Therapeutics by cytotoxic metabolite accumulation: pemetrexed causes ZMP accumulation, AMPK activation, and mammalian target of rapamycin inhibition

Abstract

Pemetrexed represents the first antifolate cancer drug to be approved by the Food and Drug Administration in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non-small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis that becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP.

Figure 1. Reversal of CEM cell growth inhibition by AICA indicated that the second target of pemetrexed is AICART, not GART

CEM cells were treated with the indicated concentrations of pemetrexed alone (A, no add) or 6R-DDATHF (B, no add) or in the presence of TdR (5.6 μM), Hx (32 μM), AICA (320 μM), or a combination of TdR with either Hx or AICA. Drug and modifying agents were added simultaneously and drug-containing medium was changed at 48 hours. Cell growth was determined after 96 hours and cell number is expressed relative to controls without drug. Panel C depicts the folate-dependent steps of de novo purine synthesis and the site of entry of AICA and AICAR into the pathway. De novo purine synthesis consists of ten sequential enzymatic reactions starting with 1-phosphoribosyl-5-pyrophosphate (PRPP) of which two, GART and AICART, are folate-dependent. The figure depicts the substrate of the GART reaction, glycinamide ribonucleotide (GAR), and the product of this reaction formylglycinamide ribonucleotide (FGAR), as well as the salvage of AICA, AICAR, and hypoxanthine catalyzed by adenine phosphoribosyltransferase (APRT), adenosine kinase (AK), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), respectively.

Figure 2. Substrate accumulation studies support an effect of pemetrexed on AICART in intact CEM cells

(A.) Accumulation of ¹⁴C-glycine as FGAR in pemetrexed and (6R)-DDATHF-treated CEM cells. Cells were exposed to either (6R)-DDATHF or pemetrexed plus 5.6 μM thymidine for 24 hours, nucleotides extracted and FGAR separated by anion-exchange chromatography. (B.) Increased cellular ZMP pools in pemetrexed-treated but not (6R)-DDATHF-treated CEM cells. Cells were exposed to drugs for 48 hours, nucleotides extracted and ZMP separated by hplc. (C.) CEM cells were exposed to 1μM pemetrexed for the indicated periods and ZMP analyzed as in (B.) The bar shown at zero time indicated the limit of sensitivity of the assay.

Figure 3. Growth inhibition by ZMP

(A.) CEM cells were exposed to the indicated concentrations of AICAR for 24 hours, and ZMP (open circles) was then analyzed as in Fig. 2B. Cell number (filled circles) was measured electronically after 24 hours. Panel B correlates the cell number after 24 hours of growth with the intracellular ZMP pool from panel A.

Figure 4. Effects of pemetrexed on activation of AMPK and inhibition of mTOR

(A.) Schematic diagram showing activation of AMPK by either AMP or ZMP that results in inhibition of mTOR and its downstream targets. (B,C.) Western blot analysis of total and phosphorylated AMPK, ACC, Raptor, S6K1, and 4EBP1. The molecular masses of these bands were 62 kD (AMPK), 280 kD (ACC), 150 kDa (Raptor), S6K1 (70 kD), and 15–20 kD (4EBP1). Equal levels of total protein (40 μg) was loaded in each lane; use of actin as a control demonstrated equal loading between lanes in this and the following figures (not shown). Vehicle was PBS, pemetrexed was used at 1 μM, AICAR was at 250 μM, and TdR was 5.6μM; drug exposure was 48 hours.

Figure 5. Expansion of the ZMP pool by AICA enhances the effect of pemetrexed on the AMPK-mTOR pathway

Exposure to AICA for 48 hours increased cellular levels of ZMP in a dose-related manner in pemetrexed-treated but not untreated CEM cells (A). In panel B, western blot analysis and drug exposures were as in Fig. 4B; AICA was used at 320 μM.

Figure 6. Prevention of the secondary effects of pemetrexed by purines and by compound C

(A.) When CEM cells were exposed to 1 μM pemetrexed and 5.6 μM thymidine for 48 hours, hypoxanthine in the medium prevented the accumulation of ZMP, in a dose-dependent manner that correlated with protection from growth inhibition. Cell growth (filled circles) and ZMP levels (open circles) were determined as in Fig. 3. (B.) Cellular activity of AMPK was assessed by western blot analysis of phosphorylation of ACC and Raptor, and the activity of mTOR was indicated by the phosphorylation of S6K1 and 4EBP1. CEM cells were exposed to drug and modifying agents for 48 hours as in Fig. 4 and Hx was used at 32 μM; conditions for western blots were as in Fig. 4. (C.) Compound C (1 μM, 48 hour exposure) prevented the cellular activity of AMPK by pemetrexed without interfering with the phosphorylation of AMPK. Vehicle was DMSO in PBS; all other conditions were as in Fig. 4.