High Concentrations Of Adenosine Research Articles

Simultaneous inhibition of CD73 and IL-6 molecules by siRNA-loaded nanoparticles prevents the growth and spread of cancer

High concentrations of adenosine and interleukin (IL)-6 in the tumor microenvironment have been identified as one of the leading causes of cancer growth. Thus, we decided to inhibit the growth of cancer cells by inhibiting the production of adenosine and IL-6 in the tumor environment at the same time. For this purpose, we used chitosan-lactate-PEG-TAT (CLP-TAT) nanoparticles (NPs) loaded with siRNA molecules against CD73, an adenosine-producing enzyme, and IL-6. Proper physicochemical properties of the produced NPs led to high cell uptake and suppression of target molecules. Administration of these NPs to tumor-bearing mice (4T1 and CT26 models) greatly reduced the size of the tumor and increased the survival of the mice, which was accompanied by an increase in anti-tumor T lymphocyte responses. These findings suggest that combination therapy using siRNA-loaded CLP-TAT NPs against CD73 and IL-6 molecules could be an effective treatment strategy against cancer that needs further study.

Adenosine Signaling in Autoimmune Disorders.

The molecular components of the purinergic system (i.e., receptors, metabolizing enzymes and membrane transporters) are widely expressed in the cells of the immune system. Additionally, high concentrations of adenosine are generated from the hydrolysis of ATP in any “danger” condition, when oxygen and energy availability dramatically drops. Therefore, adenosine acts as a retaliatory metabolite to counteract the nucleotide-mediated boost of the immune reaction. Based on this observation, it can be foreseen that the recruitment with selective agonists of the receptors involved in the immunomodulatory effect of adenosine might represent an innovative anti-inflammatory approach with potential exploitation in autoimmune disorders. Quite surprisingly, pro-inflammatory activity exerted by some adenosine receptors has been also identified, thus paving the way for the hypothesis that at least some autoimmune disorders may be caused by a derailment of adenosine signaling. In this review article, we provide a general overview of the roles played by adenosine on immune cells with a specific focus on the development of adenosine-based therapies for autoimmune disorders, as demonstrated by the exciting data from concluded and ongoing clinical trials.

Abstract 6056: A next-gen anti-CD73 Antibody with potential to show better efficacy and safety profile than existing approach

Abstract CD73, an ecto-5'-nucleotidase, plays a major role in de-phosphorylation of the extracellular adenosine monophosphate (AMP) to adenosine, which in turn is a potent immunosuppressive metabolite that modulates the immune reaction within the tumor microenvironment (TME). High concentration of adenosine, predominantly signaling through the A2A receptor, suppress innate and adaptive immune cell responses, leading to tumor escape of immune surveillance. Recent studies have shown a significant overexpression of CD73 in solid tumors alongside it's functional upregulation by inflammatory mediators and hypoxia. Thus, inhibition of CD73 enzymatic activity and its surface expression in the TME may improve anti-tumor immune activity. To test this hypothesis, we designed and developed a fully human antibody with unique structure to recognize CD73. This antibody can block the enzymatic activity of CD73 in addition to reducing the surface expression of CD73. Both enzymatic and non-enzymatic dependent functions of CD73 were significantly dampened. The antibody demonstrates a superior anti-tumor activity over reference antibodies in in-vitro and in-vivo models. Our findings indicate that the antibody has promising potential as an effective therapy for PD-1/PD-L1 nonresponsive or refractory patients, as well as a broader patient population that may benefit from a unique mechanism of action of CD73-targeting antibody. Citation Format: Xin Gan, Lei Shi, Qianqian Shan, Fei Chen, Yun He, Yiping Rong, Qiumei Du. A next-gen anti-CD73 Antibody with potential to show better efficacy and safety profile than existing approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6056.

Targeting Metabolism of Extracellular Nucleotides via Inhibition of Ectonucleotidases CD73 and CD39.

In the tumor microenvironment, unusually high concentrations of extracellular adenosine promote tumor proliferation through various immunosuppressive mechanisms. Blocking adenosine production by inhibiting nucleotide-metabolizing enzymes, such as ectonucleotidases CD73 and CD39, represents a promising therapeutic strategy that may synergize with other immuno-oncology mechanisms and chemotherapies. Emerging small-molecule ectonucleotidase inhibitors have recently entered clinical trials. This Perspective will outline challenges, strategies, and recent advancements in targeting this class with small-molecule inhibitors, including AB680, the first small-molecule CD73 inhibitor to enter clinical development. Specific case studies, including structure-based drug design and lead optimization, will be outlined. Preclinical data on these molecules and their ability to enhance antitumor immunity will be discussed.

Abstract 5555: In vivo and in vitro characterization of RVU330 best-in-class dual A2A/A2B adenosine receptor antagonist

Abstract The immunosuppressive role of adenosine attracts attention of many researchers as an interesting target for anticancer therapy. High concentrations of adenosine detected in many tumor models affect several subpopulations of infiltrating immune cells e.g. T lymphocytes, dendritic cells and macrophages. In these cells, adenosine signals via either A2A or A2B receptor, therefore the highest efficiency in reversal of adenosine mediated immunosuppression should be achieved by dual antagonism to both receptors. Here, we present data for best-in-class dual A2A/A2B antagonist RVU330 that exerts nanomolar potency in tumor-like adenosine-rich environment. By head-to-head comparison of our in-house developed dual A2A/A2B antagonist to compounds currently pursued in clinical development, we have demonstrated the best-in-class characteristics of our molecule. RVU330 maintains sub- or low nanomolar antagonistic potency in a high adenosine conditions on both A2A and A2B receptor. Multilevel pharmacological characterization spans from receptor binding and cAMP accumulation (assays in cell lines overexpressing target receptors) to functional assays in primary human immune cells which demonstrate the reversibility of immune suppression induced by adenosine. Our compound shows superior activity in antagonizing adenosine-mediated immunosuppression in CD4+ and CD8+ T-cells (IL2 and TNFα production), monocyte derived dendritic cells - moDCs (IL12 and TNFα production, driven mainly by A2B receptors) and macrophages (suppression of VEGF release by M2). High activity in human whole blood assay demonstrates that low nanomolar concentration is sufficient to fully antagonize adenosine. Therapeutic efficacy of RVU330 has been confirmed in syngeneic in vivo mouse model. New best-in-class dual A2A/A2B adenosine receptor antagonists RVU330 combines two crucial activities which addresses distinct, cell type mediated mode of action of adenosine related immunosupression. The dual A2A/A2B antagonistic profile as well as the ability to inhibit both receptors at very high adenosine conditions are key features differentiating RVU330 from other adenosine receptor antagonist. The RVU330 is currently being evaluated in preclinical studies. Citation Format: Michał Gałęzowski, Katarzyna Dziedzic, Paulina Węgrzyn, Aniela Gołas, Magdalena Bońkowska, Karolina Grycuk, Mateusz Ogórek, Aneta Bobowska, Joanna Szeremeta-Spisak, Marcin Nowogródzki, Grzegorz Satała, Iwona Łozińska-Raj, Przemysław Wyrębek, Marcelina Dudek, Anita Janiga, Marek Wronowski, Magdalena Zastawna, Mateusz Świrski, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Mateusz O. Nowak. In vivo and in vitro characterization of RVU330 best-in-class dual A2A/A2B adenosine receptor antagonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5555.

Abstract CT152: First in human study with EOS100850, a novel potent A2A antagonist, shows excellent tolerance and clinical benefit in immune resistant advanced cancers

Abstract Adenosine is a strong immunosuppressive mediator often found at high extracellular levels in the microenvironment of most tumors. Blocking A2A receptors, predominantly expressed on tumor-infiltrating immune cells, is aimed at reversing the immunosuppressive effect of adenosine. EOS100850 is a potent and highly selective small molecule antagonist of the A2A receptor that remains active even at the high adenosine concentration found in tumors and that does not cross the blood-brain barrier. This multicenter FIH Phase I clinical trial (NCT02740985) assessed the safety, pharmacokinetic (PK), pharmacodynamic (PD), and preliminary clinical activity of EOS100850 monotherapy in subjects with refractory solid tumors. Pharmacodynamic assessments were performed in peripheral blood and tumor biopsies with simultaneous determination of plasma PK. Twenty-one adult participants with advanced malignancies were treated with EOS100850 monotherapy. Cohorts of 3 participants each were evaluated at 20 and 40 mg QD or 40 mg BID administration. Six participants were subsequently tested at doses of 80 mg BID and at the maximum administered dose of 160 mg BID. All subjects had at least 1 prior regimen (median 3). PK analysis indicated dose-proportional increase of EOS100850 plasma exposures up to 80 mg BID while PD evaluations revealed prolonged and maintained A2AR inhibition over the dosing interval. The most common adverse events (>15%) attributed to the investigational treatment included Grade 1-2 nausea, fatigue, vomiting, and liver enzyme abnormalities. No drug-related Grade 3-4 AEs and no dose-limiting toxicities were observed. As of the data cut off (15JAN20), stable disease was observed in 6 participants who remain on therapy, including head & neck cancers, endometrial cancers, and checkpoint inhibitor-refractory melanoma. Further evaluation is ongoing as monotherapy and in combination with pembrolizumab or chemotherapy in both immune checkpoint-naïve and -refractory patients. Citation Format: Laurence Buisseret, Sylvie Rottey, Johann de Bono, Manon Mossakowski, Brant Delafontaine, Thubeena Manickavasagar, Nuria Kotecki, Chiara Martinoli, Manfred Schneider, Olivier De Henau, Patricia Chevron, Joanne Lager, Jean-Pascal Machiels. First in human study with EOS100850, a novel potent A2A antagonist, shows excellent tolerance and clinical benefit in immune resistant advanced cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT152.

Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells.

A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPβS) in a 1–1000 μM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100–1000 μM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 μM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.

Adenosine Protects Zebrafish Photoreceptors from Apoptosis and Damage in a Dose‐Dependent Manner in a Light‐Induced Retinal Degeneration Model of Age‐Related Macular Degeneration

PurposeAdenosine has been shown to be neuroprotective in the CNS and in retinal ganglion cells in the inner retina. However, the neuroprotective effects of adenosine on outer retinal neurons, in particular photoreceptors, is not known. Age‐related macular degeneration (AMD) is a multifactorial disease characterized by the impairment or loss of central vision due to macular dropout and loss of photoreceptors. Adenosine has been shown to inhibit photoreceptor excitability by suppressing Ca2+ influx through voltage‐gated Ca2+ channels, which could lead to cell death. The purpose of this study was to see if adenosine would reduce photoreceptor damage and death in a light‐induced retinal degeneration (LIRD) model in pigmented zebrafish.MethodsAdult wild‐type and Tg(nrd:GFP) zebrafish (used to GFP label rod photoreceptors) were dark‐adapted for 24 hours, followed by intravitreal injections of ~ 0.5 ml of 0.1 mM, 1 mM, or 5 mM (final conc: 1.67 to 167 mM) of adenosine in both eyes. Controls were injected with saline in both eyes. All zebrafish were exposed to full spectrum high‐intensity light (≥ 20,000 lux) for four days and collected at 0 and 96 hours post dark‐adaptation. Immunohistochemical analysis was performed on vertical retinal sections in order to visualize cones, rods, and the outer nuclear layer of the retina. Retinal damage was assessed using confocal microscopy with a TUNEL assay and cell specific markers.ResultsPhotoreceptors in control retinas collected at 96 hours exhibited severe truncation or loss of the outer segments, and swelling of the cell body. Additionally, there was an increase in TUNEL‐positive cells and a significant decrease in rod and cone photoreceptors in the control retinas. Photoreceptors from retinas treated with higher concentrations of adenosine (1 mM and 5 mM) lacked swelling, exhibited less cell death, reduced truncation of the outer segments, and had cell morphology similar to retinas collected prior to light exposure. Photoreceptors from retinas treated with 0.1 mM adenosine exhibited no significant differences in cell death in comparison to control retinas at 96 hours, and had similar levels of outer segment truncation and cell body morphology. However, there was a significant reduction in the number of cones and rods lost in the 0.1 mM adenosine‐treated retinas at 96 hours in comparison to control retinas at 96 hours.ConclusionThese findings show that adenosine can prevent light‐induced structural damage to rod and cone photoreceptors, prevent photoreceptor death and loss, and provide evidence of a potential novel therapeutic target for the treatment of the “dry” form of AMD.Support or Funding InformationHMC CURE Zebrafish Pilot Grant from Penn State University College of Medicine, Pennsylvania Department of Health Tobacco CURE Fund

Adenosine Receptors as Novel Targets for the Treatment of Various Cancers.

Adenosine is a ubiquitous signaling nucleoside molecule, released from different cells within the body to act on vasculature and immunoescape. The physiological action on the proliferation of tumour cell has been reported by the presence of high concentration of adenosine within the tumour microenvironment, which results in the progression of the tumour, even leading to metastases. The activity of adenosine exclusively depends upon the interaction with four subtypes of heterodimeric G-protein-coupled adenosine receptors (AR), A1, A2A, A2B, and A3-ARs on the cell surface. Research evidence supports that the activation of those receptors via specific agonist or antagonist can modulate the proliferation of tumour cells. The first category of AR, A1 is known to play an antitumour activity via tumour-associated microglial cells to prevent the development of glioblastomas. A2AAR are found in melanoma, lung, and breast cancer cells, where tumour proliferation is stimulated due to inhibition of the immune response via inhibition of natural killer cells cytotoxicity, T cell activity, and tumourspecific CD4+/CD8+ activity. Alternatively, A2BAR helps in the development of tumour upon activation via upregulation of angiogenin factor in the microvascular endothelial cells, inhibition of MAPK and ERK 1/2 phosphorylation activity. Lastly, A3AR is expressed in low levels in normal cells whereas the expression is upregulated in tumour cells, however, agonists to this receptor inhibit tumour proliferation through modulation of Wnt and NF-κB signaling pathways. Several researchers are in search for potential agents to modulate the overexpressed ARs to control cancer. Active components of A2AAR antagonists and A3AR agonists have already entered in Phase-I clinical research to prove their safety in human. This review focused on novel research targets towards the prevention of cancer progression through stimulation of the overexpressed ARs with the hope to protect lives and advance human health.

Abstract 3261: EOS100850, a non-brain penetrant highly selective A2Areceptor antagonist, uniquely maintains high potency within the adenosine rich tumor microenvironment

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We demonstrated that A2A receptor antagonists initially designed for Parkinson’s disease but repurposed for immuno-oncology dramatically loose potency in a high adenosine environment. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant orally administered agent to treat a wide range of tumor types. EOS100850 has been discovered via structure-based drug design followed by rational medicinal chemistry to improve drug-like properties. EOS100850 demonstrated nanomolar affinity and high selectivity for human A2A receptors, with pico- to nanomolar potency on human and mouse A2A receptors. Assays were performed on A2A-overexpressing HEK293 cells and primary human T cells, using conditions mimicking normal physiological and tumor-like microenvironments. In these assays, EOS100850 potency was not affected by increasing extracellular adenosine concentrations. A unique feature of this compound is the remarkable long residence time on human A2A receptors after binding, resulting in a prolonged inhibition of A2A receptor signaling at a wide range of adenosine concentrations (up to 1000 µM). Furthermore, unlike competitor A2A receptor antagonists, EOS100850 reversed with nanomolar potency A2A receptor-dependent inhibition of cytokine secretion by T cells in tumor-like assay conditions. EOS100850 potency to inhibit A2A receptor-mediated CREB phosphorylation in T cells in human and mouse whole blood was in the low nanomolar range.One of the major metabolites of EOS100850, EOS100612, identified both in vitro and in vivo, has been synthesized and was found to be active on A2A receptors. Similar to EOS100850, EOS100612 displayed pico- to nanomolar potency, high selectivity and long residence time on A2A receptors in various in vitro assays.In conclusion, EOS100850 represents a novel non-brain penetrant best-in-class A2A receptor antagonist. It remains highly potent in environments characterized by high adenosine concentrations, such as the TME, maximizing the potential intra-tumor effect and further minimizing the risk of collateral CNS toxicity. Citation Format: Erica Houthuys, Paola Basilico, Margreet Brouwer, Theo Deregnaucourt, Michel Detheux, Gregory Driessens, Bruno Gomes, Xavier Leroy, Joao Marchante, Reece Marillier, Jakub Swiercz, Stefano Crosignani. EOS100850, a non-brain penetrant highly selective A2Areceptor antagonist, uniquely maintains high potency within the adenosine rich tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3261.

Abstract 5014: Profile of ARX001822, a highly potent, selective and orally bioavailable A2a antagonist effective in preventing adenosinergic suppression of T cell activation

Abstract Adenosine is elevated in the tumour microenvironment and plays a critical role in suppressing T cell function through high affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumour burden in syngeneic models. These effects are mimicked by small molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumours, particularly in combination with checkpoint inhibitors. However, the high levels of adenosine in the tumour microenvironment can dramatically reduce the effectiveness of competitive A2a antagonists. The challenge therefore is to identify highly potent and selective A2a antagonists which retain potency in the presence of high concentrations of adenosine and therefore have the potential to nullify the adenosinergic pathway within the tumour microenvironment ARX001822 binds A2a with high affinity (Ki=0.9 nM) and with greater than 660-fold selectivity over A1, A2b and A3. In a functional assay utilising CHO cells expressing recombinant A2a ARX001822 inhibited cAMP production in response to the selective A2a agonist CGS21680 in a competitive manner (KB= 0.3nM). Activation of A2a leads to the suppression of T cell-derived cytokine production and ARX001822 prevented this suppression even in the presence of high concentration of the adenosine receptor ligand NECA (IC50=38 nM). ARX001822 was also active in human whole blood, preventing NECA-mediated elevation of pCREB in CD8+ T cells and restoring production of interferon-γ with a potency 5-20 times higher than that of competitor molecules undergoing clinical evaluation in cancer. ARX001822 was orally bioavailable in rats and mice and was effective in inhibiting elevation of pCREB in mouse CD8+ T cells in an ex vivo pharmacodynamic assay. ARX001822 is a highly potent and selective A2a antagonist which is effective in preventing adenosinergic mediated suppression of cytokine production in the presence of high concentrations of adenosine receptor ligands and a full complement of plasma proteins. Knowledge of whole blood potency on both pCREB and interferon-γ modulation combined with the exposure required for activity in the pharmacodynamic model is helpful in estimating the clinical exposure required for A2a receptor blockade and downstream events related to modulating T cell function. Citation Format: Peter M. Finan, Roy Pettipher, Jonathan White, Viral Patel, Ben Moulton, Soraya Porres, Karolina Gherbi, Elizabeth M. Rosethorne, Steven J. Charlton, Clive McCarthy. Profile of ARX001822, a highly potent, selective and orally bioavailable A2a antagonist effective in preventing adenosinergic suppression of T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5014.

AB928, a novel dual adenosine receptor antagonist, combined with chemotherapy or AB122 (anti-PD-1) in patients (pts) with advanced tumors: Preliminary results from ongoing phase I studies.

2604 Background: AB928, a selective, small-molecule A2aR/A2bR antagonist, potently blocks the immunosuppressive effects of high adenosine concentrations in the tumor microenvironment. Preclinically, combining adenosine receptor inhibition with either chemotherapy or anti-PD-1 resulted in greater tumor control, suggesting AB928 may have additive activity when paired with either of these agents in cancer pts. Methods: Three dose-escalation (3+3 design) studies are assessing the safety, pharmacokinetics (PK), pharmacodynamics, and clinical activity of increasing doses of AB928 (75, 150, 200 mg orally once daily) in combination with: standard pegylated liposomal doxorubicin in triple-negative breast cancer (TNBC) and ovarian cancer (OC); standard mFOLFOX6 in gastroesophageal cancer (GEC) and colorectal cancer (CRC); and AB122 (240 mg every 2 weeks) in various advanced tumors. Following identification of the recommended phase 2 dose of AB928 in combination with chemotherapy or AB122 in dose escalation, the following tumor cohorts may be expanded (15-40 pts/cohort) to further test the combinations: TNBC and OC, GEC and CRC, and renal cell carcinoma. Results: As of 01Feb2019, 9 pts were treated across the 3 studies, and time on treatment ranged from 1-182 days (table). Overall, AB928 combination therapy was well tolerated. Two pts underwent post-baseline disease assessment; both had stable disease. Preliminary data indicate that AB928 PK and adenosine receptor coverage in cancer pts are similar to what was previously assessed in healthy volunteers. AB122 PK and PD-1 coverage are equally unaffected by AB928 co-administration. Updated data, including biomarker data, will be presented at the meeting. Conclusions: Early results showed a favorable safety profile of AB928 combination therapy. All 3 studies are actively recruiting pts. Clinical trial information: NCT03719326; NCT03720678; NCT03629756. [Table: see text]

Nucleosides block AICAR-stimulated activation of AMPK in skeletal muscle and cancer cells.

AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an adenosine analog, is a standard positive control for AMPK activation in cell-based assays. Some broadly used cell culture media, such as minimal essential medium α (MEMα), contain high concentrations of adenosine and other nucleosides. We determined whether such media alter AICAR action in skeletal muscle and cancer cells. In nucleoside-free media, AICAR stimulated AMPK activation, increased glucose uptake, and suppressed cell proliferation. Conversely, these effects were blunted or completely blocked in MEMα that contains nucleosides. Addition of adenosine or 2'-deoxyadenosine to nucleoside-free media also suppressed AICAR action. MEMα with nucleosides blocked AICAR-stimulated AMPK activation even in the presence of methotrexate, which normally markedly enhances AICAR action by reducing its intracellular clearance. Other common media components, such as vitamin B-12, vitamin C, and α-lipoic acid, had a minor modulatory effect on AICAR action. Our findings show that nucleoside-containing media, commonly used in AMPK research, block action of the most widely used pharmacological AMPK activator AICAR. Results of cell-based assays in which AICAR is used for AMPK activation therefore critically depend on media formulation. Furthermore, our findings highlight a role for extracellular nucleosides and nucleoside transporters in regulation of AMPK activation.

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

Opposing effects of intracellular versus extracellular adenine nucleotides on autophagy: implications for β-cell function.

AMPK-mTORC1 signaling senses nutrient availability, thereby regulating autophagy. Surprisingly, we found that, in β-cells, the AMPK activator 5-amino-4-imidazolecarboxamide ribofuranoside (AICAR) inhibited, rather than stimulated, autophagy. AICAR is an intermediate in the generation of inosine monophosphate, with subsequent conversion to other purine nucleotides. Adenosine regulated autophagy in a concentration-dependent manner: at high concentrations, it mimicked the AICAR effect on autophagy, whereas at low concentrations it stimulated autophagy through its cognate A1 receptor. Adenosine regulation of autophagy was independent of AMPK or mTORC1 activity. Adenosine kinase (ADK) is the principal enzyme for metabolic adenosine clearance. ADK knockdown and pharmacological inhibition of the enzyme markedly stimulated autophagy in an adenosine A1 receptor-dependent manner. High-concentration adenosine increased insulin secretion in a manner sensitive to treatment with the autophagy inducer Tat-beclin1, and inhibition of autophagy augmented secretion. In conclusion, high concentrations of AICAR or adenosine inhibit autophagy, whereas physiological concentrations of adenosine or inhibition of adenosine clearance by ADK stimulate autophagy via the adenosine receptor. Adenosine might thus be an autocrine regulator of autophagy, independent of AMPK-mTORC1 signaling. Adenosine regulates insulin secretion, in part, through modulation of autophagy.

Abstract 3769: Pharmacokinetic-pharmacodynamic relationship for AB928, a dual antagonist of the A2aR and A2bR adenosine receptors

Abstract INTRODUCTION: CD73 converts extracellular adenosine monophosphate (AMP), derived from adenosine triphosphate, into adenosine (ADO). ADO suppresses immune responses, including those of T cells, natural killer (NK) cells and dendritic cells, via A2aR and A2bR receptors. Activation of these receptors results in increased intracellular levels of cyclic AMP (cAMP) and phosphorylation of the cAMP response element-binding protein (CREB). Monitoring the degree of pCREB induced by activation of the Aa2R and A2bR receptors in peripheral blood allows for a specific measurement of target engagement by AB928. In mice, the pCREB assay, coupled with changes in immune cell infiltrate and tumor growth rates, allows for a fuller understanding of the drug's PK/PD relationship. METHODS: Mouse and human whole blood was stimulated with the adenosine agonist NECA (5'-N-Ethylcarboxamidoadenosine) following in vitro spike in or in vivo dosing with AB928. Multi-color phospho-flow cytometry was used to assess levels of pCREB on immune cells. AB928 levels in plasma samples were determined using LC-MS-MS after protein precipitation. RESULTS: A2aR mRNA was the most prevalent adenosine receptor in CD4+ and CD8+ T cells. NK cells also express primarily A2aR, although A2bR was also detected. Dendritic cells and CD14+ monocytes expressed both A2aR and A2bR. NECA stimulation of mouse whole blood resulted in a significant increase in the levels of pCREB within CD8+ T cells with an EC50 of 100 nM, while in human whole blood the EC50 was higher at 700 nM. AB928 (100 nM) exhibited comparable shifts in the NECA dose response between human and murine whole blood, 15 and 16-fold, respectively. Due to the high level of adenosine in the tumor microenvironment, doses of AB928 associated with plasma levels that significantly inhibited 5 μM NECA in the whole-blood pCREB assay were selected for mouse efficacy studies. At these doses, significant reductions in tumor growth were noted. In human whole blood, approximately 90 nM AB928 was required to inhibit 50% of the 5 μM NECA-induced pCREB signal. Inhibition was observed on both CD4+ and CD8+ T cells. NECA-induced pCREB elevations were observed in CD14+ cells less frequently than T cells; however, when the signal was observed, AB928 inhibited it. The human pCREB assay is being utilized to monitor the PD responses in an ongoing clinical trial. Preliminary PK/PD results from this study will be presented. CONCLUSIONS: Systemic extent of receptor (A2aR and A2bR) occupancy by the novel dual antagonist AB928 can be assessed in humans and mice based on the extent of receptor-mediated CREB phosphorylation in blood lymphocytes. AB928 retains much of its inherent potency when competing against high concentrations of adenosine under physiologically relevant conditions (i.e., whole blood). Citation Format: Lisa Seitz, Devika Ashok, Manmohan R. Leleti, Jay P. Powers, Brandon Rosen, Dillon Miles, Lixia Jin, Adam Park, Tim Park, Steve Young, Ferdie Soriano, Aimee Rieger, Ulrike Schindler, Joyson Karakunnel, Matt J. Walters. Pharmacokinetic-pharmacodynamic relationship for AB928, a dual antagonist of the A2aR and A2bR adenosine receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3769.

Abstract 3751: Inhibition of A2AR by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models

Abstract Adenosine signaling through the high affinity adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which can promote tumor growth and limit the efficacy of immune checkpoint inhibitors. AZD4635 (HTL-1071) is a potent and selective oral A2AR antagonist, currently in a Phase 1 clinical trial as a single agent and in combination with durvalumab (anti-PD-L1 Ab) in patients with solid malignancies. In functional in vitro assays, the IC50 of AZD4635 for inhibition of A2AR signaling is dependent on adenosine concentrations and ranges from 1, 10.0, 143 nM in the presence of 0.1, 1, 10 μM adenosine, respectively. However, full understanding of the impact of adenosine on AZD4635 mediated anti-tumor responses requires characterization of intratumoral adenosine concentration and spatial heterogeneity. Using a novel LC/MS based method, measurement of intratumoral adenosine concentrations in a panel of syngeneic tumor models demonstrated a wide range of adenosine levels (5-122 μM). Additionally, measurement of intratumoral adenosine by desorption electrospray ionisation - mass spectroscopy (DESI-MS) demonstrated that adenosine levels are spatially heterogeneous, with levels varying up to 50-fold among regions of a single tumor. High intratumoral adenosine concentrations were correlated with decreased intratumoral CD8 infiltration in vivo. The therapeutic benefit of A2AR blockade alone and in combination with anti-PD-L1 was evaluated in a panel of syngeneic mouse tumor models with varying adenosine concentrations. In sensitive models, inhibition of A2AR signaling with AZD4635 led to a dose dependent reduction in tumor growth alone and in combination with checkpoint inhibitors. Tumors harvested from the treated mice exhibited increases in the functional activity of T cell and myeloid subsets. These results demonstrate that AZD4635 is a potent and selective A2AR inhibitor, that blockade of A2AR signaling with an inhibitor such as AZD4635 can reduce tumor burden and enhance antitumor immunity. Citation Format: Alexandra Borodovsky, Yanjun Wang, Minwei Ye, Joseph C. Shaw, Kris Sachsenmeier, Nanhua Deng, Kelly Goodwin, James D. Clarke, Richard Goodwin, Nicole Strittmatter, Carl Hay, Vasu Sah, Lawson Deborah, Corinne Reimer, Miles Congreve, Jonathan Mason, Fiona Marshall, Paul Lyne, Richard Woessner. Inhibition of A2AR by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3751.

Abstract 1683: A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity

Abstract High levels of extracellular adenosine in the tumor microenvironment are known to play a significant role in tumor immune evasion and promote tumor growth and metastasis. We defined the receptor(s) required for mediating the effect of adenosine on immune cells within the tumor microenvionment and report the characterization of a novel Immuno-Oncology-dedicated adenosine receptor 2A antagonist that functions in the high adenosine concentration found in tumors. We first explored the expression of the four adenosine receptors in primary human immune cells. A2A receptor was the main adenosine receptor expressed by CD4 and CD8 T lymphocytes and monocytes, and the only one in mature monocyte-derived dendritic cells and NK cells. A2B receptor was poorly detected in T cells and monocytes, while A1 and A3 receptors were never detected. Given these expression patterns, we further studied A2A functions in primary human T lymphocytes and monocytes. Selective A2A agonists such as CGS-21680 strongly suppressed cytokine production by activated primary human T lymphocytes, thus highlighting that A2A is the main effector receptor of the sensing of adenosine in tumors. We further confirmed the elevated extracellular adenosine level in the tumor microenvironment in several mouse and human tumors. High adenosine levels correlated with strong tumoral expression of CD73, the enzyme that converts AMP to adenosine. Interestingly, we showed that A2A receptor antagonists designed for Parkinson’s disease dramatically lost potency in a high adenosine environment ; our data indicated that a 30-fold dose increase may be required for full target inhibition within tumors. Therefore we developed a novel and potent A2A blocker with sub-nanomolar Ki and IC50 in a cAMP assay and a more than 100-fold selectivity over other adenosine receptors. Our lead compound kept a high potency in an adenosine-rich environment and restored cytokine production even in the presence of high concentrations of A2A agonists. iTeos inhibitor also efficiently reversed AMP-mediated T cell suppression. Furthermore, our compound rescued A2A receptor agonist-induced decrease of TNFα production by primary human monocytes, and was able to potently increase CD8 T cell cytotoxicity in a cytotoxicity assay with CD8 T cells as effectors and cancer cells as targets. These results suggest that iTeos new generation of A2A receptor antagonist, designed to keep a high potency in the adenosine-rich tumor microenvironment, may offer a new therapeutic opportunity in Immuno-Oncology. Citation Format: Erica Houthuys, Margreet Brouwer, Florence Nyawouame, Romain Pirson, Reece Marillier, Theo Deregnaucourt, Joao Marchante, Jakub Swiercz, Charlotte Moulin, Vanesa Bol, Gregory Driessens, Michel Detheux, Christophe Quéva, Stefano Crosignani, Bruno Gomes. A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2017-1683

New insights into a classic aptamer: binding sites, cooperativity and more sensitive adenosine detection.

The DNA aptamer for adenosine (also for AMP and ATP) is a highly conserved sequence that has recurred in a few selections. It it a widely used model aptamer for biosensor development, and its nuclear magnetic resonance structure shows that each aptamer binds two AMP molecules. In this work, each binding site was individually removed by rational sequence design, while the remaining site still retained a similar binding affinity and specificity as confirmed by isothermal titration calorimetry. The thermodynamic parameters of binding are presented, and its biochemical implications are discussed. The number of binding sites can also be increased, and up to four sites are introduced in a single DNA sequence. Finally, the different sequences are made into fluorescent biosensors based on the structure-switching signaling aptamer design. The one-site aptamer has 3.8-fold higher sensitivity at lower adenosine concentration with a limit of detection of 9.1 μM adenosine, but weaker fluorescence signal at higher adenosine concentrations, consistent with a moderate cooperativity in the original aptamer. This work has offered insights into a classic aptamer for the relationship between the number of binding sites and sensitivity, and a shorter aptamer for improved biosensor design.

Drosophila imaginal disc growth factor 2 is a trophic factor involved in energy balance, detoxification, and innate immunity

Drosophila imaginal disc growth factor 2 (IDGF2) is a member of chitinase-like protein family (CLPs) able to induce the proliferation of imaginal disc cells in vitro. In this study we characterized physiological concentrations and expression of IDGF2 in vivo as well as its impact on the viability and transcriptional profile of Drosophila cells in vitro. We show that IDGF2 is independent of insulin and protects cells from death caused by serum deprivation, toxicity of xenobiotics or high concentrations of extracellular adenosine (Ado) and deoxyadenosine (dAdo). Transcriptional profiling suggested that such cytoprotection is connected with the induction of genes involved in energy metabolism, detoxification and innate immunity. We also show that IDGF2 is an abundant haemolymph component, which is further induced by injury in larval stages. The highest IDGF2 accumulation was found at garland and pericardial nephrocytes supporting its role in organismal defence and detoxification. Our findings provide evidence that IDGF2 is an important trophic factor promoting cellular and organismal survival.