Synergistic activity of PARP inhibition by talazoparib (BMN 673) with temozolomide in pediatric cancer models in the pediatric preclinical testing program

Abstract

Purpose: Inhibitors of PARP, an enzyme involved in base excision repair, have demonstrated single-agent activity against tumors deficient in homologous repair processes. Ewing sarcoma cells are also sensitive to PARP inhibitors, although the mechanism is not understood. Here, we evaluated the stereo-selective PARP inhibitor, talazoparib (BMN 673), combined with temozolomide or topotecan.

Experimental design: Talazoparib was tested in vitro in combination with temozolomide (0.3-1,000 μmol/L) or topotecan (0.03-100 nmol/L) and in vivo at a dose of 0.1 mg/kg administered twice daily for 5 days combined with temozolomide (30 mg/kg/daily x 5; combination A) or 0.25 mg/kg administered twice daily for 5 days combined with temozolomide (12 mg/kg/daily x 5; combination B). Pharmacodynamic studies were undertaken after 1 or 5 days of treatment.

Results: In vitro talazoparib potentiated the toxicity of temozolomide up to 85-fold, with marked potentiation in Ewing sarcoma and leukemia lines (30-50-fold). There was less potentiation for topotecan. In vivo, talazoparib potentiated the toxicity of temozolomide, and combination A and combination B represent the MTDs when combined with low-dose or high-dose talazoparib, respectively. Both combinations demonstrated significant synergism against 5 of 10 Ewing sarcoma xenografts. The combination demonstrated modest activity against most other xenograft models. Pharmacodynamic studies showed a treatment-induced complete loss of PARP only in tumor models sensitive to either talazoparib alone or talazoparib plus temozolomide.

Conclusions: The high level of activity observed for talazoparib plus temozolomide in Ewing sarcoma xenografts makes this an interesting combination to consider for pediatric evaluation.

Figure 1

A. Antitumor activity of temozolomide (30 mg/kg Dx5), talazoparib (0.25 mg/kg BID Dx5, or in combination against ‘sensitive’ Ewing sarcoma models. Combination A (temozolomide 30 mg/kg Dx5; talazoparib 0.1 mg/kg BID Dx5); Combination B (temozolomide 12 mg/kg Dx5; talazoparib 0.25 mg/kg BID Dx5). Graphs show growth of individual tumors in SCID mice. B. Antitumor activity of temozolomide (30 mg/kg Dx5), talazoparib (0.25 mg/kg BID Dx5, or in combination against ‘insensitive’ Ewing sarcoma models. Combination A (temozolomide 30 mg/kg Dx5; talazoparib 0.1 mg/kg BID Dx5); Combination B (temozolomide 12 mg/kg Dx5; talazoparib 0.25 mg/kg BID Dx5). Graphs show growth of individual tumors in SCID mice.

Figure 1

A. Antitumor activity of temozolomide (30 mg/kg Dx5), talazoparib (0.25 mg/kg BID Dx5, or in combination against ‘sensitive’ Ewing sarcoma models. Combination A (temozolomide 30 mg/kg Dx5; talazoparib 0.1 mg/kg BID Dx5); Combination B (temozolomide 12 mg/kg Dx5; talazoparib 0.25 mg/kg BID Dx5). Graphs show growth of individual tumors in SCID mice. B. Antitumor activity of temozolomide (30 mg/kg Dx5), talazoparib (0.25 mg/kg BID Dx5, or in combination against ‘insensitive’ Ewing sarcoma models. Combination A (temozolomide 30 mg/kg Dx5; talazoparib 0.1 mg/kg BID Dx5); Combination B (temozolomide 12 mg/kg Dx5; talazoparib 0.25 mg/kg BID Dx5). Graphs show growth of individual tumors in SCID mice.

Figure 2

Antitumor activity of temozolomide (30 mg/kg Dx5), talazoparib (0.25 mg/kg BID Dx5), or in combination against xenograft models sensitive to temozolomide (GBM2, Rh28, MGMT-deficient), or single agent talazoparib (KT-10), and two additional alveolar rhabdomyosarcoma xenografts (Rh30, Rh41). Combination A (temozolomide 30 mg/kg Dx5; talazoparib 0.1 mg/kg BID Dx5); Combination B (temozolomide 12 mg/kg Dx5; talazoparib 0.25 mg/kg BID Dx5). Graphs show growth of individual tumors in SCID mice.

Figure 3

Loss of PARP is associated with increased sensitivity to talazoparib (TAL) or temozolomide (TMZ)/talazoparib combinations in vivo. A. KT-10 and BT-45 xenograft tissues were harvested 4 hr and 24 hr after the ninth dose of talazoparib (TAL 0.25 mg/kg administered BID) and processed as described in Materials and Methods. B. TC-71 xenografts were harvested 6 hr post day 5 dosing with temozolomide (12 mg/kg/d), talazoparib (0.25 mg/kg BID, 9 doses) or the combination. Immunoblots were probed for PARP, cleaved PARP, γH2AX, and GAPDH (loading control). C. Tumor lysates from B (TAL 0.25 mg/kg + TMZ 12 mg/kg) were treated with DNase or heat to recover any PARP bound to DNA. D, Pharmacodynamic changes in CHLA-258 xenografts. Levels of PARP and γH2AX 6 hr post dose 1 for temozolomide (12 mg/kg) and talazoparib (TAL 0.25 mg/kg); E, Levels of PARP and γH2AX 6 hr post dose 3 for temozolomide (12 mg/kg) and dose 7 for talazoparib (0.25 mg/kg); F, Levels of PARP and γH2AX 4 hr and 24 hr post dose 5 for temozolomide (12 mg/kg) and dose 11 for talazoparib (0.25 mg/kg). GAPDH was used as a loading control (n=3).

Figure 4

Pharmacodynamic changes induced by talazoparib and temozolomide in nonresponsive xenograft lines EW-8 and EW-5. A. EW-8; B. EW-5. xenograft tissues (n=3) were harvested after 5 days temozolomide (12 mg/kg/d), talazoparib (0.25 mg/kg BID, 9 doses) or the combination and processed as described in Materials and Methods. Immunoblots were probed for PARP, cleaved PARP, γH2AX, and GAPDH (loading control). C., PARP levels in control tumors (n=3); D Talazoparib alone or the combination of temozolomide and talazoparib decreases PARP transcripts in sensitive xenograft models. PARP transcripts (normalized to GAPDH transcripts) were determined by QRT-PCR in control tumors, and tumors harvested 6 hr post dosing on day 5 treatment with temozolomide (12 mg/kg) and talazoparib (0.25 mg/kg) E. Development of TC-71 xenografts resistant to Combination B. Resistance was developed as described in Materials and Methods. The response of individual naïve parental TC-71 tumors (designated Pass R0), Pass R1 (the second drug treatment), pass R3 and pass R5 are shown. The relative tumor volume plots (right) show the growth of the median tumor for each group (gray, Control; black treated). Note in Pass 0, 10 tumor bearing mice were treated and the median tumor volume at 6 weeks was < 0.1 CM³. For other groups 5 tumor bearing mice were in the treatment groups. F. Western blot for PARP, cleaved PARP and γH2AX for TC-71 parental xenografts (untreated), control (untreated) TC-71R and treated TC-71R xenografts.