AL398, A Novel NSUN6 Inhibitor, Attenuates Epithelial-Mesenchymal Transition and Metastasis in Endometrial Cancer by Abrogating m5C-Dependent Stabilization of Snail mRNA.

Endometrial cancer (EC) constitutes a leading gynecologic malignancy for which advanced or metastatic disease presents limited therapeutic options. While RNA modifications are acknowledged as key regulatory elements in cancer, the role of 5-methylcytosine (m5C) and its modifying enzymes in EC is still largely unstudied. Here, it demonstrates that the m5C methyltransferase NSUN6 is significantly upregulated in EC tissues, and its expression correlates with higher tumor grade and unfavorable prognosis. Functional studies employing gain- and loss-of-function models revealed that NSUN6 specifically enhances EC cell migration and invasion both in vitro and in vivo, without altering proliferation or apoptosis. Mechanistically, NSUN6 drives EMT by catalyzing m5C modification on Snail mRNA, which in turn stabilizes the transcript of this key EMT transcription factor. AL398 was identified through structure-based virtual screening as a novel small-molecule inhibitor that binds potently to the NSUN6 active site and suppresses its methyltransferase activity. Treatment with AL398 effectively inhibited the NSUN6/m5C/Snail axis, reversed the EMT phenotype, and attenuated EC cell migration, invasion, and metastasis in preclinical models. Collectively, it both validates the NSUN6/m5C/Snail axis as a critical EC vulnerability and provides a proof-of-concept inhibitor, AL398, laying the groundwork for the development of first-in-class RNA methylation-targeted therapies against metastatic EC.