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

Background: Most advanced melanomas initially respond to targeted therapy but eventually relapse. Increasing evidence suggests that drug-tolerant persister cells can adopt a reversible drug-refractory state and represent a key driver of therapeutic resistance. Methods: We developed MeRLin, a lineage tracing platform that integrates cellular barcoding, single-cell transcriptomic profiling, RNA fluorescence in situ hybridization, and computational analyses to track clonal and transcriptional dynamics in a patient-derived melanoma model during prolonged targeted therapy. Longitudinal analyses enabled the characterization of clonal fates, transcriptional states, and spatial organization of persister populations. Results: Clonal dynamics showed that persister subpopulations initially responded to therapy, persisted through minimal residual disease, and expanded during tumor recurrence. Four persister-associated transcriptional states characterized by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs were associated with persister populations arising from minor pre-treatment subpopulations under sustained drug pressure. Spatial transcriptomic analyses revealed structured spatial organization of these programs and suggested coordinated autocrine and paracrine interactions among persister states. Targeted barcode RNA fluorescence in situ hybridization enabled spatial mapping of clonal identity and gene expression, revealing in situ co-localization of a dominant resistant clone marked by SLC2A1 expression. Conclusions: Together, MeRLin provides a robust framework for dissecting cancer heterogeneity and characterizing persister subpopulations. Our findings demonstrate that melanoma recurrence is associated with diverse, spatially organized persister states linked to adaptive transcriptional programs.