Coordinated DNA 5-mC and RNA m5C methylation epigenetically regulates MZF1 splice variants to drive EGFR-TKI resistance

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) severely limits clinical efficacy. Myeloid zinc finger 1 (MZF1) inhibited EGFR phosphorylation and internalization by maintaining zinc homeostasis, which suggested its potential as a therapeutic target for resensitization. The two MZF1 splice variants exhibited functional divergence. MZF1 S demonstrated significantly reduced Zn 2+ -binding capacity compared with MZF1 L owing to C 2 H 2 domain deletion. Although MZF1 S was specifically upregulated in resistant patients, MZF1 L predominated in treatment-sensitive cohorts, the mechanisms regulating their differential expression remained uncharacterized. We identified NSUN7/YBX1-mediated RNA m 5 C methylation as a critical driver of resistance. This modification promoted competitive binding of SRSF1 over SRSF3 to MZF1 pre-mRNA and enhanced MZF1 S production. Overlapping loci co-regulated by RNA m 5 C and DNA 5-methylcytosine methylation in the MZF1 5ʹ-untranslated region suggested coordinated epigenetic control of alternative splicing. Characterization of these loci revealed a UHRF1/DNMT1-NSUN7/YBX1 axis that synergistically regulated splice variant selection. Targeting this axis suppressed MZF1 S while restoring MZF1 L expression, which resensitized resistant cells to EGFR-TKIs. Our findings established a dual-layer epigenetic mechanism governing alternative splicing in drug resistance and proposed methylation-regulated splice variants as biomarkers and therapeutic targets for overcoming EGFR-TKI resistance in non-small-cell lung cancer.