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.

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO⁻), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO⁻ scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent “next-generation skin care additives” that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

Chimeric antigen receptor T (CAR-T) cell therapy has transformed the treatment of hematologic malignancies, yet its broader application, particularly in solid tumors, remains constrained by high cost, labor-intensive manufacturing, limited production capacity, and variable clinical performance, as well as barriers such as poor trafficking, antigen heterogeneity, and an immunosuppressive tumor microenvironment. In vivo CAR-T cell engineering, in which CAR-T cells are generated directly within the patient, offers a paradigm shift by eliminating the need for ex vivo cell processing and complex logistical infrastructure. Among emerging approaches, messenger RNA (mRNA)-loaded lipid nanoparticles (LNPs) have emerged as a promising and clinically tractable platform for in vivo CAR-T cell generation, enabling direct reprogramming of T lymphocytes within the patient and thereby circumventing the need for leukapheresis, viral vector production, and prolonged ex vivo culture, effectively transforming the patient into their own cell therapy factory. This review synthesizes advances in mRNA-LNP-mediated in vivo CAR-T cell generation, encompassing ionizable lipid chemistry and emerging T cell-targeted delivery strategies, including surface functionalization approaches. We discuss the implications of transient CAR expression for immune activation, safety, and therapeutic durability, alongside CAR design optimization through co-stimulatory domains and safety switches. Preclinical evidence from murine tumor models and non-human primates is integrated with current regulatory considerations, and key barriers to clinical translation are highlighted. Collectively, progress in nucleic acid delivery, synthetic immunology, and precision medicine positions in vivo mRNA-CAR-T therapy as a promising modality for oncology and beyond.

Legends for Supplementary Figures and Supplementary Tables.