Ras18-mediated Fanconi Anemia pathway activation in response to camptothecin

Ras18 介导的范可尼贫血途径响应喜树碱的激活

• 批准号: 8297266
• 负责人: Komaraiah Palle
• 金额: $ 3.31万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8297266
• 关键词: Address; Affect; Agreement; Antineoplastic Agents; Biochemical; Biochemical Genetics; Biological Assay; Camptothecin; Camptothecin Analogue; Cells; Chromatin; Comb animal structure; Combined Modality Therapy; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA repair protein; Double Strand Break Repair; Drug Delivery Systems; Enzymes; Fanconi' s Anemia; Gene Components; Genome; Goals; Human; Lead; Lesion; Malignant Neoplasms; Mediating; Molecular; Molecular Target; Mono-S; Mutagens; Nuclear Antigens; Outcome; Outcome Study; Pathway interactions; Pharmaceutical Preparations; Play; Predisposition; Proteins; Proteomics; Recovery; Recruitment Activity; Reporter; Resistance; Role; S Phase; Signal Transduction; Sister Chromatid Exchange Assay; Stress; Syndrome; System; Techniques; Testing; Therapeutic; Topotecan; Treatment Efficacy; Type I DNA Topoisomerases; Ubiquitination; Work; adduct; analog; crosslink; cytotoxic; cytotoxicity; design; homologous recombination; human TOP1 protein; inhibitor/antagonist; irinotecan; killings; mutant; neoplastic cell; new therapeutic target; novel; repaired; research study; response; success; therapeutic target; tumor; ubiquitin-protein ligase; ultraviolet irradiation

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.

描述(申请人提供)：DNA拓扑异构酶I(Top1)是人类的一种基本酶，是抗癌药物喜树碱(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抑制剂显示出广谱的抗肿瘤活性，但由于大多数原因不明，这些药物的治疗效果差异很大。DNA损伤诱导药物的细胞毒性可以通过抑制DNA损伤反应和修复反应途径来增强。我们研究的长期目标是确定Top1抑制剂诱导的损伤修复的分子机制，目的是寻找新的药物靶点，通过联合治疗有效地杀死肿瘤细胞。在这个方向上，我们的研究已经确定了E3泛素连接酶Rad18在激活FA途径修复和细胞对CPT诱导的损伤耐受中的一个新的作用。然而，在CPT诱导的DNA损伤的特定背景下，Rad18激活FA途径及其可能底物的机制尚不清楚。此外，已知FA通路被复制应激激活，但其在CPT引起的DNA损伤反应中的确切作用尚不清楚。该方案的具体目标1将集中在确定Rad18和FA通路在CPT诱导的DSB修复中的确切作用。诸如DR-GFP报告系统和姐妹染色单体交换分析等技术将被用来解决这些问题。利用不同的生化和蛋白质组学方法，具体目标2将集中于确定Rad18激活FA途径的机制，并识别可能负责CPT激活FA途径的Rad18底物。本方案的具体目标3将利用最先进的生化和DNA结合分析方法，重点确定RAD18和FA通路在CPT诱导的S期检查点细胞恢复中的作用。由于Rad18是这些通路的关键调节因子，这些拟议研究的结果将为利用Rad18作为与Top1抑制剂联合治疗的靶点奠定基础。此外，我们建议鉴定的假定的RAD18底物可能代表新的肿瘤抑制机制，并可能导致新的治疗靶点的发现。 公共卫生相关性：这项提案的长期目标是了解抗癌药物喜树碱修复DNA损伤的分子机制。这些研究的结果可能确定新的治疗靶点，通过与Top1抑制剂的联合治疗有效地杀死肿瘤细胞，并可能确定新的肿瘤抑制机制。