Epigenetic dysregulation is a significant factor contributing to cisplatin resistance in first-line chemotherapy for bladder cancer. Lactate, a metabolite in the tumor microenvironment, is involved in various physiological and pathological processes, yet its non-metabolic functions remain poorly understood. As a novel post-translational modification, lactylation serves as a bridge between lactate metabolism and tumor progression. However, there are few studies on lactylation and chemotherapy resistance in bladder cancer. Therefore, there is an urgent need to identify drugs that target lactylation regulation to enhance cisplatin sensitivity and elucidate the underlying mechanisms of such sensitization.

Cell viability assays were used to screen for epigenetic-modulating drugs that enhance cisplatin sensitivity. RNA-seq and ATAC-seq were employed to analyze the epigenetic regulatory mechanisms by which HDAC inhibitors (HDACi) potentiated cisplatin chemotherapy. Drug sensitivity was assessed through in vitro functional assays, including CCK-8, colony formation, and apoptosis assays, as well as an in vivo subcutaneous tumor model and patient-derived organoids (PDOs). Western blotting was performed to identify HDACi-regulated histone lactylation sites. ChIP-qPCR and dual-luciferase reporter assays were used to examine the transcriptional regulation of DHRS2 by histone lactylation modifications.

By screening epigenetic drugs, we identified that the HDAC inhibitor Entinostat significantly enhanced the therapeutic efficacy of cisplatin chemotherapy. In vitro and in vivo models, including bladder cancer cell lines, PDOs, and xenograft mouse models, demonstrated a strong synergistic effect between Entinostat and cisplatin. Integrated RNA-seq and ATAC-seq analysis revealed that the combination therapy led to significant downregulation of platinum resistance and homologous recombination repair pathways. We further focused on the target gene dehydrogenase/reductase member 2 (DHRS2). In vitro and in vivo functional assays confirmed that Entinostat effectively counteracted cisplatin resistance induced by DHRS2 downregulation. Mechanistically, Entinostat specifically upregulated histone H3K18 lactylation (H3K18la) levels. ChIP-qPCR demonstrated that H3K18la bond to the promoter region of DHRS2, and Entinostat treatment enhances this binding, thereby promoting DHRS2 transcriptional upregulation. Finally, we validated a positive correlation between H3K18la levels and DHRS2 expression in clinical bladder cancer samples, and patients with high DHRS2 expression exhibited better response to neoadjuvant chemotherapy.

Our study systematically screened and elucidated the cisplatin-sensitizing efficacy of HDAC inhibitor Entinostat and its underlying epigenetic regulatory mechanisms in preclinical models. Phase I clinical trial will be coucted to validate the safety, feasibility, and efficacy of this combination regimen in bladder cancer.