Dioscin suppresses the progression of diffuse large B-cell lymphoma by modulating acaca-dependent histone H3K27 acetylation and inducing mitophagy

Abstract Dioscin is a natural steroidal saponin from traditional Chinese medicine with known antitumor properties and clinical applications in liver and cardiovascular disorders. Histone acetylation plays key roles in epigenetic gene regulation of diffuse large B-cell lymphoma (DLBCL) progression, and the clinical efficacy of histone deacetylase inhibitor (HDACi) is limited in DLBCL treatment, indicating the urgent need for a novel combinatory regimen. This study was performed to explore the functional mechanism and potential clinical application of dioscin in DLBCL. We first screened anti-DLBCL drugs based on the FDA Anti-tumor Drug Library (n=1746), and revealed the remarkable antitumor effect of dioscin in DLBCL. Further experiments found that dioscin treatment significantly suppressed DLBCL cell proliferation, induced G0/G1 cell cycle arrest, and promoted apoptosis in concentration- and time-dependent manner. Moreover, in DLBCL xenograft models, mice treated with dioscin exhibited significantly delayed tumor growth. To identify the direct molecular target of dioscin in DLBCL, we performed Drug Affinity Responsive Target Stability (DARTS) assays coupled with mass spectrometry and integrated target prediction. ACACA, a key rate-limiting enzyme in lipid metabolism, was identified as a key direct target of dioscin in DLBCL. Further validation confirmed dioscin bound ACACA and downregulated the protein levels of ACACA in DLBCL. The above findings prompted us to investigate the expression pattern and biological function of ACACA in DLBCL. DLBCL tissues exhibited higher levels of ACACA compared to the control cases. Survival analysis showed that DLBCL patients with elevated ACACA levels had worse clinical outcomes. Moreover, RNA-seq analysis revealed that ACACA-related genes were enriched in biological processes related to DLBCL progression. Further experiments verified that targeting ACACA could induce cell proliferation inhibition, cell cycle arrest, and cell apoptosis. Notably, ACACA knockdown was found to elevate mitochondrial ROS levels, disrupt mitochondrial membrane potential, reduce the expression level of mitochondrial marker TOM20, and increase LC3B-TOM20 co-localization, indicating the activation of mitophagy in DLBCL. Interestingly, DLBCL cells treated with dioscin also showed activated mitophagy process. We further elucidated the molecular mechanism by which dioscin and ACACA regulate DLBCL progression, and found that both dioscin treatment and ACACA knockdown increased intracellular acetyl-CoA levels in DLBCL cells. Subsequently, nuclear protein extraction and immunoblotting showed that dioscin and ACACA knockdown increased the level of histone acetylation, particularly H3K27ac, a marker of active transcription. To assess the genome-wide effects of ACACA knockdown on chromatin state, we conducted H3K27ac ChIP-seq and revealed a genome-wide increase in signal enriched around transcription start sites. Integration with RNA-seq, Hexokinase 2 (HK2), a key glycolytic enzyme related to the PINK1-Parkin-dependent mitophagy pathway, was identified as a key downstream transcriptional target. Notably, ACACA knockdown increased HK2 mRNA levels and H3K27ac occupancy at its promoter, indicating that ACACA suppressed HK2 transcription by restricting histone acetylation. Moreover, rescue experiments revealed that benitrobenrazide, a specific HK2 inhibitor, could partially reverse the regulatory effects of ACACA knockdown and dioscin treatment on cell proliferation, apoptosis, and mitophagy. To investigate novel enhanced therapeutic strategy based on dioscin, chidamide, a selective HDACi, was selected for further drug combination study. Dioscin exhibited synergistic anti-lymphoma activity with chidamide both in vitro and in vivo. Immunofluorescence staining and nuclear protein blotting showed a marked increase in H3K27ac levels following combination treatment. In xenograft mouse models, the combination regimen of dioscin and chidamide significantly suppressed tumor growth compared to either agent alone. In conclusion, our results revealed that dioscin inhibited DLBCL progression by targeting ACACA and activating the HK2-mediated mitophagy pathway through H3K27ac, and highlighted the therapeutic synergy of combinatory dioscin/chidamide treatment for clinical utility, which underscores the potential of dioscin as a novel drug for DLBCL.