大空白 了解 C-NHEJ 机制。最后，我们假设 DNAPKcs 和 ALT-EJ 的联合破坏 导致异常的 DSB 结束处理，导致大量删除和/或持久的、未修复的 DSB，从而 导致克隆形成存活率降低和/或断裂剂致敏。综上，我们将测试整体 假设 DNAPKcs 促进准确的 EJ（即无 Indel EJ），其在此类 EJ 中的作用因缺陷而被放大 一组 KU 结合因子，对于缺乏 ALT-EJ 的细胞的基因组稳定性至关重要。