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CTDP1 Regulates FANCI and the Response to DNA Interstrand Crosslinks

CTDP1 调节 FANCI 和对 DNA 链间交联的反应

基本信息

批准号：
10117104
负责人：
Nicholas Taylor Woods
金额：
$ 33.35万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-16 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10117104
关键词：
Address Affect Antibodies BRCA1 gene Biological Assay Breast Breast Cancer Cell Breast Cancer Model Breast Cancer cell line C-terminal CHEK1 gene CRISPR/Cas technology Cell Cycle Cells Chromatin Cisplatin Clinical Complement Complex Crosslinker DNA Binding DNA Damage DNA Interstrand Crosslinking DNA Repair DNA Repair Gene DNA replication fork Development Event FANCD2 protein Fanconi Anemia pathway Fanconi anemia protein Fanconi&apos s Anemia Fibroblasts Gamma-H2AX Growth HCT116 Cells Human Impairment Knock-out Lead Lesion Malignant Neoplasms Mass Spectrum Analysis Mediating Melphalan Mitomycin C Molecular Target Nebraska Neoplasm Metastasis Patients Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Process Protein Dephosphorylation Proteins Proteome RNA Polymerase II Recovery Recycling Regulation Reporting Repression Research Resistance Resolution Resources Role S Phase Site Specificity Therapeutic Western Blotting Xenograft Model cancer cell chemotherapy colon cancer cell line crosslink experimental study high throughput screening homologous recombination improved in vivo in vivo imaging inhibitor/antagonist knock-down malignant breast neoplasm mutant novel orthotopic breast cancer overexpression phosphoproteomics prevent refractory cancer research and development response small molecule inhibitor trait tumor virtual

项目摘要

Project Summary/Abstract: CTDP1 Regulates FANCI and the Response to DNA Interstrand Crosslinks RNA polymerase II subunit A C-terminal domain phosphatase, CTDP1, is the only phosphatase in the human proteome that contains a BRCA1 C-Terminal (BRCT) domain. This modular domain only exists in a small and specialized group of proteins that mediate the DNA damage response (DDR), but the role of CTDP1 in this process has not been delineated. Analysis of the BRCT interactome has identified protein interactions that occur between CTDP1 and the Fanconi anemia (FA) protein FANCI, which is required for the resolution of DNA interstrand crosslink (ICL) lesions and resistance to ICL-inducing agents mitomycin c and cisplatin. Preliminary results indicate that CTDP1 expression promotes FANCI activation and represses cellular sensitivity to the ICL-inducing compounds mitomycin c and melphalan. Repression of CTDP1 expression is also associated with an increased accumulation of cells in S-phase of the cell cycle under both normal growth conditions and in response to ICL. CTDP1 expression is required to facilitate ICL-induced FANCI interactions with DNA repair proteins, including the DNA damage foci protein γH2AX. These results represent the first reported protein interaction between FANCI and a phosphatase that can regulate its function. Thus, the central hypothesis of this research is that CTDP1 is required for the cellular response to ICL damage through the regulation of FANCI activation, and that targeted inhibition of CTDP1 can sensitize cells to ICL-causing chemotherapeutics. The Specific Aims of this project are: 1) To characterize the impact of CTDP1-mediated FANCI phosphorylations on FA pathway response to ICL; 2) To characterize the impact of CTDP1 inhibition on ICL sensitivity in an orthotopic model of breast cancer; and 3) To identify small molecule inhibitors of CTDP1 using high-throughput screening. These experiments will utilize phosphoproteomics to characterize the temporal regulation of phosphorylation sites on FANCI regulated by CTDP1 following DNA damage. Xenograft models of breast cancer will be used to determine the effects of CTDP1 inhibition on cancer growth, metastasis, and response to cisplatin using in vivo imaging. Virtual and physical high-throughput screening of potential CTDP1 BRCT-FANCI protein interaction disrupting compounds will be completed, and selected lead compounds will be validated western blot analysis of CTDP1 regulated phosphorylation events on FANCI and RPB1. Successful completion of this research will lead to a detailed understanding of the CTDP1-mediated regulation of FANCI and the ICL DNA damage response, which could be used to sensitize crosslinker resistant cancers to DNA damaging therapies.

项目摘要/摘要：CTDP1 调节 FANCI 和对 DNA 链间的反应交联 RNA 聚合酶 II 亚基 A C 末端结构域磷酸酶 CTDP1 是 RNA 聚合酶 II 亚基 A C 端结构域磷酸酶含有 BRCA1 C 端 (BRCT) 结构域的人类蛋白质组。该模块化域仅存在于介导 DNA 损伤反应 (DDR) 的一小群专门蛋白质，但其作用 CTDP1在此过程中尚未被圈定。 BRCT 相互作用组分析已鉴定出蛋白质 CTDP1 和范可尼贫血 (FA) 蛋白 FANCI 之间发生的相互作用，FANCI 是 DNA 链间交联 (ICL) 损伤的解决和对 ICL 诱导剂丝裂霉素的耐药性 c和顺铂。初步结果表明 CTDP1 表达促进 FANCI 激活并抑制细胞对 ICL 诱导化合物丝裂霉素 c 和美法仑的敏感性。镇压 CTDP1 表达还与细胞周期 S 期细胞积累的增加相关在正常生长条件下和对 ICL 的反应下。 CTDP1表达需要促进 ICL 诱导 FANCI 与 DNA 修复蛋白（包括 DNA 损伤灶蛋白 γH2AX）相互作用。这些结果代表了 FANCI 和磷酸酶之间首次报道的蛋白质相互作用，该相互作用可以调节其功能。因此，本研究的中心假设是 CTDP1 是细胞通过调节 FANCI 激活对 ICL 损伤做出反应，并针对性抑制 CTDP1 可以使细胞对引起 ICL 的化疗药物敏感。该项目的具体目标是： 1) 描述 CTDP1 介导的 FANCI 磷酸化对 FA 途径对 ICL 反应的影响； 2）表征乳腺癌原位模型中 CTDP1 抑制对 ICL 敏感性的影响； 3) 通过高通量筛选鉴定 CTDP1 的小分子抑制剂。这些实验将利用磷酸蛋白质组学来表征磷酸化的时间调节 DNA 损伤后 FANCI 上的位点受 CTDP1 调节。乳腺癌的异种移植模型将用于确定 CTDP1 抑制对癌症生长、转移和顺铂反应的影响使用体内成像。潜在 CTDP1 BRCT-FANCI 的虚拟和物理高通量筛选蛋白质相互作用破坏化合物将被完成，选定的先导化合物将被验证了 CTDP1 调节 FANCI 和 RPB1 磷酸化事件的蛋白质印迹分析。成功完成这项研究将有助于详细了解 CTDP1 介导的 FANCI 和 ICL DNA 损伤反应的调节，可用于使交联剂敏化对 DNA 损伤疗法产生耐药性的癌症。