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Immunosuppressive cells accumulating in the tumor microenvironment constitute a formidable barrier that interferes with current immunotherapeutic approaches. A unifying feature of these tumor-associated immune and vascular endothelial cells appears to be the elevated expression of ectonucleotidase CD39, which in tandem with ecto-5'-nucleotidase CD73, catalyzes the conversion of extracellular ATP into adenosine. We glycoengineered an afucosylated anti-CD39 IgG2c and tested this reagent in mouse melanoma and colorectal tumor models. We identified major biological effects of this approach on cancer growth, associated with depletion of immunosuppressive cells, mediated through enhanced Fcγ receptor-directed (FcγR-directed), antibody-dependent cellular cytotoxicity (ADCC). Furthermore, regulatory/exhausted T cells lost CD39 expression, as a consequence of antibody-mediated trogocytosis. Most strikingly, tumor-associated macrophages and endothelial cells with high CD39 expression were effectively depleted following antibody treatment, thereby blocking angiogenesis. Tumor site-specific cellular modulation and lack of angiogenesis synergized with chemotherapy and anti-PD-L1 immunotherapy in experimental tumor models. We conclude that depleting suppressive cells and targeting tumor vasculature, through administration of afucosylated anti-CD39 antibody and the activation of ADCC, comprises an improved, purinergic system-modulating strategy for cancer therapy.

Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms. Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death. Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.

CD39/ENTPD1 hydrolyzes proinflammatory nucleotides to generate adenosine. As purinergic mediators have been implicated in intestinal inflammation, we hypothesized that CD39 might protect against inflammatory bowel disease. We studied these possibilities in a mouse model of colitis using mice with global CD39 deletion. We then tested whether human genetic polymorphisms in the CD39 gene might influence susceptibility to Crohn's disease. We induced colitis in mice using Dextran Sodium Sulfate (DSS). Readouts included disease activity scores, histological evidence of injury, and markers of inflammatory activity. We used HapMap cell lines to find SNPs that tag for CD39 expression, and then compared the frequency of subjects with high vs. low CD39-expression genotypes in a case-control cohort for Crohn's disease. Mice null for CD39 were highly susceptible to DSS injury, with heterozygote mice showing an intermediate phenotype compared to wild type (WT). We identified a common SNP that tags CD39 mRNA expression levels in man. The SNP tagging low levels of CD39 expression was associated with increased susceptibility to Crohn's disease in a case-control cohort comprised of 1,748 Crohn's patients and 2,936 controls (P = 0.005-0.0006). Our data indicate that CD39 deficiency exacerbates murine colitis and suggest that CD39 polymorphisms are associated with inflammatory bowel disease in humans.

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2 -null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2 -null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2 -null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2 -knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.

CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39 −/− mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39 −/− mice. Paradoxically, T cell–mediated allergic contact hypersensitivity was severely attenuated in CD39 −/− mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39 −/− DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC–T cell communication in antigen presentation.

Deletion of Cd39/Entpd1 Results in Hepatic Insulin Resistance Keiichi Enjyoji 1 , Ko Kotani 2 , Chandrashekar Thukral 1 , Benjamin Blumel 1 , Xiaofeng Sun 1 , Yan Wu 1 , Masato Imai 1 , David Friedman 1 , Eva Csizmadia 1 , Wissam Bleibel 1 , Barbara B. Kahn 2 and Simon C. Robson 1 1 Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 2 Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Corresponding author: Keiichi Enjyoji, kenjoji{at}bidmc.harvard.edu Abstract OBJECTIVE— Extracellular nucleotides are important mediators of inflammatory responses and could also impact metabolic homeostasis. Type 2 purinergic (P2) receptors bind extracellular nucleotides and are expressed by major peripheral tissues responsible for glucose homeostasis. CD39/ENTPD1 is the dominant vascular and immune cell ectoenzyme that hydrolyzes extracellular nucleotides to regulate purinergic signaling. RESEARCH DESIGN AND METHODS— We have studied Cd39/Entpd1-null mice to determine whether any associated changes in extracellular nucleotide concentrations influence glucose homeostasis. RESULTS— Cd39/Entpd1-null mice have impaired glucose tolerance and decreased insulin sensitivity with significantly higher plasma insulin levels. Hyperinsulinemic-euglycemic clamp studies indicate altered hepatic glucose metabolism. These effects are mimicked in vivo by injection into wild-type mice of either exogenous ATP or an ecto-ATPase inhibitor, ARL-67156, and by exposure of hepatocytes to extracellular nucleotides in vitro. Increased serum interleukin-1β, interleukin-6, interferon-γ, and tumor necrosis factor-α levels are observed in Cd39/Entpd1-null mice in keeping with a proinflammatory phenotype. Impaired insulin sensitivity is accompanied by increased activation of hepatic c-Jun NH 2 -terminal kinase/stress-activated protein kinase in Cd39/Entpd1 mice after injection of ATP in vivo. This results in decreased tyrosine phosphorylation of insulin receptor substrate-2 with impeded insulin signaling. CONCLUSIONS— CD39/Entpd1 is a modulator of extracellular nucleotide signaling and also influences metabolism. Deletion of Cd39/Entpd1 both directly and indirectly impacts insulin regulation and hepatic glucose metabolism. Extracellular nucleotides serve as “metabolokines,” indicating further links between inflammation and associated metabolic derangements. Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 20 June 2008. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted June 11, 2008. Received September 6, 2007. DIABETES

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39 -deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro . This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39 -deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

The Vascular Ectonucleotidase ENTPD1 Is a Novel Renoprotective Factor in Diabetic Nephropathy David J. Friedman 1 , Helmut G. Rennke 2 , Eva Csizmadia 3 , Keiichi Enjyoji 4 and Simon C. Robson 3 4 1 Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 2 Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 3 Transplantation Division, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 4 Gastroenterology Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Address correspondence and reprint requests to David J. Friedman, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard University, Research North 359, 99 Brookline Ave., Boston, MA 02215. E-mail: dfriedma{at}caregroup.harvard.edu . Or to Simon C. Robson, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard University, Research North 301, 99 Brookline Ave., Boston, MA 02215. E-mail: srobson{at}bidmc.caregroup.edu Abstract Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39) is the dominant vascular ectonucleotidase. By hydrolyzing ATP and ADP to AMP, ENTPD1 regulates ligand availability to a large family of P2 (purinergic) receptors. Modulation of extracellular nucleotide metabolism is an important factor in several acute and subacute models of vascular injury. We hypothesized that aberrant nucleotide signaling would promote chronic glomerular injury in diabetic nephropathy. Inducing diabetes in ENTPD1-null mice with streptozotocin resulted in increased proteinuria and more severe glomerular sclerosis compared with matched diabetic wild-type mice. Diabetic ENTPD1-null mice also had more glomerular fibrin deposition and glomerular plasminogen activator inhibitor-1 (PAI-1) staining than wild-type controls. In addition, ENTPD1-null mice showed increased glomerular inflammation, in association with higher levels of monocyte chemoattractant protein-1 (MCP-1) expression. Mesangial cell PAI-1 and MCP-1 mRNA expression were upregulated by ATP and UTP but not ADP or adenosine in vitro. The stable nucleotide analog ATPγS stimulated sustained expression of PAI-1 and MCP-1 in vitro, whereas the stable adenosine analog NECA [5′-( N -ethylcarboxamido)adenosine] downregulated expression of both genes. Extracellular nucleotide-stimulated upregulation of MCP-1 is, at least in part, protein kinase C dependent. We conclude that ENTPD1 is a vascular protective factor in diabetic nephropathy that modulates glomerular inflammation and thromboregulation. BIM, bisindolylmaleimide I ENPP1/PC-1, ectonucleotide pyrophosphatase/ phosphodiesterase-1 ENTPD1, ectonucleoside triphosphate diphosphohydrolase 1 HPLC, high-performance liquid chromotography MCP-1, monocyte chemoattractant protein-1 NECA, 5′-(N-ethylcarboxamido)adenosine PAI-1, plasminogen activator inhibitor-1 PAS, periodic acid Schiff STZ, streptozotocin Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 1 May 2007. DOI: 10.2337/db06-1593. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted April 24, 2007. Received November 14, 2006. DIABETES

Functional ENTPD1 Polymorphisms in African Americans With Diabetes and End-Stage Renal Disease David J. Friedman 1 , 2 , Matthew E. Talbert 3 , 4 , Donald W. Bowden 3 , 5 , 6 , Barry I. Freedman 5 , Yves Mukanya 7 , 8 , Keiichi Enjyoji 7 , 8 and Simon C. Robson 7 , 8 1 Renal Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; 2 Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; 3 Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 4 Program in Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 5 Department of Internal Medicine/Nephrology Division, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 6 Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina; 7 Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; 8 Transplant Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. Corresponding author: David J. Friedman, dfriedma{at}bidmc.harvard.edu . Abstract OBJECTIVE The vascular ectonucleotidase ENTPD1 protects against renal injury and modulates glucose homeostasis in mouse models. We sought to determine whether human variation in ENTPD1 influences predisposition to diabetes or diabetic nephropathy. RESEARCH DESIGN AND METHODS We analyzed ENTPD1 single nucleotide polymorphisms (SNPs) in 363 African American control subjects, 380 subjects with type 2 diabetes and end-stage renal disease (DM-ESRD), and 326 subjects with ESRD unrelated to diabetes (non–DM-ESRD). Using human cell lines, we correlated disease-associated ENTPD1 haplotypes with ENTPD1 gene expression. Finally, we studied consequences of ENTPD1 deletion in a mouse model of type 2 diabetes ( db/db ). RESULTS A common ENTPD1 two-SNP haplotype was associated with increased risk for DM-ESRD ( P = 0.0027), and an uncommon four-SNP haplotype was associated with protection against DM-ESRD ( P = 0.004). These haplotypes correlated with ENTPD1 gene expression levels in human cell lines in vitro. Subjects with high ENTPD1 -expressing haplotypes were enriched in the DM-ESRD group. By crossing ENTPD1 -null mice with db mice, we show that ENTPD1 deletion has prominent effects on metabolic syndrome traits. Specifically, deletion of ENTPD1 lowered glucose levels in control (db/−) mice with one functional leptin receptor and dramatically lowered weights in db/db mice with no functional leptin receptors. Similar effects were seen in aged ENTPD1 -null mice with normal leptin receptors. CONCLUSIONS ENTPD1 polymorphisms appear to influence susceptibility to type 2 diabetes and/or diabetic nephropathy in African Americans. Studies in human cell lines and in vivo mouse data support a potential role for ENTPD1 genetic variation in susceptibility to type 2 diabetes. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 2, 2008. Accepted December 16, 2008. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. © 2009 by the American Diabetes Association.

Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.