Alantolactone Inhibits Double Expression Lymphoma via Dual-Targeted Glycogen Synthase Kinase 3 Beta and B-Cell Lymphoma2.

Background: Dual-expression lymphoma (DEL) is an aggressive subtype with concurrent MYC and BCL2 overexpression. This disease exhibits a poor prognosis and responds poorly to standard R-CHOP therapy,highlighting the urgent need for novel treatments. Alantolactone (ALA), a natural compound, has shown anticancer potential, but its efficacy and mechanism in DEL remain unclear. This study aimed to investigate the anti-tumor effects of ALA against DEL and its underlying dual-targeting mechanism. Methods: The cytotoxic activity of ALA was assessed in lymphoma cell lines and a normal lymphocyte line. Apoptosis was evaluated by flow cytometry and Western blotting. Network pharmacology, molecular docking, dynamics simulations, and cellular thermal shift assays (CETSA) were utilized to identify and validate direct targets of ALA. The anti-tumor efficacy of ALA was further examined in a DEL xenograft mouse model using PET-CT imaging and survival analysis. Results: In the present study, the anticancer activity of ALA in DEL was explored in vivo and in vitro. ALA was shown to inhibit DEL growth in vivo with little toxicity to normal tissues. Mechanistically, ALA stabilized active glycogen synthase kinase 3β (GSK3β) (binding affinity: -15.66 kcal/mol; ΔTm +9°C), enhancing β-catenin degradation and suppressing Wnt-driven oncogenesis. Simultaneously, ALA directly bound BCL2 (-22.22 kcal/mol), triggering both intrinsic and extrinsic apoptotic pathways. Simultaneously, ALA directly bound BCL2 (binding affinity: -22.22 kcal/mol), triggering both intrinsic and extrinsic apoptotic pathways. ALA exhibited robust anti-DEL activity across preclinical models. While in vivo it significantly suppressed tumor progression (SUVmax reduction >50%, p<0.01) and extended survival (median 50 vs 80 days, p<0.01) in DEL xenografts. The therapeutic relevance was underscored by clinical correlation showing DEL patients with high GSK3β expression had superior survival outcomes. Conclusion: ALA exerts potent anti-DEL activity by simultaneously targeting GSK3β in the Wnt pathway and directly inhibiting BCL2, leading to suppressed tumor proliferation and induced apoptosis. Our findings highlight ALA as a promising multi-targeting therapeutic candidate for DEL and propose a novel strategy against this refractory lymphoma.