A unique subset of epithelial ovarian cancers with platinum sensitivity and PARP inhibitor resistance

Abstract

Platinum and PARP inhibitor (PARPi) sensitivity commonly coexist in epithelial ovarian cancer (EOC) due to the high prevalence of alterations in the homologous recombination (HR) DNA repair pathway that confer sensitivity to both drugs. In this report, we describe a unique subset of EOC with alterations in another DNA repair pathway, the nucleotide excision repair (NER) pathway, which may exhibit a discordance in sensitivities to these drugs. Specifically, 8% of high-grade serous EOC from The Cancer Genome Atlas dataset exhibited NER alterations, including nonsynonymous or splice site mutations and homozygous deletions of NER genes. Tumors with NER alterations were associated with improved overall survival (OS) and progression-free survival (PFS), compared with patients without NER alterations or BRCA1/2 mutations. Furthermore, patients with tumors with NER alterations had similar OS and PFS as BRCA1/2-mutated patients, suggesting that NER pathway inactivation in EOC conferred enhanced platinum sensitivity, similar to BRCA1/2-mutated tumors. Moreover, two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in the two most platinum-sensitive tumors, were functionally associated with platinum sensitivity in vitro. Importantly, neither NER alteration affected HR or conferred sensitivity to PARPi or other double-strand break-inducing agents. Overall, our findings reveal a new mechanism of platinum sensitivity in EOC that, unlike defective HR, may lead to a discordance in sensitivity to platinum and PARPi, with potential implications for previously reported and ongoing PARPi trials in this disease.

Figure 1

NER alterations in EOC and association with outcome. A, characteristics of NER pathway alterations in EOC tumors of TCGA dataset. B, association of tumors with NER alterations with OS. Tumors with NER alterations exhibited similar median OS (63.5 vs. 59.1 months, respectively, P =0.811) compared with BRCA1/2-mutated tumors and statistically significantly higher median OS (63.5 vs. 41.5 months, respectively, P =0.048) compared with the remaining tumors. C, association of tumors with NER alterations with PFS. Tumors with NER alterations exhibited similar median PFS (30.4 vs. 19.2 months, respectively, P =0.971) compared with BRCA1/2-mutated tumors and a trend toward statistically significantly higher median PFS (30.4 vs. 14.7 months, respectively, P =0.069) compared with the remaining tumors. For both B and C, tumors that harbored both NER and BRCA1/2 mutations (n =4) were not included in the PFS or OS analysis. Inclusion of these tumors did not significantly change the results of the PFS and OS analyses.

Figure 2

ERCC6-Q524* and ERCC4-A583T sensitize to cisplatin (CDDP). A, ERCC6-deficient fibroblasts [black solid line, ERCC6^−/− + empty vector (EV)] are hypersensitive to cisplatin. Introduction of wild-type ERCC6 (black dashed line, ERCC6^−/− + WT ERCC6) rescues the sensitivity of the deficient cells, whereas the mutant protein (red solid line, ERCC6^{− /−} + ERCC6-Q524*) does not rescue survival (left plot). IC₅₀ values and immunoblot showing expression of wild-type (blot against ERCC6) or mutant ERCC6 in ERCC6-deficient fibroblasts (anti-Flag antibody) are shown on the right. B, transient overexpression of the mutant protein ERCC6-Q524* (red solid line) in 293T cells confers sensitivity to cisplatin, compared with cells transfected with either empty vector (black solid line) or wild-type ERCC6 (black dashed line). IC₅₀ values are shown on the right. An immunoblot showing expression of wild-type or mutant ERCC6 in 293T (anti-Flag antibody) is shown on the right. C, ERCC4-deficient fibroblasts (black solid line, ERCC4^−/− + EV) are hypersensitive to cisplatin. Introduction of wild-type ERCC4 (black dashed line, ERCC4^−/− + WT ERCC4) rescues the sensitivity of the deficient cells, whereas mutant protein (green solid line, ERCC4^{− −} + ERCC4-A583T) does not significantly rescue survival (left plot). IC₅₀ values and immunoblot showing expression of wild-type or mutant ERCC4 in ERCC4-deficient fibroblasts are shown on the right. Experiments were performed in triplicate and error bars represent ± 1 SEM. Statistical analyses were performed using one-way ANOVA. Asterisks indicate statistically significant values (for A, P < 10⁻⁴ EV vs. ERCC6 and P < 10⁻³ ERCC6 vs. ERCC6-Q524*; EV vs. ERCC6-Q524*; for B, P < 10⁻⁴ EV vs. ERCC6-Q524* and ERCC6 vs. ERCC6-Q524*; EV vs. ERCC6; for C, P < 10⁻⁴ EV vs. ERCC4 and P < 10⁻³ ERCC4 vs. ERCC4-A583T; EV vs. ERCC4-A583T).

Figure 3

Inactivation of ERCC6 or ERCC4 does not impair HR efficiency and does not confer sensitivity to rucaparib and doxorubicin. A, addition of wild-type (WT) or mutant ERCC6 in ERCC6-deficient fibroblasts does not affect PARPi (rucaparib) sensitivity (left). Addition of wild-type ERCC4 to ERCC4-deficient fibroblasts does not affect PARPi sensitivity (right). B, siRNA-mediated depletion of ERCC6 or ERCC4 does not significantly affect HR as measured by a DR-GFP recombination assay in U2OS cells. BRCA2 depletion is shown as a positive control. Error bars, ± 1 SEM. Results are representative of three independent experiments. C, siRNA knockdown of ERCC4 or ERCC6 does not affect ionizing radiation-induced RAD51 foci formation in U2OS cells. Cells with ≥4 RAD51 foci were scored as positive. BRCA2-depleted cells are shown as a positive control. Positive cells were quantified 6 hours after irradiation (5 Gy; left plot). For each condition, 100 cells were counted. Error bars, ±1 SEM. Representative micrographs of irradiated cells are shown (right). D, addition of wild-type ERCC6 (black dashes) or mutant protein (red solid line, ERCC6-Q524*) in ERCC6-deficient fibroblasts does not impact sensitivity to doxorubicin (left). Addition of wild-type ERCC4 (black dashes) or mutant ERCC4 (red solid line, ERCC4-A583T) in ERCC4-deficient fibroblasts does not impact sensitivity to doxorubicin (right). Error bars, ± 1 SEM. Results are representative of three independent experiments.

Figure 4

Discordance between platinum and PARPi due to NER alterations: potential implications. A, potential implications of NER alterations for platinum and PARPi sensitivity in patients with BRCA/HR-proficient (I) and BRCA/HR-deficient (II) tumors. I, if tumors with NER alterations are HR proficient, i.e., they do not harbor concurrent HR alterations that would confer sensitivity to PARPis, they are expected to be platinum sensitive but not PARPi sensitive. II, if tumors with NER alterations are BRCA/HR deficient they will initially be sensitive to both platinum and PARPis. However, if these tumors develop resistance to PARPi via reversion of the HR defect (e.g., via secondary BRCA1/2 mutations), they are predicted to retain some sensitivity to subsequent platinum therapy due to the underlying NER alterations. B, expression of BRCA1 (BRCA1 + si Ctrl, red solid line) rescues the cisplatin hypersensitivity of BRCA1^−/−MDA-MB-436 cells [empty vector (EV) + si ERCC6, black dashed line]. However, BRCA1-restored cells with ERCC6-depletion (BRCA1 + si ERCC6, red dashed line) partially retain sensitivity to cisplatin compared with BRCA1-restored line without ERCC6-depletion (BRCA1 + si Ctrl, red solid line; left plot). Sensitivity is measured by 3-day viability. Error bars, ± 1 SEM. IC₅₀ values are shown as a measure of the relative cisplatin sensitivity of MDA-MB-436 cell lines (right plot). An immunoblot showing knockdown efficiency of ERCC6 and reexpression of BRCA1 in MDA-MB-436 cells is shown on the right. C, add-back of BRCA1 cDNA (BRCA1 + si Ctrl, red solid line) restores PARPi sensitivity of BRCA1^−/− MDA-MB-436 cells (EV + si ERCC6, black dashed line). Concurrent depletion of ERCC6 (BRCA1 + si ERCC6) in this cell line does not impact PARPi sensitivity (left plot). Sensitivity is measured by 3-day viability. Error bars, ± 1 SEM. IC₅₀ values are shown as a measure of the PARPi sensitivity of MDA-MB-436 cell lines (right).