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

摘要靶向DNA依赖性蛋白激酶（DNAPKcs）催化亚基的激酶抑制剂 正在开发使肿瘤对致染色体断裂剂（染色体断裂）如放射疗法敏感。 DNAPKcs激酶抑制剂M3814（EMD Serono）正在进行临床试验，其他新的有效抑制剂已经 被举报。阐明DNAPKcs在染色体断裂修复中的作用是重要的，因为它将 能够识别其中的背景（例如肿瘤遗传背景和治疗方案）， DNAPKcs激酶抑制剂是最有效的，从而开发生物标志物来预测肿瘤反应。 长期以来，DNAPKcs被认为对促进辐射抗性是重要的，但其在染色体上的作用， 断裂修复仍然是难以捉摸的。也就是说，DNAPKcs与典型的非同源的 DNA双链断裂（DSB）修复的末端连接（C-NHEJ）途径，但不是所有C-NHEJ都需要 事件（例如，对于V（D）J重组信号EJ而言，在很大程度上是不确定的）。此外，直到最近， 检测染色体DSB的C-NHEJ的测定法，除了V（D）J重组，还没有可用的，由于 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的一个特殊标志。也就是说，我们报道了几种C-NHEJ因子（即XLF， KU 70和XRCC 4）是平端DSB（由Cas9核酸酶诱导）之间的EJ所需的，所述平端DSB是 连接而不引起插入/缺失突变（indel），即准确/无Indel EJ。根据初步数据，我们 显示DNAPKcs是No Indel EJ部分所需的，但其作用在具有XLF的细胞中显著放大 功能降低突变（亚型），包括对KU结合重要的XLF基序突变 异二聚体和DNA。基于这些发现，我们将确定DNAPKcs在染色体EJ中的作用， 断裂剂抗性在目标1中，我们假设XLF和DNAPKcs协同作用，以确保NO Indel EJ，并且这种协同作用对于断裂剂抗性是至关重要的。我们还将研究这些因素之间的相互作用 DNA复制中的两个因素，并定义了XLF和DNAPKcs的关键结构域，这两个结构域对每一个都很重要。 功能协调发展的在目标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是否协同作用， 仅用XLF，但也用四种KU结合因子（CYREN/MRI、PAXX、APLF和WRN）中的每一种，用于无插入缺失 EJ、断裂剂抗性和KU对DNA损伤的强募集。这些研究将填补我们的一个主要空白， 了解C-NHEJ机制。最后，我们证实了DNAPKcs和ALT-EJ的联合破坏， 导致异常的DSB末端加工，导致大的缺失，和/或持久的未修复的DSB，从而 导致克隆形成存活率降低和/或断裂原致敏。总之，我们将测试整体 假设DNAPKcs促进准确的EJ（即无插入缺失EJ），其在这种EJ中的作用被缺陷放大 在一组KU结合因子中，并且它对于ALT-EJ缺陷细胞中的基因组稳定性至关重要。

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.