Abstract 235: Targeting NOS sensitizes the NRAS-mutant melanomas to MEK inhibition and induces immunogenic cell death

Abstract Background: Activating NRAS mutations occur in 15-25% of melanomas; however, this subtype is highly aggressive and refractory to existing therapeutic approaches. Consequently, the identification of novel molecular mechanisms is of critical clinical importance. Melanomas harbor hyperactive nitric oxide synthases (NOS) that strongly correlate with poor prognosis. Here, we investigated the role of nitrosylation, a nitric oxide-induced posttranslational modification, in regulating the oncogenic MEK/ERK signaling pathway and Immunogenic Cell Death (ICD) in NRAS-mutant melanomas. Methods: We employed various cellular and mouse models to investigate the effect of combinatorial inhibition of NOS and MEK in NRAS-mutant melanomas. We also profiled global nitrosylation by mass-spectrometry and validated the nitrosylation status of various MEK-ERK regulators and ICD markers using Biotin-switch method. Using site-directed mutagenesis, we characterized potential nitrosylation sites on the MEK and ERK proteins. Additionally, we characterized the nitrosylated forms of potential regulators of ICD andanti-melanoma immunity using cell-based assays and immunogenic mouse models. The immunomodulatory role of nitrosylation was further characterized using the dendritic cells(DCs): CD8+ T-cell cocultures. Results: By inhibiting nitrosylation, we sensitized the NRAS-mutant melanomas to MEK inhibitors. We observed a strong correlation between global de-nitrosylation of MEK-ERK cascade proteins, and that of death-associated molecular patterns (DAMPs) which induce ICD.We also identified potential nitrosylation sites on MEK and ERK proteins with a pivotal role in melanoma cell proliferation. Further, we observed elevated nitrosylation and induction of oncogenic proteins upon MEKi acquired resistance, which was abrogated upon NOS inhibition. Using an immunocompetent mouse model, we demonstrated marked restriction of tumor growth in Nras-mutant melanoma upon inhibition of nitrosylation and MEK. Both in vivo and in vitro models showed extracellular release of ICD hallmark DAMPs, such as calreticulin, phospho-eIF2α, and HMGB1, which is a pre-requisite for ICD induction. This release triggered the activation of dendritic cells, leading to the proliferation and activation of a functional CD8+ T-cell repertoire in the tumor microenvironment. Conclusions: We conclude that nitrosylation hyperactivates the MEK-ERK pathway and suppresses the anti-melanoma immune response, thereby promoting tumor progression and Meki resistance. Inhibiting nitrosylation not only enhances MEKi efficacy but also induces ICD. Further investigations on underlying mechanism(s) will identify novel therapeutic vulnerabilities against the yet untreatable, NRAS-mutant melanoma. Citation Format: Jyoti Srivastava, Vipin Kumar Yadav, Rachel V. Jimenez, Bin Fang, Alvaro de Mingo Pulido, John M. Koomen, Kenneth Y. Tsai, Keiran SM Smalley, Paulo C. Rodriguez, Sanjay Premi. Targeting NOS sensitizes the NRAS-mutant melanomas to MEK inhibition and induces immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 235.