PURPOSE The therapeutic benefit of temozolomide (TMZ) in glioblastoma (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of TMZ resistance in GBM. EXPERIMENTAL DESIGN We developed an in vivo GBM model of TMZ resistance and used paired parental and TMZ resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. RESULTS Analysis of paired parental and resistant lines demonstrated upregulation of MGMT expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative resistant samples, chromatin immunoprecipitation demonstrated an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased di-methylation (H3K9-me2) in GBM12 and GBM14. In contrast, TMZ resistance development in GBM22 was not linked to MGMT expression and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12 TMZ resistant line, MGMT re-expression was accompanied by increased recruitment of SP1, C-JUN, NF-kB and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with TMZ and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of TMZ resistance by MGMT over-expression as compared to treatment with TMZ alone. CONCLUSION This study demonstrates, for the first time, a unique mechanism of TMZ resistance development driven by chromatin mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM.

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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.

Cancer cells can resist the effects of DNA-damaging therapeutic agents via utilization of DNA repair pathways, suggesting that DNA repair capacity (DRC) measurements in cancer cells could be used to identify patients most likely to respond to treatment. However, the limitations of available technologies have so far precluded adoption of this approach in the clinic. We recently developed fluorescence-based multiplexed host cell reactivation (FM-HCR) assays to measure DRC in multiple pathways. Here we apply a mathematical model that uses DRC in multiple pathways to predict cellular resistance to killing by DNA-damaging agents. This model, developed using FM-HCR and drug sensitivity measurements in 24 human lymphoblastoid cell lines, was applied to a panel of 12 patient-derived xenograft (PDX) models of glioblastoma to predict glioblastoma response to treatment with the chemotherapeutic DNA-damaging agent temozolomide. This work showed that, in addition to changes in O6-methylguanine DNA methyltransferase (MGMT) activity, small changes in mismatch repair (MMR), nucleotide excision repair (NER), and homologous recombination (HR) capacity contributed to acquired temozolomide resistance in PDX models and led to reduced relative survival prolongation following temozolomide treatment of orthotopic mouse models in vivo. Our data indicate that measuring the combined status of MMR, HR, NER, and MGMT provided a more robust prediction of temozolomide resistance than assessments of MGMT activity alone.