Quantitative functional profiling of ERCC2 mutations deciphers cisplatin sensitivity in bladder cancer.

Tumor gene alterations can serve as predictive biomarkers for therapy response. The nucleotide excision repair (NER) helicase ERCC2 carries heterozygous missense mutations in approximately 10% of bladder tumors, and these may predict sensitivity to cisplatin treatment. To explore the clinical actionability of ERCC2 mutations, we assembled a multinational cohort of 2,012 individuals with bladder cancer and applied the highly quantitative CRISPR-Select assay to functionally profile recurrent ERCC2 mutations. We also developed a single-allele editing version of CRISPR-Select to assess heterozygous missense variants in their native context. From the cohort, 506 ERCC2 mutations were identified, with 93% being heterozygous missense variants. CRISPR-Select pinpointed deleterious, cisplatin-sensitizing mutations, particularly within the conserved helicase domains. Importantly, single-allele editing revealed that heterozygous helicase-domain mutations markedly increased cisplatin sensitivity. Integration with clinical data confirmed that these mutations were associated with improved response to platinum-based neoadjuvant chemotherapy. Comparison with computational algorithms showed substantial discrepancies, highlighting the importance of precision functional assays for interpreting mutation effects in clinically relevant contexts. Our results demonstrate that CRISPR-Select provides a robust platform to advance biomarker-driven therapy in bladder cancer and supports its potential integration into precision oncology workflows.