The NADase enzyme CD38: an emerging pharmacological target for Systemic Sclerosis, Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Abstract

Purpose of review:

Here we review recent literature on the emerging role of nicotinamide adenine dinucleotide (NAD⁺) metabolism and its dysfunction via the enzyme CD38 in the pathogenesis of rheumatologic diseases. We evaluate the potential of targeting CD38 to ameliorate NAD⁺-related metabolic imbalance and tissue dysfunction in the treatment of systemic sclerosis (SSc), systemic lupus erythematous (SLE), and rheumatoid arthritis (RA).

Recent findings:

In this review we will discuss emerging basic, pre-clinical and human data that point to the novel role of a CD38 in dysregulated NAD-homeostasis in SSc, SLE and RA. In particular, recent studies implicate increased activity of CD38, one of the main enzymes in NAD⁺-catabolism, in the pathogenesis of persistent systemic fibrosis in SSc, and increased susceptibility of SLE patients to infections. We will also discuss recent studies that demonstrate that a cytotoxic CD38 antibody can promote clearance of plasma cells involved in the generation of RA antibodies.

Summary:

Recent studies identify potential therapeutic approaches for boosting NAD to treat rheumatologic diseases including SSc, RA, and SLE, with particular attention to inhibition of CD38 enzymatic activity as a target. Key future directions in the field include the determination of the cell-type specificity and role of CD38 enzymatic activity versus CD38 structural roles in human diseases, as well as the indicators and potential side effects of CD38-targeted treatments.

Introduction

Excessive morbidity and mortality in systemic fibrotic and inflammatory conditions remains a great concern for rheumatic diseases (1, 2). Although therapeutic options for some rheumatologic diseases continue to evolve, there remain many gaps in the understanding and optimal management of these diseases.

It is well accepted that diseases such as systemic scleroderma (SSc), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) are multifactorial and involve the interaction between genetics, environmental factors, immune cells, and stromal cells (1-4). However, the precise mechanisms underlying the pathophysiology of these diseases have not been completely understood.

One aspect that traditionally has not been considered in the pathogenesis of these diseases is the potential role of intermediary and energy metabolism. However, research on the molecular processes underlying rheumatic and immune mediated diseases in the last years have increasingly implicated the importance of intermediary metabolism in fibrosis, immune function and immune modulation.

Interestingly, recent data indicate that dysregulation of metabolism of the oxidation-reduction nucleotide nicotinamide adenine dinucleotide (NAD⁺) may play a key role in the pathophysiology of age-related health decline as well as multiple diseases states including SSc and SLE (5-14). Understanding the precise mechanisms that lead to dysregulation of NAD metabolism may contribute to the development of NAD-targeted therapies for fibrosis and autoimmunity. In particular, the widely expressed enzyme CD38 is the main NADase in mammalian tissues, and plays a key role in immune metabolism (5, 8, 9). Emerging data point to the possibility that CD38 may have an important role in the pathogenesis of SSc, SLE and RA (12, 13, 15, 16), and thus CD38-targeted therapies may represent a novel effective approach. However, much remains to be discovered before these approaches can be safely incorporated in our therapeutic arsenal.

Tissue NAD⁺-decline in the pathophysiology of human diseases.

Intracellular NAD+ levels decline in tissues during the aging process, and may also be a common mechanism of cell and tissue dysfunction in several acute and chronic disease states (5-13) (Table 1). Recent data from both animal models and human studies extend the role of cellular-tissue NAD+ decline to acute kidney injury, fetal malformations, mitochondrial myopathies, and rheumatic diseases such as SSc and SLE (10, 12, 13, 15-20). Furthermore, it has been proposed in both pre-clinical models and human studies that therapies aiming at increasing cellular NAD levels in vivo (so-called NAD-boosting therapies) could have beneficial effects in these diseases (10, 17, 21, 22) (Table 2).

Table 1:

Diseases whose pathophysiology has been associated with NAD/CD38 dysmetabolism in pre-clinical and/or human studies

Pre-clinical studies	References	Human studies	References
Hearing loss	(53, 54)	Fetal malformation (VACTERL association)	(10)
Obesity and diabetes	(46, 55-57)	Acute Kidney Injury	(18, 20)
Kidney disease	(58)	Pellagra	(23, 24)
Heart disease	(59-62)	Mitochondrial myopathies	(17)
Non-alcoholic and Alcoholic fat liver disease	(46, 63, 64)
Muscular dystrophy	(65)
Mitochondrial myopathies	(66)
Neurodegeneration-related disease and Stroke	(6, 28, 31, 67, 68)
Aging, progeroid syndromes and Lifespan	(5-8, 69-71)
Cataract, Genetic macular degeneration	(72, 73)

Table 2:

Strategies to promote NAD+-boosting.

Type of NAD+-booster	Compound Name	Mechanism of Action	References
NAD precursors	Niacin Nicotinamide Nicotinamide Riboside	Vitamin B3 derivative NAD precursors	(21)
NAD Analogous Covalent Inhibitors	Ara-F-NMN phosphoester/C48	Competitive Inhibition of NADase activity	(9, 74)
Flavonoids Non-covalent inhibitors	Apigenin	Competitive Inhibition of NADase activity	(9, 75)
	Luteolinidin
	Kuromanin
Flavonoids Non-covalent inhibitors	Rhein/K-Rhein	Uncompetitive Inhibition of NADase activity	(9, 76)
4-amino-Quinolines Non-covalent inhibitors	78c	Uncompetitive Inhibition of NADase activity	(11)
	1ah		(9, 77)
	1ai
Antibodies (IgG mAB)	Isatuximab	Cytotoxic Effect/Clearance of CD38+ cells and Allosteric Inhibition of NADase activity	(35, 78)
Antibodies (IgG mAB)	Daratumumab	Cytotoxic Effect/Clearance of CD38+ cells	(35, 79)
	TAK-079		(35, 80)
	MOR-202		(35, 78)

The role of NAD⁺ in cellular function and pathophysiology

NAD(P) is a co-factor derived from vitamin B3 that is crucial for oxy-reduction reactions including glycolysis, fat acid oxidation/synthesis, and many others (5). In addition, NAD⁺ is also a substrate for enzymes involved in DNA-damage repair such as PARPs, as well as for epigenetic and metabolism regulatory enzymes such as the Sirtuins (NAD-dependent deacetylases) (5). Therefore, NAD⁺ has many functions and dysregulation of its homeostasis can lead to impairment in a broad range of fundamental cellular processes (5). In fact, pellagra, a disease resulting from severe vitamin B3 deficiency, leads to a decrease in tissue NAD-levels and consequent disruption in NAD-dependent cellular functions (23, 24). Pellagra is associated with dysfunction of multiple organ systems including the skin, gastrointestinal, immune and the central nervous system (25). Interestingly, recent studies indicate that a “functional” pellagra (leading to tissue and cellular NAD⁺-decline) is a common feature of many pathologic conditions.

Thus, it has been proposed that NAD-depleted states could be pharmacologically approached by NAD+-boosting therapy (10, 21, 22). Our expanding understanding of NAD-metabolism has led to the characterization of both anabolic and catabolic pathways maintaining NAD homeostasis that can be targeted pharmacologically (Figure 1). These approaches include the vitamin B3 niacin and its derivatives, as well as inhibitors of the NAD-degrading enzymes CD38, PARPs and SARM1 (8, 9, 21) (Table 2).

Figure 1:

Scheme showing NAD synthesis and degradation pathways and possible interventions in NAD+-Boosting Therapies. smCD38i= small molecule CD38 inhibitors

The pharmacology of NAD metabolism and CD38 and their emerging pathogenic role in SSc and SLE

Emerging human studies confirm that NAD+-boosting with high doses of vitamin B3 may have beneficial effects in a variety of disease states (10, 17, 21, 22). A recent study demonstrated for the first time that in patients with mitochondrial myopathies, levels of NAD decline in both muscle and peripheral blood cells (17). Furthermore, the authors demonstrated that administration of vitamin 1 g B3 (Niacin) to these patients for several months leads to correction of NAD-decline and metabolomics profiles (17). Importantly, the authors also observed an improvement in muscle function and a decrease in liver fat accumulation, indicating that NAD-boosting with Niacin may provide an effective therapy for mitochondrial myopathy (17). Niacin has been used in humans for many decades, and except for flushing and rare cases of clinical relevant liver dysfunction, it is generally well tolerated and safe (26). In fact, Niacin has previously been proposed as a potential treatment for patients with Raynaud's disease (27). Another NAD precursor, Nicotinamide, has also been extensively used in humans (28). Significantly, Nicotinamide was shown to be effective in the chemoprevention of non-melanoma skin cancer in high risk patients, as well as in patients with increased risk for developing acute kidney injury (20, 29).

NAD-boosting in animals can also be achieved by supplementation with other forms of vitamin B3, including Nicotinamide riboside (NR) and Nicotinamide Mono Nucleotide (NMN), and by their reduced forms (21). However, whether these novels vitamin B3 derivatives are therapeutically superior to either Niacin or Nicotinamide has not yet been demonstrated. Furthermore, beneficial effects of high doses of vitamin B3 derivatives or of any NAD-boosting approach in patients with rheumatologic diseases have not been reported.

Therapeutic NAD+-boosting can also be achieved by inhibition of the NAD-consuming enzymes (8, 21). These include CD38, PARPs and SARM1 (5, 8, 21). SARM1 appears to be localized mostly in the neural compartment and may play a role in nerve injury during Wallerian degeneration (30, 31). On the other hand, PARPs are widely expressed poly-ADP-ribosyl polymerases involved in DNA-damage repair, and PARP inhibitors have been approved for the treatment of human cancers (32, 33). Finally, CD38 (cluster of differentiation 38) is a multifunctional enzyme that degrades NAD and modulates cellular NAD homeostasis (34). This ecto-NADase, most highly expressed on immune cells, has been implicated in several physiological and pathological states including aging, obesity, diabetes, heart disease, asthma, and inflammatory and infectious conditions (34). Recently, several classes of pharmacological tools targeting CD38 have been developed. These include enzymatic activity inhibitors such as natural products like Apigenin and Quercetin, small molecules such as thiazoloquin(az)olin(on)es, and specific inhibitory CD38 antibodies (9, 11). Moreover, CD38 has also been identified as a cell-surface marker in hematologic cancers such as multiple myeloma (MM), and the cytotoxic anti-CD38 antibody Daratumumab has been approved by FDA as therapy for MM (9, 35). Interestingly, recent studies indicate that CD38 may also play a role in dysfunction of NAD+-metabolism in both SSc and SLE (12, 13). Therefore, it has been proposed that therapeutically targeting CD38 or using an NAD-boosting approach could ameliorate fibrosis in SSc and decrease susceptibility to infections in patients with SLE (12, 13). Despite the fact that the optimal approach to target CD38 and NAD metabolism dysfunction in rheumatologic conditions remains to be fully elucidated, it is possible that in some of these, a cytotoxic antibody may be the best approach, while in others a CD38 enzymatic inhibitor may be superior. Notably, future research is needed to validate the role and cell type specificity of CD38 in the pathophysiology of these diseases, and to investigate the role of CD38-targeted therapy in pre-clinical studies.

CD38-mediated NAD metabolism in SSc.

Fibrosis is a dysregulated and prolonged repair process in which an excessive deposition of extracellular matrix (ECM) components - among which collagen is the most predominant - occurs due to a severe or repetitive injury (1, 36). Unchecked fibrosis due to persistent myofibroblast activation leads to disruption of tissue architecture and ultimately to organ failure (1, 37). Fibrotic responses occur in a setting of chronic inflammation, which is defined as an immune response that persists for at least several months (38). Synchronous multiple organs fibrosis is a unique and defining characteristic of SSc and accounts for the morbidity and mortality in this disease (1, 36). The persistent accumulation of activated myofibroblasts is a defining feature of any kind of fibrosis (38, 39). However the factor(s) responsible for inducing the transition of different progenitor cells into myofibroblasts as well as their permanent activated state are disease-specific and largely unknown. In fact, the mechanisms that lead to fibrosis and tissue dysfunction in SSc are very complex and not fully understood (40). Recently, it was suggested that decrease in the function of the NAD-dependent deacetylases SIRT1 and SIRT3 could play a pathogenic role in SSc via loss of their repressive function regulating the pro-fibrotic TGF-β-SMAD pathway (41). Further, it was proposed that the loss of SIRT1 activity in SSc patients is mediated by a NAD-deficient state caused by an increased expression of the NAD-catabolic enzyme CD38 in their tissues (12). In fact, analysis of a public transcriptome dataset including 68 patients with SSc and 22 healthy controls identified increased CD38 mRNA expression in patients with diffuse cutaneous SSc compared to both healthy control, and patients with limited cutaneous SSc (12). More importantly, CD38 mRNA expression positively correlated with both molecular markers of fibrosis and with the modified Rodnan skin score (MRSS) (12). Furthermore, in pre-clinical models of SSc, boosting NAD⁺ levels by either administration of a CD38 inhibitor or supplementation with the vitamin B3 derivative, nicotinamide riboside (NR), ameliorated both lung and skin fibrosis (12). These effects were further augmented by the combination of the small molecule CD38 inhibitor and NR (12). The pathogenic role of CD38 in systemic fibrosis was further demonstrated by genetic deletion of CD38 (12). CD38 knockout mice were protected against bleomycin-induced skin and lung fibrosis compared to wild type animals (12). Mechanistically, CD38 inhibition prevented the TGF-β induced stimulation collagen synthesis of and alpha-smooth muscle actin cytoskeletal stress fiber formation (12) (Figure 2). Another enzyme involved in NAD-catabolism is NNMT. NNMT is also highly expressed in skin biopsies from patients with SSc, and may play a role in the cellular mechanisms of this disease (42). However, the pathogenic role of NNMT has not yet been explored in either animal models or patients with SSc. Interestingly, NNMT has been recently implicated in the induction of cancer-associated fibroblasts (CAF) in ovarian cancer (42). In this study it was demonstrated that in ovarian cancer stromal cells, NNMT expression correlated with tissue fibrosis and poor outcomes, and was both necessary and sufficient for the function of CAF to support cancer growth and metastasis (42). Thus, aberrant NNMT expression or activity appears to be linked to multiple forms of pathological fibrosis. However, whether NNMT could also have a role in myofibroblast activation in SSc and thus represent a potential therapeutic target is an open question.

Figure 2:

Scheme showing mechanisms of CD38 inhibition and their activated molecular pathways in the pharmacologic approach of different rheumatologic diseases. smCD38i= small molecule CD38 inhibitors.

CD38 and the risk of infection in SLE patients.

SLE is a generalized auto-immune disease where dysfunctional T and B lymphocytes, and innate immune cells promote tissue injury and damage (2, 43). Immune dysfunction in SLE patients can also lead to a decreased capacity to fight both bacterial and viral infections (4). In fact, infections remain a major cause of morbidity and mortality in this disease (2). Increased susceptibility to infections in SLE is multifactorial and can be related to disease activity, environmental factors, and immune suppressive therapies (2, 4).

A recent study identified a dysfunctional population of endogenous T Cells that participate in increased infection susceptibility in SLE patients, and highlighted the fundamental pathogenic role of the NADase CD38 in these cells (13). The authors observed elevated CD38 expression on CD8+ cytotoxic T cells in a subset of SLE patients that exhibited increased infection frequency (Figure 2) (13). These Cd8+T cells demonstrated dysfunctional expression of several genes required for their cytotoxic activity (13). These included granzyme A, granzyme B, perforin, and interferon-gamma (IFN-γ) (13). The authors further demonstrated that deregulated expression of these genes in CD8⁺CD38^hi T cells was mediated by a mechanism driven by CD38 NAD⁺-degrading enzymatic activity (13). In particular, this was mediated by the CD38-NAD⁺-SIRT1 axis described over 15 years ago (44).

The precise immunosuppressive role of the CD38-NAD⁺-SIRT1 axis in SLE T cells appeared to be mediated by a change in the acetylation of the enzyme EZH2. EZH2 is a methyl transferase that promotes epigenetic regulation of several genes (45). When acetylated, EZH2 is active and suppresses gene expression via the enzymatic trimethylation of histone H3K27(45). However, when de-acetylated, EZH2 is inactive and cannot suppress gene expression (13). One of the deacetylase enzymes involved in this process is the NAD-dependent deacetylase SIRT1 (13). We have extensively demonstrated that the NADase CD38, by decreasing levels of NAD an obligate substrate for sirtuins, can suppress the activity of SIRT1 (11, 44, 46). Thus, in SLE patients, susceptibility to infections mediated by CD8⁺ CD38^hi T cells appears to be driven by cellular NAD⁺-decline induced by CD38, and a subsequent decrease in SIRT1-mediated deacetylation of EZH2 (13) (Figure 2). Ultimately, this leads to EZH2 acetylation and subsequent activation, causing suppression of the cytotoxic functions of CD8⁺ CD38^hi T cells (13).

Thus, this study proposed that the increased infection risk observed in SLE, which is promoted by dysfunctional cytotoxic T cells, may be ameliorated by inhibition of CD38-NADase activity leading to an increase in NAD levels, SIRT1 activation, and subsequent inhibition of the methyl transferase EZH2 (13). However, since it has been previously proposed that CD38 plays an active role in fighting bacterial infections in rodent macrophages and neutrophils (47) it is currently unclear whether inhibition of CD38 would decrease or increase the rate of infections in SLE patients. Resolving this interesting conundrum requires additional research.

The role of CD38 in antibody-mediated autoimmune diseases: targeting CD38 in plasma cells as a therapy for RA and SLE

Autoantibody-producing plasma cells (PCs) play a key role in the pathogenesis of antibody-mediated autoimmune diseases including RA and SLE (48). In general, autoantibodies participate in the pathophysiology of these diseases directly targeting the tissue or via formation of immune complexes (49). In RA, PCs are usually found in synovial biopsies, while in SLE patients, they are found in nephritic kidneys, leading to production of autoantibodies (16). Because these autoreactive PCs are long-lived and mostly resistant to conventional immunosuppressive treatments, they are interesting targets to be investigated as a new therapy for RA and SLE (50, 51). It has been previously demonstrated, using integrative analysis of synovial biopsies and PBMC in patients with RA and SLE, that CD38 is highly expressed in PCs in the peripheral blood compared to other immune cell populations (16). Interestingly, it has also been shown that PCs are reduced in MM patients treated with Daratumumab (15). Therefore, It has been postulated that Daratumumab could have an important effect in antibody-mediated autoimmune diseases via inducing cytotoxicity in autoreactive PCs (15) (Figure 2). A recent study evaluated autoantibodies in serum from MM patients, who received Daratumumab on monotherapy or combined with the programmed death-1 (PD-1) inhibitor Nivolumab (15). In six out of 41 patients in this study, autoantibodies were detected before treatment (15). In 5 out of these 6 patients Daratumumab treatment resulted in a sustained reduction of autoantibody titers, indicating the drug effectiveness in deplete autoreactive PCs (15). It has been demonstrated that the reduction of autoantibody levels is associated with clinical improvement as well as their reappearance with disease relapse (52). However, the analysis of autoantibodies levels may not be enough to investigate the effect of this anti-CD38 antibody in patients with autoimmune diseases. Therefore, additional studies are needed. Nonetheless, this study undoubtedly sets a new milestone to support the potential role of CD38 as a therapeutic target in patients with RA and SLE or other types of autoantibody-dependent autoimmune disorders.

Conclusion

NAD⁺ dysmetabolism has emerged as a common cause of tissue and cellular dysfunction in many conditions including aging, and acute and chronic diseases such as acute kidney injury and mitochondrial myopathy (5-14, 17, 18, 20). The role of NAD dysmetabolism has been investigated in pre-clinical animals models in many other conditions; these include age-related frailty, hearing loss, heart diseases, metabolic syndrome, and progeroid states just to mention a few (5-11, 14). Recently the pathogenic role of NAD dysmetabolism has been expanded to both fibrotic and autoimmune/inflammatory rheumatologic diseases including SSc, SLE and RA (12, 13, 15, 16, 19). The role of the CD38 has been explored in animals and/or human studies in these conditions (12, 13, 15, 16). Further studies will help to define the potential role of NAD-boosting and CD38 targeted therapies for these conditions. Importantly, extensive pre-clinical and clinical studies will be required to determine the safety and efficacy of these approaches in human diseases.