YTHDF1 targets the chemotherapy response by suppressing NOTCH1-induced stemness in colorectal cancer

N⁶-methyladenosine (m⁶A) modification of mRNAs is a predominant epigenetic regulatory mechanism in tumor initiation and progression. Cancer stem cells (CSCs) are the key drivers of colorectal cancer (CRC) initiation and chemotherapy resistance. Here, we found that the m⁶A reader YT521-B homologous domain family, member 1 (YTHDF1), promotes CRC stemness, tumorigenesis, and chemotherapy resistance. YTHDF1 protein expression was positively correlated with CD133 and LGR5 expression in human CRC tissues (N = 184, P < 0.001 for both markers). YTHDF1 promoted m⁶A-dependent self-renewal in CSCs and patient-derived organoids and increased the tumor-initiating potential in vivo. Lgr5-specific Ythdf1-KI mice presented accelerated Apc^Min/+ (P < 0.05) and AOM/DSS (P < 0.05)-induced colorectal tumorigenesis, whereas Lgr5-specific Ythdf1 knockout in Apc^Min/+ mice inhibited tumorigenesis (P < 0.01). Integrative multiomic profiling revealed NOTCH1 as a downstream target. YTHDF1 binds m⁶A-modified NOTCH1, promoting its translation and enhancing NOTCH signaling. NOTCH1 knockdown or blockade by the γ-secretase inhibitor DAPT abolished YTHDF1-mediated tumorigenesis in Ythdf1 knock-in mice (P < 0.01). YTHDF1 promoted resistance to oxaliplatin and 5-fluorouracil in CSCs by inhibiting apoptosis and DNA damage. AOM/DSS-treated Ythdf1 knock-in mice presented increased resistance to oxaliplatin (P < 0.001) and 5-fluorouracil (P < 0.05). Translationally, in vivo targeting of YTHDF1 via VNP-encapsulated siYTHDF1 or salvianolic acid C inhibited tumor growth (P < 0.05 for both treatments) and increased treatment efficacy when VNP was combined with oxaliplatin (P < 0.05, SAC: P < 0.01) or 5-fluorouracil (P < 0.05 for both treatments). In conclusion, YTHDF1 promotes stemness and chemoresistance in CRC via NOTCH1 activation. Targeting YTHDF1 is a promising strategy to improve the outcome of chemotherapy in CRC.