ERCC2 Helicase Domain Mutations Confer Nucleotide Excision Repair Deficiency and Drive Cisplatin Sensitivity in Muscle-Invasive Bladder Cancer

Abstract

Purpose: DNA-damaging agents comprise the backbone of systemic treatment for many tumor types; however, few reliable predictive biomarkers are available to guide use of these agents. In muscle-invasive bladder cancer (MIBC), cisplatin-based chemotherapy improves survival, yet response varies widely among patients. Here, we sought to define the role of the nucleotide excision repair (NER) gene ERCC2 as a biomarker predictive of response to cisplatin in MIBC.

Experimental design: Somatic missense mutations in ERCC2 are associated with improved response to cisplatin-based chemotherapy; however, clinically identified ERCC2 mutations are distributed throughout the gene, and the impact of individual ERCC2 variants on NER capacity and cisplatin sensitivity is unknown. We developed a microscopy-based NER assay to profile ERCC2 mutations observed retrospectively in prior studies and prospectively within the context of an institution-wide tumor profiling initiative. In addition, we created the first ERCC2-deficient bladder cancer preclinical model for studying the impact of ERCC2 loss of function.

Results: We used our functional assay to test the NER capacity of clinically observed ERCC2 mutations and found that most ERCC2 helicase domain mutations cannot support NER. Furthermore, we show that introducing an ERCC2 mutation into a bladder cancer cell line abrogates NER activity and is sufficient to drive cisplatin sensitivity in an orthotopic xenograft model.

Conclusions: Our data support a direct role for ERCC2 mutations in driving cisplatin response, define the functional landscape of ERCC2 mutations in bladder cancer, and provide an opportunity to apply combined genomic and functional approaches to prospectively guide therapy decisions in bladder cancer.See related commentary by Grivas, p. 907.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

J. Bellmunt is a consultant/advisory board member for Merck, Pfizer, AstraZeneca, Pierre Fabre, and Roche. D.F. Bajorin reports receiving commercial research grants from Bristol-Myers Squibb, Merck, and Novartis; reports receiving speakers bureau honoraria from Merck; and is a consultant/advisory board member for Merck, Genentech, and Pfizer. J.E. Rosenberg is listed as a coinventor on a patent regarding ERCC2 mutation to select patients for platinum-based chemotherapy that is owned by Memorial Sloan Kettering Cancer Center. E.M. Van Allen reports receiving commercial research grants from Novartis and Bristol-Myers Squibb; reports receiving speakers bureau honoraria from Illumina; holds ownership interest (including patents) in Genome Medical, Synapse, and Tango Therapeutics; and is a consultant/advisory board member for Genome Medical, Invitae, and Tango Therapeutics. K.M. Mouw is a consultant/advisory board member for EMD Serono and Pfizer. No potential conflicts of interest were disclosed by the other authors.

©2018 American Association for Cancer Research.

Figures

Figure 1.

Frequency and distribution of ERCC2 mutations. A, Frequency of ERCC2 mutations by tumor type across 10,919 tumors sequenced using MSK-IMPACT (15). Only tumor types with ≥100 samples are shown, and tumors with an MSI score ≥10 (i.e, hypermutated tumors) are excluded. B, Location of ERCC2 mutations identified in published and ongoing prospective clinical cohorts.

Figure 2.

UV and cisplatin sensitivity assays identify functionally deleterious ERCC2 mutations. A, WT or mutant ERCC2-complemented cells were plated on 24-well glass slides and treated with UV irradiation. At 5 or 150 minutes following UV exposure, cells were fixed and treated with FLAG-tagged DDB2 proteoprobe ("Materials and Methods"). DDB2 proteoprobe binding to (6,4)-photoproducts is visualized using an anti-FLAG antibody (red). ERCC2 expression is confirmed using an anti-Myc-ERCC2 antibody (green). Nuclei are stained with DAPI (blue). R683W is a common germline pathogenic allele (33), D312N is a common nonpathogenic germline allele (34), and G607A is a mutation identified in cisplatin-responsive tumor. B, Cisplatin sensitivity curves for WT and mutant ERCC2-complemented cell lines.

Figure 3.

Summary of UV and cisplatin sensitivity for ERCC2 mutations across three clinical cohorts. A, Black bars show the fraction of DDB2 foci repaired at 150 minutes, and gray bars show cell viability in 2 μmol/L cisplatin relative to WT ERCC2. The majority of ERCC2 mutations confer UV and cisplatin sensitivity; however, a subset of mutations has sensitivity profiles similar to WT ERCC2. B, There was strong correlation between UV and cisplatin sensitivity (P < 0.001).

Figure 4.

Distribution and functional effects of clinically observed ERCC2 mutations. A, Location of somatic ERCC2 mutations across three clinical cohorts. Mutants that fail to rescue UV and cisplatin sensitivity (<35% WT activity) are shown in red. All other mutants had activity ≥85% of WT ERCC2 and are shown in yellow. Clinical cisplatin response data were available for the DFCI/MSK and FCCC cohorts, and the three ERCC2 mutations identified in cisplatin nonresponders are highlighted in boxes. D312N (underlined) is a common nonpathogenic germline allele in the general population, whereas R683W (underlined) is a common pathogenic germline allele in patients with xeroderma pigmentosum. B, Location of clinically observed somatic mutations in the ERCC2 protein (PDB ID: 5IVW). Deleterious mutations (red) cluster at the interface between helicase domains, whereas nondeleterious mutations (yellow) reside in nonhelicase domains or are on the surface of the protein. Q758 was not ordered in the crystal structure and is therefore not shown.

Figure 5.

An ERCC2 mutation confers NER deficiency and cisplatin sensitivity in a bladder cancer cell line. A, The DDB2 proteoprobe assay was performed on the bladder cancer cell lines KU19-19 (WT ERCC2) and KE1, a cell line derived from KU19-19 that has an ERCC2 M483_T484 in-frame deletion ("Materials and Methods" Supplementary Fig. S5). B, KU19-19 cells efficiently repair UV-induced DNA damage, whereas damage persists in KE1 cells (P = 0.003). C, KE1 cells also display increased cisplatin sensitivity compared with the parental KU19-19 cell line (P = 0.02). D, Cell-cycle profiles of KU19-19 and KE1 cell lines before and after cisplatin treatment. The KE1 cell line has a significantly higher sub-G1 fraction following cisplatin treatment than the KU19-19 cell line (P = 0.017). E, The cisplatin sensitivity of KE1 cells can be rescued by reexpression of WT ERCC2 (P = 0.03 for KE1 vs. KE1 + WT ERCC2).

Figure 6.

An ERCC2 mutation confers cisplatin sensitivity in an orthotopic bladder cancer model but does not sensitize to ionizing radiation. A, KU19-19 (ERCC2 WT) or KE1 (ERCC2 mutant) cells were implanted into the bladders of nude mice (4 mice/cell line), and mice were treated with cisplatin or saline. Representative images from posttreatment day 14 are shown. B, There was no difference in tumor size between the KU19-19 and KE1 models in the absence of cisplatin; however, KE1 tumors shrink markedly following cisplatin treatment. C, No significant difference in donogenic survival following ionizing radiation was noted between the KU19-19 and KE1 cell lines (solid lines). However, there was a significant difference in survival following combined treatment with cisplatin and ionizing radiation (dashed lines; P = 0.05 for interaction between cisplatin and radiation in KU19-19 vs. KE1).