Tumors carrying BRAF-mutations over-express NAMPT that is genetically amplified and possesses oncogenic properties.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways. METHODS: Exploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose-response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself. RESULTS: The first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT. CONCLUSIONS: Overall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.

The Extracellular NADome Modulates Immune Responses.

The term NADome refers to the intricate network of intracellular and extracellular enzymes that regulate the synthesis or degradation of nicotinamide adenine dinucleotide (NAD) and to the receptors that engage it. Traditionally, NAD was linked to intracellular energy production through shuffling electrons between oxidized and reduced forms. However, recent data indicate that NAD, along with its biosynthetic and degrading enzymes, has a life outside of cells, possibly linked to immuno-modulating non-enzymatic activities. Extracellular NAD can engage puriginergic receptors triggering an inflammatory response, similar - to a certain extent - to what described for adenosine triphosphate (ATP). Likewise, NAD biosynthetic and degrading enzymes have been amply reported in the extracellular space, where they possess both enzymatic and non-enzymatic functions. Modulation of these enzymes has been described in several acute and chronic conditions, including obesity, cancer, inflammatory bowel diseases and sepsis. In this review, the role of the extracellular NADome will be discussed, focusing on its proposed role in immunomodulation, together with the different strategies for its targeting and their potential therapeutic impact.

NAMPT and NAPRT: Two Metabolic Enzymes With Key Roles in Inflammation.

Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT) are two intracellular enzymes that catalyze the first step in the biosynthesis of NAD from nicotinamide and nicotinic acid, respectively. By fine tuning intracellular NAD levels, they are involved in the regulation/reprogramming of cellular metabolism and in the control of the activity of NAD-dependent enzymes, including sirtuins, PARPs, and NADases. However, during evolution they both acquired novel functions as extracellular endogenous mediators of inflammation. It is well-known that cellular stress and/or damage induce release in the extracellular milieu of endogenous molecules, called alarmins or damage-associated molecular patterns (DAMPs), which modulate immune functions through binding pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), and activate inflammatory responses. Increasing evidence suggests that extracellular (e)NAMPT and eNAPRT are novel soluble factors with cytokine/adipokine/DAMP-like actions. Elevated eNAMPT were reported in several metabolic and inflammatory disorders, including obesity, diabetes, and cancer, while eNAPRT is emerging as a biomarker of sepsis and septic shock. This review will discuss available data concerning the dual role of this unique family of enzymes.

NAMPT Over-Expression Recapitulates the BRAF Inhibitor Resistant Phenotype Plasticity in Melanoma.

Serine-threonine protein kinase B-RAF (BRAF)-mutated metastatic melanoma (MM) is a highly aggressive type of skin cancer. Treatment of MM patients using BRAF/MEK inhibitors (BRAFi/MEKi) eventually leads to drug resistance, limiting any clinical benefit. Herein, we demonstrated that the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) is a driving factor in BRAFi resistance development. Using stable and inducible NAMPT over-expression systems, we showed that forced NAMPT expression in MM BRAF-mutated cell lines led to increased energy production, MAPK activation, colony-formation capacity, and enhance tumorigenicity in vivo. Moreover, NAMPT over-expressing cells switched toward an invasive/mesenchymal phenotype, up-regulating expression of ZEB1 and TWIST, two transcription factors driving the epithelial to mesenchymal transition (EMT) process. Consistently, within the NAMPT-overexpressing cell line variants, we observed an increased percentage of a rare, drug-effluxing stem cell-like side population (SP) of cells, paralleled by up-regulation of ABCC1/MRP1 expression and CD133-positive cells. The direct correlation between NAMPT expression and gene set enrichments involving metastasis, invasiveness and mesenchymal/stemness properties were verified also in melanoma patients by analyzing The Cancer Genome Atlas (TCGA) datasets. On the other hand, CRISPR/Cas9 full knock-out NAMPT BRAFi-resistant MM cells are not viable, while inducible partial silencing drastically reduces tumor growth and aggressiveness. Overall, this work revealed that NAMPT over-expression is both necessary and sufficient to recapitulate the BRAFi-resistant phenotype plasticity.

NAD-Biosynthetic and Consuming Enzymes as Central Players of Metabolic Regulation of Innate and Adaptive Immune Responses in Cancer.

Cancer cells, particularly in solid tumors, are surrounded by non-neoplastic elements, including endothelial and stromal cells, as well as cells of immune origin, which can support tumor growth by providing the right conditions. On the other hand, local hypoxia, and lack of nutrients induce tumor cells to reprogram their metabolism in order to survive, proliferate, and disseminate: the same conditions are also responsible for building a tumor-suppressive microenvironment. In addition to tumor cells, it is now well-recognized that metabolic rewiring occurs in all cellular components of the tumor microenvironment, affecting epigenetic regulation of gene expression and influencing differentiation/proliferation decisions of these cells. Nicotinamide adenine dinucleotide (NAD) is an essential co-factor for energy transduction in metabolic processes. It is also a key component of signaling pathways, through the regulation of NAD-consuming enzymes, including sirtuins and PARPs, which can affect DNA plasticity and accessibility. In addition, both NAD-biosynthetic and NAD-consuming enzymes can be present in the extracellular environment, adding a new layer of complexity to the system. In this review we will discuss the role of the "NADome" in the metabolic cross-talk between cancer and infiltrating immune cells, contributing to cancer growth and immune evasion, with an eye to therapeutic implications.