Withaferin A inhibits EBV-driven lymphomagenesis through multiple mechanisms, including EBNA1 degradation Open Access

• Withaferin A induces oxidative stress, NF-κB suppression, and EBNA1 loss in EBV-positive B cells.

• Withaferin A blocks EBV lymphomagenesis in vitro and in vivo via disruption of multiple pathways.

Epstein-Barr virus (EBV) infects over 90% of the global population and drives multiple aggressive B cell malignancies, including Burkitt lymphoma (BL), diffuse large B cell lymphoma (DLBCL), and Hodgkin lymphoma. Standard chemoimmunotherapy regimens can be highly effective, yet EBV-positive lymphomas sometimes exhibit poorer responses, higher resistance, and worse survival compared with EBV-negative counterparts. This reflects the virus's ability to drive immune evasion, alter cell death pathways, and exploit host immune dysfunction, underscoring the potential value of EBV-directed strategies. Withaferin A (WA), a steroidal lactone with known anti-cancer and anti-inflammatory properties, was evaluated for its efficacy against EBV-associated B cell non-Hodgkin lymphomas (B-NHLs). Across a panel of lymphoma cell lines, WA demonstrated selective cytotoxicity toward EBV-positive B-NHLs in part through proteasome-dependent degradation of EBV nuclear antigen 1 (EBNA1) and subsequent loss of viral episomes, alongside additional effects on cellular stress and survival pathways. Mechanistic studies revealed that WA collapses antioxidant defenses, drives oxidative stress, and suppresses NF-κB signaling, creating a multi-pronged disruption of viral and host survival pathways. In primary B cell models and a cord-blood humanized mouse model of EBV-driven lymphomagenesis, WA inhibited B cell transformation, reduced splenomegaly and tumor burden, and significantly prolonged survival, without evidence of increased viral replication. These findings establish WA as a potent preclinical candidate that selectively targets vulnerabilities unique to EBV-transformed B cells, supporting further optimization and evaluation for EBV-positive B cell malignancies.