CD73 Inhibitor Research Articles

Combining High-Z Sensitized Radiotherapy with CD73 Blockade to Boost Tumor Immunotherapy.

Radiation therapy (RT) has the capacity to induce immunogenic death in tumor cells, thereby potentially inducing in situ vaccination (ISV) to prime systemic antitumor immune responses. However, RT alone is often faced with various limitations during ISV induction, such as insufficient X-ray deposition and an immunosuppressive microenvironment. To overcome these limitations, we constructed nanoscale coordination particles AmGd-NPs by self-assembling high-Z metal gadolinium (Gd) and small molecular CD73 inhibitor AmPCP. Then, AmGd-NPs could synergize with RT to enhance immunogenic cell death, improve phagocytosis, and promote antigen presentation. Additionally, AmGd-NPs could also gradually release AmPCP to inhibit CD73's enzymatic activity and prevent the conversion of extracellular ATP to adenosine (Ado), thereby driving a proinflammatory tumor microenvironment that promotes DC maturation. As a result, AmGd-NPs sensitized RT induced potent in situ vaccination and boosted CD8+ T cell-dependent antitumor immune responses against both primary and metastatic tumors, which could also be potentiated by immune checkpoint inhibitory therapy.

Transcriptional control of pancreatic cancer immunosuppression by metabolic enzyme CD73 in a tumor-autonomous and -autocrine manner

Cancer cell metabolism contributes to the establishment of an immunosuppressive tumor microenvironment. Aberrant expression of CD73, a critical enzyme in ATP metabolism, on the cell surface results in the extracellular accumulation of adenosine, which exhibits direct inhibitory effects on tumor-infiltrating lymphocytes. However, little is known about the influence of CD73 on negative immune regulation-associated signaling molecules and transduction pathways inside tumor cells. This study aims to demonstrate the moonlighting functions of CD73 in immunosuppression in pancreatic cancer, an ideal model characterized by complex crosstalk among cancer metabolism, immune microenvironment, and immunotherapeutic resistance. The synergistic effect of CD73-specific drugs in combination with immune checkpoint blockade is observed in multiple pancreatic cancer models. Cytometry by time-of-flight analysis shows that CD73 inhibition reduces tumor-infiltrating Tregs in pancreatic cancer. Tumor cell-autonomous CD73 is found to facilitate Treg recruitment, in which CCL5 is identified as a significant downstream effector of CD73 using integrated proteomic and transcriptomic analyses. CD73 transcriptionally upregulates CCL5 through tumor cell-autocrine adenosine–Adora2a signaling-mediated activation of the p38–STAT1 axis, recruiting Tregs to pancreatic tumors and causing an immunosuppressive microenvironment. Together, this study highlights that CD73–adenosine metabolism transcriptionally controls pancreatic cancer immunosuppression in a tumor-autonomous and -autocrine manner.

Human soluble CD39 displays substrate inhibition in a substrate-specific manner

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.

The CD73 immune checkpoint promotes tumor cell metabolic fitness.

CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remain largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism, and provides the rationale for previously unforeseen combination therapies incorporating CD73 inhibition.

Targeting CD73 to Overcomes Resistance to First-Generation EGFR Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

In patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC), EGFR tyrosine kinase inhibitors (TKIs) improve response rate and survival. However, most patients eventually develop resistance. This study aimed to identify the role of CD73 in EGFR-mutant NSCLC and explore whether CD73 inhibition may serve as a therapeutic strategy in NSCLC patients with acquired resistance to EGFR-TKIs. We evaluated the prognostic role of CD73 expression in EGFR-mutant NSCLC using tumor samples from a single institution. We silenced CD73 in EGFR-TKI-resistant cell lines using short hairpin RNA (shRNA) targeting CD73 and also transfected a vector alone as a negative control. Using these cell lines, cell proliferation and viability assays, immunoblot assays, cell cycle analysis, colony-forming assays, flow cytometry, and apoptosis analysis were performed. High expression of CD73 was associated with shorter survival in patients with metastatic EGFR-mutant NSCLC treated with first-generation EGFR-TKI. CD73 inhibition synergistically inhibited cell viability with first-generation EGFR-TKI treatment compared with the negative control. When CD73 inhibition and EGFR-TKI treatment were combined, G0/G1 cell cycle arrest was induced through the regulation of p21 and cyclin D1. In addition, the apoptosis rate was increased in CD73 shRNA-transfected cells treated with EGFR-TKI. High expression of CD73 adversely affects the survival of patients with EGFR-mutant NSCLC. The study demonstrated that inhibiting CD73 in EGFR-TKI-resistant cell lines resulted in increased apoptosis and cell cycle arrest, which overcame the acquired resistance to first-generation EGFR-TKIs. Further research is needed to determine whether blocking CD73 plays a therapeutic role in EGFR-TKI-resistant patients with EGFR-mutant NSCLC.

Human gingival tissue‐derived mesenchymal stem cells inhibit proliferation and invasion of rheumatoid fibroblast‐like synoviocytes via the CD39/CD73 signaling pathway

AbstractBackgroundGingiva‐derived mesenchymal stem cells (GMSCs) suppress immune and inflammatory responses in experimental arthritis models. Here, we determined whether GMSCs suppress the proliferation and invasion of rheumatoid arthritis fibroblast‐like synoviocytes (RA FLSs).MethodsSurface markers of GMSCs were analyzed by flow cytometry. mRNA expression of matrix metalloproteinases and pro‐inflammatory cytokine in RA FLSs was measured by quantitative polymerase chain reaction (PCR). RA FLS proliferation was analyzed by a 5‐ethynyl‐2′‐deoxyuridine assay. To explore the molecular mechanisms, we assessed the effect of GMSCs on RA FLS proliferation by adding indoleamine‐2,3‐dioxygenase (IDO), CD39, or CD73 inhibitors. The invasion was analyzed by a transwell assay. The anti‐inflammatory effects of GMSCs were assessed in a type II collagen‐induced arthritis (CIA) mouse model.ResultsCompared with human dermal fibroblast, GMSCs displayed a higher expression of CD39. Interleukin (IL)‐8, IL‐6, MMP‐1, MMP‐3, MMP‐13, and monocyte chemoattractant protein‐1 mRNA were all decreased in RA FLSs after incubation with GMSCs. GMSCs significantly reduced RA FLS proliferation in a dose‐dependent manner in vitro, which was partly dependent on CD39/CD73 signaling. GMSCs significantly impeded the invasive capacity of RA FLSs in a dose‐dependent manner in vitro. GMSCs infusion delayed arthritis onset and reduced arthritis scores in the CIA model.ConclusionGMSCs are effective to inhibit the aggressive behavior of RA FLSs and treat experimental arthritis, implying their therapeutic potential in RA patients.

Purine metabolic pathway regulates regulatory T cell stability and function

Abstract Regulatory T cell (Treg) lymphatic migration and maintenance of transcription factor Foxp3 expression are required for suppressor function and allograft protection, and migrated Tregs express ectonucleotidase CD39 hi. Purine metabolism is implicated in Treg phenotype but the precise functions of purine metabolites on Treg function and stability have remained unclear. Sorted Foxp3+ Tregs were used to test Treg stability, migration, and cellular viability in a transendothelial migration (TEM) in vitro model. Intracellular metabolome of Tregs was analyzed by mass spectrometry, and differentially expressed metabolites between Tregs and exTregs were identified and tested. Foxp3 hiTregs displayed higher suppressive function and migration across LECs compared to Foxp3 loexTregs, but cellular viability was similar. Tregs showed differential metabolic profiles compared to exTregs, and the top 16 most differentially expressed metabolites involved the tricarboxylic acid cycle, polyamines, and purine metabolism. Ten metabolites including nicotinamide and inosine monophosphate (IMP) were downregulated, while six metabolites including putrescine, cadaverine, and N-acetylglycine were upregulated in Tregs compared to exTregs. A purine ectonucleotidase CD73 inhibitor decreased Treg conversion to exTregs and adenosine increased Treg migration and suppressive function concomitant with decreased exTreg conversion. Polyamine metabolites showed only subtle effects on exTreg conversion and migration. These results suggest that purine metabolism is a potent regulator that maintains Treg Foxp3 expression and suppressive function during Treg TEM and thus, could be a promising target for therapeutic interventions. NIH grant RO1 A1062765

Development of a Concise Process for the Synthesis of the Azaindazole Core of the CD73 Inhibitor AB680

AB680 is a highly potent small-molecule CD73 inhibitor discovered and developed by Arcus Biosciences, currently in clinical trials for the treatment of pancreatic cancer. Herein, we report a concise synthesis of 4,6-dichloro-1H-pyrazolo[3,4-b]pyridine 3, which is the central azaindazole core of AB680. The process consists of four synthetic operations and three isolations, including a PMB-protected pyrazole formation, a telescoped two-step cyclization and aromatization sequence, and finally a one-pot PMB deprotection and chlorination to furnish 3. This chemistry was successfully scaled up to provide >200 g of 3 in 44% overall yield and 97.6% HPLC purity. Overall, the route developed toward 3 represents an efficient construction of an azaindazole core, which is a synthetically challenging yet prevalent structural motif.

Abstract LB316: A next-generation KRASG12C inhibitor ABSK071 demonstrated broad synergy with other therapeutic agents in KRASG12C mutated cancer models

Abstract Background: KRAS is frequently mutated in human cancers, including pancreatic (~90%), colorectal (~35%), and lung cancer (~25%). The KRASG12C mutation (single amino acid substitution of cysteine for glycine at position 12) accounts for ~14% of lung cancer, ~4% of colorectal cancer, and ~2% of pancreatic cancer. Currently, two covalent KRASG12C inhibitors, namely sotorasib (AMG-510) and adagrasib (MRTX-849), have been approved as monotherapy to treat locally advanced or metastatic NSCLC with KRASG12C mutation through accelerated approval process. Despite the beneficial effects of KRASG12C inhibitors in clinic for certain patients, the limited antitumor efficacy in most patients and potential drug resistance are major concerns. A next-generation inhibitor with better inhibitory activity may improve anti-tumor efficacy. Combination with other therapeutic agents may also improve the single-agent activity of KRASG12C inhibitors. These approaches could both overcome the limitations of sotorasib and adagrasib and provide additional benefits to patients. Materials and methods: KRASG12C inhibitory activity of ABSK071 was examined in vitro and in vivo in comparison with sotorasib and adagrasib. To identify potential targeted agents synergizing with ABSK071, we tested a set of inhibitors that targeting signaling components of KRAS pathways, including EGFRi, FGFRi, SHP2i and SOS1i, in combination with ABSK071 in cellular experiments using KRASG12C mutant cell lines. Cell growth inhibition was measured and synergistic effect was analyzed. In vivo efficacy study was also conducted to confirm the synergistic anti-tumor effect in animal models. A set of immuno-oncology reagents, including anti-PD-1/L1, CSF-1R inhibitor (ABSK021) and CD73 inhibitor (ABSK051) were also tested in combination with ABSK071 in vivo using mouse syngeneic models. Results: ABSK071 demonstrated much stronger inhibitory activity than sotorasib and adagrasib against a variety of cell lines harboring KRASG12C, as well as significantly better in vivo anti-tumor efficacy in xenograft models that were less sensitive to sotorasib. Synergistic effects on cell growth inhibition were observed in vitro with ABSK071 in combination with several agents including cetuximab, afatinib, AZD4547, TNO-155, RMC-4630 and BI3406. Superior anti-tumor activities were observed in vivo when these agents were combined with ABSK071 than in combination with sotorasib. In mouse syngeneic models harboring KrasG12C mutations, ABSK071 also demonstrated synergy with anti PD-1/L1 antibody or other IO reagents. Conclusions: ABSK071 is a next-generation KRASG12C inhibitor with greater activity and anti-tumor efficacy in vitro and in vivo. It also demonstrated broad synergistic effects with a large set of targeted agents and immuno-oncology agents, indicating its strong potential in combinatory therapy in treating a wider range of KRASG12C-dependent cancers. Citation Format: Haiyan Ying, Fushen Guo, Juan Peng, Jie Wang, Jie Zhang, Manqi Liu, Zhixuan Zhu, Zhui Chen. A next-generation KRASG12C inhibitor ABSK071 demonstrated broad synergy with other therapeutic agents in KRASG12C mutated cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB316.

Abstract 1101: Combination therapy to overcome radiotherapy induced immunosuppression and improve tumor control

Abstract Background: The relationship between radiation therapy, immunosuppressive myeloid populations and adenosine is not well characterized. Here we test the hypothesis that radiation-induced adenosine signaling promotes tumor growth and causes suppression of T cell function and that therapeutic targeting may improve response to radiotherapy. Methods: Orthotopic 4T1 cells were implanted in Balb/c mice and tumors were irradiated using small animal radiation research platform (SARRP, Xstrahl; 220 kV) with 3 fractions of 8 Gy and/or treated with adenosine receptor 2A and 2B (A2AR/A2BRi) inhibitor, CD73 inhibitor (CD73i), and/or checkpoint inhibitor, aPD-1. Tumor response was assessed by tumor volume and the immune response characterized by flow cytometry. Results: Intratumoral immune response was evaluated 72 hours post-irradiation. Radiotherapy induced an expansion of myeloid cells particularly monocytic cells (13.5% vs 7% of CD45+ cells) with a corresponding contraction of lymphocytes. Monocytes from irradiated tumors had increased expression of most of the key members of the adenosine signaling pathway including enpp1 (2.5% vs 0.6%), CD73 (10.3% vs 4.8%), CD39 (10.7% vs 4.4%), A2aR (2.8% vs 0.6%) and A2bR (2.4% vs 0.3%), compared to untreated controls. In the absence of tumor irradiation, the combination of A2AR/A2BRi & CD73i with αPD-1 showed no improvement in tumor control compared to A2AR/A2BRi & CD73i alone or untreated controls. However, in the setting of tumor irradiation, we measured a significant reduction in 4T1 tumor volume when aPD-1 was added to A2AR/A2BRi & CD73i, compared to A2AR/A2BRi & CD73i alone (156mm^3 vs 250mm^3). The addition of αPD-1 to A2AR/A2BRi & CD73i and tumor irradiation resulted in decrease in monocytic cells (9.4% vs 15% of CD45+ cells) and expression of CD39 (7.4% vs 11%), CD73 (7.5% vs 11%), A2aR (2.2% vs 7%) and A2bR (2.7% vs 9%) in monocytic compartment, compared to radiation and A2AR/A2BRi & CD73i. Further, there was a corresponding expansion of Ki67+ proliferating (52% vs 36%) and IFN-γ producing CD8+ T cells (3.3% vs 2%) after treatment with radiation, A2AR/A2BRi & CD73i, and αPD-1, suggesting improved T cell effector function. Conclusion: Radiation-induced adenosine signaling facilitates immunosuppressive changes in the TME. In the immunotherapy insensitive 4T1 mouse model, adenosine blockade induces checkpoint inhibition sensitivity through alleviation of immunosuppression, thus improving tumor control. Citation Format: Shruti Bansal, Matthew Chaimowitz, Julia Ann, Patrick McCann, Jason Piersaint, Claudia Aiello, Namita Sen, Julia Kogan, Catherine Spina. Combination therapy to overcome radiotherapy induced immunosuppression and improve tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1101.

Abstract 5258: Preclinical testing of CD73 inhibitor AB680 for pancreatic cancer immunoprevention

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a profoundly immunosuppressive microenvironment. Innovative therapeutic strategies are urgently needed to stop the progression of precancerous lesions into aggressive PDAC, which remains a lethal malignancy. The goal of this research project is to test immunopreventive strategies by targeting the 5’ ectonucleotidase enzyme, CD73, one of the gatekeeper enzymes responsible for conversion of adenosine monophosphate (AMP), to an immunosuppressive metabolite, adenosine, in the tumor microenvironment (TME). We hypothesize that inhibition of CD73 will prevent pancreatic intraepithelial neoplasia (PanIN) formation and progression to PDAC by reversing adenosine directed immunosuppression. This research explores immunopreventive strategies aimed to restore tumor immune surveillance to prevent cancer initiation or progression. Materials and methods: We used two models: a syngeneic model of PDAC using cells derived from KrasG12D;Trp53R172H/+;PdxCre (KPC) mice and a KrasG12D;PdxCre (KC) genetically engineered mouse model (GEM) of PDAC. Oral gavage of AB680 (small molecule CD73 inhibitor) was given three days/week at 10mg/kg starting the day after KPC injections and tumor sizes were measured weekly. In the GEM model, the same treatment regimen began when the mice were between 6 and 9 weeks old and were euthanized either between 15 and 20 weeks of age or around 27 weeks of age, and pancreas tissue was harvested. Histology was analyzed and 6 fields per mouse were quantified using ImageJ. Results: As we have described (Singh, et al, bioRxiv), in the syngeneic model, there was a significant reduction in tumor growth and significant increase in activated CD8-positive T cells, dendritic cells, and macrophages from AB680 treated mice. The intratumoral adenosine levels were significantly decreased in AB680 treated mice compared to vehicle treated mice. In the KC GEM model, we quantified significantly fewer early PanIN lesions (p=0.0328), a trend in decreased advanced PanIN (p=0.0641), and significant decrease in PDAC (p=0.0058) in the AB680 treated mice when compared to the vehicle treated mice. We quantified abundance of collagen deposition as a marker of fibrosis and observed significantly decreased collagen (p<0.0001) in AB680 treated KC mice. In addition, we quantified abundance of CK19+ lesions and observed a significant decrease in CK19+ lesions in AB680 treated mice (p=0.0061) compared to vehicle treated mice. Conclusion: Inhibiting CD73 restructures TME and reduces PanIN incidence and progression to PDAC. CD73 inhibition may be a candidate immunoprevention strategy in pancreatic cancer. [Supported by NCI 75N91019D00021/75N91020F00002] Citation Format: Lincoln N. Strickland, Erika Y. Faraoni, Nicolette R. Mardik, Lana Vornik, Michelle I. Savage, Shizuko Sei, Mark S. Miller, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitor AB680 for pancreatic cancer immunoprevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5258.

Abstract 504: BPI-472372: a potent and orally bioavailable small molecule inhibitor of CD73 for cancer immunotherapy

Abstract CD73, also known as ecto-5'-nucleotidase, plays a major role in converting extracellular adenosine monophosphate (AMP) to immunosuppressive adenosine. It is frequently overexpressed across a variety of cancers. High adenosine level in the tumor microenvironment, predominantly signaling through the A2a/A2b receptors, suppresses innate and adaptive immune responses and mediates tumor immune evasion. CD73 overexpression is correlated with poor prognosis in several tumor types. Inhibition of CD73 to reduce adenosine production is a promising therapeutic approach for the treatment of cancer. Both monoclonal antibodies and small molecule inhibitors are being explored to target CD73. Small molecule CD73 inhibitor with good balance of activity and ADME properties are rare. Here we present BPI-472372, a potent, selective, and orally bioavailable small molecule CD73 inhibitor. It strongly inhibits both soluble and cell-bound CD73, with no off-target activity on other related nucleotidases (NTPDases 1/2/3/5), nor on a large panel of enzymes, receptors, and ion channels. BPI-472372 reversed AMP-induced CD4+ T cell suppression in a dose-dependent manner. In vivo, BPI-472372 exhibited anti-tumor effect in MC38 syngeneic mouse model with no visible adverse effect. Moreover, enhanced tumor growth inhibition activity was observed when BPI-472372 was combined with durvalumab (anti-PD-L1) in humanized PD-L1 syngeneic MC38 model. BPI-472372 exhibited desirable drug-like properties including high permeability, low hepatocytes clearance, and adequate oral exposure across multiple pre-clinical species. GLP toxicity study indicated that BPI-472372 was well tolerated at high dose following 4 weeks repeated administration in both mouse and dog. Taken together, BPI-472372 is a potent and orally bioavailable small molecule CD73 inhibitor with single agent anti-tumor efficacy, favorable ADME properties, and a wide therapeutic index. Citation Format: Han Han, Xiaofeng Yang, Ying Zhao, Jin Wang, Xiang Yang, Xuepeng Ju, Jiayu Zhao, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Jing Guo, Cheng Yang, Hong Lan, Hao Wu, Lieming Ding, Jiabing Wang. BPI-472372: a potent and orally bioavailable small molecule inhibitor of CD73 for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 504.

Feedback facilitation by adenosine A2A receptors of ATP release from mouse hippocampal nerve terminals

The adenosine modulation system is mostly composed by inhibitory A1 receptors (A1R) and the less abundant facilitatory A2A receptors (A2AR), the latter selectively engaged at high frequency stimulation associated with synaptic plasticity processes in the hippocampus. A2AR are activated by adenosine originated from extracellular ATP through ecto-5’-nucleotidase or CD73-mediated catabolism. Using hippocampal synaptosomes, we now investigated how adenosine receptors modulate the synaptic release of ATP. The A2AR agonist CGS21680 (10-100 nM) enhanced the K+-evoked release of ATP, whereas both SCH58261 and the CD73 inhibitor α,β-methylene ADP (100 μM) decreased ATP release; all these effects were abolished in forebrain A2AR knockout mice. The A1R agonist CPA (10-100 nM) inhibited ATP release, whereas the A1R antagonist DPCPX (100 nM) was devoid of effects. The presence of SCH58261 potentiated CPA-mediated ATP release and uncovered a facilitatory effect of DPCPX. Overall, these findings indicate that ATP release is predominantly controlled by A2AR, which are involved in an apparent feedback loop of A2AR-mediated increased ATP release together with dampening of A1R-mediated inhibition. This study is a tribute to María Teresa Miras-Portugal.

Radiofrequency Irradiation Attenuated UVB-Induced Skin Pigmentation by Modulating ATP Release and CD39 Expression.

Hyperpigmentation stimulated by ultraviolet (UV)-induced melanin overproduction causes various cosmetic problems. UV radiation's activation of the cyclic adenosine monophosphate (cAMP)-mediated cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway is the main pathway for melanogenesis. However, the secretion of adenosine triphosphate (ATP) from keratinocytes due to UV radiation also leads to melanogenesis. Adenosine, converted from ATP by CD39 and CD73, can activate adenylate cyclase (AC) activity and increase intracellular cAMP expression. cAMP-mediated PKA activation results in dynamic mitochondrial changes that affect melanogenesis via ERK. We evaluated whether radiofrequency (RF) irradiation could decrease ATP release from keratinocytes and suppress the expression of CD39, CD73, and A2A/A2B adenosine receptors (ARs) and the activity of AC and downregulate the PKA/CREB/MITF pathway, which would eventually decrease melanogenesis in vitro in UV-irradiated cells and animal skin. Our results indicate that RF decreased ATP release from UVB-irradiated keratinocytes. When conditioned media (CM) from UVB-irradiated keratinocytes (CM-UVB) were administered to melanocytes, the expressions of CD39, CD73, A2A/A2BARs, cAMP, and PKA increased. However, the expression of these factors decreased when CM from UVB and RF-irradiated keratinocytes (CM-UVB/RF) was administered to melanocytes. The phosphorylation of DRP1 at Ser637, which inhibits mitochondrial fission, increased in UVB-irradiated animal skin and was decreased by RF irradiation. The expression of ERK1/2, which can degrade MITF, was increased using RF treatment in UVB-irradiated animal skin. Tyrosinase activity and melanin levels in melanocytes increased following CM-UVB administration, and these increases were reversed after CD39 silencing. Tyrosinase activity and melanin levels in melanocytes were decreased by CM-UVB/RF irradiation. In conclusion, RF irradiation decreased ATP release from keratinocytes and the expressions of CD39, CD73, and A2A/A2BARs, which decreased AC activity in melanocytes. RF irradiation downregulated the cAMP-mediated PKA/CREB/MITF pathway and tyrosinase activity, and these inhibitory effects can be mediated via CD39 inhibition.

Synthesis and structure–activity relationships of ticlopidine derivatives and analogs as inhibitors of ectonucleotidase CD39

Ticlopidine is an antithrombotic prodrug of the thienotetrahydropyridine family. For platelet inhibition it has to undergo oxidative ring-opening by cytochrome P450 enzymes. The resulting thiol reacts with a cysteine residue of the purinergic P2Y12 receptor on thrombocytes resulting in covalent receptor blockade. Ticlopidine in its intact, not-metabolized form was previously shown to inhibit ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, also known as cluster of differentiation (CD) 39). CD39 catalyzes the extracellular hydrolysis of ATP via ADP to AMP, which is further hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine. CD39 inhibition has been proposed as a novel strategy to increase the extracellular concentration of antiproliferative ATP, while decreasing immunosuppressive and cancer-promoting adenosine levels. In the present study, we performed an extensive structure–activity relationship (SAR) analysis of ticlopidine derivatives and analogs as CD39 inhibitors followed by an in-depth characterization of selected compounds. Altogether 74 compounds were synthesized, 41 of which are new, not previously described in literature. Benzotetrahydropyridines, in which the metabolically labile thiophene is replaced by a benzene ring, were discovered as a new class of allosteric CD39 inhibitors.

Increased Synaptic ATP Release and CD73-Mediated Formation of Extracellular Adenosine in the Control of Behavioral and Electrophysiological Modifications Caused by Chronic Stress.

Increased ATP release and its extracellular catabolism through CD73 (ecto-5'-nucleotidase) lead to the overactivation of adenosine A2A receptors (A2AR), which occurs in different brain disorders. A2AR blockade blunts mood and memory dysfunction caused by repeated stress, but it is unknown if increased ATP release coupled to CD73-mediated formation of extracellular adenosine is responsible for A2AR overactivation upon repeated stress. This was now investigated in adult rats subject to repeated stress for 14 consecutive days. Frontocortical and hippocampal synaptosomes from stressed rats displayed an increased release of ATP upon depolarization, coupled to an increased density of vesicular nucleotide transporters and of CD73. The continuous intracerebroventricular delivery of the CD73 inhibitor α,β-methylene ADP (AOPCP, 100 μM) during restraint stress attenuated mood and memory dysfunction. Slice electrophysiological recordings showed that restraint stress decreased long-term potentiation both in prefrontocortical layer II/III-layer V synapses and in hippocampal Schaffer fibers-CA1 pyramid synapses, which was prevented by AOPCP, an effect occluded by adenosine deaminase and by the A2AR antagonist SCH58261. These results indicate that increased synaptic ATP release coupled to CD73-mediated formation of extracellular adenosine contributes to mood and memory dysfunction triggered by repeated restraint stress. This prompts considering interventions decreasing ATP release and CD73 activity as novel strategies to mitigate the burden of repeated stress.

ATP and its metabolite adenosine cooperatively upregulate the antigen-presenting molecules on dendritic cells leading to IFN-γ production by T cells

Dendritic cells (DCs) present foreign antigens to T cells via the major histocompatibility complex (MHC), thereby inducing acquired immune responses. ATP accumulates at sites of inflammation or in tumor tissues, which triggers local inflammatory responses. However, it remains to be clarified how ATP modulates the functions of DCs. In this study, we investigated the effects of extracellular ATP on mouse bone marrow-derived dendritic cells (BMDCs) as well as the potential for subsequent T cell activation. We found that high concentrations of ATP (1mM) upregulated the cell surface expression levels of MHC-I, MHC-II, and co-stimulatory molecules CD80 and CD86 but not those of co-inhibitory molecules PD-L1 and PD-L2 in BMDCs. Increased surface expression of MHC-I, MHC-II, CD80, and CD86 was inhibited by a pan-P2 receptor antagonist. In addition, the upregulation of MHC-I and MHC-II expression was inhibited by an adenosine P1 receptor antagonist and by inhibitors of CD39 and CD73, which metabolize ATP to adenosine. These results suggest that adenosine is required for the ATP-induced upregulation of MHC-I and MHC-II. In the mixed leukocyte reaction assay, ATP-stimulated BMDCs activated CD4 and CD8T cells and induced interferon-γ (IFN-γ) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigen-presenting and co-stimulatory molecules but not that of co-inhibitory molecules in BMDCs. Cooperative stimulation of ATP and its metabolite adenosine was required for the upregulation of MHC-I and MHC-II. These ATP-stimulated BMDCs induced the activation of IFN-γ-producing T cells upon antigen presentation.

Abstract PD2-07: Tumor CD73 regulates zoledronate induced TIL accumulation into triple negative breast cancer

Abstract Background: CD73 is a membrane-bound protein that by extracellular adenosine production, regulates immune cell function. Low CD73 expression is associated with better prognosis among triple-negative breast cancer (TNBC) patients. Adjuvant bisphosphonates (BPs, such as zoledronate) improve survival among post-menopausal breast cancer patients, but the mechanism for this is unknown. Since BPs also affect immune cells, we studied 1) whether zoledronate-treatment has an effect on tumor infiltrating lymphocytes (TILs) and 2) whether tumor CD73 expression affects these responses in TNBC tumors. Methods: 4T1 mouse mammary cells, stably expressing non-targeting shRNA (sh-NT, control cells) or sh-CD73 were inoculated into the mammary fat pads of Balb/c mice. The mice were treated with i.p. vehicle or zoledronate (ZOL) each 4th day for total of six times. Additionally, to deplete B-cells, the mice were treated with an anti-CD20 or control IgG antibody. Tumor sizes were assessed with a caliper, TILs were studied with immunostainings. Blood lymphocytes were analyzed with Flow cytometry. Results: Sh-CD73 cells formed significantly smaller tumors, than sh-NT cells. ZOL significantly inhibited the growth of both sh-CD73 and sh-NT tumors, but at sacrifice, this effect of ZOL was more significant against sh-CD73 tumors. ZOL increased the % of CD19+/CD21+ and CD19+/23+ in blood of both mice groups, and this effect was inhibited by anti-CD20 antibody. Although neither treatment alone had an effect, the % of CD3+/CD8+ cells in blood were increased in both mice groups by the combination of ZOL and anti-CD20-antibody. ZOL induced accumulation of B220+, CD4+ and CD8+ TILs into both tumor groups. These TIL effects were reduced with anti-CD20 antibody treatment. Anti-CD20 antibody alone inhibited significantly sh-CD73 tumor growth. Anti-CD20 antibody did not interfere with ZOL inhibition of tumor growth in either group. Conclusions: ZOL has inflammatory effects on blood lymphocytes and TILs, which may be affected by tumor expression of immune system regulating proteins, such as CD73. B cell depletion may further prevent tumor growth, in the context of CD73 inhibition. Further studies are needed to investigate whether B-cell inhibition affects ZOL-induced tumor growth inhibition. Citation Format: Nataliia Petruk, Arafat Siddiqui, Sina Tadayon, Arja Jukkola, Jorma Määttä, Pieta Mattila, Jouko Sandholm, Katri S. Selander. Tumor CD73 regulates zoledronate induced TIL accumulation into triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-07.

Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

Recently, a link between the biological activity of CD73 in solid tumors and multidrug resistance protein (MRP) has been proposed. Cisplatin (CP) is the most widely used anticancer agent to treat advanced and recurrent cervical cancer (CC). However, multidrug resistance protein-1 (MRP1) is overexpressed in approximately 85% of these tumors and has been strongly associated with cisplatin resistance (CPR). In this study, we examine the involvement of CD73 and the interaction of adenosine (ADO) with its receptors (ARs) in MRP1 expression in CC cells. We found that ADO positively modulates MRP1 expression in CC cells in a dose-dependent manner. The inhibition of CD73 expression with a CD73-targeted siRNA and A2AR blockade with the selective antagonist ZM241385 significantly decreased MRP1 expression and the extrusive capacity of CC cells, making them significantly more sensitive to CP treatment than cancer cells treated with MK-751, a specific MRP1 inhibitor. These results suggest CD73 inhibition or blocking ADO signaling through A2AR could be strategies to reverse CPR in patients with advanced or recurrent CC, which is characterized by very low response rates to CP (10%-20%).

Estrogen downregulates CD73/adenosine axis hyperactivity via adaptive modulation PI3K/Akt signaling to prevent myocarditis and arrhythmias during chronic catecholamines stress

BackgroundDuring myocardial damage, the sex hormone estrogen and CD73, the main enzyme that converts AMP into adenosine, are cardioprotective molecules. However, it is unclear how these two molecules work together to provide cardioprotection. The current study aimed to elucidate the interaction between estrogen and CD73 under chronic stress.MethodsOvariectomy and SHAM operations were done on FVB wild-type (WT) female mice. Two weeks after the operation, the mice were treated with daily isoproterenol (10 mg/kg/day) injections for 14 days. The effect of E2 on relevant cardiac injury biomarkers (BNP, ANP), myocardial morphology (cardiomyocyte surface area), electrocardiography, CD73 protein expression and activity, and macrophage (CD86 + and CD206 +) infiltrations were assessed. In vitro, H9C2 cells were treated with 1 nM of estrogen and 10 mM APCP (CD73 inhibitor α, β-methylene adenosine-5'-diphosphate), 10 µM isoproterenol and 20 µm LY294002 (PI3K inhibitor) for 24 h and western blot was done to elucidate the mechanism behind the effect of estrogen on the CD73/adenosine axis.ResultsEstrogen deficiency during chronic catecholamine stress caused myocardial injury, thereby triggering the hyperactivity of the CD73/adenosine axis, which aggravated myocarditis, adverse remodeling, and arrhythmias. However, estrogen normalizes CD73/Adenosine axis via the upregulation of PI3K/Akt pathways to prevent adverse outcomes during stress. In vivo results showed that the inhibition of PI3K significantly decreased PI3K/Akt pathways while upregulating the CD73/adenosine axis and apoptosis.ConclusionEstrogen’s pleiotropy cardioprotection mechanism during stress includes its normalization of the CD73/Adenosine axis via the PI3K/Akt pathway.9z1eijmQFGaVTY6LcoepL6Video