Expression Of Equilibrative Nucleoside Transporter Research Articles

Abstract 734: Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression

Abstract The profound benefit of immune checkpoint blockade for cancer therapy is restricted to limited subsets of patients with specific cancers. Many factors contribute to primary and acquired resistance to immune checkpoint blockade, including local accumulation of immunosuppressive metabolites such as the nucleoside adenosine and downstream adenosine receptor signaling. Pharmacological inhibition of adenosine generation and signaling are active areas of clinical investigation. Here we report a novel mechanism whereby adenosine suppresses anti-cancer immune responses by intracellular accumulation. We show that human T cells readily take up adenosine via equilibrative nucleoside transporter 1 (ENT1), suppressing proliferation and effector function by inhibition of de novo pyrimidine nucleotide synthesis in T cells. ENT1 expression increases upon T cell activation, suggesting uptake of extracellular adenosine is a mechanism to limit T cell responses in high adenosine environments. Quantitative mass spectrometry imaging reveals adenosine concentrations in human tumors up to the range of 100 µM - levels significantly greater than previously demonstrated and consistent with suppression of T cell responses. Deletion of ENT1 leads to potent control of tumor growth in syngeneic mouse models including KPC, a poorly immunogenic model of pancreatic ductal adenocarcinoma, and is associated with increased CD8+ T cell frequency, proliferation and cytokine production within tumors. Furthermore, ENT1 expression was observed by flow cytometry on human tumor-infiltrating CD8+ T cells. We have discovered and characterized EOS301984, a potent ENT1 antagonist that is currently being evaluated in patients with advanced solid tumors. EOS301984 blocks intracellular adenosine transport and restores pyrimidine levels in T cells activated in the presence of adenosine, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes in adenosine-rich environments. Finally, in a humanized mouse model of triple negative breast cancer resistant to anti-PD-1 blockade (MDA-MB-231), combination of EOS301984 with nivolumab synergistically led to control of tumor growth. Thus, ENT1 inhibition represents a novel approach to augment anti-cancer immune responses through restoring pyrimidine nucleotide synthesis in T cells suppressed by adenosine. Citation Format: Theodore J. Sanders, Christopher S. Nabel, Margreet Brouwer, Annelise L. Hermant, Lucas Chaible, Jean-Philippe Deglasse, Angelique Pabois, Wilfried Cathou, Aurore Smets, Michael Deligny, Joao Marchante, Quentin Dubray, Marie-Claire Lettelier, Chiara Martinoli, Reece Marillier, Olivier De Henau, Yvonne McGrath, Matthew G. Vander Heiden, Erica Houthuys. Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 734.

Visualization research on ENT1/NIS dual-function gene therapy to reverse drug resistance mediated by MUC1 in GEM-resistant pancreatic cancer

PurposeTo use bifunctional target genes to increase the intracellular transport of gemcitabine (GEM) to reverse chemotherapy resistance and to simultaneously use reporter gene imaging to localize therapeutic genes. The therapeutic effect was evaluated by [18F]FLT PET/CT to visualize the effect of gene therapy. MethodsA viral gene vector containing the pancreatic cancer-targeting promoter MUC1 for specific transcription of equilibrative nucleoside transporter 1 (ENT1) and NIS (nuclide transport channel) was employed. [125I]NaI uptake tests and [131I]NaI SPECT imaging were performed to verify the function of NIS and the target function of MUC1. The correlation between [18F]FLT uptake and GEM resistance were assessed, and the influence ENT1 and thymidine kinase 1 (TK1) expression on [18F]FLT micro-PET/CT was measured, which provides a theoretical basis for the use of [18F]FLT micro-PET/CT to evaluate the efficacy of gene therapy. ResultsFirst, functions of gene therapy were confirmed: ENT1 reversed the drug resistance of GEM-resistant pancreatic cancer cells by increasing GEM intracellular transport; MUC1 drove NIS target gene expression in pancreatic cancer; and therapeutic genes could be localized using [131I]NaI SPECT reporter gene imaging. Second, the [18F]FLT uptake ratio was affected by drug resistance and GEM treatment. The mechanism underlying this effect was related to ENT1 and TK1. Increased expression of ENT1 inhibited the expression of TK1 after GEM chemotherapy to reduce the uptake of [18F]FLT. Finally, micro-PET/CT indicated that the SUVmax of [18F]FLT could predict survival time. SUVmax exhibited an increasing trend in resistant pancreatic cancer but a trend of inhibition after upregulation of ENT1, which was more significant after GEM treatment. ConclusionsBifunctional targeted genes can localize therapeutic genes through reporter gene imaging, reverse the drug resistance of GEM-resistant pancreatic cancer and be visually evaluated through [18F]FLT micro-PET/CT.

IFN‐stimulated metabolite transporter ENT3 facilitates viral genome release

An increasing amount of evidence emphasizes the role of metabolic reprogramming in immune cells to fight infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that the expression of equilibrative nucleoside transporter 3 (ENT3, encoded by solute carrier family 29 member 3, Slc29a3) is part of the innate immune response, which is rapidly upregulated upon pathogen invasion. The transcription of Slc29a3 is directly regulated by type I interferon‐induced signaling, demonstrating that this metabolite transporter is an interferon‐stimulated gene (ISG). Suprisingly, we unveil that several viruses, including SARS‐CoV‐2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of Slc29a3 expression is sufficient to significantly decrease viral replication in vitro and in vivo. Our study reveals that ENT3 is a pro‐viral ISG co‐opted by some viruses to gain a survival advantage.

Expression of equilibrative nucleoside transporters and activity regulation by hydrogen peroxide in cultured astrocytes

Expression of equilibrative nucleoside transporters and activity regulation by hydrogen peroxide in cultured astrocytes

Drug transporters expression on PBMC membrane is associated with tacrolimus intracellular concentration

Measuring tacrolimus (TAC) its whole blood concentrations (Cmin) is mandatory in liver and kidney transplant recipients (LTR and KTR). However, while having adequate Cmin, some patients still experience rejection/adverse events, highlighting the limitation of the actual monitoring. As TAC exerts its effect inside T cells, monitoring intracellular instead of whole blood concentrations is an appealing strategy. Some drug transporters expressed at the cells membrane could be major factors of TAC diffusion into peripheral blood mononuclear cells (PBMC). The aim of the study was to explore the relationship between TAC diffusion into PBMC and cell membrane expression of four drug transporters (P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP-2), Concentrative nucleoside transporter 3 (CNT-3) and Equilibrative nucleoside transporter 1(ENT-1)). Sixty stable LTR and KTR were included in the study. TAC Blood and PBMC concentrations were determined by liquid chromatography coupled with tandem mass spectrometry. P-gp, MRP-2, CNT-3 and ENT-1 expression at the PBMC surface was evaluated using flux cytometry. Correlation between TAC PBMC/blood ratios and transporters expressions were explored using correlation tests. TAC PBMC/blood ratios were not different between LTR and KTR and the results were then analyzed altogether. P-gp, CNT-3 and ENT-1 expressions show positive correlation with TAC PBMC/blood ratio. Correlation was stronger for ENT-1 (R2 = 0.31, p < 0.0001) than for P-gp and CNT-3 (R2 = 0.17, p = 0.001 and R2 = 0.14, p = 0.003, respectively). MRP-2 expression was not correlated with PBMC/blood ratio ( Fig. 1 ). In this study, TAC diffusion into PBMC increased as a function of membrane expression of ENT-1, CNT-3 and P-gp. ENT-1 expression seems to be the strongest driver of TAC diffusion, which is consistent with its role as an influx transporter. Findings for P-gp might be explained by an induction of its expression when intracellular TAC concentration increases. ENT-1 and CNT-3 influx effects on TAC diffusion would then outweigh the P-gp efflux effect.

EMT-Induced Gemcitabine Resistance in Pancreatic Cancer Involves the Functional Loss of Equilibrative Nucleoside Transporter 1.

Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.

Abstract PO-062: EMT-induced gemcitabine resistance in pancreatic cancer involves the functional loss of equilibrative nucleoside transporter 1

Abstract Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a significant loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, basolateral membrane localization and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, basolateral localization and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed immunohistochemical analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface co-expression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT. Citation Format: Brenna Weadick, Debasis Nayak, Avinash K. Persaud, Sau Wai Hung, Radhika Raj, Wei Chen, Junan Li, Terence M. Williams, Rajgopal Govindarajan. EMT-induced gemcitabine resistance in pancreatic cancer involves the functional loss of equilibrative nucleoside transporter 1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-062.

ENT1 inhibition attenuates apoptosis by activation of cAMP/pCREB/Bcl2 pathway after MCAO in rats

Background and purposeThe present study was designed to investigate the potential role and the mechanism of equilibrative nucleoside transporter 1 (ENT1) on neuronal apoptosis and neurological deficits after middle cerebral artery occlusion (MCAO) in rats. MethodsOne hundred and thirty-four male Sprague-Dawley rats were subjected to two hours of MCAO followed by reperfusion. The time course of the expression level of ENT1 and phosphorylation of CREB were detected by western blot and immunofluorescence staining. Another set of animals were administrated with NBTI, the ENT1 inhibitor, by daily intraperitoneal injection starting at 0.5 h post-MCAO, infarction volume and neurological deficits were measured both at 24 h and 72 h post MCAO. We further explored the neuroprotection machenism by using H89, cAMP dependent protein kinase inhibitor, the expression of Bcl-2, Bax, phosphorylated CREB and Cleaved caspase-3 were quantified by Western blot, neuronal apoptosis were analyed by TUNEL staining. ResultsThe endogenous expression of ENT1 were significantly increased and peaked at 12 h after MCAO. High-dose of NBTI (15 mg/kg) reduced brain infarction volume and improved neurologic deficits both at 24 h and 72 h post MCAO. Moreover, NBTI significantly increased the level of CREB phosphorylation and extracellular adenosine concentration, and decreased the neuronal apoptosis 24 h after MCAO. NBTI treatment reduced the expression of Bax and cleaved caspase-3, while up-regulated Bcl-2 compared with vehicle group. These effects were abolished by H89 pretreatment. ConclusionsENT1 inhibition prevented neuronal apoptosis and improves neurological deficits through cAMP/PKA/CREB/Bcl-2 signaling pathway after MCAO in rats. ENT1 might be an effective target in the treatment strategy for ischemic stroke.

Are ENT1/ENT1, NOTCH3, and miR-21 Reliable Prognostic Biomarkers in Patients with Resected Pancreatic Adenocarcinoma Treated with Adjuvant Gemcitabine Monotherapy?

Evidence on equilibrative nucleoside transporter 1 (ENT1) and microRNA-21 (miR‑21) is not yet sufficiently convincing to consider them as prognostic biomarkers for patients with pancreatic ductal adenocarcinoma (PDAC). Here, we investigated the prognostic value of ENT1/ENT1, miR-21, and neurogenic locus homolog protein 3 gene (NOTCH3) in a well-defined cohort of resected patients treated with adjuvant gemcitabine chemotherapy (n = 69). Using a combination of gene expression quantification in microdissected tissue, immunohistochemistry, and univariate/multivariate statistical analyses we did not confirm association of ENT1/ENT1 and NOTCH3 with improved disease-specific survival (DSS). Low miR-21 was associated with longer DSS in patients with negative regional lymph nodes or primary tumor at stage 1 and 2. In addition, downregulation of ENT1 was observed in PDAC of patients with high ENT1 expression in normal pancreas, whereas NOTCH3 was upregulated in PDAC of patients with low NOTCH3 levels in normal pancreas. Tumor miR‑21 was upregulated irrespective of its expression in normal pancreas. Our data confirmed that patient stratification based on expression of ENT1/ENT1 or miR‑21 is not ready to be implemented into clinical decision-making processes. We also conclude that occurrence of ENT1 and NOTCH3 deregulation in PDAC is dependent on their expression in normal pancreas.

Contribution of adenosine and ATP to the carotid body chemosensory activity in ageing.

Adenosine and ATP are excitatory neurotransmitters involved in the carotid body (CB) response to hypoxia. During ageing the CB exhibits a decline in its functionality, demonstrated by decreased hypoxic responses. In aged rats (20-24months old) there is a decrease in: basal and hypoxic release of adenosine and ATP from the CB; expression of adenosine and ATP receptors in the petrosal ganglion; carotid sinus nerve (CSN) activity in response to hypoxia; and ventilatory responses to ischaemic hypoxia. There is also an increase in SNAP25, ENT1 and CD73 expression. It is concluded that, although CSN activity and ventilatory responses to hypoxia decrease with age, adjustments in purinergic metabolism in the CB in aged animals are present aiming to maintain the contribution of adenosine and ATP. The possible significance of the findings in the context of ageing and in CB-associated pathologies is considered. During ageing the carotid body (CB) exhibits a decline in its functionality. Here we investigated the effect of ageing on functional CB characteristics as well as the contribution of adenosine and ATP to CB chemosensory activity. Experiments were performed in 3-month-old and 20- to 24-month-old male Wistar rats. Ageing decreased: the number of tyrosine hydroxylase immune-positive cells, but not typeII cells or nestin-positive cells in the CB; the expression of P2X2 and A2A receptors in the petrosal ganglion; and the basal and hypoxic release of adenosine and ATP from the CB. Ageing increased ecto-nucleotidase (CD73) immune-positive cells and the expression of synaptosome associated protein 25 (SNAP25) and equilibrative nucleoside transporter 1 (ENT1) in the CB. Additionally, ageing did not modify basal carotid sinus nerve (CSN) activity or the activity in response to hypercapnia, but decreased CSN activity in hypoxia. The contribution of adenosine and ATP to stimuli-evoked CSN chemosensory activity in aged animals followed the same pattern of 3-month-old animals. Bilateral common carotid occlusions during 5, 10 and 15s increased ventilation proportionally to the duration of ischaemia, an effect decreased by ageing. ATP contributed around 50% to ischaemic-ventilatory responses in young and aged rats; the contribution of adenosine was dependent on the intensity of ischaemia, being maximal in ischaemias of 5s (50%) and much smaller in 15s ischaemias. Our results demonstrate that both ATP and adenosine contribute to CB chemosensory activity in ageing. Though CB responses to hypoxia, but not to hypercapnia, decrease with age, the relative contribution of both ATP and adenosine for CB activity is maintained.

A single episode of binge alcohol drinking causes sleep disturbance, disrupts sleep homeostasis, and down-regulates equilibrative nucleoside transporter 1.

Binge alcohol drinking, a risky pattern of alcohol consumption, has severe consequences toward health and well-being of an individual, his family, and society. Although, binge drinking has detrimental effects on sleep, underlying mechanisms are unknown. We used adult male C57BL/6J mice and exposed them to a single, 4-h session of binge alcohol self-administration, in stress-free environment, to examine neuronal mechanisms affecting sleep. We first verified binge pattern of alcohol consumption. When allowed to self-administer alcohol in a non-stressful environment, mice consumed alcohol in a binge pattern. Next, effect of binge drinking on sleep-wakefulness was monitored. While sleep-wakefulness remained unchanged during drinking session, significant increase in non-rapid eye movement (NREM) sleep was observed during 4h of active period post-binge, followed by increased wakefulness, reduced sleep during subsequent sleep (light) period; although the timing of sleep onset (at lights-on) remained unaffected. Next, electrophysiological and biochemical indicators of sleep homeostasis were examined using sleep deprivation-recovery sleep paradigm. Mice exposed to binge drinking did not show an increase in cortical theta power and basal forebrain adenosine levels during sleep deprivation; NREM sleep and NREM delta power did not increase during recovery sleep suggesting that mice exposed to binge alcohol do not develop sleep pressure. Our final experiment examined expression of genes regulating sleep homeostasis following binge drinking. While binge drinking did not affect adenosine kinase and A1 receptor, expression of equilibrative nucleoside transporter 1 (ENT1) was significantly reduced. These results suggest that binge alcohol consumption-induced down-regulation of ENT1 expression may disrupt sleep homeostasis and cause sleep disturbances. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Abstract 4059: KPC34: a co-drug that combines a DNA damaging agent with a targeted therapy for the treatment of AML

Abstract AML occurs in the elderly with ~18,000 new US cases in 2014. Mortality is an estimated 10,050 US deaths in 2014. Elderly patient prognosis is a dismal 6.6%, 5 year survival rate. AML treatment involves induction chemotherapy with Ara-C followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Chemoresistance occurs by several mechanisms including reduced activation of Ara-C via down-regulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. The low expression of both dCK and ENT-1 correlates with a poor prognosis in patients with acute leukemias. Recurrent disease is more difficult to treat successfully. Elderly patients often have trouble tolerating repeated chemotherapy treatments. Activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms has been detected in AML. The activity of these isoforms promotes leukemic proliferation and survival by ERK activation and BCL-2. KPC34 is a novel synthetic phospholipid/deoxycytidine analog conjugate. KPC34's novel properties result in improved pharmacokinetics. It overcomes chemoresistance to Ara-C by bypassing ENT-1 uptake and dCK activation. KPC34 is administered orally. It crosses the blood-brain barrier, targeting CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety inhibits PKC activity. The study’s hypothesis is that KPC34 is active against AML in vivo. KPC34 is highly cytotoxic to a variety of leukemic cell lines in vitro, with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). In order to assess the activity of KPC34 in vivo, we injected NSG mice with OCI-AML-3 cells expressing luciferase. Following engraftment, mice were treated with KPC34, Ara-C, or water on days 1, 4 and 7. A significant prolongation of survival was observed in the KCP34-treated mice (median survival 44.5 days vs. 25 days for Ara-C-treated mice, p=0.0033, by log-rank test). Retreatment of the mice with additional KPC34 resulted in a median survival time of 78.5 days (p=0.0028, single KPC34 course vs. 2 additional treatment courses). KPC34 + doxorubicin is superior to the treatment of mice with Ara-C + doxorubicin. In a PDX murine model, KPC 34 showed a reduction in leukemic burden in the liver and spleen. KPC34 is well tolerated in vivo. KPC34 is effective against multiple models of naïve and chemoresistant leukemia. It has two distinct anti-neoplastic mechanisms. Oral administration improves efficacy over i.v. dosing. These data demonstrate KPC34 efficacy in multiple preclinical models of leukemia, and support its continued translation to the clinic. This co-drug has the potential to replace Ara-C-based therapies as a single agent/combo in naïve AML, a single agent in relapsed leukemia, and it reduces the CNS leukemic burden. Citation Format: Gregory L. Kucera, Peter Alexander, Kristin Pladna, Timothy S. Pardee. KPC34: a co-drug that combines a DNA damaging agent with a targeted therapy for the treatment of AML [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 4059. doi:10.1158/1538-7445.AM2017-4059

Equilibrative nucleoside transporter 1 expression in primary human hepatocytes is highly variable and determines uptake of ribavirin.

Aims Ribavirin is a nucleoside analogue and remains a necessary component of both interferon-based and directly acting anti-viral regimens for the treatment of hepatitis C virus infection. The achievable concentration of ribavirin within hepatocytes is likely to be an important determinant of therapeutic outcome. Invitro expression levels of equilibrative nucleoside transporter 1 (ENT1) has been shown to be a predictor of treatment response in patients receiving nucleoside-based chemotherapeutic agents. We therefore investigated whether a similar relationship existed between ENT1 expression and ribavirin uptake in freshly isolated primary hepatocytes. Methods Primary hepatocytes were cultured on collagen-coated plates and exposed to ribavirin. Parallel samples were taken for high-performance liquid chromatography to assess ribavirin uptake and for quantitative polymerase chain reaction to evaluate ENT1 expression. Similar assays were performed on the human hepatoma cell line (Huh7). ENT1 gene sequence was analysed by cloning of polymerase chain reaction amplified complementary DNA followed by direct sequencing. Results There was a strong direct correlation between expression of ENT1 in primary hepatocytes and ribavirin uptake at 24 hr. Huh7 cells expressed ENT1 at similar levels to the majority of primary hepatocytes, but did not take up ribavirin. Sequencing revealed that ENT1 in Huh7 cells is wild type. Conclusions In this study, we clearly demonstrate that ribavirin uptake in primary human hepatocytes is variable and correlates with ENT1 expression. This variation in ENT1 expression may account for differences in response rate in patients receiving ribavirin-based anti-hepatitis C virus therapy.

Low expression of equilibrative nucleoside transporter 1 is associated with poor prognosis in chemotherapy-naïve pT2 gallbladder adenocarcinoma patients.

Equilibrative nucleoside transporter 1 (ENT1) is the major transporter of the chemotherapeutic drug gemcitabine, the current therapy for advanced gallbladder cancer (GBC). ENT1 expression has been proposed as a predictive marker for gemcitabine-treated pancreatic cancer patients. The aim of study was to explore the value of ENT1 measurement in chemotherapy-naïve patients with advanced GBC. Immunohistochemistry for ENT1 was performed on 214 GBC samples from patients who had never undergone co-adjuvant or neo-adjuvant chemotherapy. Advanced GBC cases were divided into groups with low or high ENT1 expression. Kaplan-Meier tests were used for survival analyses. The Cox regression method was used to assess the association of ENT1 expression with overall survival (OS). Low ENT1 expression was associated with younger patient age (P = 0.03) and moderate-to-poor histological differentiation (P = 0.01). pT2 patients with low ENT1 expression had shorter median survival (17.3 versus 28.7 months) and lower OS (17.3% versus 33.3%, P < 0.05) than patients with high ENT1 expression. Low ENT1 expression was an independent prognostic factor for OS (P = 0.036). ENT1 is a prognostic marker for pT2 GBC patients. Additional studies are needed to determine whether ENT1 has predictive value for gemcitabine response in GBC.

Persistent Hepatitis C Virus Infection Impairs Ribavirin Antiviral Activity through Clathrin-Mediated Trafficking of Equilibrative Nucleoside Transporter 1

Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-λ1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection. The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has not been established. This study provides a potential mechanism for why RBV antiviral activity is impaired in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy to increase RBV antiviral activity against HCV infection. Therefore, it is anticipated that this work would generate a great deal of interest, not only among virologists but also among the general public.

The Anti-overactive Bladder Activity of KW-7158 Is Mediated by Blocking Equilibrative Nucleoside Transporter-1

KW-7158 is a novel therapeutic candidate for treating overactive bladder (OAB) with a unique mode of action: suppression of sensory afferent nerves. However, the molecular target of this compound remains unknown. We herein report the identification of the KW-7158 target to be equilibrative nucleoside transporter-1 (ENT1). A membrane protein expression library of ca. 7000 genes was expressed in a dorsal root ganglion cell line, which we had previously generated, and subjected to screening for binding with a fluorescent derivative that retains high binding activity to the target. The screening revealed that only cells transfected with an ENT1 expression vector exhibited significant binding. We next performed [(3)H]KW-7158 binding experiments and an adenosine influx assay and found that KW-7158 binds to and inhibits ENT1. To further demonstrate the pharmacological relevance, we evaluated other known ENT1 inhibitors (nitrobenzylthioinosine, dipyridamole, draflazine) in an in vitro bladder strip contraction assay and the rat spinal cord injury OAB model. We found that all of the inhibitors exhibited anti-OAB activities, of which the potencies were comparable to that of adenosine influx inhibition in vitro. These studies demonstrated that the pharmacological target of KW-7158 is ENT1, at least in the rat OAB model. Our results will aid understanding of the precise mechanism of action of this drug and may also shed new light on the use of the adenosine pathway for the treatment of OAB.

The Novel Phospholipid Gemcitabine Conjugate KPC34 Has Activity In Acute Leukemia

Abstract Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are cancers of the bone marrow that in adults, occur mainly in the elderly. About 19,000 Americans develop these diseases annually and the 5 year survival rate is 30-40%. The prognosis for elderly patients is even worse with a 5 year survival rate of 10%. Treatment for AML and ALL involves induction chemotherapy using the deoxycytidine analog cytarabine followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Recurrent disease is much more difficult to treat successfully and elderly patients often have trouble tolerating repeated chemotherapy treatments. Chemoresistance can occur by several mechanisms including reduced activation of cytarabine via downregulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. Low expression of both dCK and ENT-1 correlate with poor prognosis in patients with acute leukemias. Another contributor to cytarabine resistance is the interaction of leukemia cells with bone marrow stroma. This interaction confers protection to leukemia cells from cytarabine by inhibiting apoptosis and ENT-1 function. Additionally, activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms α, β1, and β2 have been detected in both AML and ALL and promote leukemic proliferation and survival by activation of ERK and the anti-apoptotic protein BCL-2. The development of novel therapeutic agents or modifying existing drugs may be able to overcome chemoresistance in AML and ALL and improve clinical outcomes. KPC34 is a novel phospholipid conjugate of the deoxycytidine analog gemcitabine. Gemcitabine acts similarly to cytarabine and is administered IV to treat various tumors and lymphomas, but is rarely used for leukemias. Compared to cytarabine, gemcitabine treatment inhibits DNA replication more effectively, prevents repair of damaged DNA, and uniquely inhibits ribonucleotide reductase. KPC34's novel properties result in improved pharmacokinetics compared to gemcitabine and may be able to overcome leukemic chemoresistance to cytarabine by bypassing the need for ENT-1 uptake and dCK activation. In addition, KPC34 can be administered orally, is predicted to cross the blood-brain barrier, allowing it to target CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety is predicted to inhibit the activity of PKC. In order to determine if KPC34 has activity against acute leukemias we tested it against a variety of human and murine leukemic cell lines in vitro. KPC34 was highly cytotoxic to with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). Exposure of leukemia cells to KPC34 resulted in the induction of apoptosis as assessed by annexin V and PI assays. Human leukemic cell lines treated with KPC34 had dose-dependent reduction of colony formation, where normal donor hematopoietic stem cells did not. To test the ability of KPC34 to overcome stroma-mediated chemoresistance, SUPB15 cells were incubated in the presence or absence of a bone marrow stroma cell line for 48 hrs. The cells were then treated with 10 nM cytarabine or KPC-34. No significant difference was seen between either treatment without stroma, while KPC34 significantly increased apoptosis in the presence of stroma (p value ≤ 0.001, by Student’s t test). In order to assess the activity of KPC34 in vivo, we injected C57/BL6 mice with a syngeneic, murine Ph+ pre-B cell ALL, luciferase expressing cell line. When engraftment was established by bioluminescence imaging, mice were treated with 25 mg/kg KPC34 or water by oral gavage on days 1, 4 and 7. Treatment resulted in a significant prolongation of survival with treated mice having a median survival of 26 days compared to 13 days for controls (p value of 0.0042, by log-rank test). These data demonstrate the activity of KPC34 in multiple preclinical models of leukemia and support its continued translation to the clinic. Disclosures: Kucera: Wake Forest Baptist Health: KPC 34 Patent, KPC 34 Patent Patents &amp; Royalties.

Ceftriaxone Treatment Affects the Levels of GLT1 and ENT1 As Well As Ethanol Intake in Alcohol-Preferring Rats

Studies have demonstrated that deletion of equilibrative nucleoside transporter 1 (ENT1) is associated with reduced glutamate transporter 1 (GLT1) level, and consequently increased ethanol intake. In this study, we measured changes in GLT1 and ENT1 levels in prefrontal cortex (PFC), and nucleus accumbens (NAc) core and shell associated with alcohol drinking in alcohol-preferring (P) rats. We examined, then, whether ceftriaxone (CEF) would affect both GLT1 and ENT1 levels in these brain regions. P rats were given 24-h concurrent access to 15 and 30% ethanol, water, and food for 5 weeks. On Week 6, P rats received 100 mg/kg CEF (i.p.) or a saline vehicle for five consecutive days. Ethanol intake was measured daily for 8 days starting on the first day of injections. We found a significant reduction in daily ethanol intake in CEF-treated group, starting on Day 2 of injections. Western blot for GLT1 and binding assay for ENT1 revealed downregulation of GLT1 level, whereas ENT1 levels were increased in the NAc core and NAc shell, respectively, but not in the PFC in saline vehicle group. Importantly, CEF treatment reversed these effects in both NAc core and shell. These findings provide evidence for potential regulatory effects of CEF on both GLT1 and ENT1 expression in reducing ethanol intake.

Characterization of the Vascular Phenotype of the Equilibrative Nucleoside Transporter 1 Knockout Mouse

Adenosine is important in the regulation of vascular tone and the Equilibrative Nucleoside Transporter 1 (ENT1) is a regulator of adenosine concentrations. Thus, changes in ENT1 expression and activity may alter vascular tone regulation. We therefore examined the cardiovascular phenotype of ENT1‐knockout (KO) as compared to wild type (WT). Heart rates (HRs) and blood pressures (BPs) of KO and WT littermate male mice were obtained. Vascular reactivity was assessed in aortic ring segments obtained from 3 and 6 month old KO and WT mice. Morphological assessment of vascularized tissues was performed using H&amp;E staining. BPs under awake and anesthetised conditions were not significantly different between KO and WT. However, awake HRs were significantly different (KO:570±22 vs. WT:674±22 beats; p&lt;0.01). However, anesthetised HRs were not. KCl‐mediated significantly greater constriction (&gt;;30%) in KO rings compared to WT. However, no significant differences between the two genotypes were observed for the different receptor agonists. H&amp;E staining yielded evidence of a decreased luminal area as well as thinner aortic wall in KO mice at the age of 3 months. The enhanced effect of KCl in KO aortic rings suggests a physiological difference in the regulation of the vasoconstriction response. Decreased lumen and aortic wall thickness suggests a compensatory mechanism due to reduced HR in KO mice. These studies suggest a vascular phenotype associated with a loss of ENT1. This model will aid in unraveling the role of adenosine in vascular regulation.

Characterization of the Vascular Phenotype of the Equilibrative Nucleoside Transporter 1 Knockout Mouse

Adenosine is important in the endogenous regulation of vascular tone. Equilibrative Nucleoside Transporter 1 (ENT1) is an established regulator of adenosine concentrations in vascular tissues. Thus, changes in ENT1 expression and/or activity may alter vascular tone regulation. We therefore examined the cardiovascular phenotype of ENT1‐knockout (KO) mice as compared to wild type (WT) mice. Heart rates and blood pressures of KO and WT littermate male mice were obtained. Vascular reactivity was assessed in aortic ring segments obtained from 3 and 6 month old KO and WT mice. KCl‐induced depolarization led to significantly greater constriction (~32% increase independent of tension applied or age) in KO rings compared to WT. However, no significant differences between the two genotypes were observed using a series of receptor‐selective constrictors. Morphological assessment of H&amp;E stained vascularized tissues yielded no significant difference in luminal area or arterial wall thickness between WT and KO mice at the age of 3 months. The enhanced effect of KCl in KO aortic rings suggests a physiological difference in the regulation of depolarization‐induced, but not receptor‐mediated, vasoconstriction response in aorta. Further assessment of this model will aid in our understanding of the role of adenosine in vascular regulation.