The effect of cancer-associated missense mutations in XPA on nucleotide excision repair activity and platinum-based chemotherapy response

项目总结/摘要 核苷酸切除修复（NER）是修复由基于铂（Pt）的金属氧化物形成的DNA加合物的主要方法。 化疗，如顺铂。根据NER基因的突变对癌症患者进行分层， NER活性和细胞对铂基化疗药物的敏感性代表了一种有前途的精准医学 战略为了抓住这一机会，本建议的长期目标是：（i）制定一个强有力的 可以从患者基因组数据中获得经验证的表型见解的管道，以及（ii）基准测试 在必需的NER支架蛋白，着色性干皮病互补组A（XPA）上的管道。一 将开发一种计算方法来预测XPA中的错义突变，这些突变通过以下方式破坏NER活性： 整合蛋白质稳定性建模、与已知致病突变的接近度、二级结构 预测和进化保护。XPA中预测的有害突变将得到功能验证 通过体外和基于细胞的NER活性测定，以及通过用顺铂处理后的细胞存活测定。 对于XPA中一个经验证的突变子集，NER功能障碍和顺铂敏感性的机制将是 使用多种生物物理和结构方法测定，包括DNA结合亲和力分析， 二级结构和稳定性、三维结构以及与NER切口前复合体的相互作用 proteins.完成这项提案将提供机制的见解，如何一个子集的错义突变 在来自肿瘤基因组数据的XPA中，破坏NER活性并最终使细胞对顺铂敏感。准确 选择对基于铂的化疗治疗的良好反应者与不良反应者有可能改善患者 生活质量和总体生存率，允许对反应差的患者更早实施替代策略， 减少不必要的护理费用。此外，这一提议将为一项强有力的战略奠定基础 能够从基因组数据中获得对细胞表型的机械见解，可以扩展到所有 核心NER途径蛋白或应用于其他临床相关途径。重要的是，完成这项工作 提案将使申请人具备独特的多功能，多学科技能，这将大大提高 申请人作为未来独立调查员的潜力和职业生涯。选定机构的支持， 赞助商，与共同赞助团队和合作者定期举行进度会议，强大的多层次 指导和参加全国会议将确保申请人接受全面的培训 在这个奖学金期间的经验。

PROJECT SUMMARY/ABSTRACT Nucleotide excision repair (NER) is the primary method to repair DNA adducts formed by platinum (Pt)-based chemotherapies, such as cisplatin. Stratifying cancer patients based on mutations in NER genes that disrupt NER activity and sensitize cells to Pt-based chemotherapeutics represents a promising precision medicine strategy. To address this opportunity, the long-term objectives of this proposal are (i) to develop a robust pipeline that can derive validated, phenotypic insights from patient genomic data, and (ii) benchmark this pipeline on the essential NER scaffold protein, Xeroderma Pigmentosum Complementation Group A (XPA). A computational approach will be developed to predict missense mutations in XPA that disrupt NER activity by integrating protein stability modeling, proximity to known disease-causing mutations, secondary structure prediction, and evolutionary conservation. Predicted deleterious mutations in XPA will be functionally validated by in vitro and cell-based NER activity assays, as well as by cell survival assays after treatment with cisplatin. The mechanism of NER dysfunction and cisplatin sensitivity for a subset of validated mutations in XPA will be determined using multiple biophysical and structural approaches, including analyses of DNA binding affinity, secondary structure and stability, three-dimensional structure, and interactions with NER pre-incision complex proteins. Completion of this proposal will provide mechanistic insights into how a subset of missense mutations in XPA from tumor genomic data disrupt NER activity and ultimately sensitize cells to cisplatin. Accurately selecting good- versus poor-responders to Pt-based chemotherapy treatment has potential to improve patient quality of life and overall survival, allow for earlier implementation of alternative strategies for poor-responders, and decrease unnecessary costs of care. In addition, this proposal will lay the foundation for a robust strategy capable of deriving mechanistic insights into cell phenotype from genomic data, that can be extended to all core NER pathway proteins or applied to other clinically-relevant pathways. Importantly, completion of this proposal will equip the applicant with a uniquely versatile, multidisciplinary skillset that will greatly enhance the applicant’s potential and career as a future independent investigator. Support from the selected institution and Sponsor, regular progress meetings with the co-sponsoring team and collaborators, strong multi-tiered mentoring, and participation at national conferences will ensure the applicant receives a well-rounded training experience for the duration of this fellowship.