Transitional states in NRAS-mutated melanoma preceding drug resistance

Patients diagnosed with advanced-stage melanoma carrying NRAS-activating mutations face a dismal prognosis, as reflected by their short progression-free survival. While targeted therapy involving MEK1/2 and CDK4/6 co-inhibition has shown partial effectiveness in NRAS-mutant melanoma patients and has advanced to clinical trials, the molecular processes underlying acquired resistance to these drugs remain largely unknown. A triple therapeutic regimen involving inhibition of MAPK, CDK4/6, and PI3K-Akt-mTOR signalling pathways has shown promising results in impairing NRASmut melanoma progression. However, this treatment is accompanied by toxicity, highlighting the importance of identifying alternative strategies to improve efficacy and overcome the resistance of MEK and CDK4/6 inhibitors. Thus, the work performed in the framework of this doctoral thesis aimed to depict cell state transitions that propel NRASmut melanoma cells to become drug-resistant and reconstruct the cell state transcriptional trajectories associated with this phenomenon. First, we monitored cell growth and proliferation to determine the sensitivity of NRAS-mutant melanoma cell lines to MEK1/2 and CDK4/6 co-inhibition. Two main cellular responses have been observed, depending on the dynamics of the cells under drug exposure, namely fast and slow drug adaptation, and progression along the trajectory of resistance development. Next, we performed time-series single-cell RNA sequencing of distinct NRASmut melanoma cell lines over prolonged MEK/CDK4/6 co-targeting. The development of single-cell technologies has facilitated a deeper understanding of the heterogeneous transcriptional background and cell state plasticity observed in BRAFmut melanoma and to a lesser extent in NRASmut melanoma. To the best of our knowledge, this study represents the first endeavour to address the response and resistance to MEK/CDK4/6 co-inhibition at the level of single NRASmut melanoma cells. This approach enabled us to characterise cell populations that were sensitive to targeted therapy, as well as those that were intrinsically resistant or developed resistance over time. Upon early drug exposure, we detected slow-proliferating melanoma cells enriched in genes related to transmembrane transport, epithelial-mesenchymal transition (EMT), and adhesion, among others. Once melanoma cells resist inhibitory stress and resume proliferation, they transit towards a state, which is highly enriched in an interferon response gene signature. We then focused on supporting the inhibitory potential of drugs in transitional states. Activation of the transmembrane transport marker purinergic receptor P2RX7 improved the tumouricidal effects of dual MEK/CDK4/6 inhibition. Overall, this study gives a snapshot of melanoma transitional mechanisms towards resistance to targeted drugs and contributes to the establishment of novel treatment approaches for NRAS-mutant tumours