Identification of a synthetic lethal relationship between nucleotide excision repair (NER) deficiency and irofulven sensitivity in urothelial cancer. Cisplatin-based chemotherapy is a first-line treatment for muscle-invasive and metastatic urothelial cancer, but only a subset of patients respond to therapy. Approximately 15% of bladder urothelial tumors have a somatic missense mutation in the nucleotide excision repair (NER) gene ERCC2, which confers increased sensitivity to cisplatin-based chemotherapy. However, a significant subset of patients are ineligible to receive cisplatin-based therapy due to medical contraindications, and no NER-targeted approaches are available for platinum-ineligible or platinum-refractory ERCC2mutant cases. Here, we identify a novel synthetic lethal relationship between tumor NER deficiency and sensitivity to irofulven, an abandoned anti-cancer agent previously shown to have only modest activity in Phase I/II trials in biomarker-unselected populations. Irofulven specifically targets cells with inactivation of the transcription-coupled NER (TC-NER) pathway and leads to robust responses in vitro and in vivo, including in models with acquired cisplatin resistance, while having minimal effect on cells with intact NER. We develop and validate a composite mutational signature of ERCC2deficiency in bladder cancer that is strongly associated with cisplatin response in bladder cancer patients and is also associated with cisplatin and irofulven sensitivity in preclinical models. Although developed in ERCC2-mutant cases, the composite mutational signature is also associated with cisplatin response in cases that lack an ERCC2 mutation, and may therefore serve as an additional tool to identify patients likely to respond to NER-targeting agents including cisplatin and irofulven.