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.
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.
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.
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.
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The Terry Fox Research Institute and Foundation, Alberta Cancer Foundation, Genome Canada, Alberta Innovates Health Solutions, and the family of Clark H. Smith supported this work.
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Supplementary file1 (TIF 24678 kb) Figure S1. Western blot analysis of MLH1, MSH6, MSH2, PMS2 and MGMT protein expression in the parental U251N line and 18 TMZ- resistant sub-clones. Compared to parental sample levels, expression of MSH6, MSH2 and PMS2 was decreased in some sub-clones, while MLH1 expression remained similar. MGMT induction was observed in one resistant sub-line. “+” denotes the positive control. “P” denotes the parental U251N sample.
Supplementary file2 (TIF 24677 kb) Figure S2. Cropped chromatograms showing MMR mutations observed in U251N TMZ-resistant sub-clones as outlined in Table: (A) Colony 15 MLH1 mutation (B) Colony 6 MSH6 mutation #1 (C) Colony 6 MSH6 mutation #2 (D) Colony 6 MSH6 mutation #3 (E) Colony 7 MSH6 mutation (F) Colony 9 MSH6 mutation #1 (G) Colony 9 MSH6 mutation #2 (H) Colony 12 MSH6 mutation (I) Colony 12 MLH1 mutation and (J) Colony 1 MSH2 exon 1 deletion. All missense mutations presented with the wild-type and mutant nucleotide, suggesting allelic heterozygosity or heterogeneity among sampled cells.
Supplementary file3 (TIF 24677 kb) Figure S3. Uncropped versions of the Western blots Data presented in Figure S1 of (A) MLH1 (B) PMS2 (C) MSH6 (D) MGMT (E) MSH2 and (F) pADPr protein expression in TMZ-resistant U251N sub-clones. Molecular weight ladders could not be pictured on indicated (*) blots.
Supplementary file4 (TIF 24676 kb) Table S1. Mutational status of MMR repair genes in TMZ resistant colonies. Mutation status of MSH2, MSH6, MLH1 and PMS2 in the parental U251N line and a subset of TMZ-resistant sub-clones introduced above. Most sub-clones harbored one or more mutations in at least one MMR gene. One silent mutation and 2 single-nucleotide polymorphisms were observed in the PMS2 gene of the parental U251N line and all derivative sub-clones. WT = wild type. All amino acids and nucleotides are denoted by standard symbols.
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Yuan, A.L., Meode, M., Tan, M. et al. PARP inhibition suppresses the emergence of temozolomide resistance in a model system. J Neurooncol (2020). https://doi.org/10.1007/s11060-020-03561-1