Bi-directional regulation between NAD/NAMPT and IFN-γ/PD-L1 axes via BRD4/IRF1 and mitochondrial respiration in metastatic cutaneous melanoma

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in NAD biosynthesis and is frequently upregulated in tumors, including melanoma, where it drives disease progression and resistance to targeted therapies. Beyond its metabolic role, NAMPT also exerts immune-modulatory functions, acting as a cytokine-like mediator that shapes tumor–host interactions and influences the efficacy of immunotherapy. Building on recent evidence indicating a connection between the pro-inflammatory cytokine interferon-gamma (IFN-γ) signaling and NAD metabolism, we sought to explore this poorly understood relationship in melanoma context. Analysis of the TCGA melanoma cohort revealed a significant correlation between NAMPT expression and IFN-γ signaling, including specific genes belonging to this pathway, such as IRF1, STAT1, and CD274/PD-L1. The correlation between NAMPT and PD-L1 was also confirmed at the protein level in more than 100 cases included in a melanoma tissue microarray (TMA). Functionally, IFN-γ treatment (100ng/ml for 24h) of three MM cell lines led to increased NAMPT transcription and intracellular expression, as well as increased release. Molecularly, as previously demonstrated for IFN-γ-inducible PD-L1, also NAMPT expression upon IFN-γ signaling activation is regulated at epigenetic/transcriptional level via bromodomain protein BRD4/IRF1 complex, as revealed using BRD4 inhibitors and chromatin immunoprecipitation (ChIP)-RT-PCR experiments. In the second part of our results, we showed a bidirectional regulation between NAD/NAMPT axis and IFN-γ signaling. NAMPT inhibition, as well as genetic silencing, reduced IFN-γ signaling activation, as highlighted by RNAseq data and confirmed by biochemical validation. This reciprocal regulation involved the modulation of mitochondrial respiration, specifically mitochondrial Complex I. Analysis of the melanoma TCGA cohort revealed a significant correlation between NAMPT expression, IFN signaling and several mitochondrial Complex I subunit genes. Consistently, activation of IFN-γ signaling in melanoma cell lines induced an increase in oxygen consumption rate, which was completely abrogated by NAD/NAMPT-dependent inhibition of complex I activity, indicating a direct metabolic crosstalk.