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

项目摘要 肌肉浸润性膀胱癌(MIBCs)是一种侵袭性较强的膀胱癌 与高死亡率有关，尽管进行了密集的多模式治疗。DNA损伤剂，如 顺铂在MIBC和许多其他实体肿瘤的治疗中发挥关键作用，但已被证实的生物标志物 对DNA损伤治疗缺乏反应。 最近，核苷酸的核心成员ERCC2的体细胞突变之间的关联 切除修复(NER)途径和对以顺铂为基础的化疗的改善反应在 MIBC，代表了肿瘤DNA修复途径之间关联的首批有效例子之一 对DNA损伤剂的改变和反应。初步的功能分析表明，观察到的 ERCC2突变导致NER能力丧失；然而，这种关联的功能基础 跨肿瘤和临床背景尚不清楚。这项提议旨在描述突变在人类免疫系统中的作用 ERCC2等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 肿瘤缺乏强有力的、有效的分析来测试所观察到的突变的功能影响。第一个目标 将使用一种新的高通量、基于荧光的显微镜分析来测量 ERCC2突变支持细胞NER的能力。该检测将应用于所有ERCC2突变 在几个MIBC队列中观察到，结果将在现有治疗的背景下进行解释 反应和患者结局数据，以确定ERCC2突变和 建立一个框架来预测ERCC2突变的功能效应和治疗意义。 尽管携带ERCC2突变的MIBCs对以顺铂为基础的药物有更好的反应 化疗，ERCC2杂合子突变增加敏感性的机制尚不清楚。这个 第二个目标将利用细胞和生化方法的组合来剖析突变的影响。 关于ERCC2蛋白的功能、细胞特性以及对现有和新兴的MIBC疗法的敏感性。在……里面 此外，将通过引入ERCC2突变来研究ERCC2突变对肿瘤发生的影响 转化为正常的人类尿路上皮细胞系，并与其他已知的MIBC驱动程序突变相结合。 第三个目标将调查ERCC2(或其他DNA修复基因)突变的假设 确定MIBC患者的子集，这些患者是保留膀胱治疗的理想候选者 顺铂为主的放化疗(CRT)。ERCC2和其他1000个癌基因的靶向测序 将在两个使用CRT治疗的MIBC患者的大型队列中进行。这些分析可能会进一步 确定ERCC2在MIBC中作为生物标志物的作用，并可能对理解ERCC2的作用具有广泛的意义 DNA修复途径在各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.