Pemetrexed (PMX) antitumor activity is a consequence of primary inhibition of thymidylate synthase (TS) with much weaker inhibition of glycinamide ribonucleotide transformylase (GARFT) required for de novo purine synthesis. Methylthioadenosine phosphorylase (MTAP) salvages purines by releasing adenine from methylthioadenosine, a byproduct of the polyamine synthetic pathway, and therefore has the potential for antagonizing the antipurine effect of PMX. It has been suggested that the deletion of the MTAP gene from the majority of mesothelioma tumors enhances the activity of PMX. To investigate the role of MTAP in PMX activity, the impact of a highly potent transition-state inhibitor of this enzyme (MT-DADMe-Immucillin A (ImmA) - Ki = 86 pM) was assessed in two human mesothelioma cell lines: NCI-H28 which is MTAP+ and NCI-H2052 which is MTAP-. The relative inhibition of TS and GARFT was determined by the use of nucleoside protection. TS was found to be the primary target in both cell-lines (IC50s for H28 and H2052 were ∼100 nM and ∼40 nM respectively; there was no protection with addition of hypoxanthine alone). In the presence of thymidine, the IC50s for H28 and H2052 increased to ∼3 μM and ∼0.9 μM respectively. Full protection from PMX cytotoxicity at and beyond these concentrations required both thymidine and hypoxanthine. Hence, the antipurine effect mediated by GARFT inhibition occurred at 20-30 fold higher PMX concentrations than suppression of TS. ImmA did not alter the IC50 for PMX in the absence of thymidine. However, in the presence of thymidine, ImmA decreased the PMX IC50 by 3-4 fold in MTAP+ H28 cells without an effect in MTAP- H2052 cells. In a separate experiment in MTAP+ Hela cells, Imm A resulted in a 2-fold decrease in PMX IC50 in the presence of thymidine, but there was no effect in the absence of thymidine. Transfection of MTAP into H2052 MTAP- cells increased the PMX IC50 in the presence of thymidine with at least partial reversal with ImmA. However, there was essentially no difference noted in the absence of thymidine. These data suggest that purine salvage mediated by MTAP has no effect on PMX activity in the absence of thymidine, consistent with the primary role of TS in the action of PMX. Even under conditions in which purine inhibition is the primary site of action, suppression of MTAP has only a small effect on PMX activity. Hence, MTAP deletion in mesothelioma is unlikely to play an important role in the clinical activity of PMX in this disease.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]