ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts

Abstract

Background: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ.

Methods: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888.

Results: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis.

Conclusion: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.

Fig 1. Average viability of Group I BTICs (n = 3, ± standard deviation or SD).

Lines were treated with DMSO, ABT-888 (10 μM), TMZ (50 μM), or the combination of TMZ (50 μM) and ABT-888 (10 μM). Eight days after treatment cell viability was inferred using the alamarBlue^® assay. The unpaired t-test was applied to assess differences, as shown. Viability was unaffected by ABT-888 alone, whereas TMZ decreased viability in all lines but BT073 (B). The combination decreased viability in two of five BTICs (ns = p > 0.05; ** = p < 0.01; **** = p < 0.0001).

Fig 2. Average viability of Group II BTICs (n = 3, ± SD).

Assessments and statistics were performed as in Fig 1. Viability was unaffected by ABT-888 or TMZ alone, except for BT075 (B). The combination of TMZ and ABT-888 significantly decreased viability in all BTICs (ns = p > 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Fig 3. Average viability of Group III BTICs (n = 3, ± SD).

Assessments and statistics were performed as in Fig 1. Viability was unaffected by ABT-888 alone and by TMZ alone, except for BT147 (D). The combination decreased viability in all BTIC lines (ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Fig 4. Average viability of BTICs in Group I vs. II vs. III (n = 3, ± SD).

The unpaired t-test was applied to assess differences. On average, Group I was TMZ sensitive whereas Groups II and III were not. The addition of ABT-888 potentiated TMZ cytotoxicity in Groups II and III, but had no such potentiating effect in Group I (ns = p > 0.05; *** = p < 0.001).

Fig 5. Kaplan-Meier survival curves of mice treated with DMSO, TMZ, or TMZ and ABT-888.

Mice were implanted with BTICs from Groups I, II and III. Mice bearing BT067 (A), a Group I line, lived significantly longer after TMZ, but had no further survival benefit from the addition of ABT-888 to TMZ. Mice bearing BT030 (B), a Group II line, did not live longer after TMZ alone, but lived significantly longer after the addition of ABT-888 to TMZ. Mice bearing BT143 and BT088 (C, D), methylated lines from Group III, lived significantly longer after TMZ and longer still after the addition of ABT-888 to TMZ. Mice bearing the BT147 (E), a Group III unmethylated line, did not live significantly longer after TMZ, but had significantly longer survival after the addition of ABT-888 to TMZ. The log-rank (Mantel-Cox) test was used to compare survival curves (ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Fig 6. TMZ plus ABT-888 restores DNA breaks, G2/M checkpoint activation, and synthesis arrest in MSH6 mutant lines.

(A) Alkaline comet assay representing the mean tail moments of 50 samples grown in suspension and treated with DMSO, TMZ (100 μM), ABT-888 (100 μM), or TMZ (100 μM) and ABT-888 (100 μM). Samples were stained with SYBR green and analyzed by epiflourescence microscopy; representative images are displayed. (B) Lysates taken from MSH6-wt and MSH6-kd cells were treated with DMSO, TMZ (100 μM), ABT-888 (100 μM), or TMZ (100 μM) and ABT-888 (100 μM) and a control sample collected immediately previous to treatment (Control = C), or 1h, 6h, 1d, and 3d after drug exposure. Western blotting was performed for total and phos-CHK1, total and phos-H2A.X, and actin (controls). (C) MSH6-wt and MSH6-kd cells were grown in suspension and exposed to DMSO, TMZ (100 μM), ABT-888 (100 μM), or TMZ (100 μM) and ABT-888 (100 μM). At days 4 and 7 after treatment cells were stained with EdU and analyzed on the BD LSRII flow cytometer.