Single-cell transcriptomics of NRAS-mutated melanoma transitioning to drug resistance: elevated P2RX7 levels indicate response to targeted therapy

While effective therapy is available for melanoma patients harbouring BRAF mutations, to date, the efficient therapeutic approaches for NRAS-mutant melanoma patients are limited. Therapeutic regimen involving combinatorial inhibition of MEK1/2 and CDK4/6 prove to be beneficial as targeted therapy for NRAS-mutant melanoma. Yet, only a subset of melanoma patients responded to this treatment, whereas acquired resistance eventually emerge in responders. Upon prolonged therapy with MEK1/2 and CDK4/6 inhibitors cells switch to fully drug-resistant or senescent phenotype in vitro. Consequently, there is a necessity to portray cell populations sensitive to targeted therapy, but also cell transitions associated with NRAS-mutant melanoma resistance. To define transcriptional states arising under indicated therapy and cell fate decisions governing resistance, we performed single-cell RNA sequencing at four distinguished time points during treatment. In this study, we identified a cell population highly enriched in calcium signalling, among others, associated with positive drug response. In particular, prolonged expression of ATP-gated ion channel P2RX7 was related to a senescent-like phenotype. Next, we described an immune-like melanoma state that acquired drug resistance and re-enter the cell cycle under treatment, as well as cell populations with intrinsic potential to evade drug pressure. NRAS-mutant melanoma cell lines were treated with a drug combination involving MEK and CDK4/6 inhibitors over a prolonged period (~1 month), whereas sampling and sequencing were performed on drug-naïve and treated cells collected at distinct time points of treatment (1, 4 and 33 days) of treatment. Single-cell RNA sequencing was conducted via Drop-Seq and the NextSeq 500 (Illumina) was used for library preparation.