Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts antitumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis
Lina Wang, Lefan Zhu, Cong Liang, Xiang Huang, Ziqin Liu, Jihui Huo, Ying Zhang, Yifan Zhang, Lili Chen, Hongzhi Xu, Xiaoxing Li, Lixia Xu, Ming Kuang, Chi Chun Wong, Jun Yu
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.