The immunomodulatory ballet of tumour-derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD-L1 pathway in cancer.

Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5-year overall survival rate of around 50%, stagnant for decades. A tumour-induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil-to-lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD-L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC-derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro-tumour phenotype of NØ. UMSCC47-TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD-L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro-tumour phenotype, dependent on ADO receptors (P1R), increasing CD170high subpopulation, CD73 and PD-L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD-L1 expression. Co-culture experiments with HNSCC cells demonstrated that TEX-modulated NØ increase the CD73/PD-L1 axis, through Cyclin D-CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX-mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies.

Inhibition of Vascular Smooth Muscle Cell Proliferation by ENPP1: The Role of CD73 and the Adenosine Signaling Axis.

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.

IFN-γ-Preconditioned Human Gingival-Derived Mesenchymal Stromal Cells Inhibit Plasmacytoid Dendritic Cells via Adenosine.

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.

Enhancing hepatocellular carcinoma management: prognostic value of integrated CCL17, CCR4, CD73, and HHLA2 expression analysis.

PURPOSE: Hepatocellular carcinoma (HCC) is a critical global health concern, with existing treatments benefiting only a minority of patients. Recent findings implicate the chemokine ligand 17 (CCL17) and its receptor CCR4 as pivotal players in the tumor microenvironment (TME) of various cancers. This investigation aims to delineate the roles of CCL17 and CCR4 in modulating the tumor's immune landscape, assessing their potential as therapeutic interventions and prognostic markers in HCC. METHODS: 873 HCC patients post-radical surgery from 2008 to 2012 at Zhongshan Hospital, Fudan University were retrospectively examined. These individuals were stratified into a training cohort (n = 354) and a validation cohort (n = 519). Through immunohistochemical analysis on HCC tissue arrays, the expressions of CCL17, CCR4, CD73, CD47, HHLA2, and PD-L1 were quantified. Survival metrics were analyzed using the Cox model, and a prognostic nomogram was devised via R software. RESULTS: The investigation confirmed the presence of CCL17 and CCR4 within the cancerous and stromal compartments of HCC tissues, associating their heightened expression with adverse clinical markers and survival outcomes. Notably, the interplay between CD73 and CCR4 expression in tumor stroma highlighted a novel cellular entity, CCR4 + CD73 + stromal cells, impacting overall and relapse-free survival. A prognostic nomogram amalgamating these immunological markers and clinical variables was established, offering refined prognostic insights and aiding in the management of HCC. The findings suggest that reduced CCR4 and CCR4 + CD73 + cell prevalence may forecast improved outcomes post-TACE. CONCLUSION: This comprehensive evaluation of CCR4, CCL17, and associated markers introduces a nuanced understanding of the HCC immunological milieu, proposing CCR4 + CD73 + stromal cells as critical to HCC pathogenesis and patient stratification.

Altered Expression of Astrocytic ATP Channels and Ectonucleotidases in the Cerebral Cortex and Hippocampus of Chronic Social Defeat Stress-Susceptible BALB/c Mice.

The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5'nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.

Discovery of Novel Non-Nucleoside Inhibitors Interacting with Dizinc Ions of CD73.

High extracellular concentrations of adenosine triphosphate (ATP) in the tumor microenvironment generate adenosine by sequential dephosphorylation of CD39 and CD73, resulting in potent immunosuppression to inhibit T cell and natural killer (NK) cell function. CD73, as the determining enzyme for adenosine production, has been shown to correlate with poor clinical tumor prognosis. Conventional inhibitors as analogues of adenosine 5'-monophosphate (AMP) may have a risk of further metabolism to adenosine analogues. Here, we report a new series of malonic acid non-nucleoside inhibitors coordinating with zinc ions of CD73. Compound 12f was found to be a superior CD73 inhibitor (IC50 = 60 nM) by structural optimization, and its pharmacokinetic properties were investigated. In mouse tumor models, compound 12f showed excellent efficacy and reversal of immunosuppression in combination with chemotherapeutic agents or checkpoint inhibitors, suggesting that it deserves further development as a novel CD73 inhibitor.

Posttranslational protein modifications as gatekeepers of cancer immunogenicity.

Triple-negative breast cancer (TNBC) presents a formidable challenge in oncology due to its aggressive phenotype and the immunosuppressive nature of its tumor microenvironment (TME). In this issue of the JCI, Zhu, Banerjee, and colleagues investigated the potential of targeting the OTU domain-containing protein 4 (OTUD4)/CD73 axis to mitigate immunosuppression in TNBC. They identified elevated CD73 expression as a hallmark of immunosuppression in TNBC. Notably, the CD73 expression was regulated by OTUD4-mediated posttranslational modifications. Using ST80, a pharmacologic inhibitor of OTUD4, the authors demonstrated the restoration of cytotoxic T cell function and enhanced efficacy of anti-PD-L1 therapy in preclinical models. These findings underscore the therapeutic potential of targeting the OTUD4/CD73 axis in TNBC.

Enhanced immunosuppressive capability of mesenchymal stem cell-derived small extracellular vesicles with high expression of CD73 in experimental autoimmune uveitis.

BACKGROUND: Autoimmune uveitis is an inflammatory disease triggered by an aberrant immune response. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) are emerging as potential therapeutic agents for this condition. CD73, an ectoenzyme present on MSC-sEVs, is involved in mitigating inflammation by converting extracellular adenosine monophosphate into adenosine. We hypothesize that the inhibitory effect of MSC-sEVs on experimental autoimmune uveitis (EAU) could be partially attributed to the surface expression of CD73. METHODS: To investigate novel therapeutic approaches for autoimmune uveitis, we performed lentiviral transduction to overexpress CD73 on the surface of MSC-sEVs, yielding CD73-enriched MSC-sEVs (sEVs-CD73). Mice with interphotoreceptor retinoid-binding protein (IRBP)-induced EAU were grouped randomly and treated with 50 µg MSC-sEVs, vector infected MSC-sEVs, sEVs-CD73 or PBS via single tail vein injection. We evaluated the clinical and histological features of the induced mice and analyzed the proportion and functional capabilities of T helper cells. Furthermore, T-cells were co-cultured with various MSC-sEVs in vitro, and we quantified the resulting inflammatory response to assess the potential therapeutic benefits of sEVs-CD73. RESULTS: Compared to MSC-sEVs, sEVs-CD73 significantly alleviates EAU, leading to reduced inflammation and diminished tissue damage. Treatment with sEVs-CD73 results in a decreased proportion of Th1 cells in the spleen, draining lymph nodes, and eyes, accompanied by an increased proportion of regulatory T-cells (Treg cells). In vitro assays further reveal that sEVs-CD73 inhibits T-cell proliferation, suppresses Th1 cells differentiation, and enhances Treg cells proportion. CONCLUSION: Over-expression of CD73 on MSC-sEVs enhances their immunosuppressive effects in EAU, indicating that sEVs-CD73 has the potential as an efficient immunotherapeutic agent for autoimmune uveitis.

Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 µM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

2-Substituted-4,7-dihydro-4-ethylpyrazolo[1,5-a]pyrimidin-7-ones alleviate LPS-induced inflammation by modulating cell metabolism via CD73 upon macrophage polarization.

Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5'-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[l,5-a]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. Our data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFß1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. Our data demonstrate that pyrazolo[l,5-a]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[l,5-a]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages.

Murine and Human-Purified very Small Embryonic-like Stem Cells (VSELs) Express Purinergic Receptors and Migrate to Extracellular ATP Gradient.

Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries.

CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner.

BACKGROUND: Clinical and experimental studies have shown that the myocardial inflammatory response during pathological events varies between males and females. However, the cellular and molecular mechanisms of these sex differences remain elusive. CD73/adenosine axis has been linked to anti-inflammatory responses, but its sex-specific cardioprotective role is unclear. The present study aimed to investigate whether the CD73/adenosine axis elicits sex-dependent cardioprotection during metabolic changes and myocarditis induced by hypobaric hypoxia. METHODS: For 7 days, male and female mice received daily injections of the CD73 inhibitor adenosine 5'- (α, ß-methylene) diphosphate (APCP) 10 mg/kg/day while they were kept under normobaric normoxic and hypobaric hypoxic conditions. We evaluated the effects of hypobaric hypoxia on the CD73/adenosine axis, myocardial hypertrophy, and cardiac electrical activity and function. In addition, metabolic homeostasis and immunoregulation were investigated to clarify the sex-dependent cardioprotection of the CD73/adenosine axis. RESULTS: Hypobaric hypoxia-induced cardiac dysfunction and adverse remodeling were more pronounced in male mice. Also, male mice had hyperactivity of the CD73/adenosine axis, which aggravated myocarditis and metabolic shift compared to female mice. In addition, CD73 inhibition triggered prostatic acid phosphatase ectonucleotidase enzymatic activity to sustain adenosine overproduction in male mice but not in female mice. Moreover, dual inhibition prostatic acid phosphatase and CD73 enzymatic activities in male mice moderated adenosine content, alleviating glycolytic shift and proinflammatory response. CONCLUSION: The CD73/adenosine axis confers a sex-dependent cardioprotection. In addition, extracellular adenosine production in the hearts of male mice is influenced by prostatic acid phosphatase and tissue nonspecific alkaline phosphatase.

Regulatory T cell-derived exosome mediated macrophages polarization for osteogenic differentiation in fracture repair.

Refractory fracture presents an intractable challenge in trauma treatment. Selective polarization of macrophages as well as the recruitment of osteogenic precursor cells play key roles in osteogenic differentiation during fracture healing. Here we constructed regulatory T cell (Treg)-derived exosomes (Treg-Exo) for the treatment of fracture. The obtained exosomes displayed a spheroid shape with a hydrated particle size of approximately 130 nm. With further purification using CD39 and CD73 antibody-modified microfluidic chips, CD39 and CD73 specifically expressing exosomes were obtained. This kind of Treg-Exo utilized the ectonucleotidases of CD39 and CD73 to catalyze the high level of ATP in the fracture area into adenosine. The generated adenosine further promoted the selective polarization of macrophages. When interacting with mesenchymal stem cells (MSCs, osteogenic precursor cells), both Treg-Exo and Treg-Exo primed macrophages facilitated the proliferation and differentiation of MSCs. After administration in vivo, Treg-Exo effectively promoted fracture healing compared with conventional T cell-derived exosome. To further improve the delivery efficacy of exosomes and integrate multiple biological processes of fracture healing, an injectable hydrogel was fabricated to co-deliver Treg-Exo and stromal cell-derived factor 1 alpha (SDF-1α). With the dual effect of Treg-Exo for macrophage polarization and SDF-1α for MSC recruitment, the multifunctional hydrogel exerted a synergistic effect on fracture repair acceleration. This study provided a promising therapeutic candidate and synergistic strategy for the clinical treatment of fracture.

synNotch-programmed iPSC-derived NK cells usurp TIGIT and CD73 activities for glioblastoma therapy.

Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT - CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such "decoy" receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

Renal ischemia and reperfusion impact the purinergic signaling in a vascular bed distant from the injured site.

AIMS: Acute kidney injury (AKI) is a public health problem and represents a risk factor for cardiovascular diseases (CVD) and vascular damage. This study aimed to investigate the impact of AKI on purinergic components in mice aorta. MAIN METHODS: The kidney ischemia was achieved by the occlusion of the left kidney pedicle for 60 min, followed by reperfusion for 8 (IR8) and 15 (IR15) days. Renal function was assessed through biochemical assays, while gene expression levels were evaluated by RT-qPCR. KEY FINDINGS: Analyses of renal parameters showed renal remodeling through mass loss in the left kidney and hypertrophy of the right kidney in the IR15 group. Furthermore, after 15 days, local inflammation was evidenced in the aorta. Moreover, the aorta purinergic components were significantly impacted by the renal ischemia and reperfusion model, with increases in gene expression of the pro-inflammatory purinoceptors P2Y1, P2Y2, P2Y6, and P2X4, potentially contributing to the vessel inflammation. The expression of NTPDase2 and ecto-5'-nucleotidase were also significantly increased in the aorta of the same group. In addition, both ATP and AMP hydrolysis were significantly increased in the aorta from IR15 animals, driving the entire purinergic cascade to the production of the anti-inflammatory adenosine. SIGNIFICANCE: In short, this is the first time that inflammation of the aorta due to AKI was shown to have an impact on purinergic signaling components, with emphasis on the adenosinergic pathway. This seems to be closely implicated in the establishment of vascular inflammation in this model of AKI and deserves to be further investigated.

Cytosolic 5'-Nucleotidase III and Nucleoside Triphosphate Diphosphohydrolase 1 Dephosphorylate the Pharmacologically Active Metabolites of Gemcitabine and Emtricitabine.

Gemcitabine (dFdC) and emtricitabine (FTC) are first-line drugs that are used for the treatment of pancreatic cancer and human immunodeficiency virus, respectively. The above drugs must undergo sequential phosphorylation to become pharmacologically active. Interindividual variability associated with the responses of the above drugs has been reported. The molecular mechanisms underlying the observed variability are yet to be elucidated. Although this could be multifactorial, nucleotidases may be involved in the dephosphorylation of drug metabolites due to their structural similarity to endogenous nucleosides. With these in mind, we performed in vitro assays using recombinant nucleotidases to assess their enzymatic activities toward the metabolites of dFdC and FTC. From the above in vitro experiments, we noticed the dephosphorylation of dFdC-monophosphate in the presence of two 5'-nucleotidases (5'-NTs), cytosolic 5'-nucleotidase IA (NT5C1A) and cytosolic 5'-nucleotidase III (NT5C3), individually. Interestingly, FTC monophosphate was dephosphorylated only in the presence of NT5C3 enzyme. Additionally, nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) exhibited enzymatic activity toward both triphosphate metabolites of dFdC and FTC. Enzyme kinetic analysis further revealed Michaelis-Menten kinetics for both NT5C3-mediated dephosphorylation of monophosphate metabolites, as well as NTPDase 1-mediated dephosphorylation of triphosphate metabolites. Immunoblotting results confirmed the presence of NT5C3 and NTPDase 1 in both pancreatic and colorectal tissue that are target sites for dFdC and FTC treatment, respectively. Furthermore, sex-specific expression patterns of NT5C3 and NTPDase 1 were determined using mass spectrometry-based proteomics approach. Based on the above results, NT5C3 and NTPDase 1 may function in the control of the levels of dFdC and FTC metabolites. SIGNIFICANCE STATEMENT: Emtricitabine and gemcitabine are commonly used drugs for the treatment of human immunodeficiency virus and pancreatic cancer. To become pharmacologically active, both the above drugs must be phosphorylated. The variability in the responses of the above drugs can lead to poor clinical outcomes. Although the sources of drug metabolite concentration variability are multifactorial, it is vital to understand the role of nucleotidases in the tissue disposition of the above drug metabolites due to their structural similarities to endogenous nucleosides.

Schizophrenia risk proteins ZNF804A and NT5C2 interact in cortical neurons.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.

Intercellular crosstalk shapes purinergic metabolism and signaling in cancer cells.

CD73-derived adenosine suppresses anti-cancer immunity, and CD73 inhibitors are currently evaluated in several clinical trials. Here, we have assessed enzyme kinetics of all key purinergic ectoenzymes in five cancer cell lines (Hodgkin lymphoma, multiple myeloma, pancreas adenocarcinoma, urinary bladder carcinoma, and glioblastoma) under normoxia and hypoxia. We found that adenosine metabolism varied considerably between individual cancer types. All cell lines investigated exhibited high ecto-adenosine deaminase (ADA) activity, which critically influenced the kinetics of adenosine accumulation. Combining kinetics data with single-cell RNA sequencing data on myeloma and glioblastoma cancerous tissue revealed that purine metabolism is not homogeneously organized, but it differs in a cancer type-specific fashion between malignant cells, stromal cells, and immune cells. Since purine metabolism in cancerous tissue is most likely spatially heterogeneous and differs between the various cell types, diffusion distances in the microenvironment as well as ADA activity may be important variables that influence the level of bioactive adenosine.

Ecto-5'-nucleotidase (CD73): an emerging role as prognostic factor in allergic sensitization.

Over the years, the importance of the epithelium in the assessment of allergic sensitization and development of allergic diseases has increased. Sensitization to allergens appears to be influenced by genetic and external environmental factors. However, not all subjects exposed to environmental factors that damage epithelial cells suffer from allergic diseases. On this basis, identifying the signaling pathways that characterize the different phenotypes and endotypes of allergy is of high priority for a successful personalized therapy. Ecto-5'-nucleotidase/CD73 is a membrane-bound enzyme responsible for extracellular adenosine accumulation from AMP derived, in turn, from the hydrolysis of extracellular ATP. Current knowledge suggests that CD73 expression and enzymatic activity at epithelial barriers would be of fundamental importance to control the first defense against allergens, by preserving both physical and immunological epithelial barrier functions. Here, we highlight evidence for a crucial role of CD73 in features of allergic sensitization and the potential of this enzyme as prognostic marker and target of therapeutic intervention.

Expression of ADK-S and ADK-L Isoforms and Their Association with CD39/CD73/A2aR in Colorectal Cancer.

The level of mRNA of the long (L) and short (S) isoforms of adenosine kinase (ADK) was analyzed in patients with colorectal cancer (CRC). ADK is required to convert adenosine (ADO) to AMP. It was shown that tumor and normal colon tissues (n=13) do not differ in the level of ADK-S and ADK-L mRNA. At the same time, the level of ADK-S mRNA (tumor: p=0.0214, normal: p=0.005) in the colon tissue was lower than in the blood of CRC patients (n=20), and the level of ADK-L mRNA (tumor: p=0.007, normal: p=0.024), on the contrary, was higher. A negative correlation was found between the level of ADK-S mRNA and the level of A2aR mRNA in both tumor and normal tissues (p=0.018 and p=0.0014, respectively). In the tumor tissue, a positive correlation was shown between ADK-L and CD73 mRNA levels (p=0.017). The obtained data indicate the association ADK with the expression of CD39/CD73/A2aR in CRC patients. In this regard, the effect of recombinant ADK on the expression of CD39 and CD73 ectonucleotidase by T cells in vitro was analyzed. In a culture of peripheral blood mononuclear cells isolated from the blood of 5 healthy donors, ADK did not abolish the inhibitory effect on the expression of CD39 and CD73 by CD8+T cells in the presence of a high concentration of ATP (a source for ADO). Effects on CD39+CD4+, CD73+CD4+T cells and CD39+ Treg cells were also not found.