Mutational signature-based identification of DNA repair deficient gastroesophageal adenocarcinomas for therapeutic targeting

Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in cancer. HR-deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of HR deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Using whole exome and genome sequencing data, we found that a significant subset of GEA, but very few colorectal adenocarcinomas, show evidence of HR deficiency by mutational signature analysis (HRD score). High HRD gastric cancer cell lines demonstrated functional HR deficiency by RAD51 foci assay and increased sensitivity to platinum chemotherapy and PARP inhibitors. Of clinical relevance, analysis of three different GEA patient cohorts demonstrated that platinum treated HR deficient cancers had better outcomes. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by two photoproduct repair assays. Single-cell RNA-sequencing revealed that, in addition to inducing apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, validated by intracellular GSH assay. Overall, our study provides preclinical evidence that a subset of GEAs harbor genomic features of HR and NER deficiency and may therefore benefit from platinum chemotherapy and PARP inhibitors. NUGC3 cells were treated with the IC50 concentration of Cisplatin, Oxaliplatin, 5-FU and DMSO for 2 days. Single-cell RNA-sequencing was used to analyze the mechanism of increased sensitivity of NER-deficient gastric cancer to cisplatin.

同源重组（Homologous recombination, HR）与核苷酸切除修复（nucleotide excision repair, NER）是癌症中最易发生功能失活的两类DNA修复通路。HR缺陷型乳腺癌、卵巢癌、胰腺癌及前列腺癌对铂类化疗药物与PARP抑制剂（Poly ADP-ribose polymerase inhibitor）响应良好。然而，胃食管腺癌（gastric and esophageal adenocarcinoma, GEA）中HR缺陷的发生频率仍缺乏诊断与功能层面的验证。本研究借助全外显子组测序与全基因组测序数据，通过突变特征分析（mutational signature analysis）计算HRD评分（HRD score），结果显示存在显著比例的GEA样本呈现HR缺陷特征，但结直肠腺癌中此类样本极为罕见。高HRD评分胃癌细胞系经RAD51灶形成实验（RAD51 foci assay）验证存在功能性HR缺陷，且对铂类化疗药物及PARP抑制剂的敏感性显著升高。就临床相关性而言，对三个独立的GEA患者队列开展分析后发现，接受铂类化疗的HR缺陷型癌症患者预后更佳。一株对顺铂（cisplatin）具有高敏感性的胃癌细胞系虽表现为HR功能正常（HR proficiency），但通过两种光产物修复实验验证存在NER缺陷。单细胞RNA测序（single-cell RNA-sequencing）分析表明，顺铂处理除诱导细胞凋亡外，还可在NER缺陷型胃癌细胞中触发铁死亡（ferroptosis），该现象经细胞内谷胱甘肽（glutathione, GSH）实验得到验证。综上，本研究提供了临床前证据，证实存在一类GEA样本携带HR与NER缺陷的基因组特征，因此可从铂类化疗及PARP抑制剂治疗中获益。本研究中NUGC3细胞经顺铂、奥沙利铂（oxaliplatin）、5-氟尿嘧啶（5-fluorouracil, 5-FU）及二甲基亚砜（dimethyl sulfoxide, DMSO）的半最大效应浓度（half maximal inhibitory concentration, IC50）处理2天。单细胞RNA测序被用于解析NER缺陷型胃癌对顺铂敏感性升高的分子机制。