Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts antitumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis

Abstract

Background & AimsRNA N6-Methyladenosine (m6A) reader protein YTHDF1 has been implicated in cancer; however, its role in hepatocellular carcinoma (HCC), especially in nonalcoholic steatohepatitis-associated HCC (NASH-HCC), remains unknown. Here, we investigated the functional role and molecular mechanism of YTHDF1 on NASH-HCC and its interplay with tumor immune microenvironment.MethodsHepatocyte specific Ythdf1 overexpression mice was subjected to dietary models of NASH-HCC. Tumor infiltrating immune cells were profiled with single-cell RNA sequencing (scRNA-seq), flow cytometry, and immunostaining. The molecular target of YTHDF1 was elucidated with RNA-sequencing, m6A-sequencing, YTHDF1 RNA immunoprecipitation (RIP) sequencing, proteomics, and ribosome-profiling. Ythdf1 in NASH-HCC models was targeted by lipid nanoparticles (LNP)-encapsulated siYthdf1.ResultsYTHDF1 is overexpressed in tumor tissues than adjacent peri-tumor tissues from NASH-HCC patients. Liver specific Ythdf1 overex pression drives tumorigenesis in dietary models of spontaneous NASH-HCC. scRNA-seq and flow cytometry unraveled that Ythdf1 induced accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed cytotoxic CD8+ T cell function. Mechanistically, Ythdf1 expression in NASH-HCC cells induced the secretion of IL-6, which mediated MDSCs recruitment and activation, leading to CD8+ T cell dysfunction. Integrated m6A-seq, RIP-seq, and ribo-seq have identified EZH2 mRNA as a key YTHDF1 target. YTHDF1 binds to m6A-modified EZH2 mRNA and promotes EZH2 translation. EZH2 in turn increased expression and secretion of IL-6. Ythdf1 knockout synergized with anti-PD-1 treatment to suppress tumor growth in NASH-HCC allografts. Furthermore, therapeutic targeting of Ythdf1 using LNP encapsulated siRNA significantly increased efficacy of anti-PD-1 blockade in NASH-HCC allografts.ConclusionsWe identified that YTHDF1 promotes NASH-HCC tumorigenesis via EZH2-IL-6 signaling, which recruits and activates MDSCs to cause cytotoxic CD8+ T cell dysfunction. YTHDF1 may be a novel therapeutic target to improve response to anti-PD-1 immunotherapy for NASH-HCC.