Pyrimethamine regulates m6A modification of CCND1 via METTL14/YTHDF2 to inhibit hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a dismal prognosis with limited therapeutic options, and drug repurposing is a promising anti-HCC strategy. Pyrimethamine (Pyr), an FDA-approved antifolate drug with potential antitumor activity, its molecular mechanism in HCC remains elusive. In this study, in vitro experiments (MTT, colony formation, flow cytometry) confirmed that Pyr dose-dependently inhibited Hep3B and Huh7 cell proliferation, induced DNA damage and G0/G1 cell cycle arrest, and downregulated CCND1 expression. Mechanistically, Pyr upregulated METTL14 to enhance global m6A modification, and METTL14 directly mediated the m6A modification of CCND1 mRNA; the modified CCND1 was specifically recognized by YTHDF2, leading to its mRNA degradation. Bioinformatic and clinical sample analyses verified that METTL14 was downregulated in HCC tissues and acted as a tumor suppressor in vitro. In HCC patient-derived xenograft (PDX) models, Pyr significantly suppressed tumor growth, upregulated METTL14 and YTHDF2 expression, downregulated CCND1, and exhibited no obvious organ toxicity. This study reveals that Pyr exerts anti-HCC activity by activating the METTL14/YTHDF2 axis to induce m6A-dependent CCND1 mRNA degradation and subsequent G0/G1 arrest, uncovers a novel epitranscriptomic mechanism of Pyr, and supports its repurposing as a potential therapeutic agent for HCC.