Generation Of Adenosine Research Articles

Circulating Ectonucleotidases Signal Impaired Myocardial Perfusion at Rest and Stress.

Background Ectonucleotidases maintain vascular homeostasis by metabolizing extracellular nucleotides, modulating inflammation and thrombosis, and potentially, myocardial flow through adenosine generation. Evidence implicates dysfunction or deficiency of ectonucleotidases CD39 or CD73 in human disease; the utility of measuring levels of circulating ectonucleotidases as plasma biomarkers of coronary artery dysfunction or disease has not been previously reported. Methods and Results A total of 529 individuals undergoing clinically indicated positron emission tomography stress testing between 2015 and 2019 were enrolled in this single-center retrospective analysis. Baseline demographics, clinical data, nuclear stress test, and coronary artery calcium score variables were collected, as well as a blood sample. CD39 and CD73 levels were assessed as binary (detectable, undetectable) or continuous variables using ELISAs. Plasma CD39 was detectable in 24% of White and 8% of Black study participants (P=0.02). Of the clinical history variables examined, ectonucleotidase levels were most strongly associated with underlying liver disease and not other traditional coronary artery disease risk factors. Intriguingly, detection of circulating ectonucleotidase was inversely associated with stress myocardial blood flow (2.3±0.8 mL/min per g versus 2.7 mL/min per g±1.1 for detectable versus undetectable CD39 levels, P<0.001) and global myocardial flow reserve (Pearson correlation between myocardial flow reserve and log(CD73) -0.19, P<0.001). A subanalysis showed these differences held true independent of liver disease. Conclusions Vasodilatory adenosine is the expected product of local ectonucleotidase activity, yet these data support an inverse relationship between plasma ectonucleotidases, stress myocardial blood flow (CD39), and myocardial flow reserve (CD73). These findings support the conclusion that plasma levels of ectonucleotidases, which may be shed from the endothelial surface, contribute to reduced stress myocardial blood flow and myocardial flow reserve.

Human plasmacytoid dendritic cells express the functional purinergic halo (CD39/CD73).

Plasmacytoid dendritic cells (pDCs) are a specialized DC subset mainly associated with sensing viral pathogens and high-type I interferon (IFN-I) release in response to toll-like receptor (TLR)-7 and TLR-9 signaling. Currently, pDC contribution to inflammatory responses is extensively described; nevertheless, their regulatory mechanisms require further investigation. CD39 and CD73 are ectoenzymes driving a shift from an ATP-proinflammatory milieu to an anti-inflammatory environment by converting ATP to adenosine. Although the regulatory function of the purinergic halo CD39/CD73 has been reported in some immune cells like regulatory T cells and conventional DCs, its presence in pDCs has not been examined. In this study, we uncover for the first time the expression and functionality of the purinergic halo in human blood pDCs. In healthy donors, CD39 was expressed in the cell surface of 14.0 ± 12.5% pDCs under steady-state conditions, while CD73 showed an intracellular location and was only expressed in 8.0 ± 2.2% of pDCs. Nevertheless, pDCs stimulation with a TLR-7 agonist (R848) induced increased surface expression of both molecules (43.3 ± 23.7% and 18.6 ± 9.3%, respectively), as well as high IFN-α secretion. Furthermore, exogenous ATP addition to R848-activated pDCs significantly increased adenosine generation. This effect was attributable to the superior CD73 expression and activity because blocking CD73 reduced adenosine production and improved pDC allostimulatory capabilities on CD4 + T cells. The functional expression of the purinergic halo in human pDCs described in this work opens new areas to investigate its participation in the regulatory pDC mechanisms in health and disease.

Abstract 4432: A highly differentiated A2AR inhibitor for potential use in cancer therapy

Abstract As a potent immunosuppressor adenosine is essential for maintaining tissue homeostasis and preventing an overzealous immune response during inflammation and infection. However, adenosine generated within the tumor microenvironment by the action of ectonucleotides hinders the immune reaction towards cancer cells by signaling through adenosine receptors such as high affinity A2AR expressed on immune cells. Tumors evade the immune response by usurping pathways that negatively regulate normal immune responses. Resistance to inhibition of immune checkpoint targets arises because of an upregulation in the expression of the CD39-CD73 axis and the resulting increase in production of adenosine in the tumor microenvironment. Inhibition of either adenosine generation or signaling by inhibition of A2AR along with other immune activation strategies have been shown to be effective therapeutic approaches. In view of this, we sought to discover and develop novel small molecule inhibitors that dually target A2AR and an immuno-suppressive ligand expressed in the tumor microenvironment. Aurigene’s dual inhibitors exhibit potent inhibition of both A2AR and an immuno-suppressive target of relevance in respective biochemical assays. Potent biochemical activity translated into rescue of chemokines and IL-2 in human PBMCs. Lead compounds exhibited desirable drug-like properties and excellent pharmacokinetic exposure in rodents. In a syngeneic tumor model, lead compounds demonstrated significant tumor growth inhibition. Anti-tumor efficacy correlated well with tumor drug levels and modulation of pharmacodynamic markers. In summary, we have identified first-in-class dual inhibitors with good drug like properties, which showed significant antitumor efficacy. Evaluation of these lead compounds in additional tumor models and in vivo toxicity studies will be presented. Citation Format: Chandregowda Venkateshappa, Kishore Narayanan, Rashmi Nair, Aravind AB, Ramakishore VP Putta, Jwala Nagaraj, Megha Goyal, Vijay Kamal Ahuja, Amith A Dhudashiya, Samiulla DS, Girish Daginakatte, Thomas Antony, Kavitha Nellore, Susanta Samajdar, Murali Ramachandra. A highly differentiated A2AR inhibitor for potential use in cancer therapy. [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 4432.

Abstract 1158: Inosine endows CAR T cells with features of increased stemness and anti-tumor potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that link an extracellular tumor specific domain to intracellular signaling domains. Despite of remarkable results against refractory B cell malignancies, intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. Ectoenzyme CD39 plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. Then CD73 subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via the purigenic pathway. We developed a high affinity version of the disialoganglioside (GD2)-targeting chimeric antigen receptor (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR (adenosine A2a receptor) but observed only modest changes in phenotype and transcriptomics. In contrast, overexpression of transmembrane-bound adenosine deaminase, which metabolizes adenosine to inosine, induced higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity driven by glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinical GD2 CAR T cells in inosine-containing culture media enhances their in vivo efficacy, leading to improved survival in GD2+ tumor-bearing mice. In conclusion, we propose introducing inosine during GMP cell manufacturing as a novel strategy to improve CAR T function and subsequent clinical outcomes of CAR T cell therapy. Citation Format: Dorota Klysz, Katie Freitas, Meena Malipatlolla, Stefanie Maier, Carley Fowler, Louai Labanieh, Lucille Stuani, Bence Daniel, Katalin Sandor, Peng Xu, Jing Huang, Amaury Leruste, Vimal Keerthi, Nikolaos Gkitsas, Shabnum Patel, Justin Arredondo-Guerrero, Chris Fisher, Kyle Asano, Sunny Patel, Ansuman Satpathy, Kara Davis, Elena Sotillo, Steven Feldman, Crystal Mackall. Inosine endows CAR T cells with features of increased stemness and anti-tumor potency [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 1158.

Characterization of purinergic signaling in tumor-infiltrating lymphocytes from lower- and high-grade gliomas.

Malignant gliomas are highly heterogeneous glia-derived tumors that present an aggressive and invasive nature, with a dismal prognosis. The multi-dimensional interactions between glioma cells and other tumor microenvironment (TME) non-tumoral components constitute a challenge to finding successful treatment strategies. Several molecules, such as extracellular purines, participate in signaling events and support the immunosuppressive TME of glioma patients. The purinergic signaling and the ectoenzymes network involved in the metabolism of these extracellular nucleotides are still unexplored in the glioma TME, especially in lower-grade gliomas (LGG). Also, differences between IDH-mutant (IDH-Mut) versus wild-type (IDH-WT) gliomas are still unknown in this context. For the first time, to our knowledge, this study characterizes the TME of LGG, high-grade gliomas (HGG) IDH-Mut, and HGG IDH-WT patients regarding purinergic ectoenzymes and P1 receptors, focusing on tumor-infiltrating lymphocytes. Here, we show that ectoenzymes from both canonical and non-canonical pathways are increased in the TME when compared to the peripheral blood. We hypothesize this enhancement supports extracellular adenosine generation, hence increasing TME immunosuppression.

Molecular mechanisms of inflammation in the pathogenesis of respiratory disorders in patients with pulmonary tuberculosis

Aim. To assess external respiration (ER) and its relationship with the activity of enzymes involved in purine metabolism in patients with acute and chronic forms of pulmonary tuberculosis (TB).Materials and methods. In patients with acute and chronic TB, we assessed the activity of adenosine deaminase (ADA)-1, 2 in the blood serum (eADA), mononuclear cells, and neutrophils, the concentration of ecto-5’-nucleotidase (eNT5E) in the blood serum, the level of CD26 (dipeptidyl peptidase-4, DPPIV) in the blood serum and mononuclear cells, production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) in mononuclear cells and neutrophils, as well as parameters of ER.Results. Patients with TB were found to have an increase in the concentration of eNT5E and eADA-2 activity in the blood serum, stimulated production of ROI in neutrophils, a decrease in the concentration of DPPIV (CD26) in mononuclear cells, and a fall in the production of RNI in mononuclear cells and neutrophils. In patients with chronic TB, a decrease in the activity of ADA-1 in mononuclear cells and a fall in the concentration of DPPIV (CD26) in the blood serum were noted. In patients with acute TB, a decrease in the activity of eADA-1 in the blood serum and ADA-1 in neutrophils, reduced production of ROI in mononuclear cells, and an increase in spontaneous production of ROI in neutrophils were revealed. Correlations were found between the parameters of ER and the concentration of eNT5E in the blood serum, spontaneous production of ROI in mononuclear cells and production of RNI in neutrophils in chronic TB, as well as between eADA-2 in the blood serum, ADA-1 in neutrophils, DPPIV (CD26) activity in mononuclear cells, and ROI and RNI production in mononuclear cells and neutrophils.Conclusion. The data obtained make it possible to associate regulation of external respiration with parameters of purine metabolism, in particular with the concentration and activity of enzymes responsible for generation and metabolism of adenosine, that determine its level outside cells and inside mononuclear cells and neutrophils, with expression of cofactor molecules, as well as with the duration of activation of cells in innate immunity, neutrophils, and monocytes/ macrophages, determined largely by the potential of adenosine regulation.

Syntaphilin Regulates Neutrophil Migration in Cancer.

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

Inhibition of CD39 unleashes macrophage antibody-dependent cellular phagocytosis against B-cell lymphoma.

Redirection of tumor-associated macrophages to eliminate tumor cells holds great promise for overcoming therapeutic resistance to rituximab and other antibody drugs. Here, we determined the expression of ectonucleotidases CD39 and CD73 in diffuse large B-cell lymphoma (DLBCL), and examined the impact of extracellular ATP (eATP) metabolism on macrophage-mediated anti-lymphoma immunity. Immunostaining of tissue microarray samples showed that CD39 (the ecto-enzyme for eATP hydrolysis) was highly expressed in tumors with the non-germinal center B-cell-like (non-GCB) subtype, and to a lesser extent tumors with the GCB subtype. By contrast, the expression of CD73 (the ecto-enzyme for adenosine generation) was undetectable in tumor cells. Pharmacological blockade of CD39 prevented eATP degradation and enhanced engulfment of antibody-coated lymphoma cells by macrophages in a P2X7 receptor-dependent manner, indicating that eATP fueled antibody-dependent cellular phagocytosis (ADCP) activity. Importantly, inhibition of CD39 augmented in vivo anti-lymphoma effects by therapeutic antibodies including rituximab and daratumumab. Furthermore, the addition of a CD39 inhibitor to anti-CD20 and anti-CD47 combination therapy significantly improved survival in a disseminated model of aggressive B-cell lymphoma, supporting the benefit of dual targeting CD39-mediated eATP hydrolysis and CD47-mediated "don't eat me" signal. Together, preventing eATP degradation may be a potential approach to unleash macrophage-mediated anti-lymphoma immunity.

CD73-generated extracellular adenosine promotes resolution of neutrophil-mediated tissue injury and restrains metaplasia in pancreatitis.

Pancreatitis is currently the leading cause of gastrointestinal hospitalizations in the US. This condition occurs in response to abdominal injury, gallstones, chronic alcohol consumption or, less frequently, the cause remains idiopathic. CD73 is a cell surface ecto-5'-nucleotidase that generates extracellular adenosine, which can contribute to resolution of inflammation by binding adenosine receptors on infiltrating immune cells. We hypothesized genetic deletion of CD73 would result in more severe pancreatitis due to decreased generation of extracellular adenosine. CD73 knockout (CD73-/- ) and C57BL/6 (wild type, WT) mice were used to evaluate the progression and response of caerulein-induced acute and chronic pancreatitis. In response to caerulein-mediated chronic or acute pancreatitis, WT mice display resolution of pancreatitis at earlier timepoints than CD73-/- mice. Using immunohistochemistry and analysis of single-cell RNA-seq (scRNA-seq) data, we determined CD73 localization in chronic pancreatitis is primarily observed in mucin/ductal cell populations and immune cells. In murine pancreata challenged with caerulein to induce acute pancreatitis, we compared CD73-/- to WT mice and observed a significant infiltration of Ly6G+, MPO+, and Granzyme B+ cells in CD73-/- compared to WT pancreata and we quantified a significant increase in acinar-to-ductal metaplasia demonstrating sustained metaplasia and inflammation in CD73-/- mice. Using neutrophil depletion in CD73-/- mice, we show neutrophil depletion significantly reduces metaplasia defined by CK19+ cells per field and significantly reduces acute pancreatitis. These data identify CD73 enhancers as a potential therapeutic strategy for patients with acute and chronic pancreatitis as adenosine generation and activation of adenosine receptors is critical to resolve persistent inflammation in the pancreas.

Abstract IA013: Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention

Abstract Introduction: The all stages combined five-year survival rate for pancreatic adenocarcinoma (PDA) is 11%; however, the five-year survival rate for localized PDA is 42%. These statistics highlight the importance of early prevention strategies to prevent disease progression and metastatic dissemination. Through the NCI PREVENT program, this research program explores immunoprevention strategies for PDA by targeting CD73, a gatekeeper ectoenzyme responsible for production of extracellular adenosine. We have recently shown aggressive subtypes of pancreatic intraepithelial neoplasia (PanIN) and PDA arising in ductal pancreatic epithelium have elevated CD73 and intrapancreatic adenosine indicating adenosine generation may be an early trigger of immunosuppression. We hypothesize inhibition of CD73 and adenosine generation will promote a more robust anti-tumor immune response and prevent PanIN and PDA progression. Methods: We tested three small molecule CD73 inhibitors (APCP, OP-5244, and AB680) in a syngeneic PDA mouse model by injecting 100-200k murine PDA cells derived from KrasG12D;Trp53R172H/+;Pdx:Cre (KPC) mice in the flanks of C57BL/6 female mice. Tumor sizes were measured weekly and tumor volume and mass were recorded at time of death. Dosage: APCP oral gavage (3x/week at 20mg/kg) and intraperitoneal (IP) (3x/week at 20 mg/kg). OP-5244 oral (3x/week at 25mg/kg and 10mg/kg). AB680 oral gavage (3x/week at 10mg/kg). HPLC analysis was performed for each inhibitor to quantify adenosine levels. Results: IP delivery of APCP significantly reduced tumor growth and intratumoral adenosine levels; however oral gavage delivery did not reduce tumor growth. Similarly, oral gavage delivery of OP-5244 did not reduce tumor growth. AB680 significantly reduced tumor volume and intratumoral adenosine levels and CyTOF immunoprofiling showed activated CD8+ T cells, dendritic cells, and macrophages were significantly increased in the tumors from AB680 treated mice. Conclusion: APCP IP delivery is more effective than oral gavage delivery and OP-5244 oral gavage delivery does not significantly decrease tumor growth. AB680 oral gavage delivery significantly decreases tumor growth and tumor adenosine concentrations. We observed a significant increase in infiltration of activated CD8+ T cells. AB680 shows high translational potential for preclinical testing in spontaneous GEM models. Citation Format: Lincoln Ballew, Kanchan Singh, Vidhi Chandra, Tingting Mills, Erika Y. Faraoni, Victoria Mota, Trent Clark, Lana Vornik, Michelle I. Savage, Shizuko Sei, Altaf Mohammed, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA013.

A tumor intrinsic role of CD73 in pancreatic adenocarcinoma

: CD73, a membrane-bound nucleotidase, is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and acts as a key mediator of immune escape in PDAC mainly through generation of anti-inflammatory adenosine from pro-inflammatory adenosine triphosphate (ATP) in concert with CD39. Thus, CD73 has been considered as a promising target for the immunotherapy of tumors. So far, multiple highly effective CD73 inhibitors that block its nucleotidase activity have been developed and are currently under evaluation in early phases of clinical trials against malignant tumors including PDAC. However, the clinical activity in PDAC patients in some of these clinical trials are depressing, suggesting that the mechanism of CD73 in tumor progression is more complicated than expected. Recently, a published study indicated that CD73 contributes to chemotherapeutic resistance in PDAC via a novel mechanism, which differs from its previously known activity as a nucleotidase responsible for adenosine production on cell membrane. These findings suggest that an optimized CD73 targeting strategy should also consider the endoplasmic reticulum (ER)-associated and nucleotidase-independent activities of CD73. In this commentary, we will discuss the enzyme-dependent and independent activities of CD73 in tumor microenvironment (TME) of PDAC and will highlight a new targeting strategy for inhibiting the expression of CD73 to improve the efficacy of anti-CD73 therapy.

PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis

Metastasis indicates a grave prognosis in patients with hepatocellular carcinoma (HCC). Our previous studies showed that RNA binding motif protein Y-linked (RBMY) is potentially a biomarker for poor survival in HCC patients, but its role in metastasis is largely unclear. A total of 308 male patients with primary HCC were enrolled. RBMY expression was traced longitudinally by immunostaining from the manifestation of a primary HCC tumor to the formation of a distant metastasis, and its upstream regulators were screened with a protein microarray. Aseries of metastasis assays in mouse models and HCC cell lines were performed to explore new functional insights into RBMY. Cytoplasmic expression of RBMY was associated with rapid distant metastasis (approximately 1 year after resection) and had a predictive power of 82.4% for HCC metastasis. RBMY conferred high migratory and invasive potential upon phosphorylation by the provirus integration in Moloney 1 (PIM1) kinase. Binding of PIM1 to RBMY caused mutual stabilization and massive translocation of RBMY from nuclei to mitochondria, thereby preventing mitochondrial apoptosis and augmenting mitochondrial generation of adenosine triphosphate/reactive oxygen species to enhance cell motility. Depletion of RBMY suppressed Snail1/zinc finger E-box binding homeobox transcription factor 1-mediated epithelial-mesenchymal transition and dynamin-related protein 1-dependent mitochondrial fission. Inactivation and knockout of PIM1 down-regulated the expression of RBMY. In nude mice, cytoplasmic RBMY promoted liver-to-lung metastasis by increasing epithelial-mesenchymal transition, mitochondrial proliferation, and mitochondrial fission, whereas nuclear-restricted RBMY impeded the mitochondrial switch and failed to induce lung metastasis. This study showed the regulation of HCC metastasis by PIM1-driven cytoplasmic expression of RBMY and suggested a novel therapeutic target for attenuating metastasis.

Evidence that cervical cancer cells cultured as tumorspheres maintain high CD73 expression and increase their protumor characteristics through TGF-β production.

Recently, a link between the biological activity of CD73 and tumorigenicity in solid tumors has been proposed. We previously reported that the generation of adenosine (Ado) by the activity of CD73 in cervical cancer (CC) cells induces transforming growth factor-beta 1(TGF-β1) production to maintain CD73 expression. In the present study, we analyzed the participation of TGF-β1 in CD73 expression and the development of protumoral characteristics in CaSki CC cells cultured as tumorspheres (CaSki-T) and in monolayers (CaSki-M). Compared with those in CaSki-M cells, CD73 expression and Ado generation ability were significantly increased in CaSki-T cells. CaSki-T cells exhibited enrichment in the CSC-like phenotype due to increases in the expression levels of stem cell markers (CD49f, CK17, and P63; OCT4 and SOX2), greater sphere formation efficiency (SFE), and an increase in the percentage of side population (SP) cells. Interestingly, compared with CaSki-M cells, CaSki-T cells produced a greater amount of TGF-β1 and presented a marked protumor phenotype characterized by a significant decrease in the expression of major histocompatibility complex class-I (MHC-I) molecules, an increase in the expression of multidrug resistance protein-I (MRP-I) and vimentin, and an increase in the protein expression levels of Snail-1 and Twist, which was strongly reversed with TGF-β1 inhibition. These results suggest that the presence of TGF-β1-CD73-Ado feedback loop can promote protumoral characteristics in the CC tumor microenvironment.

Radiofrequency ablation in combination with CD73 inhibitor AB680 reduces tumor growth and enhances anti-tumor immunity in a syngeneic model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma presents a 5-year overall survival rate of 11%, placing an imperative need for the discovery and application of innovative treatments. Radiofrequency ablation represents a promising therapy for PDA, as studies show it induces coagulative necrosis and a host adaptive immune response. In this work we evaluated the effects of RFA treatment in vivo by establishing a syngeneic mouse model of PDA and performing tumor ablation in one flank. Our studies revealed RFA acutely impaired PDA tumor growth; however, such effects were not sustained one week after treatment. Adenosine (ADO) pathway represents a strong immunosuppressive mechanism that was shown to play a role in PDA progression and preliminary data from ongoing clinical studies suggest ADO pathway inhibition may improve therapeutic outcomes. Thus, to investigate whether ADO generation may be involved in tumor growth relapse after RFA, we evaluated adenosine-monophosphate (AMP), ADO and inosine (INO) levels by HPLC and found they were acutely increased after treatment. Thus, we evaluated an in vivo CD73 inhibition in combination with RFA to study ADO pathway implication in RFA response. Results showed combination therapy of RFA and a CD73 small molecule inhibitor (AB680) in vivo promoted sustained tumor growth impairment up to 10 days after treatment as evidenced by increased necrosis and anti-tumor immunity, suggesting RFA in combination with CD73 inhibitors may improve PDA patient response.

LBA17 Primary endpoint results of SYNERGY, a randomized phase II trial, first-line chemo-immunotherapy trial of durvalumab, paclitaxel, and carboplatin with or without the anti-CD73 antibody oleclumab in patients with advanced or metastatic triple-negative breast cancer (TNBC)

Overexpression of the ectoenzyme CD73 is involved in the generation of immunosuppressive adenosine in the tumor microenvironment and is associated with poor prognosis in patients (pts) with TNBC. Blocking CD73 with oleclumab may enhance the antitumor response to the combination of an anti-PD-L1 with chemotherapy in TNBC.

Disruption of CD73-Derived and Equilibrative Nucleoside Transporter 1–Mediated Adenosine Signaling Exacerbates Oxygen-Induced Retinopathy

Retinopathy of prematurity (ROP) is characterized by pathologic angiogenesis in retina, and it remains a leading cause of blindness in children. Although enhanced extracellular adenosine is markedly increased in response to retinal hypoxia, adenosine acting at the A1 and A2A receptors oppositely affects pathologic angiogenesis. In the oxygen-induced retinopathy (OIR) model of ROP, we demonstrated herein that pharmacologic and genetic inactivation of CD73 (the key 5'-ectonucleotidase for extracellular generation of adenosine) did not affect normal retinal vasculature development but exacerbated intravitreal neovascularization at postnatal day (P) 17 and delayed revascularization at P21 of OIR. This exacerbated damage to retinal vessels by CD73 inactivation was associated with increased cellular apoptosis and microglial activation but decreased astrocyte function at P17 of OIR. Furthermore, pharmacologic blockade of equilibrative nucleoside transporter 1/2 (ENT1/2; bidirectional transport for controlling the balance of intracellular and extracellular adenosine) by 6-nitrobenzylthioinosine aggravated pathologic angiogenesis at P17 of OIR. Last, pharmacologic blockade of ENT1/2 and genetic inactivation of CD73 also aggravated avascular areas at the hyperoxia phase (P12) of OIR. Thus, disruption of CD73-derived extracellular adenosine or ENT1/2-mediated transport of adenosine flux across membrane aggravated the damage to retinal vessels. These findings support that adenosine is an endogenous protective regulator that limits oxygen-induced retinopathy, and enhancing extracellular adenosine signaling represents a novel neuroprotection strategy for ROP by targeting CD73 and ENT1/2 activities.

Identification and characterization of two novel functional CD39 splice variants that negatively modulate the proinflammatory response in the Japanese flounder Paralichthys olivaceus

Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a key regulator of inflammatory responses by scavenging proinflammatory extracellular ATP (eATP) and promoting the generation of immunosuppressive adenosine. In addition to the previously identified CD39 homolog (designated CD39a), here we identified two novel functional CD39 splice variants (termed CD39b and CD39c) from teleost fish Japanese flounder (Paralichthys olivaceus). Transcriptional expression analysis revealed that all three CD39 splicing variants were coexpressed in Japanese flounder head kidney macrophages (HKMs) and a variety of normal tissues but with differential tissue/immune cell expression preferences. Immune challenge experiments showed that the expression of CD39 splice variants was modulated by both in vitro inflammatory stimulation and in vivo bacterium Edwardsiella piscicida infections. However, even though all CD39 splice variants were functional cell surface-expressed proteins and shared generally similar domain structures, their varied cytoplasmic N-terminal sequences resulted in a varied capability for hydrolysis of eATP and attenuation of eATP/P2XR-mediated proinflammatory cytokine expression. Pharmacological inhibition of endogenous CD39 activity in Japanese flounder HKMs or overexpression of CD39 splice variants in Japanese flounder FG-9307 cells further revealed the role of CD39 variants in down-regulating LPS-induced proinflammatory cytokine production. In addition, luciferase reporter analysis demonstrated that the CD39 splice variant promoters had varied transcriptional activities and that Sp1 was a potential transcription factor for CD39c transcription regulation. Taken together, we report for the first time that functional CD39 splice variants provide additional means to downregulate eATP- and LPS-induced proinflammatory response(s) in fish.

Inhibition of adenosine deaminase activity reverses resistance to the cytotoxic effect of high adenosine levels in cervical cancer cells

Adenosine (ADO) generation in the tumor microenvironment (TME) plays important roles in the promotion of tumor growth, invasion, and metastasis and in suppression of the antitumor immune response. Recently, adenosine deaminase (ADA) activity in the TME has been proposed to be a compensatory mechanism against toxic accumulation of ADO in cancerous tissues. In the present study, the expression and functional activity of ADA in cervical cancer (CeCa) tumor cells were analyzed: C33A (HPV-), CaSki (HPV + ), and HeLa (HPV + ) cells. CeCa tumor cells, as well as activated T lymphocytes (ATLs), which were used as a positive control, showed different ADA contents in the membrane and intracellularly and a strong ability to convert ADO into inosine (INO). Treatment of tumor cells with EHNA, a specific ADA inhibitor, decreased the viability of CeCa tumor cells in a dose-dependent manner. In C33A (EHNA half maximal inhibitory concentration (IC50) = 374 μM), CaSki (EHNA IC50 = 273.6 μM), and HeLa (EHNA IC50 = 252.2 μM) cells, EHNA strongly reversed the resistance of tumor cells to the cytotoxic effect of high concentrations of ADO; 38.82 ± 3.1%, 47.18 ± 4.7%, and 71.63 ± 6.9% of the cells were apoptotic, and 40 ± 4.8%, 52 ± 5.3% and 70 ± 6.8% of the cells had mitochondrial membrane damage, respectively. In ATLs (EHNA IC50 = 391.8 μM) treated with EHNA, 32.4 ± 4.4% were apoptotic, and 32 ± 4.3% had mitochondrial membrane damage. These results suggest that the presence and activity of ADA in CeCa tumor cells can provide protection against the cytotoxic effect of high ADO contents in the TME. Therefore, the inhibition of ADA could be a strategy for the treatment of CeCa.

IL-4 prevents adenosine-mediated immunoregulation by inhibiting CD39 expression.

The ectonucleotidase CD39 functions as a checkpoint in purinergic signaling on effector T cells. By depleting eATP and initiating the generation of adenosine, it impairs memory cell development and contributes to T cell exhaustion, thereby causing defective tumor immunity and deficient T cell responses in older adults who have increased CD39 expression. Tuning enzymatic activity of CD39 and targeting the transcriptional regulation of ENTPD1 can be used to modulate purinergic signaling. Here, we describe that STAT6 phosphorylation downstream of IL-4 signaling represses CD39 expression on activated T cells by inducing a transcription factor network including GATA3, GFI1, and YY1. GATA3 suppresses ENTPD1 transcription through prevention of RUNX3 recruitment to the ENTPD1 promoter. Conversely, pharmacological STAT6 inhibition decreases T cell effector functions via increased CD39 expression, resulting in the defective signaling of P2X receptors by ATP and stimulation of A2A receptors by adenosine. Our studies suggest that inhibiting the STAT6 pathway to increase CD39 expression has the potential to treat autoimmune disease while stimulation of the pathway could improve T cell immunity.

Abstract 2074: ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with multiple myeloma

Abstract Adenosine accumulation within the tumor microenvironment can severely limit antitumor immunity by promoting the expansion of immunosuppressive cell types and impairing immune cell function, including T cells, natural killer cells and dendritic cells. The generation of adenosine from adenosine monophosphate (AMP) requires the activity of a cell surface ecto-5’-nucleotidase, CD73, expressed on the surface of various cell types including immune cells. CD73 overexpression is observed in many tumor types and correlates with unfavorable clinical outcomes. Given the essential role of CD73 in generating adenosine, inhibition of CD73 is a promising therapeutic strategy. Recent clinical proof of concept data demonstrated a significant improvement in progression free survival for non-small cell lung cancer patients upon targeting CD73 with the oleclumab anti-CD73 antibody in combination with anti-PDL1, relative to anti-PDL1 checkpoint inhibitor treatment alone.We developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73, that is highly selective for CD73 and exhibits picomolar potency in biochemical assays, completely blocking adenosine production from AMP. In ex vivo assays, ORIC-533 displayed greater potency in restoring immunosuppressed CD8+ T cell proliferation and activation compared to oleclumab, as well as greater potency versus small molecule inhibitors of CD73 and the adenosine receptor. In multiple myeloma (MM) patients, elevated adenosine levels in the bone marrow (BM) niche correlate with progression of multiple myeloma through a CD73-mediated pathway. Moreover, we recently reported a functional role of CD73 signaling in BM of MM patients, indicating that CD73 inhibition may represent a unique vulnerability and treatment strategy for MM (Ray et al., abstract #2675, ASH 2021). We tested the impact of CD73 inhibition in bone marrow aspirates from patients with relapsed or refractory MM, using an autologous ex vivo assay system comprised of the multiple myeloma BM microenvironment. CD73 inhibition stimulated the activation of plasmacytoid dendritic cells and T cell activation in the context of the MM BM milieu. Moreover, ORIC CD73 inhibitor as a single agent overcame immune suppression and triggered significant lysis and cell death of multiple myeloma cells by autologous T-cells in the bone marrow microenvironment. Taken together, these results demonstrate that the ORIC CD73 inhibitor potently inhibits the adenosine pathway, which restores anti-tumor immunity and therefore holds potential for patients with MM. Based upon these data, ORIC-533 is being studied as a single agent in a Phase 1 clinical trial in patients with relapsed or refractory multiple myeloma. Citation Format: Melissa R. Junttila, Arghya Ray, Robert Warne, Xi Chen, Ting Du, Dena Sutimantanapi, Jae H. Chang, Brian Blank, Jared Moore, Chudi O. Ndubaku, Tatiana Zavorotinskaya, Pratik Multani, Omar Nadeem, Dharminder Chauhan, Kenneth C. Anderson, Lori S. Friedman. ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2074.