Srivastava Jyoti, Yadav Vipin K, Jimenez Rachel V, Phadatare Pravin R, Inamdar Ninad A, Young Montana M, Bacchiocchi Antonella, Halaban Ruth, Fang Bin, Pulido Alvaro de Mingo, Tsai Kenneth Y, Smalley Keiran S M, Koomen John M, Rodriguez Paulo C, Premi Sanjay
Department of Tumor Microenvironment and Metastasis, Moffitt Cancer Center and Research Institute, Tampa, Florida.
Department of Immunology, Moffitt Cancer Center and Research Institute, Tampa, Florida.
Cancer Res. 2025 Jun 16;85(12):2268-2287. doi: 10.1158/0008-5472.CAN-24-0693.
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.
Nitrosylation-regulated molecular mechanisms present a vulnerability that can be exploited to sensitize NRAS-mutant melanomas to existing targeted therapies while enhancing antitumor immunity.
激活型NRAS突变存在于所有黑色素瘤的15%至25%中。然而，这种亚型对包括免疫疗法和RAS抑制剂在内的现有疗法仍然难治；因此，确定创新的治疗策略至关重要。我们研究了亚硝基化（一种一氧化氮诱导的翻译后修饰）在黑色素瘤进展和治疗耐药中的作用。抑制亚硝基化使NRAS突变型黑色素瘤对靶向MEK抑制剂（MEKi）敏感，导致ERK-MAPK通路持续下调，同时NRAS、MEK、ERK、RSK1和DUSPs伴随去亚硝基化。使用质谱进行的全局亚硝基化蛋白质组分析揭示了多种ERK调节因子的亚硝基化。功能获得和功能丧失研究证实亚硝基化与ERK激活之间存在正相关。ERK和MEK蛋白含有潜在的亚硝基化位点，其突变消除了它们的磷酸化并抑制细胞生长。亚硝基化蛋白质组还包含损伤相关分子模式（DAMP），即已知可诱导免疫原性细胞死亡（ICD）的因子。值得注意的是，亚硝基化抑制与MEKi联合在免疫活性小鼠模型中显著抑制NRAS突变型黑色素瘤生长。这伴随着MEK-ERK信号下调以及钙网蛋白、磷酸化eIF2α和HMGB1等DAMP的细胞外释放，证实了ICD诱导。此外，该组合显著增加了肿瘤微环境中CD8 + T细胞、树突状细胞和巨噬细胞的数量，这在树突状细胞和T淋巴细胞的共培养中得到验证。总之，当前研究表明，亚硝基化抑制使NRAS突变型黑色素瘤对靶向MEKi诱导的细胞死亡敏感，并导致非亚硝基化（活性）DAMP的释放，这些DAMP通过ICD诱导强大的抗黑色素瘤免疫反应。这些发现突出了目前无法治疗的NRAS突变型黑色素瘤亚型中潜在的治疗弱点。
亚硝基化调节的分子机制呈现出一种弱点，可被利用来使NRAS突变型黑色素瘤对现有靶向疗法敏感，同时增强抗肿瘤免疫力。
