iNOS is a key mediator of anti-PD-1 melanoma therapy response

Background Inducible nitric oxide synthase (iNOS) and its product nitric oxide (NO) were historically linked to poor melanoma outcomes, yet recent evidence shows NO supports anti-tumor immunity. This study examines how iNOS shapes anti–PD-1 efficacy, particularly through interferon signaling. Methods B16-D5 melanoma tumors were implanted in wild-type (WT) and iNOS knockout (KO) mice to compare tumor growth and response to anti–PD-1 therapy. Flow cytometry, apoptosis assays, and RNA sequencing assessed NO production, PD-L1 expression, and interferon-related gene activation. In vitro , melanoma cells were treated with NO donors (DETA NONOate, SNAP) to assess proliferation and apoptosis. Peripheral blood mononuclear cells from 27 melanoma patients receiving anti–PD-1 therapy were analyzed with multiparameter flow cytometry to correlate NO-associated immune subsets with progression-free survival (PFS). Results Tumors grew significantly faster in iNOS KO mice, and anti–PD-1 therapy had no effect, demonstrating that iNOS-derived NO contributes to treatment efficacy. NO donors inhibited melanoma proliferation and induced apoptosis in vitro . Transcriptomic analysis showed anti–PD-1 upregulated interferon pathway genes (STAT1, IRF1, IFNB1) in WT but not iNOS KO mice. In patients, a NO-producing dendritic-cell subset (DAF-FM + CD11c + ) was associated with improved PFS (hazard ratio 0.453; 95% CI = 0.270-0.992; p=0.048), indicating a NO-dependent enhancement of interferon-driven immune activity. Conclusion iNOS-derived NO is necessary for effective anti–PD-1 immunotherapy in melanoma, promoting interferon signaling and immune activation. Loss of iNOS impairs tumor control and immune responsiveness, supporting NO as a potential biomarker and therapeutic adjunct to be explored while challenging assumptions about its deleterious role in melanoma.