Histone methyl-transferase G9a inhibition boosts the efficacy of immune checkpoint inhibitors in experimental hepatocellular carcinoma

Background and Aims Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy. Yet, their efficacy in hepatocellular carcinoma (HCC) remains limited, partly due to tumor-intrinsic mechanisms of immune evasion. This study focused on the identification of potential epigenetic drivers of immune resistance in HCC evaluating the therapeutic potential of targeting the histone methyltransferase G9a (EHMT2). Approach and Results We analyzed G9a expression across multiple human HCC cohorts and found that elevated G9a levels were inversely correlated with the most relevant immune-related gene expression signatures predictive of ICI responsiveness. Using HCC cell lines and orthotopic models implemented in immunocompetent mice, we assessed the effects of pharmacologic inhibition of G9a with two innovative epigenetic inhibitors, CM272 and EZM8266. G9a blockade enhanced tumor cell immunogenicity by restoring IFNγ responsiveness, increasing MHC-I surface expression, and promoting chemokine-mediated (CXCL10) recruitment of T cells. Mechanistically, G9a inhibition induced a viral mimicry response through derepressing endogenous retroviral elements and the accumulation of cytosolic double-stranded RNA. In vivo , G9a inhibition synergized with anti–PD-1 therapy to suppress tumor growth, significantly enhancing CD8⁺ T cell infiltration. Notably, in a clinically-relevant post-hepatectomy HCC recurrence model, the combination therapy overcame immune resistance. Conclusions G9a functions as a central epigenetic barrier to antitumor immunity in HCC. Pharmacologic G9a inhibition reprograms the tumor microenvironment, enhances immunogenicity, and sensitizes tumors to ICIs. These findings provide strong preclinical rationale for integrating G9a-targeted therapies with immunotherapy, particularly in perioperative settings.