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.

Key Points: CLL lymphocytes show high intracellular and extracellular NAMPT levels, further increased upon activation. eNAMPT prompts differentiation of CLL monocytes into M2 macrophages that sustain CLL survival and reduce T-cell proliferation.

Because of its relatively indolent clinical course, chronic lymphocytic leukemia (CLL) offers a versatile model for testing novel therapeutic regimens and drug combinations. Nicotinamide is the main NAD⁺ precursor and a direct inhibitor of four classes of enzymes, including the sirtuins. SIRT1, the main member of the sirtuin family, inactivates p53 by deacetylating a critical lysine residue. In this study, we showed that CLL cells express high levels of functional SIRT1, which is inhibited by exogenous nicotinamide. This agent blocks proliferation and promotes apoptosis selectively in leukemic cells that express wild-type (wt) p53. Nicotinamide modulates the p53-dependent genes p21, NOXA, BAX, and Mcl-1, indicating an activation of the p53 pathway and of caspase-3. DNA-damaging chemotherapeutics, such as etoposide, activate a functional loop linking SIRT1 and p53 through the induction of miR-34a. When leukemic cells are simultaneously exposed to nicotinamide and etoposide, we observe a significant increase in miR-34a levels with a concomitant inhibition of SIRT1. Furthermore, p53 acetylation levels are higher than with either agent used alone. Overall, treatment with both nicotinamde and etoposide shows strongly synergistic effects in the induction of apoptosis. We therefore concluded that nicotinamide has the dual property of inhibiting SIRT1 through a noncompetitive enzymatic block (p53 independent) and at the same time through miR-34a induction (p53 dependent). These observations suggested the therapeutic potential of nicotinamide, a novel, safe, and inexpensive drug, to be used in addition to chemotherapy for CLL patients with wt p53. Cancer Res; 71(13); 4473–83. ©2011 AACR.

Background Programmed cell death ligand 1 (PD-L1) is a cell surface molecule that plays a critical role in suppressing immune responses, mainly through binding of the PD-1 receptor on T lymphocytes. PD-L1 may be expressed by metastatic melanoma. However, its clinical and biological significance remains unclear. Here, we investigated whether expression of PD-L1 in metastatic melanoma identifies a biologically more aggressive form of the disease, carrying prognostic relevance. Patients and Methods PD-L1 expression was analyzed by immunohistochemistry using two different antibodies in primary tumors and paired metastases from 81 melanoma patients treated at a single institution. Protein expression levels were correlated with PD-L1 mRNA, BRAF mutational status and clinical outcome. PD-L1⁺ and PD-L1⁻ subsets of the A375 cell line were stabilized in vitro and compared using gene expression profiling and functional assays. Results were confirmed using xenograft models. Results PD-L1 membrane positivity was detected in 30/81 (37%) of patients. By multivariate analysis, Breslow thickness and PD-L1 membrane positivity were independent risk factors for melanoma-specific death [PD-L1 5% cutoff (HR 3.92, CI 95% 1.61-9.55 p< 0.003), PD-L1 as continuous variable (HR 1.03, CI 95% 1.02-1.04 p< 0.002)]. PD-L1 expression defined a subset of the BRAF-mutated A375 cell line characterized by a highly invasive phenotype and by enhanced ability to grow in xenograft models. Conclusions PD-L1 is an independent prognostic marker in melanoma. If confirmed, our clinical and experimental data suggest that PD-L1⁺ melanomas should be considered a disease subset with distinct genetic and morpho-phenotypic features, leading to enhanced aggressiveness and invasiveness.

BRAF inhibitors (BRAFi) improve survival in metastatic melanoma patients (MMP) but the duration of clinical benefit is limited by development of drug resistance. Here, we investigated whether the expression of programmed death-ligand 1 (PD-L1) and the density of tumor-infiltrating mononuclear cells (TIMC) predict the occurrence of resistance, hence affecting the clinical outcome in BRAFi-treated MMP. PD-L1 expression (cutoff 5%) was analyzed by immunohistochemistry with two different antibodies in BRAF^V600-mutated formalin-fixed and paraffin-embedded samples from 80 consecutive MMP treated with BRAFi at a single institution. TIMC were evaluated by conventional hematoxylin and eosin staining. Forty-six and 34 patients received vemurafenib and dabrafenib, respectively. Membranous expression of PD-L1 was detected in 28/80 (35%) of patients. At multivariate analysis, absence of tumoral PD-L1 staining [odd ratio (OR) 10.8, 95% confidence interval (CI) 2.7–43.3, P < 0.001] and the presence of TIMC (OR 6.5, 95% CI 1.7–24.3, P < 0.005) were associated with a better response to treatment. Median progression-free survival (PFS) and overall survival were 10 and 15 months, respectively. By multivariate assessment, PD-L1 expression [hazard ratio (HR) 4.3, 95% CI 2.1–8.7, P < 0.0001] and absence of TIMC (HR 2.5, 95% CI 1.4–4.7, P < 0.002) correlated with shorter PFS. PD-L1 overexpression (HR 6.2, 95% CI 2.8–14.2, P < 0.0001) and absence of TIMC (HR 3.1, 95% CI 1.5–6.5, P < 0.002) were independent prognostic factors for melanoma-specific survival. Our results provide the first proof-of-principle evidence for the predictive and prognostic relevance of PD-L1 immunohistochemical expression and density of immune cell infiltration in BRAF^V600-mutated MMP treated with BRAFi.