Inhibition Of Adenosine A2A Receptor Research Articles

CD8+ T cells sustain antitumor response by mediating crosstalk between adenosine A2A receptor and glutathione/GPX4.

Antitumor responses of CD8+ T cells are tightly regulated by distinct metabolic fitness. High levels of glutathione (GSH) are observed in the majority of tumors, contributing to cancer progression and treatment resistance in part by preventing glutathione peroxidase 4-dependent (GPX4-dependent) ferroptosis. Here, we show the necessity of adenosine A2A receptor (A2AR) signaling and the GSH/GPX4 axis in orchestrating metabolic fitness and survival of functionally competent CD8+ T cells. Activated CD8+ T cells treated ex vivo with simultaneous inhibition of A2AR and lipid peroxidation acquire a superior capacity to proliferate and persist in vivo, demonstrating a translatable means to prevent ferroptosis in adoptive cell therapy. Additionally, we identify a particular cluster of intratumoral CD8+ T cells expressing a putative gene signature of GSH metabolism (GMGS) in association with clinical response and survival across several human cancers. Our study addresses a key role of GSH/GPX4 and adenosinergic pathways in fine-tuning the metabolic fitness of antitumor CD8+ T cells.

Adenosine A2A receptor antagonist KW6002 protects against A53T mutant alpha-synuclein-induced brain damage and neuronal apoptosis in Parkinson's disease mice by restoring autophagic flux

BackgroundProtein misfolding and inclusion body aggregation caused by α-Syn mutations in the brain often cause neurodegeneration and cognitive impairment, among which the A53T point mutation is more common. Inhibition of adenosine A2A receptor (A2AR) can alleviate the pathological symptoms of brain dysfunction caused by A53T-α-Syn protofibrils, but the mechanism of action is still unclear. AimThis studies aimed to investigate the potential therapeutic role of the A2AR inhibitor KW6002 in a mouse model of brain synucleinopathy. MethodsA53T-α-Syn fibre precursor cell nuclear protein was injected into the bilateral prefrontal cortex of mice to establish a synucleinopathy animal model, and the A2AR inhibitor KW6002 (5 mg/kg) was injected intraperitoneally to intervene. ResultThe intracerebral injection of A53T-α-Syn protofibrils triggers the formation of inclusion bodies in the brain, leading to astrocyte activation, an increased number of apoptotic cells, and suppression of autophagic flux. The administration of KW6002 significantly reversed these phenomena. In vitro experiments revealed that A53T-α-Syn protofibrils inhibited HT-22 autophagy in mouse hippocampal neuronal cells, whereas KW6002 increased cellular autophagic flux, upregulated the expression of LAMP2A and Hsc70 proteins and inhibited the expression of SQSTM1 protein. The present study suggests that KW6002 reduces the level of α-Syn phosphorylation by inhibiting A2AR protein, at the same time, enhances the autophagic flux of neuronal cells, resulting in the degradation of A53T-α-Syn protofibrils and thus reducing the neuronal toxicity and apoptosis induced by A53T-α-Syn protofibrils. ConclusionKW6002 has a significant protective effect on neuronal injury induced by A53T-α-Syn.

Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma

Inhibition of adenosine A2A receptor (A2AR) is a promising approach for cancer immunotherapy currently evaluated in several clinical trials. We here report that anti-obesogenic and anti-inflammatory functions of A2AR, however, significantly restrain hepatocellular carcinoma (HCC) development. Adora2a deletion in mice triggers obesity, non-alcoholic steatohepatitis (NASH), and systemic inflammation, leading to spontaneous HCC and promoting dimethylbenzyl-anthracene (DMBA)- or diethylnitrosamine (DEN)-induced HCC. Conditional Adora2a deletion reveals critical roles of myeloid and hepatocyte-derived A2AR signaling in restraining HCC by limiting hepatic inflammation and steatosis. Remarkably, the impact of A2AR pharmacological blockade on HCC development is dependent on pre-existing NASH. In support of our animal studies, low ADORA2A gene expression in human HCC is associated with cirrhosis, hepatic inflammation, and poor survival. Together, our study uncovers a previously unappreciated tumor-suppressive function for A2AR in the liver and suggests caution in the use of A2AR antagonists in patients with NASH and NASH-associated HCC.

Knockdown of adenosine A2A receptors in hippocampal neurons prevents post-TBI fear memory retrieval

The formation of fear memory is crucial in emotional disorders such as PTSD and anxiety. Traumatic brain injury (TBI) can cause emotional disorders with dysregulated fear memory formation; however, their cross-interaction remains unclear and hurdled the treatment against TBI-related emotional disorders. While adenosine A2A receptor(A2AR) contributes to the physiological regulation of fear memory, this study aimed to evaluate the A2AR role and possible mechanisms in post-TBI fear memory formation using a craniocerebral trauma model, genetically modified A2AR mutant mice, and pharmacological A2AR agonist CGS21680 and antagonist ZM241385. Our finding showed (i) TBI enhanced mice freezing levels (fear memory) at seven days post-TBI; (ii) The A2AR agonist CGS21680 enhanced the post-TBI freezing levels; conversely, the A2AR antagonist ZM241385 reduced mice freezing level; further (iii) Genetic knockdown of neuronal A2AR in the hippocampal CA1, CA3, and DG regions reduced post-TBI freezing levels, while A2AR knockout in DG region yielded the most reduction in fear memory; finally, (iv) AAV-CaMKII-Cre virus-mediated DG deletion of A2AR on excitatory neurons led to a significant decreased freezing levels post-TBI. These findings indicate that brain trauma increases fear memory retrieval post-TBI, and A2AR on DG excitatory neurons plays a crucial role in this process. Importantly, inhibition of A2AR attenuates fear memory enhancement, which provides a new strategy to prevent fear memory formation/enhancement after TBI.

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2–3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A2AR), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF—A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner.

Abstract 4517: Targeting Notch1 via adenosine A2A receptor to modulate tumor immunity

Abstract One of the hallmarks of cancer is the ability to evade the host immune system and this is achieved through different mechanisms. Several studies have showed that the accumulation of adenosine in the tumor microenvironment suppresses T-cell functions, thus helping the tumor to evade the immune system. Finding a way to counteract adenosine-mediated immune suppression might greatly enhance the host endogenous immune response against cancer and the efficacy of adoptive immunotherapies. To achieve this, we need to understand how adenosine regulates T-cell functions. In a collaborative project, our group recently showed that stimulation of adenosine A2A receptor (A2AR) reduces T-cell receptor (TCR)-signaling and consequently Notch1 activation and expression in CD8+ T-cells, which are critical to cancer immunity. This suggests that A2AR stimulation suppresses CD8+ T-cell function through inhibition of TCR-induced Notch1, which is required for the activation and function of CD8+ T-cells. Based on these observations, we hypothesized that while stimulation of A2AR suppresses CD8+ T-cells, inhibition of A2AR should protect CD8+ T-cells from the adenosine-mediated immune suppression. Notch1 is likely to be critical in this process since it was shown that ectopic expression of Notch1 intracellular domain prevents the adenosine-mediated immune suppression in CD8+ T-cells. Therefore, we aim to investigate the effect of A2AR inhibition on TCR activation and Notch1 to evaluate the A2AR-Notch axis as a novel immunotherapeutic target. Our data show that pharmacological inhibition of A2AR with a selective antagonist induces tumor cell death and increases the number of CD8+ T-cells in tumor-derived spheroids from a mouse triple-negative breast cancer (TNBC) model. The effect of A2AR inhibition appears to be immune-mediated since the same agent did not induce cell death in tumor-derived spheroids from immunocompromised mice. Along the same lines, we show that, inhibition of A2AR restored Notch1 activation and proliferation in primary murine CD8+ T-cells, but did not affect Notch1 and proliferation in TNBC cell lines. This effect is likely to be strictly dependent on Notch since A2AR inhibition failed to rescue CD8+ T-cell proliferation from the suppressive effect of gamma-secretase inhibition. It is not yet known how A2AR regulates Notch1, however, our latest preliminary results suggest that A2AR stimulation might promote the endosomal degradation of Notch1, whereas, A2AR inhibition might switch Notch1 fate from endosomal degradation to activation. Further investigation is needed to establish how A2AR regulates Notch1 and whether A2AR inhibition can protect CD8+ T-cell function in vivo in the tumor microenvironment. Overall our current data provides a rationale for the evaluation of A2AR antagonists as a Notch-modulating immunotherapy. Citation Format: Giulia Monticone, Fokhrul M. Hossain, Deniz A. Ucar, Samarpan Majumder, Claudia Sorrentino, Paulo C. Rodriguez, Rosa A. Sierra, Antonio Pannuti, Stephen Hatfield, Barbara A. Osborne, Lisa M. Minter, Silvana Morello, Lucio Miele. Targeting Notch1 via adenosine A2A receptor to modulate tumor immunity [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 4517.

Novel Adenosine Analog, N6-(4-Hydroxybenzyl)-Adenosine, Dampens Alcohol Drinking and Seeking Behaviors.

Adenosine signaling is associated with ethanol-related behaviors. We previously found that adenosine A2A receptor (A2AR) activation dampens ethanol drinking behaviors in equilibrative nucleoside transporter 1 (ENT1) knockout mice, and A2AR inhibition augments reward-seeking behavior in wild-type mice. The novel adenosine analog N6-(4-hydroxybenzyl)-adenosine (NHBA), which is isolated from the rhizomes of Gastrodia elata, activates A2AR and inhibits ENT1. Here, we examined the effects of NHBA on ethanol drinking in the two-bottle choice test and operant ethanol seeking behaviors. We selected mice exhibiting high ethanol drinking behavior in the two-bottle choice test. NHBA (0.1 mg/kg, i.p.) reduced ethanol drinking behavior in a limited-access 3-hour drinking session in high-consumption ethanol drinking mice, and NHBA (0.1 mg/kg, i.p.) did not alter locomotor activity in the open-field test. Operant conditioning with 10% ethanol and 10% sucrose (10E10S) reward increased zone entries and time spent in the ethanol zone, while NHBA (0.1 mg/kg, i.p.) dampened ethanol zone preference in the Y-maze. Furthermore, NHBA (0.1 mg/kg, i.p.) devalued 10E10S and 10% ethanol (10E) reward after operant conditioning with 10E10S and 10E. Taken together, NHBA through A2AR activation and ENT1 modulation may dampen ethanol drinking and seeking behaviors, suggesting that NHBA is a potential therapeutic agent for treating alcohol use disorder. SIGNIFICANCE STATEMENT: Our work highlights that A2AR activation and ENT1 inhibition by a novel adenosine analog isolated from Gastrodia elata, N6-(4-hydroxybenzyl)-adenosine, decreases ethanol drinking and seeking behaviors. We suggest that NHBA is a potential therapeutic agent to treat alcohol use disorder.

Inhibition of the Adenosine A2A Receptor Mitigates Excitotoxic Injury in Organotypic Tissue Cultures of the Rat Cochlea.

The primary loss of cochlear glutamatergic afferent nerve synapses due to noise or ageing (cochlear neuropathy) often presents as difficulties in speech discrimination in noisy conditions (hidden hearing loss (HHL)). Currently, there is no treatment for this condition. Our previous studies in mice with genetic deletion of the adenosine A2A receptor (A2AR) have demonstrated better preservation of cochlear afferent synapses and spiral ganglion neurons after noise exposure compared to wildtype mice. This has informed our current targeted approach to cochlear neuroprotection based on pharmacological inhibition of the A2AR. Here, we have used organotypic tissue culture of the Wistar rat cochlea at postnatal day 6 (P6) to model excitotoxic injury induced by N-methyl-d-aspartate (NMDA)/kainic acid (NK) treatment for 2 h. The excitotoxic injury was characterised by a reduction in the density of neural processes immediately after NK treatment and loss of afferent synapses in the presence of intact sensory hair cells. The administration of istradefylline (a clinically approved A2AR antagonist) reduced deafferentation of inner hair cells and improved the survival of afferent synapses after excitotoxic injury. This study thus provides evidence that A2AR inhibition promotes cochlear recovery from excitotoxic injury, and may have implications for the treatment of cochlear neuropathy and prevention of HHL.

Adenosine A2A receptor inhibition restores the normal transport of endothelial glutamate transporters in the brain

Excitatory amino acid transporters (EAATs) on cerebral vascular endothelial cells play an important role in maintaining glutamate homeostasis in the brain. The dysfunction of endothelial EAATs is an important reason for the dramatically elevated brain glutamate levels after brain injury, such as traumatic brain injury (TBI). The adenosine A2A receptor (A2AR) plays an important role in regulating the brain glutamate level after brain injury; however, researchers have not clearly determined whether this role was related to its ability to regulate endothelial EAATs. Activation of A2AR in vitro not only decreased the PKA- and glutamate level-dependent strengthening of the interaction between NKA-α1 and the FXYD1 subunit and the subsequent decrease in the activity of Na+/K+-ATPases (NKAs) but also enhanced its interaction with EAATs and ultimately aggravated the reverse transport function of endothelial EAATs under oxygen-glucose deprivation (OGD) conditions. Conversely, inhibition of A2AR restored the normal transport of EAAT. Moreover, A2AR inhibition increased NKA activity and decreased its interaction with EAATs in isolated brain capillaries after TBI, further confirming its role in endothelial EAATs in vivo. Based on our results, A2AR played an important role in regulating endothelial EAAT function, and strategies that restore the normal transport of endothelial EAATs through the inhibition of A2AR might serve as an effective treatment for brain injury.

Abnormal striatal plasticity in a DYT11/SGCE myoclonus dystonia mouse model is reversed by adenosine A2A receptor inhibition

Striatal dysfunction is implicated in many movement disorders. However, the precise nature of defects often remains uncharacterized, which hinders therapy development. Here we examined striatal function in a mouse model of the incurable movement disorder, myoclonus dystonia, caused by SGCE mutations. Using RNAseq we found surprisingly normal gene expression, including normal levels of neuronal subclass markers to strongly suggest that striatal microcircuitry is spared by the disease insult. We then functionally characterized Sgce mutant medium spiny projection neurons (MSNs) and cholinergic interneurons (ChIs). This revealed normal intrinsic electrophysiological properties and normal responses to basic excitatory and inhibitory neurotransmission. Nevertheless, high-frequency stimulation in Sgce mutants failed to induce normal long-term depression (LTD) at corticostriatal glutamatergic synapses. We also found that pharmacological manipulation of MSNs by inhibiting adenosine 2A receptors (A2AR) restores LTD, again pointing to structurally intact striatal circuitry. The fact that Sgce loss specifically inhibits LTD implicates this neurophysiological defect in myoclonus dystonia, and emphasizes that neurophysiological changes can occur in the absence of broad striatal dysfunction. Further, the positive effect of A2AR antagonists indicates that this drug class be tested in DYT11/SGCE dystonia.

Abstract 5579: Strategic inhibition of adenosine A2A receptor (A2AR) by CPI-444 improves CD8+:Treg ratios and enhances T-cell killing of a HER-2/neu expressing murine tumor

Abstract It has been demonstrated that a higher CD8+ to CD4+FoxP3+ T regulatory cells (Tregs) ratio coincides with improved therapeutic outcomes for patients receiving immunotherapies in the clinic. The last several years of research have drawn attention to many such therapeutics and strategies. Adenosine is an abundant extracellular signaling molecule in the tumor microenvironment (TME) of many cancers. Signal transduction through the GPCR A2AR enhances the immunosuppressive activity of Tregs while simultaneously attenuating tumor-specific CD4+/CD8+ T cells. We have evaluated the appropriate coordination and dosing for a small-molecule inhibitor of A2AR in a Her-2/neu expressing murine model of breast cancer. We have demonstrated its capacity to augment existing therapeutic strategies in this tolerant model. To test our hypotheses on the ability of the small molecule to work in concert with existing therapeutics, we tested the role of CPI-444 when given with a T cell-inducing vaccine. Neu-expressing mammary tumor bearing HER-2/neu transgenic (Neu-N) mice were administered combinations of low-dose cyclophosphamide (Cy) to deplete Tregs, followed one day later with a granulocyte-macrophage colony-stimulating factor (GM-CSF) and neu-expressing whole cell vaccine (GVAX). The day following administration of GVAX, mice received adoptively transferred high-avidity naïve neu-specific CD8+ T cells intravenously one day after vaccination. Administration of specific components in this strategy (i.e. Cy, GVAX, adoptive transfer) were altered to gain insight into the mechanistic effects of CPI-444 in vivo through analysis of tumor progression, tumor clearance, and flow cytometric analysis of the TIL. Mice treated with the A2AR inhibitor or the vehicle control were administered each by oral gavage daily for 14 days (survival) or until 4 days post-adoptive transfer (TIL) at the peak infiltrate time. Of the strategies tested, Cy, followed by concomitant administration of GVAX with 100mg/kg of CPI-444 for 14 days, and a single adoptive therapy treatment provided a 55% overall survival (OS) compared with 0%-20% OS in vehicle controls (P<0.005). Further, we have observed both broad and specific changes in the T cell compartment of the tumor-infiltrating leukocyte (TIL) population. Broadly we have observed a refinement in the T cell portion of the TIL with decreased numbers of T cells overall, but an increase in specific prognostic ratios, namely a relative increase in favorability of the CD8+:Treg ratio when compared to controls (P<0.05). We hypothesize that strategic combination therapy of CPI-444 with existing therapeutics can refine the local immune profile to enhance favorable outcomes in vivo. CPI-444 is currently involved in numerous clinical trials; this work may provide rationale for the expansion of these trials into new cancer types. Citation Format: Blake A. Scott, Todd Armstrong, Stephen Willingham, Ian McCaffery, Elizabeth M. Jaffee. Strategic inhibition of adenosine A2A receptor (A2AR) by CPI-444 improves CD8+:Treg ratios and enhances T-cell killing of a HER-2/neu expressing murine tumor [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 5579. doi:10.1158/1538-7445.AM2017-5579

Abstract 320: Inhibition of adenosine A2A receptor (A2AR) by CPI-444 enhances CD8+ T cell killing of a HER-2/neu expressing murine tumor

Abstract Immunotherapies such as checkpoint inhibitors that block immune suppressive mechanisms are transforming the way some cancers are treated. The identification of additional mechanisms that suppress immune cell function is stimulating the search for other immune modulating agents that are additive or synergistic with current immune checkpoint inhibitors or are capable of overcoming mechanisms of resistance and tolerance. Extracellular adenosine and its signaling through A2AR increases the activity of regulatory T cells (Tregs), and attenuates tumor-specific CD4+/CD8+ T cells. In this study we evaluated the role of a small-molecule inhibitor of A2AR in enhancing antitumor immunity against murine Her-2/neu (neu)-expressing breast cancer. We examined whether an A2AR inhibitor would enhance the activity of cancer specific CD8+ T cells when given with a T cell inducing vaccine. To test this, neu-expressing mammary tumor bearing HER-2/neu transgenic (Neu-N) mice were treated with low dose cyclophosphamide (Cy) to deplete Tregs followed one day later with a granulocyte-macrophage colony-stimulating factor (GM-CSF) and neu-expressing whole cell vaccine (GVAX). Mice were treated with the A2AR inhibitor, CPI-444, by oral gavage daily for twelve days. Mice also received adoptively transferred high-avidity naïve neu-specific CD8+ T cells intravenously one day after vaccination. Mice were monitored for tumor progression, and tumor-infiltrating lymphocytes (TIL) were harvested from additional mice at multiple time points and analyzed for T cell activation by flow cytometry. Inflammatory changes in the tumor microenvironment were assessed by immunohistochemistry. We found that T cells adoptively transferred to mice together with Cy, a neu-targeted vaccine and CPI-444 are more effective at reducing established tumors and delaying tumor progression when compared with mice treated with Cy, vaccine, adoptively transferred T cells and vehicle control. Of particular note, mice receiving the same treatment but with CPI-444 in lieu of vehicle had a significant mean difference in tumor area of 6.446mm2 (P<0.05) when compared to the vaccine and vehicle control cohort. When compared to the tumor alone cohort, mice receiving the full therapeutic regimen had a significant mean difference in tumor area of 13.59mm2 (P<0.001). Mechanistic studies are underway to assess the activity of the adoptively transferred T cells in the TIL. These studies may provide the rationale to test an A2AR inhibitor in combination with adoptively transferred T cells and/or vaccines in patients with cancer. Citation Format: Blake A. Scott, Todd Armstrong, Elizabeth M. Jaffee. Inhibition of adenosine A2A receptor (A2AR) by CPI-444 enhances CD8+ T cell killing of a HER-2/neu expressing murine tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 320.