Methotrexate inhibits the TEL-JAK2–STAT pathway through a folate metabolism-dependent mechanism in Ba/F3 cells

Methotrexate (MTX) is a folate antagonist that inhibits dihydrofolate reductase (DHFR), thereby depleting intracellular reduced folate pools. Previous studies have reported that MTX suppresses the JAK-STAT signaling pathway; however, the contribution of folate antagonism to this effect versus potential direct kinase inhibition remains poorly understood. In this study, we investigated the effects of MTX on the signaling activity of TEL-JAK2, a constitutively active fusion protein associated with T-cell acute lymphoblastic leukemia. Ba/F3 cells expressing TEL-JAK2 were treated with MTX or 5-fluorouracil (5-FU), a DNA synthesis inhibitor that targets thymidylate synthase without depleting the upstream folate pool. Both agents effectively reduced cell proliferation and viability while inducing apoptosis. Notably, MTX suppressed the phosphorylation of TEL-JAK2 and its downstream effectors, STAT3 and STAT5. The MTX-induced suppression of the TEL-JAK2-STAT axis was fully reversed by supplementation with folinic acid. Furthermore, genetic ablation of DHFR using CRISPR/Cas9 recapitulated these effects, whereas overexpression of DHFR attenuated MTX-induced apoptosis and suppression of the TEL-JAK2-STAT axis, confirming that disruption of folate metabolism leads to the suppression of the TEL-JAK2-STAT axis. In contrast, 5-FU had no effect on the phosphorylation of TEL-JAK2, STAT3, or STAT5, indicating that the inhibition is not a secondary consequence of DNA synthesis impairment. These findings demonstrate that MTX inhibits the TEL-JAK2-STAT pathway through a folate-dependent mechanism, revealing a novel link between folate metabolism and oncogenic JAK-STAT signaling.