Identification of a Synthetic Lethal Relationship between Nucleotide Excision Repair Deficiency and Irofulven Sensitivity in Urothelial Cancer

Abstract

Purpose: Cisplatin-based chemotherapy is a first-line treatment for muscle-invasive and metastatic urothelial cancer. Approximately 10% 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 is ineligible to receive cisplatin-based therapy due to medical contraindications, and no NER-targeted approaches are available for platinum-ineligible or platinum-refractory ERCC2-mutant cases.

Experimental design: We used a series of NER-proficient and NER-deficient preclinical tumor models to test sensitivity to irofulven, an abandoned anticancer agent. In addition, we used available clinical and sequencing data from multiple urothelial tumor cohorts to develop and validate a composite mutational signature of ERCC2 deficiency and cisplatin sensitivity.

Results: We identified a novel synthetic lethal relationship between tumor NER deficiency and sensitivity to irofulven. 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 also found that a composite mutational signature of ERCC2 deficiency was strongly associated with cisplatin response in patients and was also associated with cisplatin and irofulven sensitivity in preclinical models.

Conclusions: Tumor NER deficiency confers sensitivity to irofulven, a previously abandoned anticancer agent, with minimal activity in NER-proficient cells. A composite mutational signature of NER deficiency may be useful in identifying patients likely to respond to NER-targeting agents, including cisplatin and irofulven.See related commentary by Jiang and Greenberg, p. 1833.

Figure 1:

NER-deficient cell lines are sensitive to irofulven in vitro and in vivo. A. Chemical structure of irofulven. B. Cell viability assays demonstrate increased irofulven sensitivity in NER-deficient cell lines compared with their isogenic NER-proficient counterparts. C. Irofulven dose-response for KE1 (ERCC2 mutant, NER deficient) xenografts. The black arrow denotes the time of first irofulven injection. D. KE1 xenograft weights were significantly lower in 0.5 and 1.0 mg/kg irofulven-treated mice than in untreated mice. * p-value 0.01-0.05, ** p value 0.001< p<0.01, and *** p-value <0.001.

Figure 2:

Irofulven sensitivity is conferred by TC-NER or common NER gene defects and persists in cisplatin-resistant models. A. SiRNA-mediated depletion of the TC-NER gene, ERCC6, or the common NER gene, ERCC3, results in significantly higher irofulven sensitivity compared with depletion of the GG-NER gene, DDB2, as measured by crystal violet staining (top) or viability assay (bottom). B. Immunoblot analysis showing RNA polymerase (RBP1) levels following irofulven treatment in NER-proficient KU19-19 and NER-deficient KE1 cells (top) and immunoblot analysis showing accumulation of phospho-H2AX, cleaved PARP, and cleaved caspase in irofulven-treated KE1 cells. C. A cisplatin-resistant derivative of the NER-deficient MDA-MB-468 cell line demonstrates decreased sensitivity to cisplatin, but remains sensitive to irofulven. D. Immunoblot analysis showing that ERCC4 (XPF) protein expression remains absent in the cisplatin-resistant MDA-MB-468 line (lane 3), similar to the parental cisplatin-sensitive line (lane 2) and consistent with persistent NER deficiency. Lane 1 (positive control) shows an MDA-MB-468 line engineered to stably express WT ERCC4. * p-value 0.01-0.05, ** p value 0.001< p<0.01, and *** p-value <0.001.

Figure 3:

Development of the ERCC2mut logistic regression–based classifier and validation of the association between ERCC2mut scores and ERCC2 mutation status in three independent bladder cancer cohorts. Samples with an ERCC2 mutation are shown in green and WT ERCC2 samples are shown in gray. A. Nine mutational features were significantly associated with ERCC2 mutation status in TCGA bladder cancer WES cohort and were used to develop the composite ERCC2mut score. B. ERCC2mut signature scores are strongly associated with ERCC2 mutation status in TCGA bladder cancer cohort (p<2.2×10⁻¹⁶). A value of ≥0.70 maximally separates ERCC2-mutant from WT cases and is denoted by the red horizontal dash-dotted line. In each validation cohort, ERCC2 mutants are highly enriched among patients with a high ERCC2mut score (≥0.70). C: DFCI/MSK cohort (n=50), p=3.3×10⁻⁴. D: BGI cohort (n=98), p=7.5×10⁻⁵. E: Philadelphia cohort (n=48), p=7.9×10⁻⁴. P-values were calculated using Fisher exact test.

Figure 4:

ERCC2mut signature scores are associated with cisplatin response, including among WT ERCC2 cases. Cisplatin responders are colored in orange and nonresponders are in black. ERCC2 mutant cases are denoted by green asterisks. A. ERCC2mut signature scores for all cases in the DFCI/MSKCC cohort (n=50). B. ERCC2mut signature scores for all cases in the Philadelphia cohort (n=48). C. ERCC2mut signature scores for WT ERCC2 cases in the DFCI/MSKCC cohort (n=41); high ERCC2mut signature scores (≥0.70) are significantly associated with cisplatin response (p=0.02). D. OS for patients with WT ERCC2 tumors in the DFCI/MSKCC cohort. OS was significantly longer for WT ERCC2 patients with ERCC2mut signature scores ≥0.70 (p=0.046). Positive predictive value (PPV) and negative predictive value (NPV) of the ERCC2mut composite signature were calculated for each cohort. P-values were calculated by the Fisher exact test.

Figure 5:

NER deficiency drives the ERCC2mut composite mutational signature. A. Separate clonal populations of NER-proficient KU19–19 or NER-deficient KE1 cells were cultured in parallel for approximately 30 generations and single cells were then isolated, expanded, and harvested. B. WGS and mutational signature analysis revealed significantly higher ERCC2mut scores in the NER-deficient KE1 samples compared with the NER-proficient KU19–19 samples.