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Translational Cancer Mechanisms and Therapy

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

Sabine Topka, Zoe Steinsnyder, Vignesh Ravichandran, Kaitlyn Tkachuk, Yelena Kemel, Chaitanya Bandlamudi, Mogens Winkel Madsen, Helena Furberg, Ouathek Ouerfelli, Charles M. Rudin, Gopa Iyer, Steven M. Lipkin, Semanti Mukherjee, David B. Solit, Michael F. Berger, Dean F. Bajorin, Jonathan E. Rosenberg, Barry S. Taylor, Elisa de Stanchina, Joseph Vijai and Kenneth Offit
DOI: 10.1158/1078-0432.CCR-20-3322 Published April 2021

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

This article is featured in Highlights of This Issue, p. 1825

Translational Relevance

In this study, we identified a subset of patients with cancer harboring germline or somatic aberrations within the nucleotide excision repair (NER) pathway, which has not been characterized previously. There are currently no approved NER-specific therapeutic strategies that target this group of patients. We demonstrate that recurrent heterozygous mutations in NER pathway genes, ERCC2 and ERCC3, as observed in these patients, can confer significant sensitivity in vitro and in vivo to the previously developed anticancer drug, irofulven. This study provides a molecularly targeted, preclinical approach to cancers with mutations in NER pathway genes demonstrating preferential sensitivity to the drug irofulven alone, or in combination with other agents. Similar to the impact of poly (ADP-ribose) polymerase (PARP) inhibitors on the landscape of treatment for homologous recombination–deficient tumors, irofulven and related compounds could significantly improve treatment outcomes for patients with NER-deficient tumors.

Introduction

Germline mutations in DNA repair genes are a common cause of hereditary cancer predisposition. Defects in genes that regulate double-strand break (DSB) repair/homologous recombination (HR), such as BRCA1/2, increase risk for several cancers (1–5), and confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (6). Alterations in mismatch repair (MMR) genes are associated with increased cancer risk for colorectal, gastric, endometrial, and other cancers (7), and the associated microsatellite instability (MSI)-high phenotype is predictive of response to immune checkpoint inhibitors (8). Cancer risk associated with mutations in nucleotide excision repair (NER) genes is less well understood and currently there are no targeted therapies that are FDA approved specifically for patients with germline or somatic mutations in NER pathways genes.

The ATP-dependent DNA helicases, ERCC2 and ERCC3, are part of the transcription factor IIH (TFIIH) complex, which is involved in RNA polymerase II–mediated transcription and plays a crucial role in the process of NER. NER is divided into two subpathways, transcription-coupled NER (TC-NER) and global genomic NER (GG-NER; ref. 9). Following recognition of DNA damage by either TC-NER or GG-NER sensors, repair is performed by a common mechanism involving unwinding of DNA at the damage site by helicases ERCC2 and ERCC3, incision by endonucleases ERCC1/4/5, and subsequent error-free gap filling and ligation. Biallelic mutations in the genes encoding the core NER factors ERCC1–5 give rise to inherited syndromes associated with increased cancer risk (10, 11).

Candidate gene studies have suggested common SNPs in ERCC2 as a potential risk factor for increased melanoma and bladder cancer risk (12, 13). We recently showed that the ERCC3 p.R109X variant confers partial loss of function and increased breast cancer predisposition (14). On the basis of these observations, other truncating ERCC3 mutations (somatic or germline) are predicted to confer haploinsufficiency as well. Somatic ERCC2 mutations are present in approximately 10% of bladder cancers, and found at lower frequency in a range of other cancer types, and have been demonstrated to correlate with increased cisplatin sensitivity (15). Thus, NER gene mutations can act as predictive biomarkers that indicate an increased risk to develop cancers for carriers of pathogenic variants in these genes and, therefore, can be helpful for early cancer detection when carriers are subjected to increased cancer screening. At the same time, they can also be used as prognostic biomarkers, informing physicians about increased sensitivity of tumors harboring variants in these genes to drugs targeting DNA repair deficiencies.

The fungal sesquiterpene illudin S exhibited toxicity against cells deficient for NER pathway components (16). We have previously shown that heterozygous mutations in ERCC3 confer increased sensitivity to illudin S (14). Illudin S exhibited enhanced cytotoxicity in multidrug resistant and leukemia cells, but was not suitable for clinical use because of toxicities observed in animal studies (17). Hence, efforts were undertaken to develop semisynthetic derivatives of illudin S, such as the acylfulvenes, with better therapeutic indices, that could potently induce apoptosis in tumor cells at lower doses (18). The illudin S derivative, irofulven, has shown very promising antitumor activity in both cell lines and xenografts, especially in solid tumors (19). Illudin S and irofulvan create DNA lesions that are specifically recognized by TC-NER, but are ignored by GG-NER sensors (20). However, whether irofulven has selectivity for tumor cells with heterozygous mutations in ERCC2/ERCC3 or other common NER or TC-NER pathway genes has not been characterized.

Here, we take a first step toward estimating the prevalence of putative functionally significant monoallelic NER pathway mutations in a large dataset of patients with cancer. We then investigate a potential treatment option targeted for an NER-deficient patient population by assessing preclinical efficacy of irofulven in heterozygous ERCC2- or ERCC3-mutant cells in vitro and in isogenic cell line- or patient-derived xenografts (PDX). We elucidate the increased efficacy of irofulven as a mechanistic consequence of hypomorphic NER function and delineate the deregulated cellular signaling pathways downstream conferring cytotoxicity. Building on these data, we identified combination therapies that can act synergistically. Overall, our data support irofulven-based combinations as a precision therapy for tumors with NER mutations.

Materials and Methods

Tumor-normal sequencing and interrogation of germline and somatic mutational datasets

All germline and tumor sequencing performed in this study was completed as part of a research study. Paraffin-embedded tumor and blood samples from patients were obtained and sequenced using the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform, a capture-based next-generation sequencing (NSG) assay capable of identifying mutations, copy-number alterations, and select gene fusions involving 341 cancer-associated genes in the first iteration and 468 in the more recent iteration, as described previously (21, 22). For the anonymized cases, sequence data were assigned a unique study identifier and irretrievably delinked from personal identifiers before variant calling. Germline variants from the .bam file of the constitutional DNA with mapping and base quality scores of >20 were called using GATK Haplotypecaller2. Only variants with 50× depth of sequencing were included in further analysis. For germline data, only heterozygous variants with <1% population frequency in the Genome Aggregation Database (gnomAD) were included in the analysis. Subsequent pathogenicity filters were applied and included the following criteria. Variants reported as benign and likely benign in ClinVar were excluded and we used CAVA (23), VEST (24), and our in-house algorithm, PathoMAN (25), for variant annotation and in silico prediction to filter for pathogenic and likely pathogenic variants. In addition, variants observed in the homozygous state were excluded, because these are less likely to confer loss of function if they are tolerated in the homozygous state within these critical genes. Variants showing a moderate or high impact in CAVA and PathoMAN, as well as a VEST score of >0.6, were included in the final analysis set. Somatic variants were derived from Memorial Sloan Kettering Cancer Center (MSKCC) cBioPortal (26, 27). Variants were filtered to exclude those annotated as likely neutral through the OncoKB knowledge base (28) and only variants with a functional impact score > 2.5 as determined by the MutationAssessor tool (29) were included in the analysis dataset.

Cell culture and treatments

Human mammary epithelial (HMLE) cells harboring the heterozygous ERCC3 R109X variant have been generated by our group using CRISPR/Cas9 as described previously (14). Similarly, the ERCC2 missense variants, E79D and Y24C, were introduced into the HMLE cell line, and heterozygous and homozygous clones were expanded and validated by PCR and Sanger sequencing. An isogenic pair of KU1919 human bladder carcinoma cells of wild-type (WT) and ERCC2 compound heterozygous mutant (L485fs/T484_L485del) was kindly provided by the Solit laboratory at MSKCC (New York, NY). For cell viability assays, these cell lines were treated with various compounds as described and cell viability was assessed at the indicated timepoints following treatment by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega).

Western blotting

Protein lysates were prepared in RIPA Buffer (Pierce) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Samples were run on 4%–12% gradient Bis-Tris SDS PAGE Gels (Invitrogen), transferred onto Polyvinylidene Difluoride Membranes (Bio-Rad), and probed with antibodies against ERCC3 (ARP37963_P050; 1:2,500; Aviva Systems Biology), ERCC2 (ab54676; 1:1,000; Abcam), phospho-Histone H2A.X (Ser139) (1:1,000; Cell Signaling Technology), phospho-Chk1 (Ser345) (1:1,000; Cell Signaling Technology), and GAPDH (V-18; 1:2,000; Santa Cruz Biotechnology). Horseradish peroxidase–conjugated secondary antibodies were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Flow cytometry

For flow cytometric analysis, 24 hours after seeding, cells were either left untreated or were treated with the indicated doses of irofulven for 2 hours. Treated cells were subsequently washed with PBS and supplemented with drug-free medium. The cells were harvested at different timepoints after treatment and fixed with 4% PFA for 10 minutes at room temperature. Cells were briefly chilled on ice and ice-cold methanol was added to a final concentration of 90%. The cells were incubated on ice for 30 minutes and stored at −20°C until further processing. For immunostaining, the cells were washed twice with PBS/0.5% BSA and incubated with anti-phospho-Histone H2A.X (Ser139) antibody (Alexa Fluor 488 conjugate, 1:250; Cell Signaling Technology) for 1–2 hours at room temperature, washed again, and incubated with Alexa Fluor 488–conjugated secondary antibody for 45 minutes at room temperature. For each condition, a minimum number of 2 × 104 cells were recorded and data from at least two replicate experiments were analyzed using the FlowJo software (V10.5.3). An example of the gating strategy can be found in Supplementary Fig. S1.

Xenograft experiments

Animal studies were approved by the MSKCC Institutional Animal Care and Use Committee (New York, NY) and Research Animal Resource Center. NSG (The Jackson Laboratory) and athymic (nu/nu; Envigo Laboratories) mice were used for in vivo studies and were cared for in accordance with institutional guidelines. PDXs were generated as follows: 6-week-old NSG female mice were implanted subcutaneously with specimens freshly collected from patients at MSK Hospital (New York, NY) under an approved Institution Review Board biospecimen protocol, as described previously (30). Tumors developed within 2–4 months and were expanded into additional mice by serial transplantation. The generated PDXs were subjected to high-coverage NGS with the MSK-IMPACT assay (Supplementary Table S1). Isogenic cell line xenografts were generated by injecting 10 × 106 hRAS-V12–driven ERCC3 WT or heterozygous R109X-mutant HMLE cells together with Matrigel (50:50) subcutaneously in 6-week-old athymic female mice. In all instances, once tumors reached an average volume of 100 mm3, mice (8–10 mice/group) were randomized to receive either vehicle or irofulven 3.5 mg/kg i.p. for 5 consecutive days. Cisplatin was given at a dose of 3.5 mg/kg i.p. once weekly to avoid excessive toxicities. The treatment cycle was repeated every 21 days for a total of three cycles. Mice were observed daily throughout the treatment period for signs of morbidity/mortality. Tumors were measured twice weekly using calipers, and volume was calculated using the formula: length × width2 × 0.52. Body weight was also assessed twice weekly. Treatment was terminated when either significant toxicities were observed, tumors had completely regressed, or when mice had to be sacrificed because of large tumor burden. Upon completion of the experiments, the tumors were excised, flash frozen, and stored at −80°C until further analyses.

RNA sequencing and data analysis

Fresh-frozen tumor tissues were subsequently preserved in RNAlater (Invitrogen) and RNA was extracted using the RNeasy Midi Kit (Qiagen). After RiboGreen quantification and quality control by Agilent BioAnalyzer, 0.99–1 μg of total RNA underwent ribosomal depletion and library preparation using the TruSeq Stranded Total RNA LT Kit (Illumina, catalog no., RS-122-1202) according to instructions provided by the manufacturer with eight cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a 100-bp/100-bp paired-end run, using the HiSeq 3000/4000 SBS Kit (Illumina). The output data (FASTQ files) were mapped to the human genome, converted to BAM format, and expression count matrix was generated from the mapped reads using HTSeq (https://htseq.readthedocs.io/en/release_0.11.1/). Read counts were normalized for individual transcript lengths, log transformed, and differential gene expression analysis was performed using the R package, DeSeq2. Significant results were filtered by Padjusted < 0.05 and mean expression > 100 reads. Significantly differentially expressed genes were subjected to pathway analysis using the gene set enrichment analysis (GSEA) software (31). The Broad Institute's Molecular Signatures Database (MSigDB) was used to retrieve significant pathways listed in the curated (C2) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and hallmark (H) gene sets (32). Enrichment plots were generated using GSEA 4.1.0 for all significant pathways and determined by FDR q value (<0.25), as suggested by GSEA for exploratory discovery. The matrix and raw data for RNA sequencing (RNA-seq) reported in this article have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE14476.

Results

Functionally disruptive NER pathway gene mutations may be present in a significant subset of patients with cancer

The MSK-IMPACT assay is an FDA-authorized diagnostic test designed to detect somatic and germline alterations in more than 400 cancer-associated genes (21). We leveraged the prospective MSK-IMPACT initiative to define the spectrum of NER gene mutations in a cohort of more than 40,000 patients with advanced/metastatic cancers. Somatic mutational data were available for all patients, whereas fully curated sample-matched germline mutational data were available for approximately 20,000 patients. NER pathway members, ERCC2, ERCC3, ERCC4, and ERCC5 were included in our sequencing panel. These four proteins, together with ERCC1 (which forms a heteroduplex with ERCC4), are major pleiotropic NER factors. Another reason to include ERCC4 and ERCC5 in this analysis was that both earlier studies, as well as a new report carried out with the irofulven precursor drug, illudin S, indicate that ERCC1/4 and ERCC5 deficiency can sensitize cells to this class of drugs as well (16, 33).

We filtered all observed variants within these four genes to generate final tumor and normal datasets including only variants predicted to impair gene function. ERCC3 p.R109X, a hypomorphic mutation, is one of the most common germline variants in the NER pathway and was observed across a range of different cancer types. As expected, missense variants made up the largest part of the alterations detected. There is evidence from both hereditary syndromes, involving ERCC2–5, and functional studies, mainly interrogating ERCC2 missense variants, that a significant fraction of these variants may impair gene function. Therefore, we decided to include missense variants in our analysis, but applied several filtering methods to exclude the ones unlikely to affect gene function. Filtering steps are depicted in Fig. 1; briefly, we excluded intronic and synonymous variants, as well as variants with a minor allele frequency of more than 1%. To predict pathogenicity within the remaining set of variants, we leveraged various databases and in silico predictions to exclude variants predicted to be benign/tolerated (for detailed description, see Materials and Methods section). These, together with high-confidence pathogenic/likely pathogenic (P/LP) variants, which include truncating (nonsense and frameshift), canonical splice site, and start-loss/stop-loss variants, were included in our final dataset, as shown in Fig. 1. All variants with additional information and pathogenicity prediction scores are included in Supplementary Table S2 (germline dataset) and Supplementary Table S3 (somatic dataset). Together with predicted likely pathogenic missense variants, putative pathogenic germline alterations in the NER pathway genes, ERCC2, ERCC3, ERCC4, and ERCC5, were seen at a frequency of about 5%–10% within all cancer types examined, with the highest overall mutation burden observed in ERCC2 (Fig. 1A). For a more conservative estimate, we created an additional subset of variants including high-confidence P/LP variants and only missense variants that are linked to development of NER deficiency syndromes in the homozygous/compound heterozygous state or ones that have been functionally validated (11, 15, 34, 35). These variants, on average were observed in 2.3% of patients across cancer types (0.98%–3.81%; Supplementary Table S4).

Figure 1.
Figure 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.

Within the MSK-IMPACT tumor dataset, we identified >1,200 patients whose tumors had potentially disruptive somatic alterations (mutations and deletions) in ERCC2–5 (Fig. 1B). Notably, somatic (combined P/LP and candidate likely pathogenic missense) mutations and deletions of ERCC2–5 were found in approximately 10% of patients each with bladder and uterine cancers, and >5% of patients with bowel or skin cancers (Fig. 1B). Highest confidence P/LP variants, deletions, as well as NER deficiency syndrome–associated and functionally validated missense variants were observed in 1.3% of patients on average across cancer types (0.17%–5.3%; Supplementary Table S5). While LOH in the tumor was observed in three cases with germline mutations of ERCC3, overall, the rate of LOH was not significantly different in ERCC2–5 mutation carriers compared with noncarriers across 30,000 patients for whom LOH data were available (Supplementary Table S6). In addition, in a subset of patients with truncating ERCC3 germline variants, only 6.5% showed a second somatic deleterious alteration in an NER pathway gene (ERCC2–5). In sum, somatic gene mutations that could impair NER pathway function were found in a subset of patient's tumors, and, thus, effective precision therapies targeting vulnerabilities in NER-deficient tumors could have significant clinical impact on this patient population.

Cells carrying a hypomorphic ERCC3 mutation show significant sensitivity to irofulven

To delineate the functional effects of NER pathway mutations and to explore agents as potential therapeutic drugs that can exploit NER deficiency, we generated isogenic cellular models carrying specific hypomorphic mutations identified in our cancer patient cohort. We had previously engineered nontransformed HMLE cells to harbor a heterozygous (c.325 C>T, p.R109*) ERCC3 mutation using CRISPR/Cas9-based homology-directed repair (14). We used the isogenic cell line pair to identify compounds with increased toxicity to the ERCC3-mutant cells. We initially tested a range of compounds, which have either been shown to be active in various DNA repair–deficient cells and organisms (cisplatin, melphalan, mitomycin C, olaparib, and irofulven) or compounds previously described to affect ERCC3 function (triptolide and spironolactone). Among these seven compounds, the most significant dose-dependent difference in sensitivity between the ERCC3 WT and R109X heterozygous–mutant cells was observed with irofulven (Fig. 2A) and spironolactone (Fig. 2G). Strikingly, at the lowest concentration of irofulven tested (150 nmol/L), WT cells showed no reduction in viability, whereas the cell viability of the R109X-mutant cells was reduced by almost 40% (IC50WT, 370 nmol/L and IC50MUT, 160 nmol/L). A small, but significant, increase in sensitivity of the mutant cell line was observed in response to cisplatin (Fig. 2B; IC50WT, 6.8 μmol/L and IC50MUT, 5.3 μmol/L) and mitomycin C (Fig. 2C; IC50WT, 363 nmol/L and IC50MUT, 216 nmol/L), but compared with irofulven, the overall difference was small and/or restricted to a narrow dose range. No significant differences in sensitivity between ERCC3 WT and -mutant cells were observed using the bifunctional alkylating agent, melphalan (Fig. 2D; IC50WT, 5.9 μmol/L and IC50MUT, 5.5 μmol/L) and the PARP inhibitor, olaparib (Fig. 2E; IC50WT, 39.2 μmol/L and IC50MUT, 37.5 μmol/L). Triptolide, a diterpenoid epoxide with anti-inflammatory and immunosuppressive activities, has been shown to covalently bind human ERCC3 and inhibit its DNA-dependent ATPase activity (36). We found that triptolide treatment significantly lowered ERCC3 transcript levels (Supplementary Fig. S2), but not protein levels, in both WT and R109X-mutant cells (Fig. 2H), and showed only mild differential toxicity in the isogenic cell line pair (Fig. 2F; IC50WT, 11 μmol/L and IC50MUT, 7.4 μmol/L). Spironolactone, a widely used mineralocorticoid receptor antagonist, has been described to induce E1 ubiquitin ligase–mediated degradation of ERCC3. We observed that R109X-mutant cells were more sensitive than ERCC3 WT cells to spironolactone in vitro (Fig. 2G; IC50WT, 35.4 μmol/L and IC50MUT, 14.5 μmol/L) and confirmed loss of ERCC3 expression 2 hours after spironolactone treatment (Fig. 2H). Because baseline levels of ERCC3 were significantly lower in the ERCC3 R109X–mutant cells compared with WT cells, they were depleted more rapidly of this essential protein when exposed to spironolactone. However, in contrast to irofulven, prolonged spironolactone exposure was required to exert its effect in vitro (Supplementary Fig. S3A). We also did not detect increased in vivo efficacy of spironolactone as a single agent in ERCC3 R109X–mutant xenografts at doses considered safe for use in mice (Supplementary Fig. S3B). Thus, spironolactone is unlikely to be clinically useful as a targeted anticancer therapy in NER-deficient cells.

Figure 2.
Figure 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 with WT cells. B–D, Sensitivity to other DNA cross-linking agents is only marginally increased in the R109X mutant. E, No difference was observed in response to olaparib. The effect of drugs previously shown to directly affect ERCC3 was assessed, showing a small differential response for triptolide (inhibitor of ERCC3 DNA-dependent ATPase activity; F), while a substantial differential response was observed after treatment with spironolactone (inducing proteasomal degradation of ERCC3; G). H, Western blotting showing the effect of irofulven (IRO), spironolactone (SP), and triptolide (TPL) on ERCC3 protein levels. Only spironolactone affects ERCC3 protein levels, leading to a strong reduction at doses as low as 1 μmol/L in the mutant cells. Bladder cancer cells harboring a compound heterozygous ERCC2 mutation show significantly higher sensitivity to irofulven (I) and cisplatin (L) compared with WT cells. HMLE cells harboring ERCC2 missense variants E79D (J and M) or Y24C (K and N) show significantly higher sensitivity to irofulven compared with WT cells. For the E79D mutation, significance is indicated only for the comparison between WT and heterozygous mutant, the difference between WT and homozygous mutant cells was highly significant (P ≤ 0.0001) at all doses tested. Data represent 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).

Truncating and missense mutations in ERCC2 confer strong sensitivity to irofulven

To interrogate response of additional NER pathway genes to irofulven, we generated CRISPR/Cas9-edited HMLE cell lines harboring specific somatic heterozygous (c.237 G>T; p.E79D) or homozygous (c.237 G>T; p.E79D and c.71 A>G; p.Y24C) missense variants in ERCC2 (Supplementary Fig. S4). Both mutations were observed multiple times within our patient cohort and were absent from gnomAD. In addition, an isogenic human bladder cancer cell line (KU1919) pair was used, because somatic ERCC2 mutations are most common in bladder cancers (37). The KU1919-mutant cell line was engineered to harbor a compound heterozygous ERCC2 mutation (c.1451 C>A/c.1452insG; p.T484K/L485fs*15 and c.1449delACGCTG; p.T484_L485del). The ERCC2-mutant KU1919 cells showed reduced proliferation as compared with WT cells, which was not observed in the HMLE ERCC2/3-mutant cells (Supplementary Fig. S5). When comparing drug sensitivities in the KU1919 cell line pair, the ERCC2-mutant cells showed greater sensitivity to both irofulven and cisplatin (Fig. 2I and L). Strikingly, irofulven (IC50WT, 399 nmol/L and IC50MUT, 98 nmol/L) conferred a stronger selective toxicity against the mutant cells as compared with cisplatin (IC50WT, 3.2 μmol/L and IC50MUT, 1 μmol/L). Especially in the lower dose range (IC50 and below), concentrations at which the WT cells were relatively unaffected, irofulven significantly (P < 0.0001; Fig. 2) reduced growth of the mutant cells. At the WT IC25 concentration (a concentration that reduced viability of WT cells by 25%), irofulven inhibited the growth of the mutant cells by about 90%, whereas cisplatin led to less than 70% reduction in cell number. Moreover, irofulven displayed higher potency, with antitumor effect observed in the nanomolar range compared with cisplatin, which exerted cytotoxic effects at micromolar concentrations. HMLE cells expressing the ERCC2 c.237 G>T (p.E79D) missense variant were strongly sensitive to irofulven (IC50WT, 373 nmol/L; IC50HET, 238 nmol/L; and IC50HOM, 16 nmol/L) and cisplatin (IC50WT, 7.2 μmol/L; IC50HET, 4.4 μmol/L; and IC50HOM, 0.65 μmol/L), with a stronger differential toxicity to irofulven again observed in mutant cells (Fig. 2J and M). Here, the WT IC25 dose decreased the number of E79D heterozygous–mutant cells by more than 50% (with irofulven) as compared with less than 40% (with cisplatin). Similarly, the homozygous ERCC2 c.71 A>G (p.Y24C) variant was highly sensitive to both irofulven and cisplatin (Fig. 2K and N). When comparing the two ERCC2 mutants, the ERCC2 E79D mutation seemed to more strongly perturb ERCC2 function, as indicated by increased drug sensitivity in comparison with the Y24C-mutant cells. For both irofulven and cisplatin, the IC50 concentration for the homozygous E79D cells was significantly lower than that required to achieve a 50% reduction in cell viability of the homozygous Y24C cell line (IC50IRO, 139 nmol/L and IC50CIS, 1.86 μmol/L).

ERCC2- and ERCC3-mutant cells have impaired ability to repair irofulven-induced DNA damage

To assess DNA damage levels and biochemical response to irofulven treatment, we determined expression of DNA damage and cell-cycle checkpoint markers via Western blotting. During continuous exposure to a sublethal dose of irofulven (HMLE, 150 nmol/L and KU1919, 100 nmol/L) over time, both ERCC2- and ERCC3-mutant cell lines exhibited greater baseline levels of DNA damage as indicated by increased phosphorylation of H2AX in untreated cells (Fig. 3). The engineered HMLE-mutant cells showed stronger activation of DNA damage and checkpoint signaling after different durations of exposure to irofulven (Fig. 3A). This effect was observed in both the heterozygous and homozygous ERCC2 E79D–mutant HMLE cell lines as well (Fig. 3B). The parental ERCC2 WT KU1919 bladder cancer cell line was resistant to irofulven and showed no substantial increase in DNA damage even with prolonged exposure to irofulven, whereas the isogenic ERCC2-deficient KU1919 cells demonstrated significantly increased DNA damage and p53 activation after prolonged treatment with irofulven (Fig. 3C). DNA damage was examined by flow cytometry at various timepoints after a 2-hour exposure to irofulven (HMLE, 150 nmol/L and KU1919, 100 nmol/L) and subsequent recovery after removal of the drug (Fig. 3D and E; Supplementary Fig. S6). In the nontransformed HMLE cells, we observed an initial increase in γH2AX that was similar in both WT and ERCC2/3-mutant cells, but a reduction in this marker of DNA damage was delayed in ERCC2/3-mutant cells. This effect was most pronounced at 18 hours posttreatment, when WT cells showed a more than 50% reduction of γH2AX, but ERCC2/3 mutants retained more than 70% of γH2AX (Fig. 3D and E). In the bladder cancer cells, short exposure with a sub-IC50 dose of irofulven did not robustly induce H2AX phosphorylation in the WT background, whereas the ERCC2-mutant cell line showed a maximal increase in γH2AX levels 12 hours after the drug was removed from the cells, which did not return to baseline levels by 24 hours after drug exposure (Fig. 3F). In addition to impaired removal of γH2AX, we also observed impaired recovery of RNA synthesis in the ERCC2-mutant cells and differences in RNA polymerase II dynamics in both ERCC2- and ERCC3-mutant cell lines upon irofulven treatment (Supplementary Fig. S7).

Figure 3.
Figure 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 (IRO). Levels of H2AX, p-Chk-1, and ERCC2/3 were assessed in isogenic cell line pairs treated over different length of exposure to a dose of 150 (HMLE) or 100 nmol/L (KU1919) irofulven. Western blot analyses showed 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 γH2AX were assessed by flow cytometry. ERCC2/3-mutant cells show reduced reduction of γH2AX-positive cells over time, indicating a decrease in DNA repair efficiency. Bar graphs show average from two biological replicate experiments recording a minimum of 2 × 104 cells per condition. Significance was determined by two-way ANOVA test to measure effect in mutant cells compared with WT cells at all timepoints (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

Irofulven significantly inhibits the growth of ERCC2- and ERCC3-mutant tumors

To assess response of NER mutants to irofulven in vivo in both naturally arisen and genetically defined tumors, we used a PDX model, as well as isogenic cell line–derived xenografts. A PDX model from a patient with small cell lung cancer was used, harboring a heterozygous truncating mutation in ERCC2 p.E85* (c.253G>T). We used a previously established dose of 3.5 mg/kg irofulven that did not lead to sustained weight loss in mice when administered consecutively for 5 days (Supplementary Fig. S8). Cisplatin was used at the same dose of 3.5 mg/kg and administered once per week to avoid renal toxicity. Both irofulven and cisplatin treatment suppressed tumor growth over the first two treatment cycles, while after the third cycle, tumors in the cisplatin-treated groups progressed (mean tumor volume, 2,065 mm3) and irofulven-treated tumors showed sustained growth suppression (mean tumor volume, 496 mm3; Fig. 4A). To establish an ERCC3-mutant mouse model with which we could study the antitumor effects of irofulven in vivo, as HMLE cells do not form tumors in mice, the parental and ERCC3 R109X isogenic cells were modified to overexpress an oncogenic HRAS mutant (G12V). Tumor volumes in the vehicle-treated mice measured over the course of the experiment showed no significant difference in the ability of the two cell lines to form tumors (Fig. 4B). The R109X tumors initially grew slower, but reached larger tumor volumes by the time of sacrifice. Mice receiving irofulven exhibited initial tumor growth suppression in both the WT and mutant groups after the first round of treatment. During the second treatment cycle, tumors in the ERCC3 WT group exhibited an average tumor volume significantly larger than mutant tumors (WTIRO = 190.4 mm3 and R109XIRO = 23.9 mm3; P = 0.0027). At the start of the third treatment cycle, 3 mice within the R109X tumor group had a complete response with the remaining mice still exhibiting stable growth suppression. In contrast, WT tumors resumed growth at a similar rate as initially observed in the vehicle group. At time of sacrifice, most mice treated with irofulven in the WT group showed progression of disease (n = 7; 78%), 2 mice (22%) showed partial responses, and no complete responses were observed (Fig. 4B). In contrast, in the ERCC3 R109X group, we observed only one case of stable disease (10%), six partial responses (60%), and in three cases (30%) mice showed complete tumor regression.

Figure 4.
Figure 4.

Growth suppression of ERCC2- and ERCC3-mutant tumors in response to irofulven and discovery of pathways deregulated in ERCC3-mutant tumors. A, PDXs 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.

RNA-seq identifies a network of potentially irofulven-sensitive genes and provides insights into codependencies

RNA-seq was performed on eight tumors, each from the ERCC3 WT and R109X vehicle–treated groups, and seven tumors, each from the irofulven-treated groups. Genes differentially expressed between groups were identified and subsequently overrepresented pathways were determined using the KEGG pathway and hallmark gene sets from the MSigDB (Supplementary Table S7). First, we compared transcriptomic profiles between the WT and R109X vehicle groups to assess the effect of the R109X mutation alone on global transcript expression (Fig. 5A; Supplementary Fig. S9). ERCC3 R109X–mutant tumors showed a significantly increased expression of transcripts involved in major DNA repair pathways, most significantly in base excision repair (BER) and MMR. In addition, we observed increased expression of genes involved in DNA replication, metabolism, cell-cycle checkpoint signaling, and the proteasome pathway. Among the most significantly downregulated pathways were the TGFβ pathway and protein secretion, the latter indicating increased ER stress. Next, we compared each of the vehicle groups with their respective irofulven-treated groups. In the WT background, irofulven treatment led to a significant increase in expression of genes involved in DNA repair pathways, especially BER and HR, as well as DNA replication, metabolism, and cell-cycle checkpoint signaling (Fig. 5B; Supplementary Fig. S10). These pathways already showed a strong upregulation in the R109X-mutant tumors even without irofulven exposure and no significant increase in these pathways was observed in the R109X tumors following treatment with irofulven (Fig. 5C; Supplementary Fig. S11). Instead, in the mutant background, irofulven treatment resulted in increased expression of genes involved in pathways including the p53/apoptosis pathway, TGFβ and MAPK signaling pathways, and ribosomal biogenesis. Pathways significantly downregulated following irofulven treatment included protein secretion in both ERCC3 WT and -mutant groups and cell-cycle checkpoint signaling specifically in the R109X-mutant tumors. Deregulation of the immune network was seen in both WT and mutant tumors following irofulven treatment. Interestingly, irofulven treatment had the opposite effect on the MAPK signaling pathway depending on the genotype, with WT tumors showing downregulation of KRAS signaling, while this pathway was upregulated in the mutant tumors following irofulven treatment.

Figure 5.
Figure 5.

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

Irofulven acts synergistically with PARP inhibition, platinum, and inhibition of ER-associated degradation

On the basis of our observation that tumors treated with irofulven showed an upregulation of genes related to DNA repair pathways other than NER, we decided to examine the effect of irofulven in combination with the PARP inhibitor, olaparib. We found that the two compounds acted synergistically as defined by the Chou–Talalay method (38) over a broad concentration range in ERCC3-mutant cells [combination index (CI), 0.46–0.84] and showed synergistic to additive effects in WT cells (CI, 0.43–1.1; Fig. 6A; Supplementary Table S8). The combination of irofulven and cisplatin acted in a weakly synergistic to additive manner in both WT (CI, 0.84–1) and mutant (CI, 0.93–1.1) cells (Fig. 6B; Supplementary Table S9). The combination of irofulven with an inhibitor of ER-associated protein degradation (ERAD), eeyarestatin I, showed synergistic activity across a broad dose range in both WT (CI, 0.71–0.94) and mutant (CI, 0.71–0.92) cells (Fig. 6C; Supplementary Table S10). In bladder cancer cells, we found the combinations of irofulven and cisplatin acted synergistically. When using the Chou–Talalay method, synergism was observed specifically in ERCC2 mutants, while the combination was determined to have an additive effect in WT cells. In addition, we used the Loewe score to determine and display synergy over the wide range of combinations used in a 3-dimensional model, which determined overall synergistic activity of cisplatin and irofulven in both cell lines, with a higher synergy score for the ERCC2-mutant cell line (Fig. 6D; Supplementary Table S11).

Figure 6.
Figure 6.

Drug combinations with potential synergistic activity in ERCC2/3 mutants. A, Olaparib acts synergistically in combination with irofulven (IRO) specifically in the ERCC3-mutant background. B, Combination treatment with cisplatin increases sensitivity of both ERCC3-mutant and WT cells to irofulven in an additive manner. C, Combinations of irofulven and eeyarestatin I (EeI) show moderate synergism in ERCC3-mutant cells and moderately synergistic to additive effects in WT 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 WT bladder cancer cells. Representative cooperativity screens and Loewe plots are shown.

Discussion

The occurrence and impact of alterations in the NER pathway on cancer risk have recently been described, and we and others have shown that both germline and somatic mutations in NER pathway genes are observed in patients with cancer across multiple cancer types (21, 39–42). Currently, platinum constitutes the preferred therapy for patients with urothelial tumors bearing mutations in ERCC2 (15, 43). Here, we demonstrate that another chemotherapeutic agent, irofulven, could constitute a precision therapeutic against tumors of diverse types but bearing shared genetic defects in NER pathways. Irofulven shows high selective toxicity in cells and tumors with hypomorphic mutations in the NER genes ERCC2 and ERCC3, surpassing effects of currently used chemotherapeutics, while exhibiting lower toxicities to WT cells. Our results are consistent with prior studies indicating vulnerability of ERCC2/3-deficient cells to precursors of irofulven (16). Early studies were performed on rodent cell lines harboring chemically induced biallelic frameshift mutations. The current findings build upon those early observations and significantly extend their impact toward the larger number of patients with cancer, observed to harbor putatively damaging monoallelic NER gene mutations. Experiments in human normal mammary epithelial and bladder carcinoma cell lines engineered to carry clinically observed heterozygous NER mutations, as well as relevant tumor models show that those mutations, in different cellular backgrounds, conferred selective sensitivity to this class of drugs in vitro and in vivo. Our results are in line with a separate, nonoverlapping study by Börcsök and colleagues (44) demonstrating significant irofulven sensitivity in a set of in vitro and in vivo models with deficiencies in common NER or TC-NER factors. Importantly, Börcsök and colleagues also show that NER-mutant cells with acquired resistance to cisplatin retain sensitivity to irofulven, which would make irofulven an attractive candidate therapeutic even for patients that demonstrate acquired resistance to platinum therapy. Irofulven, and other sesquiterpenes, have been reported to act through a unique mechanism of action, inducing a type of DNA damage that requires an intact TC-NER pathway to be resolved (20, 45). Irofulven has also been shown to exhibit potent antitumor activity across a range of cancer cell lines, including those with multidrug-resistant phenotypes, as well as cell lines resistant to platinum and taxane drugs (46). Because of these properties, between 1999 and 2006, multiple clinical trials evaluated the efficacy of irofulven among genotypically unselected patients with various tumor types; generally mixed responses and some reports of early toxicities in these trials limited subsequent investigations (47). While the overall response rate in treated patients in these historic trials was not superior to comparison drugs, a small subset of patients showed substantial responses following irofulven. On the basis of our observations in isogenic cell line and xenograft models, we predict that patients harboring mutations in ERCC2 or ERCC3 will demonstrate a high probability of response to irofulven and related compounds.

Analysis of a large cohort of patients with cancer showed that ERCC2 and ERCC3, as well as ERCC4 and ERCC5, encoding for NER-specific helicases and incision nucleases, are mutated both somatically and in the germline of patients with cancer, thus pointing to a subset of patients that could benefit from irofulven or irofulven analog therapy. Because ERCC2 somatic mutations already constitute a biomarker for bladder cancer treatment with platinum drugs, irofulven may constitute a precision drug option for a subset of patients, specifically cisplatin nonresponders. Irofulven may also provide a potential first-line treatment in this and other tumor contexts, because it showed high activity in NER pathway–mutant mammary, as well as bladder cancer–derived cells and in xenograft models, but reduced toxicity in WT cells compared with cisplatin.

Mechanistically, it has been shown that acylfulvenes alkylate DNA and form DNA adducts that disrupt DNA and RNA synthesis, arrest cells in G1–S-phase, and induce apoptosis (48). These findings are in agreement with our transcriptome analysis showing an increase in expression of genes involved in those pathways following irofulven treatment. The increase in cell-cycle checkpoint signaling already observed in untreated ERCC3-mutant cells warrants further exploration. Our observation that the TGFβ pathway is downregulated in the R109X-mutant cells is consistent with a recent study showing decreased TGFβ signaling in NER-deficient cells (49). Our data also suggest the potential for using irofulven in combination with PARP inhibitor treatment, a synergism that could lead to an improved response. Both in vitro drug combination experiments, as well as RNA-seq data results point to dependence of NER mutants on other repair pathways, especially BER. It is important to note here that the six major DNA repair pathways in humans share common proteins and work closely together to resolve complex lesions and maintain genome integrity. A stronger than previously appreciated cooperation has recently been described between NER and BER in the repair of various types of lesions (50). The HR and nonhomologous end joining (NHEJ) pathways are activated as a result of both directly and indirectly formed DNA DSBs, the latter resulting when DNA insults cannot be resolved by the other DNA repair mechanisms. Because of high cooperativity between DNA repair pathways' alterations in a pathway other than the one targeted by a specific drug can lead to resistance. This has been shown in the case of PARP inhibition (PARPi) where resistance is often caused by BRCA1/2 reversion mutations, but can also arise due to loss of NHEJ pathway activity (51). Similarly, increased activation of either the NER, HR, or MMR pathways has been associated with cisplatin resistance (52). Overall, this indicates that combinations of agents targeting different DNA repair pathways could be very efficient and help to overcome drug resistance. Combinations of PARPi and platinum agents have been explored in multiple phase I–III clinical trials, albeit with limited success because overlapping toxicities required dosing of both agents to be reduced (53). Because these trials were not performed on patient cohorts selected for biomarkers of tumor DNA repair deficiency in which even a combination of these drugs at reduced doses could generate successful results, we propose testing combinations such as PARPi and irofulven in PDX models from NER-deficient patients and, if successful, in subsequent precision medicine combination trials, such as NCI-ComboMATCH. Such studies can provide further evidence of the efficacy of targeted combination therapy in a specific genetic background. Our RNA-seq data also suggest an increased dependence of ERCC3-mutant cells on the unfolded protein response pathway, as indicated by a significant reduction in protein secretion, which is augmented in the presence of irofulven. Our in vitro combination experiments have also shown synergistic activity of an irofulven combination with ERAD inhibition, in both ERCC3 WT and -mutant cells. Thus, overall, our drug combination experiments indicate that the success of this therapeutic strategy will depend on both cellular, as well as specific genetic background.

The observed perturbed DNA repair ability of ERCC2 and ERCC3 heterozygous–mutant cells further indicates that these pathogenic variants, present in the germline or in tumors without homozygous mutations or LOH, act as hypomorphs. We have previously suggested that heterozygous ERCC3-mutant cells, like other components of DNA damage repair pathways (e.g., H2AX, BLM, and CHEK1), may act via dosage dependency and mechanisms such as haploinsufficiency, leading to tumorigenesis (14). These data suggest a model whereby the resulting functional impairment does not appear to affect basal transcription, but affects DNA repair under conditions of prolonged or increased genotoxic stress. Such prolonged stress may give rise to additional oncogenic mutations leading to cancer formation, as well as increased intratumoral genetic instability.

Overall, we identified many patients with cancer that harbor alterations in NER core pathway members ERCC2–5. Although the NER pathway consists of more than 40 members, ERCC2–5 genes are the major pleiotropic members of the pathway and most frequently altered in syndromic patients (11). As we observed similar rates of LOH among NER-proficient and -deficient patients, we would not assume that alterations in these genes act as classical tumor suppressors. Instead, mutations in NER pathways may play a role in the initial steps of cancer formation, but may act in concert with other deficiencies. While we observed both low rates of LOH and low incidence of cooccurrence of somatic NER gene mutations in germline carriers of pathogenic NER variants, more than one third of tumors showed cooccurrence of pathogenic NER germline variants with likely pathogenic alterations in other DNA repair pathway genes, which could cooperate in generating overall genomic instability. In addition, complete inactivation of ERCC2/3 may be detrimental to tumor growth based on their involvement in general transcription as members of the TFIIH complex. While the mechanism of NER gene involvement in tumor initiation/progression requires further investigation, alterations affecting function of these genes appear to sensitize tumors to NER-targeting agents. Patients with both germline and somatic LoF variants would qualify for treatment with NER-targeting agents, but prior to treatment of germline mutation carriers, safety studies should be performed using mouse models harboring heterozygous germline mutations in NER genes. Although increased general toxicities have not been observed with PARPi treatment of BRCA1/2 germline carriers, the effect of irofulven has not been studied yet in this context.

Limitations of this study include the definition of NER deficiency in the patient population by applying a predictive model in which putative pathogenic mutations are classified largely through in silico prediction tools. Especially for missense variants, additional functional models will be needed to classify candidate variants. However, missense variants in the core NER genes ERCC2–5 are frequently reported among patients with hereditary syndromes; in the case of ERCC2, functional studies have shown that many of these missense variants are deleterious. After applying several filtering steps for increased stringency, selected missense variants were reported, including the ones reported in the in vitro experiments here. The resulting data suggest that a defined subset of patients could benefit from treatment exploiting NER deficiency, a hypothesis that needs further exploration and validation. Such validation will require additional large datasets across tumor types, as well as expanding the assessment of irofulven sensitivity to NER pathway members other than ERCC2 and ERCC3. To identify potential candidates for a “basket” type clinical trial, orthogonal approaches to determine NER deficiency, such as tumor profile or functional assays, would be helpful. In addition, our current studies have been done on a limited set of in vitro and in vivo models and may not be transferrable to all types of mutations and tissues. Therefore, it will be of interest to assess a larger panel of PDX models derived from different tissue types and NER pathway deficiencies. Although multiple clinical trials were initiated with irofulven, including one at our institution in the early 2000s (NCT00005070), tumor tissues from these trials were not available to retrospectively assess presence of NER pathway alterations in extreme responders. Finally, while cross-resistance between irofulven and cisplatin has not been observed, we cannot fully exclude the possibility of such cross-resistance occurring in a human clinical setting or with other DNA repair–targeting agents.

To define novel treatment strategies for patients with hypomorphic mutations in the ERCC2/3 complex, we propose a drug repurposing approach for irofulven, ideally in the form of a basket trial for patients showing NER pathway deficiency (54), for which patients can be selected in real-time on the basis of assessment of NER deficiencies through next-generation gene panel sequencing or combination of tissue microarray and IHC on biopsy or resection material. An additional strategy would be high-throughput screening for other compounds that selectively target NER pathways.

Authors' Disclosures

S. Topka reports a patent for use of Illudin class of alkylating agents in patients harboring mutations in the ERCC3 gene (PCT/US2018/022588) issued. M. Winkel Madsen reports to be a chief operating officer at Oncology Venture, a Danish Biotech Company. Oncology Venture has provided the compound irofulven (without any costs) that has been used in this study. Oncology Venture is currently running a clinical phase II trial in prostate cancer with irofulven. H. Furberg reports grants from NCI during the conduct of the study. O. Ouerfelli reports grants from NCI during the conduct of the study; is listed as inventor in patents filed by MSKCC; received intellectual property rights from Johnson & Johnson, Jazz Pharmaceuticals, Y-mAbs, and AngioGenex; and owns equity interests in AngioGenex (uncompensated). C.M. Rudin reports personal fees from AbbVie, Amgen, AstraZeneca, Bicycle, Celgene, Genentech/Roche, Ipsen, Janssen, Jazz, Lilly/Loxo, Pfizer, PharmaMar, Syros, Vivotek, Bridge Medicines, Earli, and Harpoon outside the submitted work. G. Iyer reports personal fees and other from Mirati Therapeutics and Janssen and other from Seagen, Inc outside the submitted work. D.B. Solit reports personal fees from Pfizer, Lilly Oncology, Loxo Oncology, BridgeBio, and Illumina outside the submitted work. M.F. Berger reports personal fees from Roche outside the submitted work. J.E. Rosenberg reports personal fees and other from Seattle Genetics, Astellas, AstraZeneca, Roche/Genentech, and QED Therapeutics, as well as personal fees from Chugai, Eli Lilly, Merck, BMS, EMD Serono/Pfizer, BioClin, Adicet Bio, Fortress Biotech, Western Oncolytics, GlaxoSmithKline, Janssen Oncology, Boehringer Ingelheim, and Mirati outside the submitted work; Dr. Rosenberg reports a patent for ERCC2 to predict cisplatin sensitivity pending to DFCI. B.S. Taylor reports grants from Genentech, Inc. and personal fees from Boehringer Ingelheim and Loxo Oncology at Lilly outside the submitted work. J. Vijai reports a patent for PCT/US2018/022588 issued to MSKCC. K. Offit reports a patent for PCT/US2018/022588 for use of Illudin class of alkylating agents in patients harboring mutations in ERCC3 pending to MSKCC. No disclosures were reported by the other authors.

Authors' Contributions

S. Topka: Conceptualization, formal analysis, supervision, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. Z. Steinsnyder: Investigation. V. Ravichandran: Data curation, software, formal analysis, investigation. K. Tkachuk: Resources, data curation, project administration. Y. Kemel: Data curation, validation. C. Bandlamudi: Data curation, software, formal analysis, validation, investigation. M. Winkel Madsen: Resources. H. Furberg: Formal analysis, funding acquisition, validation. O. Ouerfelli: Supervision, writing–review and editing. C.M. Rudin: Resources. G. Iyer: Resources, funding acquisition, writing–review and editing. S.M. Lipkin: Funding acquisition, writing–review and editing. S. Mukherjee: Software, validation. D.B. Solit: Resources, funding acquisition, writing–review and editing. M.F. Berger: Writing–review and editing. D.F. Bajorin: Funding acquisition, writing–review and editing. J.E. Rosenberg: Writing–review and editing. B.S. Taylor: Supervision, writing–review and editing. E. de Stanchina: Conceptualization, supervision, validation, writing–review and editing. J. Vijai: Conceptualization, supervision, funding acquisition, validation, project administration, writing–review and editing. K. Offit: Conceptualization, supervision, funding acquisition, validation, project administration, writing–review and editing.

Acknowledgments

We thank Dr. Robert Benezra for providing HMLE cells and Oncology Venture for providing the drug irofulven. We also thank Sara Khalil and Hannah Lipsky for experimental assistance. We gratefully acknowledge the members of the Molecular Diagnostics Service in the Department of Pathology and the use of the Integrated Genomics Operation Core and are grateful for valuable input from Cassidy Cobbs, Neeman Mohibullah, Kety Huberman, and Agnes Viale. We further wish to acknowledge assistance from the Bioinformatics and wish to specifically thank Mono Pirun, Amy Webber, and Nicholas Socci. We thank members of the Antitumor assessment core, specifically Connor Hagen, Elizabeth Peguero, and Amanda Kulick, led by Elisa de Stanchina. The Molecular Diagnostics Service in the Department of Pathology and the Integrated Genomics Operation Core were funded by the NCI Cancer Center Support Grant (P30 CA08748), Cycle for Survival and Marie-Josée, and Henry R. Kravis Center for Molecular Oncology. The MSK Bioinformatics Core was funded, in part, through the NIH/NCI Cancer Center Support grant (P30 CA008748). The MSK Antitumor assessment core received funding through P30 CA008748 S5 and U54 OD020355-01. This work was supported by the Matt Bell Foundation (to S.M. Lipkin). We acknowledge the support of The V Foundation for Cancer Research (to H. Furberg and J. Vijai), the P50 CA221745 (NIH bladder SPORE awarded to D.F. Bajorin, D.B. Solit, K. Offit, J. Vijai, and G. Iyer), the NIH core grant (P30 CA008748 to MSKCC), and the Breast Cancer Research Foundation grant and the Kate and Robert Niehaus Foundation for providing funding to the Robert and Kate Niehaus Center for Inherited Cancer Genomics at Memorial Sloan Kettering Cancer Center (to K. Offit).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Clin Cancer Res 2021;27:1997–2010

  • Received August 21, 2020.
  • Revision received October 19, 2020.
  • Accepted November 12, 2020.
  • Published first November 16, 2020.

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Targeting Germline- and Tumor-Associated Nucleotide Excision Repair Defects in Cancer
Sabine Topka, Zoe Steinsnyder, Vignesh Ravichandran, Kaitlyn Tkachuk, Yelena Kemel, Chaitanya Bandlamudi, Mogens Winkel Madsen, Helena Furberg, Ouathek Ouerfelli, Charles M. Rudin, Gopa Iyer, Steven M. Lipkin, Semanti Mukherjee, David B. Solit, Michael F. Berger, Dean F. Bajorin, Jonathan E. Rosenberg, Barry S. Taylor, Elisa de Stanchina, Joseph Vijai and Kenneth Offit
Clin Cancer Res April 1 2021 (27) (7) 1997-2010; DOI: 10.1158/1078-0432.CCR-20-3322
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