Blocking Nitrosylation Induces Immunogenic Cell Death by Sensitizing <i>NRAS</i> -Mutant Melanoma to MEK Inhibitors
Jyoti Srivastava, Vipin Yadav, Rachel V. Jimenez, Pravin R. Phadatare, Nitin Inamdar, Montana M. Young, Antonella Bacchiocchi, Ruth Halaban, Bin Fang, Álvaro de Mingo Pulido, Kenneth Y. Tsai, Keiran S.M. Smalley, John M. Koomen, Paulo C. Rodrı́guez, Sanjay Premi
Abstract
Activating NRAS mutations occur in 15% to 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 posttranslational 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 denitrosylation 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 damage-associated molecular patterns (DAMP), 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 such as calreticulin, phospho-eIF2α, and HMGB1, confirming ICD induction. Furthermore, the combination significantly increased the repertoire of CD8+ T cells, dendritic cells, and macrophages in the tumor microenvironment, which was validated in cocultures of dendritic cells 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 antimelanoma immune response via ICD. These findings highlight potential therapeutic vulnerabilities in the currently untreatable NRAS-mutant melanoma subtype. SIGNIFICANCE: Nitrosylation-regulated molecular mechanisms present a vulnerability that can be exploited to sensitize NRAS-mutant melanomas to existing targeted therapies while enhancing antitumor immunity.