Delineation of MGMT Hypermethylation as a Biomarker for Veliparib-Mediated Temozolomide-Sensitizing Therapy of Glioblastoma

Abstract

Background: Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed.

Methods: The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction.

Results: The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines.

Conclusion: Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.

Figure 1.

Pharmacokinetics and dosing schedule evaluation. A) Brain and plasma veliparib levels were measured up to six hours after the ninth dose (12.5mg/kg), administered twice daily; presented in the graphs are the averages from five observations, and vertical bars represent standard deviation. B-D) Evaluation of different dosing schedules for placebo, temozolomide (TMZ) alone, or combined with continuous veliparib were evaluated in a single experiment (n = 10 mice per group). Veliparib was dosed at 12.5mg/kg bid Monday through Friday (M-F) for six weeks combined with various TMZ regimens. B) Standard TMZ: 50mg/kg daily M-F x 1 week, (C) dose dense TMZ: TMZ 25mg/kg daily M-F x 3 weeks, and (D) metronomic TMZ: 15mg/kg M-F x 6 weeks. E) Standard TMZ (50mg/kg x 5 days) in combination with veliparib 12.5mg/kg bid x 5 or 12 days for two 28-day cycles (n = 10 mice per group, except TMZ group had n = 9 mice). P values by log-rank are reported for the comparison of TMZ vs TMZ + veliparib in all cases, except that the P value in E compares TMZ combined with veliparib x 5 days vs TMZ with veliparib x 12 days. All statistical tests were two-sided. AUC = area under the curve; TMZ = temozolomide.

Figure 2.

Comparison of temozolomide (TMZ)/veliparib vs TMZ response in Mayo glioblastoma multiforme (GBM) patient-derived xenograft (PDX) models based on MGMT promoter methylation status. Ratio of median survival for treatment (TMZ/veliparib or TMZ alone) relative to placebo (survival ratio) and the difference of survival ratio for TMZ/veliparib minus survival ratio for TMZ alone (survival ratio difference) are presented as boxplots. A) Boxplots show the survival ratio difference based on O ⁶-methylguanine DNA methyltransferase (MGMT) methylation status for 26 PDX lines. B) Boxplots show the survival ratio for TMZ/veliparib or TMZ alone for six MGMT promoter-unmethylated PDX models and (C) 20 MGMT promoter-hypermethylated PDX GBM models. For each xenograft line, mice with established orthotopic xenografts were randomized to therapy with placebo, veliparib (12.5mg/kg bid x 5 days), TMZ alone (50mg/kg x 5 days), or the combination of TMZ/veliparib for three cycles. Mice were observed daily and killed upon reaching a moribund state. The P values denote a Wilcoxon rank-sum test for graph in (A) and a paired signed rank test for graphs in (B and C). All statistical tests were two-sided. Δ-MS ratio = survival ratio difference; MGMT = O ⁶-methylguanine DNA methyltransferase.

Figure 3.

DNA damage signaling following temozolomide (TMZ)/veliparib treatment. Western blotting analysis of DNA damage response following treatment of flank tumor xenografts for five days with either placebo, veliparib, TMZ, or TMZ/veliparib (pooled samples from 3 mice per group) as in Figure 2 in (A) GBM12 vs GBM6 and (B) GBM39 vs GBM43. Tumor samples were harvested 72 hours after the last dose of TMZ, except veliparib-treated samples in GBM12 and GBM39 were harvested at two hours as denoted by an asterisk (*). C) Mice with orthotopic GBM12 tumors were treated as in (A) and processed for immunofluorescence for γH2AX (green) and DAPI (blue) 72 hours after the last dose of TMZ. Images captured with a 20X objective on a Leica AF6000 microscope; bar = 50 µm. Chk1 = checkpoint kinase 1; Chk2 = checkpoint kinase 2; DAPI = 4’,6-diamidino-2-phenylindole; KAP1 = KRAB-associated protein 1; MGMT = O ⁶-methylguanine DNA methyltransferase; PAR = poly ADP-ribose; PARP1 = poly ADP-ribose polymerase 1.

Figure 4.

O ⁶-methylguanine DNA methyltransferase (MGMT) overexpression decreases sensitizing effects of veliparib. A) Western blot analysis of GBM12, GBM12-MGMT (GBM12 cells transduced with pSIN-MGMT-UbEm), or GBM12TMZ#3080 (temozolomide [TMZ]-resistant derivative of GBM12 with acquired MGMT expression). Assessment of efficacy of TMZ, veliparib, or the combination in orthotopic models (n = 10 mice per group) of (B) GBM12-MGMT and (C) GBM12TMZ#3080 using the dosing regimen in Figure 1B. The P values denote a log-rank test comparing survival in TMZ/veliparib vs TMZ alone. All statistical tests were two-sided. MGMT = O ⁶-methylguanine DNA methyltransferase.

Figure 5.

Influence of O ⁶-methylguanine DNA methyltransferase (MGMT) status in GBM28 sublines. A) Polymerase chain reaction (PCR) amplification of MGMT and PTEN in GBM28 xenografts after indicated passage. Lanes representing passage 9 had both MGMT and PTEN intact and are denoted as GBM28A, while lanes representing late passage 13 with MGMT deleted but PTEN intact are denoted as GBM28B. B) aCGH analysis comparing GBM28A and GBM28B. Copy number variations on chromosome 10 are shown. C and D) The efficacy of TMZ, veliparib, or the combination using the dosing regimen in Figure 2 are shown in orthotopic models (n = 10 mice per group) of (C) GBM28A (D) GBM28B. The P values denote a log-rank test comparing survival in TMZ/veliparib vs TMZ alone. All statistical tests were two-sided. kBP = kilo basepairs of DNA; MGMT = O ⁶-methylguanine DNA methyltransferase; Pass# = passage number; PTEN = phosphatase and tensin homolog.