Effective sensitization of temozolomide by ABT-888 is lost with development of temozolomide resistance in glioblastoma xenograft lines

Abstract

Resistance to temozolomide and radiotherapy is a major problem for patients with glioblastoma but may be overcome using the poly(ADP-ribose) polymerase inhibitor ABT-888. Using two primary glioblastoma xenografts, the efficacy of ABT-888 combined with radiotherapy and/or temozolomide was evaluated. Treatment with ABT-888 combined with temozolomide resulted in significant survival prolongation (GBM12: 55.1%, P = 0.005; GBM22: 54.4%, P = 0.043). ABT-888 had no effect with radiotherapy alone but significantly enhanced survival in GBM12 when combined with concurrent radiotherapy/temozolomide. With multicycle therapy, ABT-888 further extended the survival benefit of temozolomide in the inherently sensitive GBM12 and GBM22 xenograft lines. However, after in vivo selection for temozolomide resistance, the derivative GBM12TMZ and GBM22TMZ lines were no longer sensitized by ABT-888 in combination with temozolomide, and a similar lack of efficacy was observed in two other temozolomide-resistant tumor lines. Thus, the sensitizing effects of ABT-888 were limited to tumor lines that have not been previously exposed to temozolomide, and these results suggest that patients with newly diagnosed glioblastoma may be more likely to respond to combined temozolomide/poly(ADP-ribose) polymerase inhibitor therapy than patients with recurrent disease.

Figure 1

ABT-888 combined with chemo-radiation in GBM orthotopic xenografts. Mice with established orthotopic xenografts from (A) GBM12 and (B) GBM22 were randomized to therapy with the indicated combinations of temozolomide (TMZ – 33 mg/kg/day, 5 of 7 days × 2 weeks), radiation (RT – 2 Gy/day, 5 of 7 days × 2 weeks) and ABT-888 (15 mg/kg/day, 6 of 7 days × 2 weeks). Mice were followed until reaching a moribund state, and survival results are shown. Indicated p-values are comparing an indicated treatment with or without ABT-888. C) The change in relative body weight for mice from the GBM12 experiment treated with placebo, RT/TMZ, or ABT-888/RT/TMZ is shown. The gray bar indicates the duration of treatment. * - p <0.005; † - p=0.05; ‡ - p=0.28.

Figure 1

ABT-888 combined with chemo-radiation in GBM orthotopic xenografts. Mice with established orthotopic xenografts from (A) GBM12 and (B) GBM22 were randomized to therapy with the indicated combinations of temozolomide (TMZ – 33 mg/kg/day, 5 of 7 days × 2 weeks), radiation (RT – 2 Gy/day, 5 of 7 days × 2 weeks) and ABT-888 (15 mg/kg/day, 6 of 7 days × 2 weeks). Mice were followed until reaching a moribund state, and survival results are shown. Indicated p-values are comparing an indicated treatment with or without ABT-888. C) The change in relative body weight for mice from the GBM12 experiment treated with placebo, RT/TMZ, or ABT-888/RT/TMZ is shown. The gray bar indicates the duration of treatment. * - p <0.005; † - p=0.05; ‡ - p=0.28.

Figure 1

ABT-888 combined with chemo-radiation in GBM orthotopic xenografts. Mice with established orthotopic xenografts from (A) GBM12 and (B) GBM22 were randomized to therapy with the indicated combinations of temozolomide (TMZ – 33 mg/kg/day, 5 of 7 days × 2 weeks), radiation (RT – 2 Gy/day, 5 of 7 days × 2 weeks) and ABT-888 (15 mg/kg/day, 6 of 7 days × 2 weeks). Mice were followed until reaching a moribund state, and survival results are shown. Indicated p-values are comparing an indicated treatment with or without ABT-888. C) The change in relative body weight for mice from the GBM12 experiment treated with placebo, RT/TMZ, or ABT-888/RT/TMZ is shown. The gray bar indicates the duration of treatment. * - p <0.005; † - p=0.05; ‡ - p=0.28.

Figure 2

ABT-888 combined with adjuvant temozolomide in GBM orthotopic xenografts. Mice with established orthotopic xenografts from (A) GBM12 and (B) GBM22 were randomized to therapy with TMZ (66 mg/kg/day, day 1–5) administered in 1, 2 or 3 – 28 day cycles (TMZ 1, TMZ 2, and TMZ 3, respectively), or combined therapy with ABT-888 (15 mg/kg/day, day 1–6) and TMZ for 1, 2 or 3 – 28 day cycles (TMZ ABT 1, TMZ ABT 2, and TMZ ABT 3, respectively), or placebo or ABT-888 alone for 3 cycles. Indicated p-values are comparing TMZ alone to TMZ + ABT-888 for each cycle.

Figure 2

ABT-888 combined with adjuvant temozolomide in GBM orthotopic xenografts. Mice with established orthotopic xenografts from (A) GBM12 and (B) GBM22 were randomized to therapy with TMZ (66 mg/kg/day, day 1–5) administered in 1, 2 or 3 – 28 day cycles (TMZ 1, TMZ 2, and TMZ 3, respectively), or combined therapy with ABT-888 (15 mg/kg/day, day 1–6) and TMZ for 1, 2 or 3 – 28 day cycles (TMZ ABT 1, TMZ ABT 2, and TMZ ABT 3, respectively), or placebo or ABT-888 alone for 3 cycles. Indicated p-values are comparing TMZ alone to TMZ + ABT-888 for each cycle.

Figure 3

ABT-888 combined with temozolomide in xenograft lines with acquired TMZ resistance. Mice with established orthotopic xenograft from (A) GBM12TMZ, (B) GBM22TMZ, and (C) GBM39TMZ were randomized to therapy with placebo, ABT-888 alone, TMZ alone and TMZ + ABT-888, except for GBM39 in which the ABT-888 alone arm was omitted. The p-values shown correspond to the comparison between TMZ alone and TMZ + ABT-888.

Figure 3

ABT-888 combined with temozolomide in xenograft lines with acquired TMZ resistance. Mice with established orthotopic xenograft from (A) GBM12TMZ, (B) GBM22TMZ, and (C) GBM39TMZ were randomized to therapy with placebo, ABT-888 alone, TMZ alone and TMZ + ABT-888, except for GBM39 in which the ABT-888 alone arm was omitted. The p-values shown correspond to the comparison between TMZ alone and TMZ + ABT-888.

Figure 3

ABT-888 combined with temozolomide in xenograft lines with acquired TMZ resistance. Mice with established orthotopic xenograft from (A) GBM12TMZ, (B) GBM22TMZ, and (C) GBM39TMZ were randomized to therapy with placebo, ABT-888 alone, TMZ alone and TMZ + ABT-888, except for GBM39 in which the ABT-888 alone arm was omitted. The p-values shown correspond to the comparison between TMZ alone and TMZ + ABT-888.

Figure 4

PARP activity in xenograft lines: A) Endogenous PARP activity levels were evaluated in the indicated tumor lines and compared to those in normal brain. B) Inhibition of PARP activity was evaluated by western blotting for PAR. Mice with established flank tumors from GBM12 (n=6) or GBM12TMZ (n=6) were treated with Placebo (P) or ABT-888 15 mg/kg/day (A) in divided doses and killed after the final dose. Individual tumor lysates were resolved by SDS-PAGE and immunoblotted with an antibody specific for PAR polymer and subsequently for actin.

Figure 4

PARP activity in xenograft lines: A) Endogenous PARP activity levels were evaluated in the indicated tumor lines and compared to those in normal brain. B) Inhibition of PARP activity was evaluated by western blotting for PAR. Mice with established flank tumors from GBM12 (n=6) or GBM12TMZ (n=6) were treated with Placebo (P) or ABT-888 15 mg/kg/day (A) in divided doses and killed after the final dose. Individual tumor lysates were resolved by SDS-PAGE and immunoblotted with an antibody specific for PAR polymer and subsequently for actin.

Figure 5

High-dose ABT-888 therapy. Mice with established intracranial xenografts derived from the secondary TMZ resistant line, GBM14TMZ, were randomized to therapy with placebo, TMZ alone (66 mg/kg/day × 5 days), ABT-888 (15 mg/kg/day, divided dose) + TMZ or ABT-888 (40 mg/kg/day, divided dose) + TMZ. Survival curves are shown for each arm. P-values are shown for TMZ relative to placebo and for the two ABT-888 treatment arms relative to TMZ alone.