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

总结。针对DNA依赖蛋白激酶催化亚单位(DNAPKcs)的激酶抑制剂 正在开发用来使肿瘤对分裂(染色体断裂)因素敏感的药物，如放射治疗。 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 已经上报了。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 能够确定以下情况(例如，肿瘤遗传背景和治疗方案) DNAPKcs激酶抑制剂是最有效的，从而开发出预测肿瘤反应的生物标记物。 长期以来，DNAPKcs一直被认为是促进辐射抗性的重要因素，但它在染色体上的作用 断裂的修复仍然难以捉摸。也就是说，DNAPKcs与正则非同源中的因素相关联 DNA双链断裂(DSB)修复的末端连接(C-NHEJ)途径，但不是所有C-NHEJ所必需的 事件(例如，对于V(D)J重组信号Ej基本上是可有可无的)。此外，直到最近，具体的 除了V(D)J重组，还没有检测染色体DSB的C-NHEJ的方法，因为 部分冗余与替代EJ(ALT-EJ)途径。为了解决这一技术差距，我们的实验室 最近发现了C-NHEJ的一个特殊特征。也就是说，我们报告了几个C-NHEJ因子(即XLF， KU70和XRCC4)是钝端DSB(由Cas9核酸酶诱导)之间EJ所必需的 在不引起插入/缺失突变(INDELs)的情况下连接，即为了获得准确/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，但它的作用在有XLF的细胞中被显著放大 功能减退突变(亚型)，包括对结合KU重要的XLF基序突变 异源二聚体和DNA。在这些发现的基础上，我们将确定DNAPKcs在染色体EJ和 抗碎裂剂。在目标1中，我们假设XLF和DNAPKcs协同作用以确保没有 Indel EJ，这种协同作用对破碎剂的抗性至关重要。我们还将研究它们之间的相互作用 DNA复制中的两个因素，并定义了XLF和DNAPKcs的关键结构域，对其中的每一个都很重要 功能。在目标2中，我们研究了DNAPKcs与一组KU结合因子之间的相互作用，以及 Alt-EJ，对裂解原抗性和基因组稳定性的影响。我们测试DNAPKcs是否协同工作 仅与XLF结合，但也与四个KU结合因子(CYREN/MRI、PAXX、APLF和WRN)中的每一个结合，用于No Indel EJ、碎裂剂抗性和KU对DNA损伤的强势募集。这些研究将填补我们在 对C-NHEJ机制的理解。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，从而导致大量删除和/或持久的、未修复的DSB 导致克隆存活率降低和/或裂解原致敏。总而言之，我们将测试总体 假设DNAPKcs促进了准确的EJ(即没有Indel EJ)，即它在这样的EJ中的作用被缺陷放大 在一组KU结合因子中，它对ALT-EJ缺陷细胞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.