A multi-omics integrative analyses reveals CYBB-mediated apoptotic and immune dysregulation as a key target of berberine in diffuse large B-cell lymphoma.

BACKGROUND: Berberine has shown antitumor activity across multiple malignancies, yet its molecular targets and mechanisms of action in diffuse large B-cell lymphoma (DLBCL) remain incompletely defined. We integrated genetic causality, multi-omics data, and functional validation to identify berberine-related molecular pathways relevant to DLBCL. METHODS: We identify bioinformatic screening and Mendelian randomization (MR) on 760 candidate berberine-related genes; 315 with adequate instruments underwent MR for causal links to DLBCL. Follow-up analyses included pathway enrichment, Protein-Protein Interaction (PPI) networks, machine-learning prognostic modeling, molecular docking and dynamics, single-cell RNA sequencing, cell-cell communication inference, bulk transcriptomic validation, and in vitro assays. RESULTS: MR identified several berberine-related genes with putative causal associations with DLBCL. ABCA1, ITK, MAPK3, TGM2, and DAPK1 were associated with decreased DLBCL risk, whereas CYBB, RAC1, JUN, and CPT1A were associated with increased risk. Enriched pathways encompassed B-cell receptor signaling, immune checkpoints, apoptosis, and MAPK signaling. Apoptosis pathway activity correlated with favorable prognosis across cohorts. Integrative analyses prioritized CYBB as a central hub. Molecular docking and dynamics indicated stable berberine-CYBB binding. Single-cell analyses showed CYBB + B cells had distinct apoptotic, immunoregulatory, and communication features, including BTLA-TNFRSF14 signaling. Bulk deconvolution linked high CYBB to worse prognosis, proliferation, and an immunosuppressive microenvironment. Functionally, CYBB knockdown reduced DLBCL cell viability and increased apoptosis; berberine inhibited growth dose-dependently, and cellular thermal shift assays supported berberine-CYBB target engagement in cells. CONCLUSION: This integrative study identifies berberine-related genes with potential causal relevance to DLBCL and prioritizes CYBB as a functionally relevant target linked to apoptosis, immune regulation, and prognosis, highlighting the potential involvement of berberine-CYBB axis underlying berberine activity in DLBCL, although rescue experiments are needed to confirm its specificity and relative contribution.