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 3

Single allele editing CRISPR-Select to quantify functional impacts of heterozygous ERCC2 missense mutations.

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Single allele editing CRISPR-Select to quantify functional impacts of he...
(A and B) Principle of exon guide RNA editing compared with intron guide RNA editing. Following Cas9 cleavage, the ssODN repair templates are employed to introduce the mutation of interest (Mut) or a synonymous mutation (WT*) that are tracked by NGS. On the other allele, Cas9 introduces a cut, but, due to inefficiency of editing, this predominantly results in InDel events. Two cellular editing events resulting in Mut and WT* ERCC2 are depicted separated by dashed lines. (A) In exon guide RNA editing, the second allele events are frameshifts that generally are degraded by nonsense-mediated mRNA decay (NMD). (B) Using an intron guide RNA system, the second allele InDels are now in the noncoding region. This system can hence circumvent the formation of a high proportion of frameshifts and be used to mimic a heterozygous condition. (C and D) Quantification of exon and intron InDels at D2 in an exon guide RNA editing system compared with an intron guide RNA system. (E) Impact on cisplatin sensitivity of exon guide RNA and intron guide RNA for N238S and D609G variants. The normalized Mut:WT* shown corresponds to the ratio of the Mut:WT* normalized to the initial D2 timepoint. The error bars represent the standard deviation of 3 independent experiments. The statistical significance was determined using an unpaired 2-tailed t test.