Clonal dynamics reveal cancer resistance arises from adaptive programs [RNA-Seq]

Most advanced cancers initially respond to targeted therapies but eventually relapse1. Rather than acquiring new mutations, resistance is driven by drug-tolerant persister cells (DTP) that enter a reversible drug-refractory state and sustain minimal residual disease2. Here, we developed MeRLin, a high-resolution barcoding platform combining single-cell RNA sequencing, RNA fluorescence in situ hybridization, and computational analyses to track clonal and transcriptional dynamics of melanoma cells during targeted therapy. Clonal tracking reveals that dominant resistant clones arise from minor pre-treatment subpopulations. The pre-treatment melanoma populations diversify into phenotypically distinct DTP subpopulations, marked by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs associated with adaptive resistance. Spatial transcriptomics revealed the co-localization of lipid metabolism and PI3K signaling programs near the tumor boundaries, and a complex network of autocrine and paracrine interactions among DTP subpopulations. Using barcoded RNA fluorescence in situ hybridization, we identified a dominant persister subpopulation in resistant tumors marked by SLC2A1 expression. Thus, MeRLin provides a robust framework to dissect melanoma heterogeneity and uncover vulnerabilities in persister populations to improve long-term treatment efficacy. Overall design: WM4237-1 cells were spinfected with a lentiviral barcode library in the presence of polybrene (8 µg/mL). mNeptune2.5-positive cells were sorted by flow cytometry to ensure a multiplicity of infection (MOI) below 40%. Barcoded cells were then expanded and split into replicate populations, with one aliquot preserved as the original control. Replicates of barcoded WM4237-1 cells were subcutaneously injected into immunodeficient mice and monitored for tumor growth. Upon establishment, tumors were treated with BRAFi/MEKi, and samples were collected longitudinally throughout treatment until relapse.