Mara Galli

Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner

Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this key proinflammatory cytokine. The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family, as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohns disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Radiotherapy Increases the Permissiveness of Established Mammary Tumors to Rejection by Immunomodulatory Antibodies

It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(α)-CD137, α-CD40] or relieve immunosuppression [α-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of α-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when α-CD137 and α-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-γ ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.

Nicotinamide Phosphoribosyl Transferase/Pre-B Cell Colony-Enhancing Factor/Visfatin Is Required for Lymphocyte Development and Cellular Resistance to Genotoxic Stress

Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N′-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.

The Nicotinamide Phosphoribosyltransferase: A Molecular Link between Metabolism, Inflammation, and Cancer

Beyond its well-described role in cellular metabolism, intracellular nicotinamide adenine dinucleotide (NAD) levels have been shown to affect the enzymatic activity of a series of NAD-dependent enzymes, influencing biological responses such as cell survival and inflammation. Nicotinamide phosphoribosyl transferase activity has been shown to be essential for maintaining adequate intracellular NAD levels, suggesting that this enzyme may in fact play a central role in modulating the activity of a wide range of NAD-dependent enzymes. Several recent observations concur with this hypothesis and suggest that by regulating NAD availability, Nampt is able to control both cell viability and the inflammatory response. Nampt may thus represent a novel pharmacological target with valuable anti-inflammatory and antitumor properties.

Sirtuins and inflammation: Friends or foes?

Lysine acetylation/deacetylation has been recognized as an important posttranslational modification regulating numerous cellular processes. Sirtuins represent novel players in these complex regulatory circuits. These NAD-dependent lysine-deacetylases have attracted much interest based on their role in the regulation of lifespan in lower organisms, and their capacity to interfere with cell growth, proliferation and survival in response to stress. Their absolute requirement for NAD suggests that these enzymes may represent an important molecular link between metabolism and several human disorders such as diabetes and cancer. More recently, the identification of several transcription factors known to play a role in the immune system as sirtuin substrates has suggested that this family of enzymes may also play an important role in the regulation of inflammation, a pathological situation with clear links to metabolism and aging in humans. We review herein the possible links between nuclear sirtuins and the regulation of an immune response, and discuss the possible strategies that may lead to the development of novel therapeutic approaches to treat inflammation by targeting sirtuin activity.

The Curative Outcome of Radioimmunotherapy in a Mouse Breast Cancer Model Relies on mTOR Signaling

Radiotherapy is a successful treatment modality for localized cancer. Our group has been exploring radiotherapy in combination with immunotherapy (radioimmunotherapy) to enhance systemic antitumor responses. Previously, we have shown that when local radiotherapy was combined with monoclonal antibodies (mAbs) (that enable T-cell responses by engaging costimulation [anti (α)-CD137] and blocking coinhibition [α-PD-1], up to 100% of mice bearing established syngeneic AT-3 mammary tumors were cured, but single modality treatments were not curative. Here, we investigated the molecular mechanisms underlying responses to this radioimmunotherapy approach. We observed that inhibition of signaling through the mammalian target of rapamycin (mTOR) pathway during the first 10 days of treatment severely impaired the curative effect of radioimmunotherapy, at least in part by reducing MHC class I expression on tumor cells, reducing dendritic cell (DC) activation status and CD8+ T-cell function. This data indicates that the efficacy of this type of radioimmunotherapy approach involves mTOR signaling and therefore, mTOR inhibitory drugs may impede the efficacy of similar radioimmunotherapy approaches in humans.

Enhancing the antitumor effects of radiotherapy with combinations of immunostimulatory antibodies.

The development and use of combination immunotherapy-based anticancer regimens is at an early but clearly exciting stage. We now demonstrate that the antibody-based co-targeting of multiple immunostimulatory and/or inhibitory pathways can be used safely and effectively in combination with single dose or fractionated radiotherapy to cure mice bearing established mammary tumors.

Evoking durable anti-cancer responses with blocking antibodies to PD-1 and PD-L1

Inhibitory immune checkpoints play a critical role in regulating the strength and duration of immune responses in order to maintain self-tolerance and prevent autoimmunity (1). Such regulatory mechanisms are typically exploited by tumors as an immune escape mechanism and thereby pose a major obstacle to the induction of clinically relevant anti-cancer immune responses capable of controlling and/or eradicating disease (2).