Single-cell multi-omics dissection of RevitalAge Markers uncovers age-dependent immunotherapy resistance and druggable targets in melanoma

Aging-related molecular reprogramming profoundly influences melanoma progression and therapeutic sensitivity, yet underlying mechanisms remain poorly understood. We constructed a comprehensive multi-omics atlas integrating mutations, transcription, methylation, and copy number variations, utilizing the MOFA algorithm to uncover key age-driving factors. Among 20 factors, Factor6 exhibited significant negative age correlation and survival protection (HR = 0.95, P = 4.494e-05), defined as "RevitalAge Marker (RAM)". Integration of three single-cell RNA sequencing datasets revealed 4,796 RAM+ cells (predominantly endothelial cells and fibroblasts) exerting tumor-suppressive functions through VEGFA-VEGFR2 angiogenic pathways and mitochondrial ATP synthesis, while 7,229 RAM- cells (dominated by malignant cells) exhibited enhanced EMT, hypoxic adaptation, and MAPK signaling activation. RAM+ cells were governed by ZFP42 and IRF8 maintaining anti-tumor immunity, while RAM- cells controlled by ILF2 and ISL1 promoted metastasis and immune evasion, with RAM- malignant cells enriched in patients over 65 years and associated with immunotherapy resistance. Machine learning analysis of RAM-associated genes identified five core signatures (GPR143, ST3GAL4, RAB38, GMPR, FDFT1) demonstrating superior immunotherapy response prediction (AUC = 0.78-1.00). Drug sensitivity profiling revealed ST3GAL4 exhibited strong correlations with AZ628 (pan-RAF inhibitor) and RDEA119 (MEK inhibitor), which was further validated by molecular docking showing excellent binding affinities (binding energies: -8.7 and - 7.2 kcal/mol). This study provides structural evidence for targeted therapeutic strategies in ST3GAL4-overexpressing melanoma and establishes foundations for age-stratified immunotherapy.