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 hepatoma spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSCs). Stem cell-specific conditional Ythdf1 knock-in (CKI) mice treated with diethylnitrosamine showed elevated tumor burden compared with wild type mice. YTHDF1 promoted CSC 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. 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 a YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC. To decipher whether YTHDF1 modulates HCC stemness in m6A-dependent manner, we applied RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing to pinpoint downstream targets regulated by YTHDF1-m6A epigenetic axis in HCC. To investigate the molecular mechanism that silencing of YTHFD1 increased sensitivities against lenvatinib/sorafenib, we performed RNA-seq on tumor tissues from orthotopic xenograft implants treated with LNP-siYTHDF1 in combination with either lenvatinib or sorafenib.