Novel DNA damage response gene from genomic screening

项目摘要 电离辐射（IR）与人体健康之间的关系是矛盾的; IR既是一种潜在的 致癌物和用于治疗癌症的高效治疗剂。该提案利用了这一点 关系--利用罕见的、遗传上对辐射敏感的个体的基因组测序来鉴定新基因 在细胞对IR的反应中起作用，然后靶向这些基因的产物进行功能研究， 初步药物开发。由于在这些个体中发现的突变赋予了辐射敏感性 在不显著影响生存的情况下，所鉴定的基因可能构成特别有利的靶点， 设计药物使肿瘤放射敏感同时限制对正常组织的细胞毒性。 25年来，我们一直致力于阐明人类辐射敏感性的遗传学， 对表现出极端IR超敏性的个体进行研究，作为鉴定最具IR超敏性的基因的一种手段。 对细胞DNA损伤反应的显著影响。在这些研究的过程中， 患者已被转介到我们的诊断测试共济失调毛细血管扩张症，奈梅亨断裂综合征 或连接酶IV综合征。从这些患者中建立的细胞系显示出辐射超敏性 在克隆形成存活测定中，但在ATM、NBN或LIG 4中没有携带致病突变。初步 我们利用全外显子组测序的研究，在一些受试者中， 已知参与DNA损伤反应的基因，例如ERCC 6和MCPH 1。更有趣的是， 然而，有5个基因的致病突变被发现，并证实是放射增敏，但其 在已建立的DNA损伤反应途径中的作用是未知的和未预料到的。 在本申请中，我们建议使用全基因组测序来建立这些初步发现 (WGS)为了在剩余的未测序的放射敏感细胞中捕获更大部分的致病变体 线，改进的生物信息学工具来过滤这些变体，以及高通量功能筛选测定， 有效地识别它们影响的基因。功能研究将应用于我们拥有的两个新基因 因为它们显示出作为潜在的化学放射增敏靶点的前景。第一个是MTPAP， 是一种非典型的多聚腺苷酸聚合酶和末端尿苷酰转移酶，当突变或敲低时， 放射增敏的新机制，涉及破坏组蛋白稳态。第二个基因ATIC 编码催化嘌呤从头合成的最后两个步骤的双功能酶。敲减或 ATIC的化学抑制耗尽细胞ATP，改变细胞的细胞周期分布，并导致 增加DNA双链断裂和照射后受损的存活率。对于这两个分子中的每一个 我们将确定它们在电离辐射暴露的细胞反应中的作用机制，并评估 我们已经从计算机筛选中获得的小分子抑制剂的疗效， 这些辐射敏感的表型。

Project Abstract The relationship between ionizing radiation (IR) and human health is paradoxical; IR is both a potent carcinogen and a highly effective therapeutic agent for the treatment of cancers. This proposal exploits this relationship—utilizing genome sequencing of rare, genetically radiosensitive individuals to identify novel genes that act in the cellular response to IR and then targeting the products of these genes for functional studies and preliminary drug development. Since the mutations identified in these individuals confer radiation sensitivity without significantly compromising survival, the genes identified may constitute particularly favorable targets for the design of drugs intended to radiosensitize tumors while limiting cytotoxicity to normal tissue. For more than 25 years we have worked to elucidate the genetics of human radiation sensitivity, focusing on individuals displaying extremes of IR hypersensitivity as a means of identifying genes with the most significant effects on cellular DNA damage responses. Over the course of these studies, a steady stream of patients has been referred to us for diagnostic testing for Ataxia-telangiectasia, Nijmegen Breakage Syndrome or Ligase IV Syndrome. Cell lines established from some of these patients displayed radiation hypersensitivity in clonogenic survival assays but did not harbor causative mutations in ATM, NBN or LIG4. In preliminary studies we have utilized whole exome sequencing which, in some subjects, revealed deleterious alleles at genes known to be involved in DNA damage responses, such as ERCC6 and MCPH1. More intriguing, however, were 5 genes where causative mutations were found and confirmed to be radiosensitizing, but whose roles in established DNA damage response pathways were both unknown and unanticipated. In this application we propose to build upon these preliminary findings using whole genome sequencing (WGS) to capture a greater fraction of the causative variants in the remaining unsequenced radiosensitive cell lines, improved bioinformatic tools to filter those variants, and high throughput functional screening assays to efficiently identify the genes they impact. Functional studies will be applied to two of the novel genes we have identified because they show promise as potential chemoradiosensitization targets. The first of these, MTPAP, is a non-canonical poly-A polymerase and terminal uridyltransferase, which, when mutated or knocked down, radiosensitizes by a novel mechanism involving disruption of histone homeostasis. The second gene, ATIC, encodes a bifunctional enzyme that catalyzes the final two steps of de novo purine synthesis. Knockdown or chemical inhibition of ATIC depletes cellular ATP, alters the cell cycle distribution of cells and results in increased DNA double-strand breaks and impaired survival after irradiation. For each of these two molecules we will define their mechanisms of action in the cellular response to ionizing radiation exposure and evaluate the efficacy of small molecule inhibitors which we have already obtained from in silico screening to reproduce these radiosensitive phenotypes in cells in culture.