PARP inhibition suppresses the emergence of temozolomide resistance in a model system.

INTRODUCTION: Temozolomide (TMZ) is a life prolonging DNA alkylating agent active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately acquired TMZ resistance severely undermines its clinical efficacy. Using an in vitro model, we tested whether poly (ADP-ribose) polymerase-1 and -2 (PARP) inhibition could suppress the emergence of resistance to enhance the effectiveness of TMZ. METHODS: Using the MGMT-methylated GBM line U251N, in which TMZ resistance can be induced, we developed a method to rapidly recreate mechanisms of TMZ resistance seen in GBMs, including MMR mutations and MGMT re-expression. We then assessed whether TMZ resistant U251N sub-clones could be re-sensitized to TMZ by co-treatment with the PARP inhibitor ABT-888, and also whether the emergence of resistance could be suppressed by PARP inhibition. RESULTS: U251N cultures chronically exposed to TMZ developed discrete colonies that expanded during TMZ treatment. These colonies were isolated, expanded further as sub-clones, and assessed for mechanisms of TMZ resistance. Most resistant sub-clones had detectable mutations in one or more mismatch repair (MMR) genes, frequently MSH6, and displayed infrequent re-expression of MGMT. TMZ resistance was associated with isolated poly(ADP-ribose) (pADPr) up-regulation in one sub-clone and was unexplained in several others. TMZ resistant sub-clones regressed during co-treatment with TMZ and ABT-888, and early co-treatment of U251N parental cultures suppressed the emergence of TMZ resistant colonies. CONCLUSION: In a model of acquired resistance, co-treatment with TMZ and a PARP inhibitor had two important benefits: re-sensitization of TMZ resistant cells and suppression of TMZ resistance.

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

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.