ASH1L in Hepatoma Cells and Hepatic Stellate Cells Promotes Fibrosis‐Associated Hepatocellular Carcinoma by Modulating Tumor‐Associated Macrophages

Abstract Hepatocellular carcinoma (HCC) often occurs in the context of fibrosis or cirrhosis. Methylation of histone is an important epigenetic mechanism, but it is unclear whether histone methyltransferases are potent targets for fibrosis‐associated HCC therapy. ASH1L, an H3K4 methyltransferase, is found at higher levels in activated hepatic stellate cells (HSCs) and hepatoma cells. To determine the role of ASH1L in vivo, transgenic mice with conditional Ash1l depletion in the hepatocyte cell lineage ( Ash1l flox/flox Alb cre ) or HSCs ( Ash1l flox/flox GFAP creERT2 ) are generated, and these mice are challenged in a diethylnitrosamine (DEN)/carbon tetrachloride (CCl 4 )‐induced model of liver fibrosis and HCC. Depleting Ash1l in both hepatocytes and HSCs mitigates hepatic fibrosis and HCC development. Multicolor flow cytometry, bulk, and single‐cell transcriptomic sequencing reveal that ASH1L creates an immunosuppressive microenvironment. Mechanically, ASH1L‐mediated H3K4me3 modification increases the expression of CCL2 and CSF1, which recruites and polarizes M2‐like pro‐tumorigenic macrophages. The M2‐like macrophages further enhance tumor cell proliferation and suppress CD8 + T cell activation. AS‐99, a small molecule inhibitor of ASH1L, demonstrates similar anti‐fibrosis and tumor‐suppressive effects. Of pathophysiological significance, the increased expression levels of mesenchymal ASH1L and M2 marker CD68 are associated with poor prognosis of HCC. The findings reveal ASH1L as a potential small‐molecule therapeutic target against fibrosis‐related HCC.