Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs.

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

Premature senescence and cardiovascular disease following cancer treatments: mechanistic insights.

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality, especially among the aging population. The "response-to-injury" model proposed by Dr. Russell Ross in 1999 emphasizes inflammation as a critical factor in atherosclerosis development, with atherosclerotic plaques forming due to endothelial cell (EC) injury, followed by myeloid cell adhesion and invasion into the blood vessel walls. Recent evidence indicates that cancer and its treatments can lead to long-term complications, including CVD. Cellular senescence, a hallmark of aging, is implicated in CVD pathogenesis, particularly in cancer survivors. However, the precise mechanisms linking premature senescence to CVD in cancer survivors remain poorly understood. This article aims to provide mechanistic insights into this association and propose future directions to better comprehend this complex interplay.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

A stress-induced cilium-to-PML-NB route drives senescence initiation.

Cellular senescence contributes to tissue homeostasis and age-related pathologies. However, how senescence is initiated in stressed cells remains vague. Here, we discover that exposure to irradiation, oxidative or inflammatory stressors induces transient biogenesis of primary cilia, which are then used by stressed cells to communicate with the promyelocytic leukemia nuclear bodies (PML-NBs) to initiate senescence responses in human cells. Mechanistically, a ciliary ARL13B-ARL3 GTPase cascade negatively regulates the association of transition fiber protein FBF1 and SUMO-conjugating enzyme UBC9. Irreparable stresses downregulate the ciliary ARLs and release UBC9 to SUMOylate FBF1 at the ciliary base. SUMOylated FBF1 then translocates to PML-NBs to promote PML-NB biogenesis and PML-NB-dependent senescence initiation. Remarkably, Fbf1 ablation effectively subdues global senescence burden and prevents associated health decline in irradiation-treated mice. Collectively, our findings assign the primary cilium a key role in senescence induction in mammalian cells and, also, a promising target in future senotherapy strategies.

TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity.

Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolize nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies.Abbreviations: 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund's complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4',6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N6-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund's adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter.

CD38 inhibitor 78c increases mice lifespan and healthspan in a model of chronological aging.

Nicotinamide adenine dinucleotide (NAD) levels decline during aging, contributing to physical and metabolic dysfunction. The NADase CD38 plays a key role in age-related NAD decline. Whether the inhibition of CD38 increases lifespan is not known. Here, we show that the CD38 inhibitor 78c increases lifespan and healthspan of naturally aged mice. In addition to a 10% increase in median survival, 78c improved exercise performance, endurance, and metabolic function in mice. The effects of 78c were different between sexes. Our study is the first to investigate the effect of CD38 inhibition in naturally aged animals.

Dihydronicotinamide Riboside Is a Potent NAD+ Precursor Promoting a Pro-Inflammatory Phenotype in Macrophages.

Nicotinamide adenine dinucleotide (NAD) metabolism plays an important role in the regulation of immune function. However, a complete picture of how NAD, its metabolites, precursors, and metabolizing enzymes work together in regulating immune function and inflammatory diseases is still not fully understood. Surprisingly, few studies have compared the effect of different forms of vitamin B3 on cellular functions. Therefore, we investigated the role of NAD boosting in the regulation of macrophage activation and function using different NAD precursors supplementation. We compared nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) supplementation, with the recently described potent NAD precursor NRH. Our results show that only NRH supplementation strongly increased NAD+ levels in both bone marrow-derived and THP-1 macrophages. Importantly, NRH supplementation activated a pro-inflammatory phenotype in resting macrophages, inducing gene expression of several cytokines, chemokines, and enzymes. NRH also potentiated the effect of lipopolysaccharide (LPS) on macrophage activation and cytokine gene expression, suggesting that potent NAD+ precursors can promote inflammation in macrophages. The effect of NRH in NAD+ boosting and gene expression was blocked by inhibitors of adenosine kinase, equilibrative nucleoside transporters (ENT), and IκB kinase (IKK). Interestingly, the IKK inhibitor, BMS-345541, blocked the mRNA expression of several enzymes and transporters involved in the NAD boosting effect of NRH, indicating that IKK is also a regulator of NAD metabolism. In conclusion, NAD precursors such as NRH may be important tools to understand the role of NAD and NADH metabolism in the inflammatory process of other immune cells, and to reprogram immune cells to a pro-inflammatory phenotype, such as the M2 to M1 switch in macrophage reprogramming, in the cancer microenvironment.

CD38-NADase is a new major contributor to Duchenne muscular dystrophic phenotype.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration. Two important deleterious features are a Ca2+ dysregulation linked to Ca2+ influxes associated with ryanodine receptor hyperactivation, and a muscular nicotinamide adenine dinucleotide (NAD+ ) deficit. Here, we identified that deletion in mdx mice of CD38, a NAD+ glycohydrolase-producing modulators of Ca2+ signaling, led to a fully restored heart function and structure, with skeletal muscle performance improvements, associated with a reduction in inflammation and senescence markers. Muscle NAD+ levels were also fully restored, while the levels of the two main products of CD38, nicotinamide and ADP-ribose, were reduced, in heart, diaphragm, and limb. In cardiomyocytes from mdx/CD38-/- mice, the pathological spontaneous Ca2+ activity was reduced, as well as in myotubes from DMD patients treated with isatuximab (SARCLISA® ) a monoclonal anti-CD38 antibody. Finally, treatment of mdx and utrophin-dystrophin-deficient (mdx/utr-/- ) mice with CD38 inhibitors resulted in improved skeletal muscle performances. Thus, we demonstrate that CD38 actively contributes to DMD physiopathology. We propose that a selective anti-CD38 therapeutic intervention could be highly relevant to develop for DMD patients.

Benefits in cardiac function by CD38 suppression: Improvement in NAD+ levels, exercise capacity, heart rate variability and protection against catecholamine-induced ventricular arrhythmias.

CD38 enzymatic activity regulates NAD+ and cADPR levels in mammalian tissues, and therefore has a prominent role in cellular metabolism and calcium homeostasis. Consequently, it is reasonable to hypothesize about its involvement in cardiovascular physiology as well as in heart related pathological conditions. AIM: To investigate the role of CD38 in cardiovascular performance, and its involvement in cardiac electrophysiology and calcium-handling. METHODS AND RESULTS: When submitted to a treadmill exhaustion test, a way of evaluating cardiovascular performance, adult male CD38KO mice showed better exercise capacity. This benefit was also obtained in genetically modified mice with catalytically inactive (CI) CD38 and in WT mice treated with antibody 68 (Ab68) which blocks CD38 activity. Hearts from these 3 groups (CD38KO, CD38CI and Ab68) showed increased NAD+ levels. When CD38KO mice were treated with FK866 which inhibits NAD+ biosynthesis, exercise capacity as well as NAD+ in heart tissue decreased to WT levels. Electrocardiograms of conscious unrestrained CD38KO and CD38CI mice showed lower basal heart rates and higher heart rate variability than WT mice. Although inactivation of CD38 in mice resulted in increased SERCA2a expression in the heart, the frequency of spontaneous calcium release from the sarcoplasmic reticulum under stressful conditions (high extracellular calcium concentration) was lower in CD38KO ventricular myocytes. When mice were challenged with caffeine-epinephrine, CD38KO mice had a lower incidence of bidirectional ventricular tachycardia when compared to WT ones. CONCLUSION: CD38 inhibition improves exercise performance by regulating NAD+ homeostasis. CD38 is involved in cardiovascular function since its genetic ablation decreases basal heart rate, increases heart rate variability and alters calcium handling in a way that protects mice from developing catecholamine induced ventricular arrhythmias.

Critical Role of Astrocyte NAD+ Glycohydrolase in Myelin Injury and Regeneration.

Western-style diets cause disruptions in myelinating cells and astrocytes within the mouse CNS. Increased CD38 expression is present in the cuprizone and experimental autoimmune encephalomyelitis models of demyelination and CD38 is the main nicotinamide adenine dinucleotide (NAD+)-depleting enzyme in the CNS. Altered NAD+ metabolism is linked to both high fat consumption and multiple sclerosis (MS). Here, we identify increased CD38 expression in the male mouse spinal cord following chronic high fat consumption, after focal toxin [lysolecithin (LL)]-mediated demyelinating injury, and in reactive astrocytes within active MS lesions. We demonstrate that CD38 catalytically inactive mice are substantially protected from high fat-induced NAD+ depletion, oligodendrocyte loss, oxidative damage, and astrogliosis. A CD38 inhibitor, 78c, increased NAD+ and attenuated neuroinflammatory changes induced by saturated fat applied to astrocyte cultures. Conditioned media from saturated fat-exposed astrocytes applied to oligodendrocyte cultures impaired myelin protein production, suggesting astrocyte-driven indirect mechanisms of oligodendrogliopathy. In cerebellar organotypic slice cultures subject to LL-demyelination, saturated fat impaired signs of remyelination effects that were mitigated by concomitant 78c treatment. Significantly, oral 78c increased counts of oligodendrocytes and remyelinated axons after focal LL-induced spinal cord demyelination. Using a RiboTag approach, we identified a unique in vivo brain astrocyte translatome profile induced by 78c-mediated CD38 inhibition in mice, including decreased expression of proinflammatory astrocyte markers and increased growth factors. Our findings suggest that a high-fat diet impairs oligodendrocyte survival and differentiation through astrocyte-linked mechanisms mediated by the NAD+ase CD38 and highlights CD38 inhibitors as potential therapeutic candidates to improve myelin regeneration.SIGNIFICANCE STATEMENT Myelin disturbances and oligodendrocyte loss can leave axons vulnerable, leading to permanent neurologic deficits. The results of this study suggest that metabolic disturbances, triggered by consumption of a diet high in fat, promote oligodendrogliopathy and impair myelin regeneration through astrocyte-linked indirect nicotinamide adenine dinucleotide (NAD+)-dependent mechanisms. We demonstrate that restoring NAD+ levels via genetic inactivation of CD38 can overcome these effects. Moreover, we show that therapeutic inactivation of CD38 can enhance myelin regeneration. Together, these findings point to a new metabolic targeting strategy positioned to improve disease course in multiple sclerosis and other conditions in which the integrity of myelin is a key concern.

FBF1 deficiency promotes beiging and healthy expansion of white adipose tissue.

Preadipocytes dynamically produce sensory cilia. However, the role of primary cilia in preadipocyte differentiation and adipose homeostasis remains poorly understood. We previously identified transition fiber component FBF1 as an essential player in controlling selective cilia import. Here, we establish Fbf1tm1a/tm1a mice and discover that Fbf1tm1a/tm1a mice develop severe obesity, but surprisingly, are not predisposed to adverse metabolic complications. Obese Fbf1tm1a/tm1a mice possess unexpectedly healthy white fat tissue characterized by spontaneous upregulated beiging, hyperplasia but not hypertrophy, and low inflammation along the lifetime. Mechanistically, FBF1 governs preadipocyte differentiation by constraining the beiging program through an AKAP9-dependent, cilia-regulated PKA signaling, while recruiting the BBS chaperonin to transition fibers to suppress the hedgehog signaling-dependent adipogenic program. Remarkably, obese Fbf1tm1a/tm1a mice further fed a high-fat diet are protected from diabetes and premature death. We reveal a central role for primary cilia in the fate determination of preadipocytes and the generation of metabolically healthy fat tissue.

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder marked by numerous progressively enlarging kidney cysts. Mettl3, a methyltransferase that catalyzes the abundant N6-methyladenosine (m6A) RNA modification, is implicated in development, but its role in most diseases is unknown. Here, we show that Mettl3 and m6A levels are increased in mouse and human ADPKD samples and that kidney-specific transgenic Mettl3 expression produces tubular cysts. Conversely, Mettl3 deletion in three orthologous ADPKD mouse models slows cyst growth. Interestingly, methionine and S-adenosylmethionine (SAM) levels are also elevated in ADPKD models. Moreover, methionine and SAM induce Mettl3 expression and aggravate ex vivo cyst growth, whereas dietary methionine restriction attenuates mouse ADPKD. Finally, Mettl3 activates the cyst-promoting c-Myc and cAMP pathways through enhanced c-Myc and Avpr2 mRNA m6A modification and translation. Thus, Mettl3 promotes ADPKD and links methionine utilization to epitranscriptomic activation of proliferation and cyst growth.

Wnt-induced, TRP53-mediated Cell Cycle Arrest of Precursors Underlies Interstitial Cell of Cajal Depletion During Aging.

BACKGROUND & AIMS: Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC). METHODS: Mice aged 1-107 weeks, klotho mice, APCΔ468 mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription-polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference. RESULTS: The klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G1/S and G2/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G1/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity. CONCLUSIONS: Increased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers.

CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels.

Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.

Metalloproteinase PAPP-A regulation of IGF-1 contributes to polycystic kidney disease pathogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.

A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo.

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

The Multi-faceted Ecto-enzyme CD38: Roles in Immunomodulation, Cancer, Aging, and Metabolic Diseases.

CD38 (Cluster of Differentiation 38) is a multifunctional ecto-enzyme that metabolizes NAD+ and mediates nicotinamide dinucleotide (NAD+) and extracellular nucleotide homeostasis as well as intracellular calcium. CD38 is also an emerging therapeutic target under conditions in which metabolism is altered including infection, aging, and tumorigenesis. We describe multiple enzymatic activities of CD38, which may explain the breadth of biological roles observed for this enzyme. Of greatest significance is the role of CD38 as an ecto-enzyme capable of modulating extracellular NAD+ precursor availability: 1 to bacteria unable to perform de novo synthesis of NAD+; and 2 in aged parenchyma impacted by the accumulation of immune cells during the process of 'inflammaging'. We also discuss the paradoxical role of CD38 as a modulator of intracellular NAD+, particularly in tumor immunity. Finally, we provide a summary of therapeutic approaches to CD38 inhibition and 'NAD+ boosting' for treatment of metabolic dysfunction observed during aging and in tumor immunity. The present review summarizes the role of CD38 in nicotinamide nucleotide homeostasis with special emphasis on the role of CD38 as an immunomodulator and druggable target.

Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia.

PURPOSE: CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it's relevance as a therapeutic target in CLL remains understudied. EXPERIMENTAL DESIGN: We investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT. RESULTS: In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model. CONCLUSIONS: Overall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.

Targeting senescent cells alleviates obesity-induced metabolic dysfunction.

Adipose tissue inflammation and dysfunction are associated with obesity-related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug-inducible "suicide" genes driven by the p16Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra-abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity-related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity-related metabolic dysfunction and its complications.

A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline.

Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.