Blocking Nitrosylation Induces Immunogenic Cell Death by Sensitizing NRAS-Mutant Melanoma to MEK Inhibitors

Abstract Activating NRAS mutations occur in 15-25% of all melanomas. However, this subtype remains refractory to existing therapeutics, including immunotherapy and RAS inhibitors; therefore, identifying innovative treatment strategies is of utmost importance. We investigated the role of nitrosylation, a nitric oxide-induced post-translational modification, in melanoma progression and therapeutic resistance. Inhibiting nitrosylation sensitized NRAS-mutant melanomas to targeted MEK inhibitors (MEKi), leading to sustained downregulation of the ERK-MAPK pathway, along with concomitant de-nitrosylation of NRAS, MEK, ERK, RSK1, and DUSPs. Global nitrosylome profiling using mass-spectrometry revealed nitrosylation of multiple ERK regulators. Gain- and loss-of-function studies confirmed a positive association between nitrosylation and ERK activation. ERK and MEK proteins harbored potential nitrosylation sites, mutation of which abrogated their phosphorylation and inhibited cell growth. The nitrosylome also contained death-associated molecular patterns (DAMPs), factors known to induce immunogenic cell death (ICD). Notably, nitrosylation inhibition combined with MEKi markedly inhibited Nras-mutant melanoma growth in an immunocompetent mouse model. This was accompanied by downregulated MEK-ERK signaling and extracellular release of DAMPs like calreticulin, phospho-eIF2α, and HMGB1, confirming ICD induction. Furthermore, the combination significantly increased the repertoire of CD8+ T cells, dendritic cells (DCs), and macrophages in the tumor microenvironment, which was validated in co-cultures of DCs and T-lymphocytes. In conclusion, the current study demonstrates that nitrosylation inhibition sensitizes NRAS-mutant melanomas to targeted MEKi-induced cell death and causes the release of non-nitrosylated (active) DAMPs that induce a potent anti-melanoma immune response via ICD. These findings highlight potential therapeutic vulnerabilities in the currently untreatable NRAS-mutant melanoma subtype.