Targeting Germline- and Tumor-Associated Nucleotide Excision Repair Defects in Cancer

Abstract

Purpose: Nucleotide excision repair (NER) gene alterations constitute potential cancer therapeutic targets. We explored the prevalence of NER gene alterations across cancers and putative therapeutic strategies targeting these vulnerabilities.

Experimental design: We interrogated our institutional dataset with mutational data from more than 40,000 patients with cancer to assess the frequency of putative deleterious alterations in four key NER genes. Gene-edited isogenic pairs of wild-type and mutant ERCC2 or ERCC3 cell lines were created and used to assess response to several candidate drugs.

Results: We found that putative damaging germline and somatic alterations in NER genes were present with frequencies up to 10% across multiple cancer types. Both in vitro and in vivo studies showed significantly enhanced sensitivity to the sesquiterpene irofulven in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to a current standard-of-care agent, cisplatin. Hypomorphic ERCC2/3-mutant cells had impaired ability to repair irofulven-induced DNA damage. Transcriptomic profiling of tumor tissues suggested codependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies.

Conclusions: These findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs.See related commentary by Jiang and Greenberg, p. 1833.

Fig. 1:. Putative functionally significant alterations in NER genes ERCC2-5 are found in a significant subset of patients with cancer.

(A) Germline variant filtering scheme and number of patients with likely damaging ERCC2-5 germline mutations as well as alteration frequencies across different cancer types among 16,712 patients with cancer sequenced through MSK-IMPACT. (B) Somatic variant filtering process and number of patients with likely pathogenic ERCC2-5 somatic mutations or deletions as well as alteration frequencies across different cancer types in >40,000 patients with cancer sequenced through MSK-IMPACT. Highest frequency of mutations in those NER pathway members is observed in bowel, skin, uterine and bladder cancers.

Fig. 2:. ERCC3 and ERCC2 mutant cells show significant sensitivity to irofulven treatment.

(A) Cells harboring the heterozygous ERCC3 R109X mutation show significantly higher sensitivity to irofulven compared to WT cells. (B-D) Sensitivity to other DNA crosslinking agents is only marginally increased in the R109X mutant. (E) No difference was observed in response to olaparib. (F, G) The effect of drugs previously shown to directly affect ERCC3 was assessed, showing a small differential response for triptolide (F; inhibitor of ERCC3 DNA-dependent ATPase activity) while a substantial differential response was observed after treatment with Spironolactone (G; inducing proteasomal degradation of ERCC3). (H) Western blot showing the effect of irofulven, spironolactone and triptolide on ERCC3 protein levels. Only spironolactone affects ERCC3 protein levels, leading to a strong reduction at doses as low as 1μM in the mutant cells. (I, L) Bladder cancer cells harboring a compound heterozygous ERCC2 mutation show significantly higher sensitivity to irofulven (I) and cisplatin (L) compared to WT cells. (J, K, M, N) Human mammary epithelial cells harboring ERCC2 missense variants E79D (J, M) or Y24C (K, N) show significantly higher sensitivity to irofulven compared to WT cells. For the E79D mutation significance is indicated only for the comparison between wildtype and heterozygous mutant, the difference between wildtype and homozygous mutant cells are highly significant (p≤0.0001) at all doses tested. Data represents the mean of three experiments with error bars representing the SEM. Significance was determined by two-way ANOVA test. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001

Fig. 3:. NER mutant cells treated with irofulven show increased DNA damage and impaired DNA repair kinetics.

(A-C) Induction of DNA damage and checkpoint activation in response to irofulven. Levels of H2AX, p-Chk-1 and ERCC2/3 were assessed in isogenic cell line pairs cells treated over different length of exposure to a dose of 150nM (HMLE) or 100nM (KU1919) irofulven. Western blots show increased activation of H2AX and Chk-1 in ERCC2/3 mutant cells. The 48-hour timepoint for the E79D homozygous mutant is missing due to extensive cell death at this stage. (D-F) Recovery from irofulven-induced DNA damage over time. Levels of γ-H2AX were assessed by Flow cytometry. ERCC2/3 mutant cells show reduced reduction of γ-H2AX positive cells over time indicating a decrease in DNA repair efficiency. Bar graphs show average from two biological replicate experiments recording a minimum of 20,000 cells per condition. Significance was determined by two-way ANOVA test to measure effect in mutant cells compared to wildtype cells at all time points; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001

Fig. 4:. Growth suppression of ERCC2 and ERCC3 mutant tumors in response to irofulven and discovery of pathways de-regulated in ERCC3 mutant tumors.

(A) Patient-derived xenografts from an individual with small cell lung cancer harboring a truncating mutation in ERCC2; p.E85* (c.253G>T), show substantial growth suppression in response to irofulven. Tumors initially respond to treatment with cisplatin as well, but here tumors ceased to respond after the second treatment cycle (B) Irofulven mediates strongly increased and sustained growth suppression in CRISPR engineered ERCC3 heterozygous mutant xenografts.

Fig. 5:. Pathway analysis of differentially expressed genes.

(A) Significantly Up- and Down-regulated pathways within KEGG and Hallmark gene sets in xenografts from vehicle-treated ERCC3 WT/R109X compared to vehicle-treated wildtype mice (B) Significantly Up- and Down-regulated pathways within KEGG and Hallmark gene sets in wildtype xenografts from mice treated with irofulven compared to wildtype tumors from vehicle treated mice (C) Significantly Up- and Down-regulated pathways within KEGG and Hallmark gene sets in ERCC3 WT/R109X tumors from irofulven-treated mice compared to ERCC3 WT/R109X tumors from vehicle treated mice. Red arrows mark pathways related to DNA repair signaling. Normalized enrichment scores (NES) reflects the degree to which a gene set is overrepresented at the extremes (top or bottom) across a list of genes ranked by hypergeometrical score (HGS) based on differential gene expression. Higher enrichment scores indicate a shift of genes belonging to certain pathway categories towards either end of the ranked list, representing up or down regulation (positive or negative values, respectively). Pathways with FDR q-value (<0.25) were further considered for biological relevance.

Fig. 6:. Drug combinations with potential synergistic activity in ERCC2/3 mutants.

(A) Olaparib acts synergistically in combination with irofulven specifically in the ERCC3 mutant background. (B) Combination treatment with cisplatin increases sensitivity of both ERCC3 mutant and wildtype cells to irofulven in an additive manner. (C) Combinations of irofulven and eeyarestatin I show moderate synergism in ERCC3 mutant cells and moderately synergistic to additive effects in wildtype cells. Representative CI plots are shown. (D) Strong synergistic activity of irofulven and cisplatin is detected in ERCC2 mutant bladder cancer cells and weak synergism is observed in wildtype bladder cancer cells. Representative cooperativity screens and Loewe plots are shown.