Comprehensive transcriptomic characterization of two melanoma cell lines with acquired dual resistance to BRAF and MEK inhibitors.

Resistance to targeted therapy remains a major clinical challenge in the treatment of BRAF-mutant melanoma. To elucidate the molecular mechanisms underlying acquired resistance to dual BRAF and MEK inhibition, we performed transcriptomic profiling of two metastatic melanoma cell lines, Hs294T and WM9, that had become resistant to the combination of vemurafenib (a BRAF inhibitor) and cobimetinib (an MEK inhibitor). Resistant cell lines were derived through stepwise drug exposure until stable resistance was achieved in the presence of 0.4 µM vemurafenib and 0.4 µM cobimetinib. Total RNA was extracted and subjected to high-throughput sequencing using the KAPA Stranded mRNA-seq Kit and Illumina NovaSeq 6000 platform, yielding ~ 20 million paired-end reads per sample. Sequencing data were processed with standard RNA-seq pipelines, including read alignment (STAR), quantification (featureCounts), normalization, and differential expression analysis (DESeq2). Gene set enrichment analysis (GSEA) was performed using MSigDB collections. Transcription factors and kinases activity were inferred using the decoupleR package. All statistical analyses incorporated multiple-testing correction using the Benjamini-Hochberg method. Both resistant cell lines exhibited strong upregulation of extracellular matrix components, epithelial-mesenchymal transition (EMT) markers, and chemokine signaling genes, along with repression of melanocytic lineage markers. Inference of transcription factors and kinases activity uncovered line-specific regulatory rewiring, including activation of MYOCD, SMAD3, and TP53, and suppression of immune regulators such as RFX5 and RFXANK. WM9 cells exhibited broader transcriptional reprogramming and a more pronounced engagement of inflammatory and immune-related pathways. At the same time, Hs294T resistance was marked by a more restricted program focused on stromal remodeling and impaired antigen presentation. RNA-seq-based profiling of dual-resistant melanoma cell lines revealed distinct, context-specific transcriptional programs associated with acquired resistance. These findings complement prior characterization of the same models and highlight divergent adaptive strategies, providing a transcriptomic resource to inform follow-up in vitro testing of candidate combinations aimed at overcoming BRAF/MEK inhibitor resistance.