N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression

N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell-specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC.Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.