Quantitative functional profiling of ERCC2 mutations deciphers cisplatin sensitivity in bladder cancer
Judit Börcsök, Diyavarshini Gopaul, Daphne Devesa-Serrano, Clémence Mooser, Nicolas Jonsson, Matteo Cagiada, Dag R. Stormoen, Maya N. Ataya, Brendan J. Guercio, Hristos Z. Kaimakliotis, Gopa Iyer, Kresten Lindorff-Larsen, Lars Dyrskjøt, Kent W. Mouw, Zoltan Szallasi, Claus S. Sørensen
Judit Börcsök, Diyavarshini Gopaul, Daphne Devesa-Serrano, Clémence Mooser, Nicolas Jonsson, Matteo Cagiada, Dag R. Stormoen, Maya N. Ataya, Brendan J. Guercio, Hristos Z. Kaimakliotis, Gopa Iyer, Kresten Lindorff-Larsen, Lars Dyrskjøt, Kent W. Mouw, Zoltan Szallasi, Claus S. Sørensen
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Research Article Cell biology Genetics Oncology

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

Abstract

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.

Authors

Judit Börcsök, Diyavarshini Gopaul, Daphne Devesa-Serrano, Clémence Mooser, Nicolas Jonsson, Matteo Cagiada, Dag R. Stormoen, Maya N. Ataya, Brendan J. Guercio, Hristos Z. Kaimakliotis, Gopa Iyer, Kresten Lindorff-Larsen, Lars Dyrskjøt, Kent W. Mouw, Zoltan Szallasi, Claus S. Sørensen

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Figure 2

CRISPR-Select analysis establishes that helicase-domain ERCC2 mutations confer platinum sensitivity.

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CRISPR-Select analysis establishes that helicase-domain ERCC2 mutations ...
(A) CRISPR-Select workflow. iCas9-MCF10A cells are transfected with equal amount of repair templates harboring the mutation of interest (Mut) or a synonymous mutation (WT*). The WT* is used as an internal normalization control. Following CRISPR editing, most cells with a mutation of interest knocked-in on one allele will have a disruptive frameshift (fs) InDel on the other allele. Cells are harvested at day 2 (D2; initial timepoint) and the remaining cells are split into untreated or cisplatin-treated conditions and collected at D12.The region containing the Mut or WT* is deep sequenced and the Mut:WT* ratio is calculated. (B) Schematic representation of ERCC2 gene structure and position of the mutations investigated by CRISPR-Select. The mutations correspond to germline mutations selected from ClinVar and somatic missense mutations identified in bladder cancer cohorts. The conserved helicase domains of ERCC2 are depicted. (C) Impact of a known pathogenic (Y639*) and a likely benign (D312N) variant on cell fitness in TP53 WT and KO iCas9-MCF10A cell lines. The normalized Mut:WT* shown corresponds to the ratio of the Mut:WT* normalized to D2. (D and E) Impact on cisplatin sensitivity of ERCC2 variants. The normalized Mut:WT* frequencies of somatic missense mutations in (D) TP53 KO iCas9-MCF10A and (E) bladder cancer cell lines. The error bars represent the standard deviation of 3 independent experiments. The statistical significance was determined using a paired2-tailed t test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.