Investigating the effect of ERCC2 mutations on DNA repair capacity and chemo-radiotherapy response in muscle-invasive bladder cancer

项目摘要 肌层浸润性膀胱癌（MIBC）代表膀胱肿瘤的一个侵袭性子集， 与高死亡率相关，尽管进行了强化的多模态治疗。DNA破坏剂， 顺铂在MIBC和许多其他实体瘤的治疗中发挥关键作用，但经验证的生物标志物 缺乏对DNA损伤治疗的反应。 最近，ERCC 2（核苷酸的核心成员）的体细胞突变之间的关联 切除修复（NER）途径，以及对顺铂为基础的化疗反应的改善， MIBC，代表了肿瘤DNA修复途径之间关联的第一个验证实例之一， 改变和对DNA损伤剂的反应。初步的功能分析表明， ERCC 2突变导致NER能力的丧失;然而，这种关联的功能基础 在肿瘤和临床环境中的差异尚不清楚。该提案旨在描述突变在以下方面的作用： ERCC 2和其他DNA修复基因在MIBC生物学和治疗反应中的作用。 理解新的DNA修复改变的生物学相关性的限制之一是， 肿瘤的最大缺陷是缺乏稳健、有效的测定来测试观察到的突变的功能效应。第一个目标 将采用一种新的高通量，基于荧光的显微镜测定来测量 ERCC 2突变支持细胞NER的能力。该试验将应用于所有ERCC 2突变 在多个MIBC队列中观察到，将在可用治疗的背景下解释结果 缓解和患者结局数据，以确定ERCC 2突变的功能格局， 建立一个框架，用于预测ERCC 2突变的功能效应和治疗意义。 尽管携带ERCC 2突变的MIBC对顺铂治疗的反应有所改善， 在化疗中，杂合ERCC 2突变赋予敏感性的机制尚不清楚。的 第二个目标将利用细胞和生物化学方法的组合来剖析突变的影响 ERCC 2蛋白功能，细胞特性以及对已建立和新兴MIBC疗法的敏感性。在 此外，将通过引入ERCC 2突变来研究ERCC 2突变对肿瘤发生的影响 单独和与其它已知的MIBC驱动突变组合的正常人尿路上皮细胞系中。 第三个目标是研究ERCC 2（或其他DNA修复基因）突变的假设。 定义一个MIBC患者子集，这些患者是保留膀胱治疗的理想候选人， 顺铂为基础的放化疗（CRT）。ERCC 2和1000个其他癌症基因的靶向测序 将在使用CRT治疗的两个大型MIBC患者队列中进行。这些分析可能会进一步 确定ERCC 2作为MIBC生物标志物的作用，并可能对理解ERCC 2的作用具有广泛的意义。 DNA修复途径在多种肿瘤环境中的改变。

Project Summary Muscle-invasive bladder cancers (MIBCs) represent an aggressive subset of bladder tumors that are associated with high mortality rates despite intensive multimodality treatment. DNA-damaging agents like cisplatin play a key role in the treatment of MIBC and many other solid tumors, yet validated biomarkers of response to DNA-damaging therapy are lacking. Recently, an association between somatic mutations in ERCC2, a core member of the nucleotide excision repair (NER) pathway, and improved response to cisplatin-based chemotherapy was uncovered in MIBC, representing one of the first validated examples of an association between a tumor DNA repair pathway alteration and response to a DNA-damaging agent. Preliminary functional analysis suggests that the observed ERCC2 mutations result in loss of NER capacity; however, the functional underpinnings of this association across tumors and clinical contexts are not known. This proposal aims to characterize the role of mutations in ERCC2 and other DNA repair genes in MIBC biology and treatment response. One of the limitations to understanding the biological relevance of novel DNA repair alterations in tumors is the lack of robust, efficient assays to test the functional effects of observed mutations. The first aim of this application will employ a novel high-throughout, fluorescence-based microscopy assay to measure the ability of ERCC2 mutations to support cellular NER. The assay will be applied to all ERCC2 mutations observed across several MIBC cohorts, and findings will be interpreted in the context of available treatment response and patient outcome data in order to define the functional landscape of ERCC2 mutations and develop a framework for predicting functional effects and therapeutic implications of ERCC2 mutations. Although MIBCs harboring ERCC2 mutations have improved response to cisplatin-based chemotherapy, the mechanism by which heterozygous ERCC2 mutations confer sensitivity is not known. The second aim will utilize a combination of cellular and biochemical approaches to dissect the effect of mutations on ERCC2 protein function, cellular properties, and sensitivity to established and emerging MIBC therapies. In addition, the effect of ERCC2 mutations on tumorigenesis will be investigated by introducing ERCC2 mutations into a normal human urothelial cell line alone and in combination with other known MIBC driver mutations. The third aim will investigate the hypothesis that mutations in ERCC2 (or other DNA repair genes) define a subset of MIBC patients who are ideal candidates for bladder-preserving treatment with concurrent cisplatin-based chemoradiotherapy (CRT). Targeted sequencing of ERCC2 and 1000 additional cancer genes will be performed in two large cohorts of MIBC patients treated using CRT. These analyses are likely to further define a role for ERCC2 as a biomarker in MIBC and may have broad implications for understanding the role of DNA repair pathway alterations in a variety of tumor settings.