Rationale for poly(ADP-ribose) polymerase (PARP) inhibitors in combination therapy with camptothecins or temozolomide based on PARP trapping versus catalytic inhibition

Abstract

We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors exert their cytotoxicity primarily by trapping PARP-DNA complexes in addition to their NAD(+)-competitive catalytic inhibitory mechanism. PARP trapping is drug-specific, with olaparib exhibiting a greater ability than veliparib, whereas both compounds are potent catalytic PARP inhibitors. Here, we evaluated the combination of olaparib or veliparib with therapeutically relevant DNA-targeted drugs, including the topoisomerase I inhibitor camptothecin, the alkylating agent temozolomide, the cross-linking agent cisplatin, and the topoisomerase II inhibitor etoposide at the cellular and molecular levels. We determined PARP-DNA trapping and catalytic PARP inhibition in genetically modified chicken lymphoma DT40, human prostate DU145, and glioblastoma SF295 cancer cells. For camptothecin, both PARP inhibitors showed highly synergistic effects due to catalytic PARP inhibition, indicating the value of combining either veliparib or olaparib with topoisomerase I inhibitors. On the other hand, for temozolomide, PARP trapping was critical in addition to catalytic inhibition, consistent with the fact that olaparib was more effective than veliparib in combination with temozolomide. For cisplatin and etoposide, olaparib only showed no or a weak combination effect, which is consistent with the lack of involvement of PARP in the repair of cisplatin- and etoposide-induced lesions. Hence, we conclude that catalytic PARP inhibitors are highly effective in combination with camptothecins, whereas PARP inhibitors capable of PARP trapping are more effective with temozolomide. Our study provides insights in combination treatment rationales for different PARP inhibitors.

Fig. 1.

Olaparib augments the cytotoxicity of temozolomide better than veliparib and PARP deficiency. (A and B) Viability curves of the indicated cell lines after continuous treatment for 72 hours with the indicated drugs. Cellular ATP concentration was used to measure cell viability. The viability of untreated cells was set as 100%. Error bars represent the S.D. (n = 3). *CI = 0.1–0.3 and **CI < 0.1 are indicated as strong and very strong synergy between the two treatments, respectively (see Fig. 6 and Supplemental Table 1). Viability curves of wild-type (upper panels) and PARP1^−/− cells (lower panels) treated with temozolomide alone or with the indicated concentrations of olaparib (A) or veliparib (B). The concentrations of PARP inhibitors are shown beside each curve in micromolar units. (C–F) Cell cycle analyses of wild-type (C and D) and PARP1^−/− cells (E and F) 12 hours after the indicated drug treatments [temozolomide (TMZ; 100 μM), olaparib (1 μM), and veliparib (1 μM)]. Representative data are shown from independent experiments with consistent results experiments (C and E). Percentages of cells in the sub-G1, G1, S, and G2-M phases are shown (D and F). Total counts within the outer frame of (C) or (E) are set as 100%. Results are the average of three independent experiments. Statistical analyses were performed for the G2-M population. *P < 0.05; **P < 0.01. Data of average and S.D. for all phases are shown in Supplemental Table 2. BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate.

Fig. 2.

Olaparib and veliparib augment the cytotoxicity of camptothecin (CPT) comparably. (A and B) Viability curves are shown as in Fig. 1A. *CI 0.1–0.3 and **CI < 0.1 are described as strong and very strong synergy between the two treatments, respectively. (C–F) Cell cycle analyses of wild-type (C and D) and PARP1^−/− cells (E and F) 12 hours after the indicated drug treatments [CPT (20 nM), olaparib (1 μM), and veliparib (1 μM)]. Representative data are shown from multiple experiments (C and E). Percentages of cells in the sub-G1, G1, S, and G2-M phases are shown (D and F). Total counts within the outer frame of (C) or (E) are set as 100%. Results are the average of three independent experiments. Statistical analyses were performed for the G2-M population. *P < 0.05; **P < 0.01. Data of average and S.D. for all phases are shown in Supplemental Table 2. BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate.

Fig. 3.

Differential effect of olaparib and veliparib with temozolomide (A) or camptothecin (B) in human cell lines. Viability curves of human prostate cancer DU145 cells (top panels) and human glioblastoma SF295 cells (bottom panels) after continuous treatment for 72 hours with the indicated drug treatments [olaparib (1 μM) and veliparib (1 μM)]. Viability curves are shown as in Fig. 1A. *CI = 0.1–0.3 and **CI < 0.1 are described as strong and very strong synergy between the two treatments, respectively.

Fig. 4.

PARP inhibitors induce PARP-DNA complexes with temozolomide (TMZ) but not with camptothecin (CPT). Western blot of chromatin-bound fractions against anti-PARP1, anti–histone H3, and anti-PAR antibodies (A). Western blot of chromatin-bound fractions against anti-PARP1 and anti-PCNA antibodies (C). Samples were prepared from wild-type DT40 cells (left) and DU145 cells (right) treated for 30 minutes and 4 hours, respectively, with the indicated drugs. Controls without drug are shown in lanes 1 and 9. Histone H3 (A) and PCNA (C) were used as positive markers for loading control. The blots are representatives of multiple experiments. (B) Quantification of PARP-DNA complexes after the indicated treatments. Signal intensity was quantified using ImageJ software (National Institutes of Health) from four independent Western blot analyses (two blots of wild-type DT40 and two blots of DU145 cells). The intensity of the PARP1 blot divided by the intensity of the corresponding histone H3 blot was measured for each treatment, and normalized to the sample of 1 μM veliparib + 1 mM TMZ treatment. Means ± S.D. (n = 4) are shown.

Fig. 5.

PARP1 is not involved in the repair of cisplatin- and etoposide-induced lesions. Viability curves are shown as in Fig. 1A. (A and C) Viability curves of wild-type and PARP1^−/− DT40 cells treated with cisplatin (left) or etoposide (right) (top panels). (Bottom panels) Viability curves of wild-type DT40 cells in combinations with the indicated concentrations of olaparib (micromolar units beside curves) with cisplatin (left) or etoposide (right). (B and D) Viability curves of human prostate cancer DU145 cells in combination with the indicated concentrations of olaparib (micromolar units beside curves) with cisplatin (left) or etoposide (right).

Fig. 6.

Quantitative analyses of synergistic effects in the different combinations. (A) Fa-CI plots obtained from the data of Fig. 1 (A and B) for temozolomide in combination with 1 μM olaparib or 1 μM veliparib. (B) Fa-CI plots obtained from the data of Fig. 2 (A and B) for camptothecin in combination with 1 μM olaparib or 1 μM veliparib. (C and D) Fa-CI plots obtained from the data of Fig. 5A (bottom panel) for cisplatin (C) and Fig. 5C (bottom panel) for etoposide (D) in combination with 1 μM olaparib. (A–D) Shading reflects the level of synergism. CI between 0.3 and 0.7, CI between 0.1 and 0.3, and CI less than 0.1 indicate synergy, strong synergy, and very strong synergy, respectively. All data for CI are shown in Supplemental Table 1.