Ras18-mediated Fanconi Anemia pathway activation in response to camptothecin

描述（由申请人提供）：DNA拓扑异构酶I（Top1）是人体必需的酶，是抗癌剂喜树碱（CPT）及其类似物（拓扑替康和伊立替康）的分子靶标，用于治疗不同的癌症。喜树碱通过将Top1-cc转化为复制介导的双链断裂（DSB）来可逆地稳定Top1-cc。尽管Top1抑制剂显示出广谱抗肿瘤活性，但由于大多数不确定的原因，这些药物的治疗功效高度可变。DNA损伤诱导药物的细胞毒性可以通过抑制DNA损伤反应和对它们作出反应的修复途径来增强。我们研究的长期目标是确定Top1抑制剂诱导的病变修复中涉及的分子机制，目的是确定新的药物靶点，以通过联合治疗有效杀死肿瘤细胞。在这个方向上，我们的研究已经确定了一个新的作用，E3泛素连接酶Rad 18在激活FA通路的修复和细胞耐受CPT诱导的损伤。然而，Rad 18在CPT诱导的DNA损伤的特定背景下激活FA途径及其推定底物的机制尚不清楚。此外，已知FA途径被复制应激激活，但其在CPT引起的DNA损伤反应中的确切作用尚不清楚。本提案的具体目标1将集中于确定Rad 18和FA通路在CPT诱导的DSB修复中的确切作用。将使用DR-GFP报告系统和姐妹染色单体交换试验等技术来解决这些问题。具体目标2将使用各种生物化学和蛋白质组学方法，重点确定Rad 18激活FA通路的机制，并确定负责激活FA通路的假定Rad 18底物。本提案的具体目标3将集中于通过使用最先进的生物化学和DNA组合测定法来确定Rad 18和FA途径在CPT诱导的S期检查点的细胞恢复中的作用。由于Rad 18是这些途径的关键调节因子，因此这些拟议研究的结果将为利用Rad 18作为与Top1抑制剂联合治疗的治疗靶点奠定基础。此外，我们提出要鉴定的推定Rad 18底物可能代表新的肿瘤抑制机制，并可能导致发现新的治疗靶点。 公共卫生相关性：这项计划的长期目标是了解修复抗癌药物喜树碱诱导的DNA损伤的分子机制。这些研究的结果可能会发现新的治疗靶点，通过与Top1抑制剂的联合治疗有效地杀死肿瘤细胞，并可能发现新的肿瘤抑制机制。

DESCRIPTION (provided by applicant): DNA topoisomerase I (Top1) is an essential enzyme in humans and is the molecular target of the anticancer agent camptothecin (CPT), and its analogues (topotecan and irinotecan) that are used to treat different cancers. Camptothecins reversibly stabilize Top1-cc by converting them into replication mediated double strand breaks (DSB). Although Top1 inhibitors show broad spectrum antitumor activity, the therapeutic efficacies of these agents are highly variable due to mostly undefined reasons. The cytotoxicity of DNA damage inducing drugs can be enhanced by inhibiting the DNA damage response and repair pathways that respond to them. The long-term objective of our studies is to define the molecular mechanisms involved in repair of Top1-inhibitors induced lesions with the goals of identifying novel drug targets to effectively kill tumor cells by combination therapies. In this direction, our studies have identified a novel role for E3 ubiquitin ligase Rad18 in activation of FA pathway in repair and cell tolerance to CPT-induced damage. However, the mechanisms by which Rad18 activates FA pathway and its putative substrates in the specific context of CPT- induced DNA damage are unknown. Moreover, the FA pathway is known to be activated by replication stress, but its precise roles in DNA damage response to CPT are unknown. The specific aim 1 of this proposal will focus on determining the precise roles of Rad18 and FA pathway in repair of CPT-induced DSB. Techniques such as DR-GFP reporter system and sister chromatid exchange assays will be used to address these questions. Using various biochemical and proteomic approaches, the specific aim 2 will focus on determining the mechanisms by which Rad18 activates FA pathway and identifying the putative Rad18 substrates that are responsible for the activation of FA pathway in response to CPT. The specific aim 3 of this proposal will focus on determining roles of Rad18 and FA pathway in cell recovery from CPT induced S-phase checkpoint, by using state of the art biochemical and DNA combing assays. Since Rad18 is a critical regulator of these pathways, the outcomes from these proposed studies will establish a rationale for exploiting Rad18 as a therapeutic target in combination therapies with Top1 inhibitors. Moreover, the putative Rad18 substrates that we propose to identify may represent novel tumor suppressive mechanisms and can potentially lead to discovery of novel therapeutic targets. PUBLIC HEALTH RELEVANCE: The long term goal of this proposal is to understand the molecular mechanisms that repair DNA damage induced by anticancer drug camptothecin. The outcomes from these studies may identify novel therapeutic targets to effectively kill tumor cells by combination therapies with Top1 inhibitors and may identify a novel tumor suppressive mechanism.