Dihydroartemisinin inhibits histone lactylation through YAP1 to act as a ‘hot’ switch for ‘cold’ tumor in hepatocellular carcinoma

Abstract

Background

Hepatocellular carcinoma (HCC) is characterized by a ‘cold’ tumor microenvironment (TME), which limits the efficacy of immune checkpoint inhibitors (ICIs). In a previous study, we demonstrated that dihydroartemisinin (DHA) could disrupt the immunosuppressive TME and delay HCC progression.

Purpose

This study aimed to explore the precise mechanisms underlying the shift of the TME from an immunosuppressive to an immunostimulatory state.

Methods

We constructed HCC subcutaneous allograft mice model to investigate the in vivo antitumor effects of DHA. We further performed single-cell RNA sequencing (scRNA-seq) and multiplex immunohistochemistry (mIHC) to analyze the immune dynamics during DHA-induced TME transition in HCC. Using NCG mice (lacking T, B, and NK cells), BALB/c nude mice (lacking T cells), and CD8⁺ T cell-depleted mice, we assessed whether the antitumor effect of DHA in HCC depends on its immune functions and determined the key role of T cells in antitumor immunity. Additional downstream mechanisms were explored using YAP1 knockdown HCC cells and liver-specific Yap1 knockout mice through techniques such as Western blot, immunofluorescence (IF), metabolic flux analysis, co-immunoprecipitation (Co-IP), chemokine chip, and flow cytometry (FCM).

Results

We observed that DHA transforms the TME of HCC from an immune-cold to an immune-hot state by increasing the abundance and proportion of T cells, NK cells, M1-like TAMs, and DCs. Notably, DHA was found to enhance the proportion of IFN-γ⁺ CD8⁺ T cells within the TME of HCC-bearing mice, and its therapeutic effects were dependent on CD8⁺ T cells. Our results suggest that DHA could suppress histone lactylation (Kla) and acetylation (Kac) in HCC cells by inhibiting YAP1. Mechanistically, DHA reduces lactate transport, thereby decreasing lactate accumulation within the TME, lowering P300/CBP catalytic activity, and enhancing the deacetylation effect of HDAC1-3. This finding reveals a new mechanism by which DHA, through metabolic changes and epigenetic regulation, remodels the TME of HCC. Additionally, a combination strategy involving DHA and anti-PD-1 therapy demonstrated the potential to reshape the cold HCC TME via inhibition of the YAP1-lactylation positive feedback loop.

Conclusion

Our study confirms that DHA inhibits histone Kla in HCC cells through YAP1 inhibition, which shifts the TME from an immune-cold to immune-hot state. Moreover, DHA enhance the anti-HCC immune response dependent on CD8⁺ T cells, thereby sensitizing anti-PD-1 therapy. This novel finding positions DHA as a promising strategy for optimizing anti-PD-1 therapy in HCC, warranting further clinical application studies.