CD157 signaling promotes survival of acute myeloid leukemia cells and modulates sensitivity to cytarabine through regulation of anti-apoptotic Mcl-1

CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

Enzymatic and Chemical Syntheses of Vacor Analogs of Nicotinamide Riboside, NMN and NAD

It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide (VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor, vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by nicotinamide phosphoribosyltransferase (NamPT).

PROGRAMMED AXON DEATH EXECUTOR SARM1 IS A MULTI-FUNCTIONAL NAD(P)ASE WITH PROMINENT BASE EXCHANGE ACTIVITY, ALL REGULATED BY PHYSIOLOGICAL LEVELS OF NMN, NAD, NADP AND OTHER METABOLITES

SARM1 is an NAD glycohydrolase and TLR adapter with an essential, prodegenerative role in programmed axon death (Wallerian degeneration). It has low basal NADase activity that becomes strongly activated by NAD precursor NMN. Very high levels of NAD oppose this activation, competing for the same allosteric site on SARM1′s regulatory ARM domain. Injury or diseases that deplete axons of NMNAT2, an essential enzyme converting NMN to NAD, cause SARM1 activation. The resulting NAD degradation by SARM1, combined with loss of NAD synthesis by NMNAT2, causes rapid depletion of axonal NAD. This NAD loss is widely assumed to mediate axon death and is consequently a key focus for therapeutic strategies for axonopathies. However, like other NAD(P) glycohydrolases, SARM1 has additional enzyme activities whose contributions, consequences and regulation need to be fully understood. Here, we compare the multiple actions and regulation of recombinant human SARM1 with those of two other NAD(P) glycohydrolases, human CD38 and Aplysia californica ADP ribosyl cyclase. We find that SARM1 has the highest transglycosidation (base exchange) activity of these enzymes at neutral pH and with some bases this dominates NAD(P) hydrolysis and cyclisation. Moreover, like its NADase and NADPase reactions, SARM1-mediated base exchange at neutral pH is activated by increases in the NMN:NAD ratio, which we show for the first time can act in the presence of physiological levels of both metabolites. We establish that SARM1 base exchange is the most likely physiological source of calcium mobilizing agent NaADP, and potentially of other NAD(P) analogues, which could contribute to axon and cell death. We also identify regulatory effects of free pyridine bases, of NADP and of nicotinic acid riboside (NaR) on SARM1 that represent further therapeutic opportunities. These data will help to pinpoint which of the multiple functions of SARM1 is responsible for axon degeneration and how it can be optimally targeted to block axon degeneration in disease.

Proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy: potential targets in calcium regulatory network

Herein proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy was performed to achieve new potential targets for treating epileptic seizures. A total of 144 differently expressed proteins in both left and right hippocampi by two-dimensional electrophoresis coupled to matrix-assisted laser desorption-mass spectrometry were identified across the rat models of epilepsy. Based on network analysis, the majority of differentially expressed proteins were associated with Ca2+ homeostasis. Changes in ADP-ribosyl cyclase (ADPRC), lysophosphatidic acid receptor 3 (LPAR3), calreticulin, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), synaptosomal nerve-associated protein 25 (SNAP 25) and transgelin 3 proteins were probed by Western blot analysis and validated using immunohistochemistry. Inhibition of calcium influx by 8-Bromo-cADP-Ribose (8-Br-cADPR) and 2-Aminoethyl diphenylborinate (2-APB) which act via the ADPRC and LPAR3, respectively, attenuated epileptic seizures. Considering a wide range of molecular events and effective role of calcium homeostasis in epilepsy, polypharmacy with multiple realistic targets should be further explored to reach the most effective treatments.

Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle

Background Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). Methods Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. Results Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. Conclusions In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.

Complementary NAD+ Replacement Strategies Fail to Functionally Protect Dystrophin-Deficient Muscle

Background Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). Methods Using a metabolomics approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. Results Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, or improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. Conclusions In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, neither small molecule strategy is sufficient to protect or restore muscle function, undermining the therapeutic approach.

Complementary NAD+ Replacement Strategies Fail to Functionally Protect Dystrophin-Deficient Muscle

Background Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to lessen the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). Methods Using a metabolomics approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit by providing a biosynthetic precursor, nicotinamide riboside, or specifically inhibiting the NAD-degrading activity of the ADP-ribosyl cyclase, CD38. Results Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy effectively lessened muscle damage markers or improved hindlimb strength following repeated rounds of eccentric challenge and recovery. Conclusions Intramuscular NAD depletion occurs rapidly after eccentic injury, with broad pleitropic effects on the molecular phenotype of the tissue. Currently available means of protecting or replenishing the muscle NAD pool via orally administered small molecules are insufficient to functionally restore dystrophin-deficient muscle.

Complementary NAD+ Replacement Strategies Fail to Functionally Protect Dystrophin-Deficient Muscle

Background Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to lessen the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). Methods Using a metabolomics approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit by providing a biosynthetic precursor, nicotinamide riboside, or specifically inhibiting the NAD-degrading activity of the ADP-ribosyl cyclase, CD38. Results Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy effectively lessened muscle damage markers or improved hindlimb strength following repeated rounds of eccentric challenge and recovery. Conclusions Intramuscular NAD depletion occurs rapidly after eccentic injury, with broad pleitropic effects on the molecular phenotype of the tissue. Currently available means of protecting or replenishing the muscle NAD pool via orally administered small molecules are insufficient to functionally restore dystrophin-deficient muscle.

CD157: From Myeloid Cell Differentiation Marker to Therapeutic Target in Acute Myeloid Leukemia

Human CD157/BST-1 and CD38 are dual receptor-enzymes derived by gene duplication that belong to the ADP ribosyl cyclase gene family. First identified over 30 years ago as Mo5 myeloid differentiation antigen and 10 years later as Bone Marrow Stromal Cell Antigen 1 (BST-1), CD157 proved not to be restricted to the myeloid compartment and to have a diversified functional repertoire ranging from immunity to cancer and metabolism. Despite being a NAD+-metabolizing ectoenzyme anchored to the cell surface through a glycosylphosphatidylinositol moiety, the functional significance of human CD157 as an enzyme remains unclear, while its receptor role emerged from its discovery and has been clearly delineated with the identification of its high affinity binding to fibronectin. The aim of this review is to provide an overview of the immunoregulatory functions of human CD157/BST-1 in physiological and pathological conditions. We then focus on CD157 expression in hematological tumors highlighting its emerging role in the interaction between acute myeloid leukemia and extracellular matrix proteins and its potential utility for monoclonal antibody targeted therapy in this disease.