Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma

Abstract

Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.

Fig 1. EWSCs are sensitive to PARP inhibition and S-phase DNA-damaging agents.

(A) and (C) Scatter plots of IC₅₀ (μM) values on a log scale comparing drug sensitivity of EWS-FLI1-positive and wild-type (WT) EWS-FLI1-negative cell lines to (A) four PARPi and (C) camptothecin and cisplatin. The sample size (n) is indicated and each circle represents the IC₅₀ of one cell line. The red bar is the geometric mean and the drug name is depicted above each plot along with the significance of the association as determined by an unpaired two-sample t-test. (B) Long term viability assays in EWSCs were performed in the presence of vehicle (-) or increasing concentrations of four PARPi as indicated. Non-EWSC lines (U-2-OS and HeLaSF) are included for comparison. These data are representative of 3 independent experiments.

Fig 2. Olaparib induces DNA DSBs in S-phase of the cell cycle in EWSCs.

(A) ES8 cells were treated with vehicle or olaparib and stained with Hoechst (nucleus; blue) and for γH2AX (DSBs; green). Images on the left are of the 8-hour time point. The graph measures fold increase in γH2AX responders at the time points indicated. Error bars represent the standard deviation of the mean of technical triplicates. (B) ES8 cells were treated with olaparib for 16 hours following a 6-hour pre-treatment with palbociclib (CDK4/6i) or vehicle and percentage of γH2AX responders determined. (C) ES8 cells were treated with vehicle, 5μM aphidicolin (AphD), 5μM olaparib (Ola) or a 30-minute pre-treatment with aphidicolin followed by olaparib for 8 hours and percentage of γH2AX responders determined. Asterisks indicate student’s paired t-test P value *P<0.05, **P<0.01, ns = not significant, relative to control. These data are representative of 3 independent experiments.

Fig 3. DNA DSB repair by HR is functional in EWSCs.

(A) Western blot of ES8 cells treated with olaparib for the times indicated. Markers are grouped as part of ATM or ATR signaling. Tubulin served as a loading control. (B) Western blot of ES8 cells treated with camptothecin and harvested at various time points following drug washout. GAPDH served as a loading control. (C) Percentage of EdU-positive and EdU-negative ES8 cells with >5 nuclear RAD51 foci following 6-hour treatment with vehicle or olaparib (ola). (D) Olaparib log GI₅₀ (μM) of cell lines mock-transfected or transfected with CtIP or BRCA1 siRNA as indicated.

Fig 4. EWSCs are sensitive to PARP1 trapping.

(A) Relative viability of mock-transfected and PARP1 siRNA-transfected ES8 cells treated with vehicle or olaparib. Asterisks indicate student’s paired t-test P value **P<0.01, ns = not significant. (B) PARP1 expression in cells transfected with a scrambled control or a titration of PARP1_1 siRNA and their relative viability following treatment with vehicle or olaparib. (C) IC50 values of parental ES8 and PARPi-resistant OLAR5 cells to five different PARPi and the fold difference between them. (D) Western blot of PARP1 expression in ES8 and OLAR5 cells. Viability values are the mean of technical triplicates and representative of 3 independent experiments.

Fig 5. Temozolomide enhances olaparib-induced PARP1 trapping.

(A) Heatmaps (left panel) of relative viability values of ES8 cells screened against a combination of niraparib and one of three chemotherapies or MMS. High viability values are in red and low viability values in green. Graphs (right panel) show the corresponding dose response curves measuring relative viability with a separate line plotted for each concentration of the combined drug. The dose response for niraparib alone is highlighted in red. Viability values are the mean of technical duplicates. (B) Relative viability of EWSCs treated with vehicle, niraparib (0.5uM) or temozolomide (TMZ) alone (200uM), or in combination. The combination effect is highlighted in green. (C) Fold induction of caspase 3/7 activation in EWSCs following treatment with vehicle, olaparib or temozolomide alone or in combination for a total of 48 hours. A student’s paired t-test was performed and significance values are indicated. (D) Cellular sub-fractionation assay following treatment of EWSCs with vehicle (-), MMS in combination with olaparib (ola), or olaparib and temozolomide (TMZ) alone or in combination for 4 hours.

Fig 6. Temozolomide enhances PARP inhibitor sensitivity in multiple tumour types.

List of cell lines screened against a combination of olaparib and temozolomide. Whether enhancement of PARP inhibitor sensitivity with temozolomide is observed (✔) or not (✖) is indicated.