Clonal dynamics shaped by diverse drug-tolerant persister states in melanoma resistance

Most advanced melanomas initially respond to targeted therapy but eventually relapse. Rather than acquiring new mutations, resistance is driven by drug-tolerant persister cells that enter a reversible drug-refractory state. We developed MeRLin, a high-resolution lineage tracing platform integrating cellular barcoding, single-cell transcriptomics, RNA fluorescence in situ hybridization (FISH), and computational analyses to track clonal and transcriptional dynamics in patient-derived melanoma models during prolonged therapy. Clonal dynamics revealed that persister subpopulations first responded to treatment but persisted and expanded during minimal residual disease, ultimately leading to tumor recurrence. Pre-treatment melanoma populations diversified into four conserved persister states characterized by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs associated with adaptive resistance. Spatial transcriptomics showed the organization of these adaptive programs and a complex signaling network of autocrine and paracrine interactions among persister subpopulations. Barcoded RNA-FISH enabled spatial mapping of clonal identity and gene expression, revealing in situ co-localization of a dominant resistant clone with SLC2A1 expression. MeRLin provides a robust framework for dissecting cancer heterogeneity and identifying vulnerabilities in persister populations.