Withaferin A suppresses malignant progression of leukemia by reducing promoter methylation levels

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with limited effective treatment options. Withaferin A, a steroidal lactone derived from Withania somnifera , has demonstrated broad anticancer activity across various tumor models. Emerging evidence suggests its potential to modulate DNA methylation and to suppress malignant behaviors, though the underlying mechanisms have so far remained unclear. In this study, an in-depth investigation of the pharmacological effects of withaferin A in T-ALL was conducted, focusing on its epigenetic regulatory role. Cytotoxicity assays showed that withaferin A exhibited the strongest activity among its analogs, particularly against CCRF-CEM cells. Through the integration of bioinformatics, network pharmacology, and functional cell experiments, it was found that withaferin A induces G2/M cell cycle arrest, promotes apoptosis, inhibits migration, and suppresses microtubule polymerization in T-ALL cells. Luciferase reporter experiments further confirmed that withaferin A decreases methylation of the CMV promoter. Also, withaferin A alters the expression levels of P21, ITPKB, RAD21, PRR11, AFMID, CCDC34 , and CYB5R1 in CCRF-CEM cells. In conclusion, our study provides comprehensive evidence that withaferin A suppresses the malignant progression of T-ALL by modulating DNA methylation levels. The study delivers novel evidence for its role in DNA methylation and lays an experimental foundation for its potential clinical application in T-cell acute lymphoblastic leukemia.