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Therapeutics, Targets, and Chemical Biology

OCTN1 Is a High-Affinity Carrier of NucleosideAnaloguesChristina D. Drenberg1, Alice A. Gibson1, Stanley B. Pounds2, Lei Shi2,Dena P. Rhinehart3, Lie Li3, Shuiying Hu1, Guoqing Du4, Anne T. Nies5,6,Matthias Schwab5,7, Navjotsingh Pabla1,William Blum8, Tanja A. Gruber4,Sharyn D. Baker1, and Alex Sparreboom1

Abstract

Resistance to xenobiotic nucleosides used to treat acute mye-loid leukemia (AML) and other cancers remains a major obstacleto clinical management. One process suggested to participate inresistance is reduced uptake into tumor cells via nucleosidetransporters, although precise mechanisms are not understood.Through transcriptomic profiling, we determined that low expres-sion of the ergothioneine transporter OCTN1 (SLC22A4; ETT)strongly predicts poor event-free survival and overall survival inmultiple cohorts of AML patients receiving treatment with thecytidine nucleoside analogue cytarabine. Cell biological studies

confirmed OCTN1-mediated transport of cytarabine and variousstructurally related cytidine analogues, such as 20deoxycytidineand gemcitabine, occurs through a saturable process that is highlysensitive to inhibition by the classic nucleoside transporter inhi-bitors dipyridamole and nitrobenzylmercaptopurine ribonucleo-side. Our findings have immediate clinical implications giventhe potential of the identified transport system to help refinestrategies that could improve patient survival across multiplecancer types where nucleoside analogues are used in cancertreatment. Cancer Res; 77(8); 2102–11. �2017 AACR.

IntroductionAll endogenous and xenobiotic nucleosides are polar hydro-

philic compounds that are poorly membrane permeable andrequire functional transporters to enter cells. The two majorclasses of mammalian nucleoside transporters consist of equili-brative nucleoside transporters (ENT) and concentrative nucleo-side transporters (CNT). Two proteins of the former class, ENT1(SLC29A1) and ENT2 (SLC29A2),mediate the transport of purineand pyrimidine nucleosides down their concentration gradients.These transporters exhibit broad permeant selectivity and aresubdivided on the basis of their sensitivity (ENT1) or resistance(ENT2) to inhibition by nanomolar amounts of nitrobenzylmer-captopurine ribonucleoside (NBMPR; ref. 1).

One of the first pyrimidine nucleoside to be developed as ananticancer drug was cytarabine (cytosine arabinoside; Ara-C), aderivative of 20-deoxycytidine (2). Intracellular accumulation ofcytarabine depends on plasma concentrations of the drug and thecompound enters cells exclusively by transporter-mediated pro-cesses (3). Once cytarabine has entered cells, it is phosphorylatedby deoxycytidine kinase to the monophosphorylated, 50-deriva-tive Ara-CMP and subsequently by nucleotide kinases to thepharmacologically active, triphosphorylated metabolite Ara-CTP(4). Cytarabine is used clinically in the treatment of hematologicmalignancies, in particular in acute myeloid leukemia (AML),although intrinsic insensitivity and/or the development of resis-tance remains a major obstacle to successful long-term treatmentoutcome (5). Reduced uptake into leukemic cells has been pro-posed as a process underlyingmost instances of clinical resistanceto cytarabine, although the responsible mechanism remainspoorly understood (6).

Previous investigations demonstrated that low cytarabineuptake in AML cells predicts poor response to therapy (7), thatcytarabine uptake is required for its conversion to Ara-CTP byleukemic blast cells, and that myeloblasts form more Ara-CTPthan lymphoblasts because of higher nucleoside transport (8).Interestingly, cytarabine uptake in AML cells is completely inhib-ited by nanomolar concentrations of NBMPR and dipyridamole(9). Because the uptake of cytarabine in leukemic blasts is highlysensitive to NBMPR, it has been assumed and asserted for severaldecades that cytarabine enters cells through an ENT1-dependentmechanism (1). This conclusion is supported by some (10, 11)but not all statistical association studies (12–14), indicating thatincreased ENT1 mRNA abundance is correlated with increasedcellular sensitivity or clinical responsiveness to cytarabine. Moreimportantly, however, published studies to date employing het-erologous expression systems have shown that cytarabine is either

1Division of Pharmaceutics, College of Pharmacy, The Ohio State University,Columbus, Ohio. 2Department of Biostatistics, St. Jude Children's ResearchHospital, Memphis, Tennessee. 3Department of Pharmaceutical Sciences, St.Jude Children's Research Hospital, Memphis, Tennessee. 4Department of Oncol-ogy, St. Jude Children's Research Hospital, Memphis, Tennessee. 5Dr. MargareteFischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. 6Univer-sity of T€ubingen, T€ubingen, Germany. 7Department of Clinical Pharmacology,University Hospital, T€ubingen, Germany. 8Division of Hematology, The Depart-ment of Internal Medicine, The Ohio State University Comprehensive CancerCenter, The Ohio State University, Columbus, Ohio.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Sharyn D. Baker, Division of Pharmaceutics and Phar-maceutical Chemistry, College of Pharmacy, The Ohio State University, 500 W.12thAve., Columbus, OH43210. Phone: 614-685-6016; Fax: 614-688-4028; E-mail:[email protected]; and Alex Sparreboom, [email protected]

doi: 10.1158/0008-5472.CAN-16-2548

�2017 American Association for Cancer Research.

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veryweakly transportedbyENT1 (<2-fold vs. control; refs. 15, 16),or is not transported by ENT1 at all (17). Against this background,the elucidation of novel, ENT1-independent transport mechan-isms leading to antileukemic effects following cytarabine treat-ment is considered of high clinical relevance. In the current study,we report that the ability of cytarabine and several structurallyrelated nucleosides to enter cells is facilitated by the ergothioneineuptake transporter OCTN1 (SLC22A4; ETT), and that low expres-sion of OCTN1 in leukemic cells is a strong predictor of poorsurvival in multiple cohorts of patients with AML treated withcytarabine-based regimens.

Materials and MethodsAdditional details of materials andmethods are reported in the

Supplementary Methods.

Clinical association studiesGene expression data were collected from 168 pediatric

patients with de novo AML enrolled on the AML02 trial(18, 19), of whom 134 were included in the analysis [2 were notrandomized; 14were excluded due to lack of induction Iminimalresidual disease (MRD) assessment; 1 was excluded due to lack ofFLT3-ITD status; and 17 were excluded due to lack of French-American-British (FAB) classification status]. Total RNA was iso-lated from primary blast samples, as described previously(18, 19). Data on all 392 SLC (uptake) transporter probe setsfrom the Affymetrix U133Av2 expression array were extracted,log2 transformed, normalized by Z-score, visualized using Rsoftware package (R Studio v0.98.1091), and tested for statisticalassociation with overall survival (OS) and event-free survival(EFS). Of the transporter probe sets, 22 were associated with bothOS and EFS (P < 0.05), among which 14 were associated with aHR < 1 and improved survival. After exclusion of genes encodingtransporter proteins not localized to the outer membrane,SLC22A4 (OCTN1) was identified as the top-ranking gene forfurther consideration (Supplementary Table S1). The associationbetween SLC22A4 expression and OS and EFS was tested by 3different Cox regression models. The first model was only strat-ified by treatment arms, the second was adjusted by risk andstratified by treatment arms, and the third was adjusted by age,MRD, FLT3-ITD, core binding factor (CBF), FAB category AML-M7without t(1;22) and other 11q23, and stratified by treatment arm.To illustrate the association between gene expression and survivaloutcome in Kaplan–Meier plots, SLC22A4 expression values werecategorized into 3 groups basedonquantile ranking.Other cut-offpoints for grouping were considered, for example, on the basis ofpossible gaps within the dataset after plotting expression datawithin their empirical distribution function. However, no gapswere observed, suggesting that quantile ranking is an appropriatestrategy to cluster expression values. Complete remission (CR)status was designated as less than 5% of blasts in the bonemarrow, and induction failure was defined as the absence of CRwithin 3 months of the start of treatment.

External validation was performed on published data from 54pediatric patients with AML receiving cytarabine-based chemo-therapy on whom gene expression (Affymetrix U95Av2 micro-array) and clinical data (EFS and CR) were available (see Sup-plementary Table S2 in ref. 14). Gene expression values providedin the published report were transformed to percentiles such thatthe HR can be explained as a change of event rate per one

percentile change of expression from the lowest expression valueobserved. Additional external validation studies on OS and EFSinvolved gene expression analyses on the AML gene expressiondataset from The Cancer Genome Atlas (TCGA), consisting ofRNAseq data from Illumina Hi-seq analyses on 172 samples fromadult patients with de novo AML (20). The information of interestwas extracted using the cBioPortal for Cancer Genomics (21, 22).

Source of cell linesThe human embryonic kidney cell line HEK293 (Invitrogen),

HeLa, and Chinese hamster ovary (CHO; ATCC) were obtainedfrom commercial sources and used without further authenti-cation by the authors. The PK15 and PK15NTD cell lines wereprovided by Chung-Ming Tse (Johns Hopkins University, Balti-more,MD) . The AML cell line CHRF288-11 cell linewas obtainedfrom Tanja Gruber (St. Jude Children's Research Hospital, Mem-phis, TN); CMS from Yubin Ge (Karmanos Cancer Institute,Detroit, MI); M07e, MOLM-13, ML-2, and NB4 from DSMZ;MV4-11, HL-60, KG-1, THP-1, and U937 from ATCC; andOCI-AML3 from Brian Sorrentino (St. Jude Children's ResearchHospital, Memphis, TN). All cell lines were tested regularly formycoplasma contamination using a commercially available kit(Lonza). Passages were kept to a minimum and the cells were notused beyond passage 30.

Transfection of HEK293 cellsHEK293 was used for the generation of OCTN1- and ENT1-

overexpressing models, because these cells had the lowest nativeuptake of cytarabine compared with other cells commonly usedfor transfection, such as the cervical cancer cell line HeLa or CHOcells (0.94 vs. 5.4 vs. 4.6 pmol/mg/min, respectively). Interest-ingly, OCTN1 has been immune-detected in HeLa cells (23),and the relatively high uptake of cytarabine in CHO cells waspreviously connected with the number of NBMPR-binding sites(24). Reconstructed OCTN1 and ENT1 cDNAs (Origene) with aFlag-tag were subcloned into the pMIG II vector, engineeredfrom a MSCV-IRES-GFP vector. Empty vector (VC) or the vectorencoding OCTN1-Flag or ENT1-Flag were cotransfected withpEQ-Pam3(-E) and pVSVg into 293T cells using Fugene trans-fection reagent (Roche). Virus-containing medium was collectedat 72 hours and used to infect HEK293 cells in the presenceof hexadimethrine bromide (polybrene; 6 mg/mL). Seven to10 days after infection, GFP-positive cells were sorted by FACSto approximately 95% purity. Cells were maintained in DMEM(Life Technologies) supplemented with 10% FBS (Hyclone) at37�C with 5% CO2.

The expression levels ofOCTN1 in the transfectedHEK293 cellswere confirmed by RT-PCR and Western blot analysis (Supple-mentary Fig. S1A), and plasmamembrane localization ofOCTN1was demonstrated by confocal microscopy (Supplementary Fig.S1B). Additional characterization of the model system with geneexpression arrays revealed detectable transcripts in HEK293 cellsof other putative cytarabine transporter genes, including thoseencoding CNT1, CNT2, CNT3, ENT1, ENT2, ENT3, and ENT4(Supplementary Fig. S1C), as well as OCTN1-related genes,including those encoding OAT1, OAT2, OAT3, OCTN2, OCT1,OCT2, and OCT3 (Supplementary Fig. S1D). QuantitativeRT-PCRwith Transporter RT2 Profiles PCR arrays (SupplementaryFig. S1E), Western blot analysis (Supplementary Fig. S1F), andfunctional analysis (Supplementary Fig. S1G), using uridine as a

Nucleoside Transport by OCTN1

www.aacrjournals.org Cancer Res; 77(8) April 15, 2017 2103




substrate for ENT1 and carnitine as a substrate for OCTN2,confirmed that these transporters were not differentiallyexpressed in cells transfected with VC as compared with cellsoverexpressingOCTN1. Functionally, theOCTN1-overexpressedHEK293 cells were further characterized by their ability to effi-ciently accumulate the knownOCTN1 substrates, ergothioneine,a naturally occurring thiourea derivative of histidine (25),as well as 14C-labeled tetraethylammonium (TEA), a small-molecule organic cation (26). The observed transport efficienciesfor ergothioneine (62 � 8 mL/mg/min) and TEA (1.1 � 0.12mL/mg/min) were similar to those reported previously (25).Phenotypic characterization of the heterologous expressionmodel of ENT1 indicated efficient accumulation of the knownsubstrates uridine (3.3-fold vs. control cells) and gemcitabine(1.5-fold). These observations suggest that the overexpressedHEK293 cells represent a bona fide model to evaluate a possiblecontribution of OCTN1 and ENT1 to the transport of AML-directed chemotherapeutics, such as cytarabine.

Real-time PCRRNA was reverse transcribed using SuperScript III First Strand

Synthesis supermix for real-time RT-PCR (Invitrogen), accordingtomanufacturer's recommendations. Gene transcripts were quan-tified using SYBR Green PCR mastermix (Qiagen) and primersobtained from Applied Biosystems that were specific to OCTN1,ENT1, and other transporters of interest. Reactions were carriedout in triplicate, with transcripts normalized to GAPDH.

Gene-chip arraysRNA was extracted from HEK293 cells using the RNEasy Mini

Kit (Qiagen), samples were amplified, and then analyzed usingthe Affymetrix GeneChip arrays. Data on 293 solute carrier geneswere extracted, normalized by the RMA algorithm, and analyzedusing the Partek Genomics Suite 6.4 software.

Immunoblot analysisForprotein expressionof select transporters, cellswere lysed, and

crude membrane fractions were prepared using the ProteoExtractNative Membrane Protein Extraction Kit (Calbiochem), accordingto themanufacturer's protocol. Protein concentration of themem-branepreparationsweredeterminedusing thePierceBicinchoninicAcid (BCA) Protein Assay Kit (Thermo Fisher Scientific) in micro-plates. Membrane samples (20 mg) were separated by SDS-PAGEand transferred to polyvinylidene difluoride (PVDF). Western blotanalysis was performed using a-OCTN1 (1:500; Thermo FisherScientific), a-ENT1 (1:1,000, Invitrogen), a-OCTN2 (1:1,000,Invitrogen), or a-transferrin receptor (TfR1; 1:1,000; Invitrogen).Secondarya-rabbit ora-mouseantibodies (1:2,000) conjugated toperoxidase (Jackson ImmunoResearch) were used and proteinswere visualized by chemiluminescence using the SignalFire ECLReagent (Cell Signaling Technology) on an Odyssey Fc ImagingSystem (LI-COR). Immunoblots were performed a minimum oftwo times on samples collected from different experiments.

Site-directed mutagenesisThe QuikChange II XL Site-Directed Mutagenesis Kit (Agilent)

was utilized to generate the OCTN1 variant rs1050152(c.1507C>T; L503F), according to suggested methods. The muta-genesis primers were designed using the QuikChange PrimerDesign program and synthesized by Integrated DNA Technolo-

gies. The plasmid was sequenced to confirm successful mutagen-esis and then used to generate stably transfected HEK293 cells.

Cellular accumulation studiesCells were seeded at 5� 105 cells/well in 6-well plates. At 70%

confluence, cells were washed with PBS and replaced with fresh,serum-free medium containing varying concentrations of radi-olabeled or unlabeled compound. At the end of the incubationperiod, the medium was removed, and cells were washed twicewith ice-old PBS.A25-mL aliquot of the lysatewas used to estimateprotein concentration using aBCAProteinAssayKit. Radioactivitywasmeasured on a Perkin Elmer Topcount or a Beckman LS 6500liquid scintillation counter, after mixing the sample with Scin-tisafe 30% scintillation fluid (PerkinElmer). Cytarabine andphosphorylated metabolites were measured in cell extracts byLC/MS-MS, as described previously (27). Cellular accumula-tion was expressed as pmol per mg of protein (pmol/mg), as apercentage of control cells (%VC) with vector set to 100%, or asa concentration-normalized transport efficiency (mL/mg/min).At least two independent experiments were performed usingmultiple replicates.

Cytotoxicity assaysTo evaluate cytotoxicity in response to cytarabine and related

nucleoside analogues, HEK293 cells were seeded at 2,000 cells perwell in 96-well plates and cultured for 20 hours. Cells were thentreated with nucleosides of interest at increasing concentrations(0.313–100 mmol/L). Cell viability was determined using aCellTiter-Glo luminescent cell viability assay at 72 hours (Pro-mega). The concentration of drug that inhibited cell proliferationby 50% (IC50) was determined using the software programGraphPad Prism version 5.0 (GraphPad Software).

Silencing of OCTN1 expressionThe on-target plus SMART pool human SLC22A4 siRNA for

OCTN1 silencing (Dharmacon) and the Mission siRNA NegativeNontargeting Control (NT) #1 siRNA (Sigma) were used in allexperiments. OCI-AML3 cells were transfected with OCTN1siRNA or negative control siRNA using Nucleofector II andNucleofector Kit T (Amaxa), according to the manufacturer'sprotocol. Briefly, OCI-AML3 cells (3 � 106) were treated with3 mg of siRNA for each sample, and OCTN1 suppression wasevaluated using crude membrane fractions extracted 48 hourslater using ProteoExtract Native Membrane Protein Extraction Kit(Calbiochem). Next, immunoblots were performed with 10 mgmembrane samples using SDS-PAGE and then transferred toPVDF membranes. The membrane was probed using OCTN1(1:500; Thermo Fisher Scientific) or a-transferrin receptor(1:1,000; Invitrogen), and a secondary a-rabbit or a-mouse IgGconjugated to peroxidase (Jackson ImmunoResearch) was used at1:10,000 or 1:1,000. Proteins were visualized by chemilumines-cence with the Signal Fire ECL Reagent (Cell Signaling Techno-logy), and quantitated by ImageJ software. Immunoblots wereperformed a minimum of two times on samples collected fromdifferent experiments.

Cellular uptake assay in OCI-AML3 cells was also determinedafter 48-hour transfection with OCTN1 siRNA or control. Cellswere preincubated with DMSO or NBMPR (10 mmol/L) for 15minutes, followed by addition of [3H]cytarabine (1 mmol/L), anduptake assays were performed with 5-minute incubation times.

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After incubation, cells were washed three times with ice-coldPBS, lysedwith1NNaOH, andagitated for 1hour. Lysate aliquotsof 25 mL were used to estimate protein concentration using aBCA Protein Assay Kit (Thermo Fisher Scientific). Radioactivitywas measured on a liquid scintillation analyzer Tri-Carb 4810TR (Perkin Elmer) after mixing the sample with 4,000 mL ofEmulsifier-Safe scintillation fluid (Perkin Elmer). Cytarabineuptake levels were normalized to total protein levels in eachgroup, and expressed relative to the uptake observed in cells tran-sfected with the NT siRNA.

Statistical analysisData are presented as mean values and SEM, unless stated

otherwise. An unpaired two-sided Student t test or one-wayANOVA with a Tukey post hoc test were used to evaluate statisticalsignificance, using P < 0.05 as a cutoff.

Results and DiscussionIdentification of OCTN1 as a predictor of survival in AML

As the commonly held contention of ENT1 playing animportant or even exclusive role in cytarabine transport intoAML cells is poorly supported by experimental evidence, wepostulated the existence onmyeloblasts of a currently unknown,highly efficient cytarabine carrier. To identify such a new puta-

tive cytarabine uptake transporter, we performed comprehen-sive transcriptomic profiling of blast samples from patients withAML receiving cytarabine-based chemotherapy. This analysiswas based on the expectation that such an approach wouldidentify genes that, when overexpressed on AML blasts, increasemediated intracellular accumulation of cytarabine, and conse-quently associate with improved responsiveness. Using theAffymetrix U133Av2 platform, we interrogated 392 transporterprobe sets, captured on the microarray in 134 pediatric patientswith de novo AML treated on the AML02 multicenter trial(18, 19). After adjusting for known prognostic factors, includingage, risk group, and treatment arm, the gene most significantlyassociated with both OS (P ¼ 0.0074; HR, 0.56) and EFS (P ¼0.024; HR, 0.67) was SLC22A4, a gene encoding the ergothio-neine uptake transporter OCTN1 (ETT; Fig. 1A and B; Table 1).In particular, we observed that increased expression of SLC22A4(probe set ID, 205896_at) was associated with reduction in therate of relapse, resulting in an improved survival rate. Thisfinding was independently replicated in a second cohort of54 pediatric patients with AML for whom EFS data were available(P ¼ 0.0074; HR, 0.50), and who received a similar cytarabine-based treatment regimen (Fig. 1C; ref. 14). In addition, increasedexpression of SLC22A4 was associated with improved OS (P ¼0.0048) and EFS (P ¼ 0.0091) in 172 clinically annotated adultcases of de novo AML (Fig. 1D and 1E). Recent genetic association

Figure 1.

Association of OCTN1 gene expression with survival in AML. Kaplan–Meier plots show rates of OS and EFS, according to leukemic blast expression of theOCTN1 transporter gene SLC22A4, in a cohort of 134 pediatric patientswith AML (A andB), an independent cohort of 54 pediatric patientswith AML (C), and a cohortof 172 adult patients with AML (D and E), all receiving cytarabine-based chemotherapy. Gene expression ranks represent low expressors (bottom 1/3, red),intermediate expressors (middle 1/3, black), and top expressors (top 1/3, blue).






studies have revealed the molecular genetic heterogeneity of AMLand have shown that mechanisms of resistance are highly het-erogeneous (28). In this connection, it is of interest to point outthat the expressionof SLC22A4was significantly higher in patientsentering CR as compared with those experiencing inductionfailure or relapse within the first year of CR in both the AML02trial (P¼0.029) and the pediatric replication cohort (P¼0.0014).Thisfinding suggests that the observed associationofOCTN1withsurvival is unrelated to heterogeneity of the disease but ratherwithit being a predictor of treatment response.

Identification of OCTN1 as a high-affinity cytarabinetransporter

In the gene expression association study that led to the identi-fication of SLC22A4 as a predictor of survival in AML, patientsreceived combination chemotherapy involving cytarabine, dauno-rubicin, etoposide, and mitoxantrone (19). An initial examination

Table 1. Correlation of SLC22A4 expression with survival in AML

Modela P HR (95% CI)

OS1 0.0077 0.5939 (0.4048–0.8712)2 0.0033 0.5540 (0.3738–0.82123 0.0074 0.5611 (0.3677–0.8562)

EFS1 0.0184 0.6645 (0.4731–0.9333)2 <0.0001 0.2089 (0.1000–0.4368)3 0.0240 0.6694 (0.4724–0.9484)

Abbreviation: CI, confidence interval.aModel 1 is only stratified by treatment arms; Model 2 is adjusted by risk andstratifiedby treatment arms;Model 3 is adjustedbyage,MRD, FLT3-ITD, CBF,M7without t(1;22) and other 11q23, which are the factors that were found to bestatistically significant in the original association analyses (18, 19), and stratifiedby treatment arm. Note that independent of the model, SLC22A4 expression issignificantly associated with both OS and EFS outcomes, with higher expressionlevels being associated with better survival outcomes (i.e., HR significantly lessthan 1).

Figure 2.

In vitro transport of cytarabine by OCTN1. A, Characterization of the transport of various AML-directed therapeutics (concentration, 1 mmol/L; 5-minute uptake)was performed in HEK293 cells transfected with an VC or OCTN1. B, A comparison of OCTN1- and ENT1-mediated transport of cytarabine was done inHEK293 cells. C, Time-dependence of cytarabine (Ara-C) transport by OCTN1 at early time points (range, 10–300 seconds). D, Sensitivity of OCTN1-mediatedcytarabine transport to temperature, sodium, pH, and inhibitors. E and F, Concentration-dependent transport of cytarabine (1–50 mmol/L; 5-minute uptake)by OCTN1 (E), and these data shown as an Eadie–Hofstee transformation (F). Data are shown as mean values (symbols) and SEM (error bars), using 9–60observations per group. Solid lines represent a fit of the experimental data to a nonlinear maximum-effect model or linear regression. v, transport velocity;[S], substrate concentration.

Drenberg et al.





of these 4 compounds in our heterologous expression modelrevealed that cytarabine uptake was increased by 60- to 70-fold inHEK293 cells engineered to overexpress OCTN1 compared withcontrol cells, whereas the increases ranged only 1.1- to 1.5-fold forthe 3 other drugs (Fig. 2A). The weak interaction observed herebetween OCTN1 and etoposide or mitoxantrone is in line withpreviously reported uptake data (29, 30), andwith the notion thatetoposide can competitively inhibit the specific binding ofNBMPR to an unidentified cytarabine carrier on AML blasts(31). Importantly, in a comparative analysis, we found that theefficiency of cytarabine transport by OCTN1was greater than thatobserved for ENT1 (Fig. 2B) in the same HEK293 cell–basedmodel system. Subsequent studies indicated that OCTN1-medi-ated cytarabine transport was time-dependent (Fig. 2C), sensitiveto temperature, pH-dependent as observed for TEA transport byOCTN1 (32), saturable (Fig. 2D), and was associated with a

Michaelis–Menten constant (Km) of 1.95 � 0.37 mmol/L and amaximum velocity (Vmax) of 193 pmol/mg/min (Fig. 2E). Inaddition, an Eadie–Hofstee analysis revealed that the transporterinteraction with cytarabine involved a single, saturable binding-site on the OCTN1 protein (Fig. 2F). Interestingly, whereas theOCTN1-mediated transport of ergothioneine is dependent onsodium (33), no pH dependence was found for the transport ofcytarabine by OCTN1, although the absolute intracellular druguptake was decreased in the absence of sodium (Fig. 2C). How-ever, a proportionally similar decrease in cytarabine uptake wasobserved in vector control cells in the absence of sodium, indi-cating that the net OCTN1-mediated uptake is independent ofsodium. This unusual phenomenon has been reported previouslyfor pyrilamine, verapamil, and etoposide in cells transfected withOCTN2 (29), although the mechanism underlying this phenom-enon remains unclear.

Figure 3.

Inhibition of OCTN1-mediated cytarabine transport. A, Characterization of the transport of cytarabine (Ara-C; concentration, 1 mmol/L; 5-minute uptake) inthe absence or presence of putative OCTN1 inhibitors (concentration, 10–50 mmol/L) was performed in HEK293 cells, transfected with an VC or OCTN1. B and C,Influence of NBMPR on the transport of cytarabine in various cell lines (B) and primary human epidermal keratinocytes (C). D, Concentration-dependence of theinhibitory properties of NBMPR and dipyridamole on OCTN1-mediated transport of cytarabine was assessed over a 0.01–10 mmol/L concentration range (5-minuteuptake). E, The time-dependent influence of NBMPR (10 mmol/L) on the formation of the pharmacologically active metabolite cytarabine triphosphate (Ara-CTP)in cells exposed to cytarabine (10 mmol/L; 5- to 30-minute uptake) was determined by LC/MS-MS. Data are shown as mean values (bars or symbols) andSEM (error bars), using 6–15 observations per group. Solid lines represent a fit of the experimental data to an inverse nonlinear maximum-effect model(D). IC50, concentration required to inhibit OCTN1-mediated cytarabine transport by 50%; <LLQ, lower than the lower limit of quantitation of the analytic assay.A schematic of OCTN1-mediated transport, metabolism, and (in)activation of cytarabine in cells is shown in F. 1, NBMPR-sensitive transport by OCTN1; 2,deoxycytidine kinase; 3, deoxycytidine monophosphate kinase; 4, nucleoside diphosphate kinase; 5, cytoplasmic 50-nucleotidase; 6, cytidine deaminase.






Next, we found that OCTN1-mediated transport of cytara-bine was highly sensitive to a 10- to 50-fold molar excess ofcytarabine, its structurally related endogenous nucleoside, 20-deoxycytidine, or the classic nucleoside transporter inhibitors,NBMPR and dipyridamole (Fig. 3A). The transport of cytara-bine could also be inhibited by NBMPR in the Sus scrofaepithelial kidney cell line PK15 (34), Flp-In 293 cells engi-neered to overexpress OCTN1, and several AML cell lines(Fig. 3B). However, cytarabine transport was insensitive to thepresence of the known OCTN1 substrates ergothioneine andTEA, or to the known OCTN1 inhibitor cimetidine (Fig. 3A;ref. 35). The notion that compounds such as ergothioneine andTEA do not competitively inhibit cytarabine transport suggeststhe possible existence of a substrate recognition site on OCTN1for cytarabine that is distinct from that used by ergothioneineand other low molecular weight cations. This is consistent withthe observation that even high micromolar concentrations ofNBMPR did not inhibit OCTN1-mediated transport ofergothioneine in HEK293 cells or in human primary epidermalkeratinocytes (Fig. 3C), cells that are known to take up nucleo-sides such as cytarabine and clofarabine (36), and that func-tionally express OCTN1 (37). Further examination in HEK293cells revealed that the NBMPR- and dipyridamole-mediatedinhibition of cytarabine transport by OCTN1 was associatedwith low nanomolar IC50 values (Fig. 3D), which are in thesame range as levels required to inhibit ENT1 function orcytarabine uptake in human myeloblasts (38).

The formation of Ara-CTP in HEK293 cells overexpressingOCTN1 increased in a time-dependent manner, and could becompletely prevented by NBMPR (Fig. 3E), suggesting that its

mechanism of interaction involves a restriction of cytarabineaccess to the intracellular compartment (Fig. 3F). In contrast, thepresence of MK571, an inhibitor of ATP-binding cassette trans-porters involved in the cellular efflux of cytarabine and its mono-phosphorylated metabolite Ara-CMP (39), did not substantiallyinfluence the extent of radioactivity accumulating following a5-minute exposure of OCTN1-expressing HEK293 cells tocytarabine (Supplementary Fig. S2A). The lack of substantialefflux occurring during the experimental conditions appliedwas confirmed by measuring the time-course of extracellularcytarabine–derived radioactivity after loading of cells withcytarabine (Supplementary Fig. S2B).

OCTN1 expression in myeloid cells affects cytarabine uptakeBefore evaluating the contribution of OCTN1 to cytarabine

transport in AML cells, we confirmed that SLC22A4 was presenton AML blasts, and found that it was highly variable betweenpatients and among different AML subtypes (Fig. 4A). More-over, the OCTN1 protein was expressed in a majority ofcommonly used AML cell lines (Fig. 4B). We next comparedthe uptake of cytarabine in one of these cells lines with reducedOCTN1 expression after siRNA. Compared with cells trans-fected with a nontargeting control siRNA, partial silencing ofOCTN1 resulted in significantly decreased uptake of cytarabine(Fig. 4C). These results are consistent with the involvement ofOCTN1 as an uptake transporter of cytarabine that can conferdrug sensitivity in AML cells.

Interestingly, we recently reported that the presence ofa functional polymorphic germline variant in SLC22A4(rs1050152; c.1507C>T; L503F; ref. 40) is significantly correlated

Figure 4.

Influence of OCTN1 on cytarabine uptake in AML cells. A, Gene expression of OCTN1 and ENT1 (two probe sets) in primary AML blast samples from pediatricpatients used in the survival analysis shown in Fig. 1A and B, as determined by microarray analysis. Each column represents an individual primary sample, andcolumns are categorized by cytogenetic AML subtypes of prognostic relevance. B, Protein expression of OCTN1 in a panel of 12 AML cell lines. C, Cellularuptake of cytarabine (Ara-C; 1 mmol/L; 5-minute uptake) with or without NBMPR preincubation was measured in OCI-AML3 cells, 48 hours after transfection with anontargeting (NT) control siRNA or a siRNA-targeting OCTN1. Results are shown as cytarabine uptake as compared with cells transfected with NT siRNA.Data are representative of two independent experiments done in triplicate. OCTN1 protein expression was determined by Western blot analysis frommembrane extraction of OCI-AML3 cells 48 hours posttransfection, and the transferrin receptor served as loading control. The relative expression differenceof OCTN1 after RNAi is indicated by the numbers above the lanes.

Drenberg et al.





with febrile neutropenia, the major dose-limiting toxicity ofcytarabine, in a cohort of 164 pediatric patients with AMLreceiving cytarabine-based therapy (41). Expression of thismutant OCTN1 in HEK293 cells (Supplementary Fig. S3A andS3B) revealed that, compared with the reference OCTN1, thisvariant is associated with increased transport of TEA andincreased formation of Ara-CTP after exposure to cytarabine(Supplementary Fig. S3C and S3D). This finding is consistentwith the contention that OCTN1 transports cytarabine intonormal bonemarrow and blood cells where it is phosphorylated,that these phosphorylated metabolites accumulate in these cells,and produce dose-limiting hematologic toxicities.

Role of OCTN1 in cellular sensitivity to nucleosideanalogues

Previous investigations demonstrated that certain antiviralnucleosides, including azidothymidine, ganciclovir, and acy-clovir, as well as the anticancer nucleosides 2-chloro-20-deox-yadenosine (cladribine) and cytarabine itself, can be trans-ported to a moderate extent (1.5 to 4-fold vs. control cells)by other SLC22A family members that show similarity toOCTN1, such as rat OCT1 (SLC22A1; refs. 42, 43). In heterol-ogous expression models analogous to the one developed forOCTN1, we found, however, that cytarabine was not a trans-ported substrate of the human organic cation transporters

Figure 5.

Transport of nucleosides by OCTN1.A, Expression of OCTN1 was evaluatedin the NCI60 cancer cell line panel, andrank-ordered by observed correlationcoefficients (R) between geneexpression and cytotoxic potencies of118 drugs (antimetabolites are in redbold and underlined). Compounds(N¼ 18) with positive values of R > 0.25(indicated by red bar) andcorresponding to P < 0.03 are shown.B, The transport of nucleosideanalogues (concentration, 1 mmol/L;5-minute uptake; 9–30 observationsper group) by OCTN1 was furtherevaluated in transfected HEK293 cells.Bars represent normalized transportdivided by substrate concentration. a,cytidine analogues; b, adenosineanalogues; c, guanosine analogue. Thecontribution of OCTN1 to nucleoside-induced cytotoxicity was evaluated inHEK293 cells transfectedwithOCTN1 orVC after continuous drug exposure,followed by CellTiter-Glo analysis at72 hours (12 observations perconcentration, per group) withincreasing concentrations (C) and atlow (1 mmol/L), intermediate (10mmol/L), and high doses (100 mmol/L;D). Data are shown as mean values(bars or symbols) and SEM (error bars).Solid lines represent a fit of theexperimental data to an inversenonlinear maximum-effect model.� , P < 0.03; �� , P <0.003; �� P < 0.0001.






OCT1, OCT2 (SLC22A2), and OCT3 (SLC22A3; SupplementaryFig. S4A), or the carnitine transporter OCTN2 (SLC22A5; Sup-plementary Fig. S4B). This suggests that OCTN1 may be uniqueamong human SLC22A family of transporters in its ability tointeract with cytarabine.

To evaluate the selectivity of OCTN1 for the transport ofnucleoside analogues, we initially performed pharmacologicprofiling of the NCI60 cell panel, which comprises 60 differenthuman tumor cell lines, representing leukemia, melanoma, andcancers of the lung, colon, brain, ovary, breast, prostate, andkidney. The 60–cell line dose response produced by a givencompound results in a biological response pattern that can beutilized in a recognition algorithm known as COMPARE.Applying this algorithm on 118 well-characterized anticancerdrugs, Pearson correlation coefficients (R) were calculatedfor assessment of OCTN1–drug relationships. Using a cutoffR > 0.25, corresponding to P < 0.03, we found that thecytotoxicity of 18 anticancer drugs of different classes wasstatistically significantly associated with OCTN1 expression(Fig. 5A). Among these were 8 antimetabolites, includingFDA-approved nucleoside analogues (fludarabine, decitabine,and cytarabine), nucleoside dialdehyde derivative (inosinedialdehyde), and nucleobase analogues (ftorafur and fluoro-uracil). Confirmatory validation studies performed in ourtransfected HEK293 cells revealed that OCTN1 can transporta remarkably broad range of nucleosides with variable efficien-cy, primarily depending on the 20 and 50 sugar positions,including all tested antimetabolites identified as hits in ourCOMPARE analysis (Fig. 5B).

The newly identified OCTN1 substrates include endogenousnucleosides (e.g., 20-deoxycytidine) and xenobiotic cytidine ana-logues (e.g., gemcitabine), adenosine analogues (e.g., clofarabineand fludarabine), and guanosine analogues (e.g., ribavirin; Sup-plementary Fig. S5). Among the tested nonnucleoside antimeta-bolites, we found that compared with xenobiotic nucleosides,OCTN1 demonstrated modest transport of fluorouracil (6.5-foldvs. control), but this was not observed for hydroxyurea (1.1�),mercaptopurine (1.3�), methotrexate (1.1�), or a host of anti-cancer drugs from other therapeutic classes (Supplementary Fig.S6). It should be pointed out that a previous investigation thatcorrelated the expression patterns of SLC transporters (includingSLC22A4) with growth inhibition data for 1,429 compounds intheNCI60 cell line panel failed to identify cytarabine as a hit (30).The reasons underlying these discrepant findings are not entirelyclear, but they may relate to differences in the experimentalmodels and specific conditions applied to the COMPAREanalysis.

In addition to increasing accumulation of multiple xenobioticnucleosides, overexpression of OCTN1 in HEK293 cells wasassociated with increased sensitivity to cytarabine and otherrelated nucleoside analogues, used in the treatment of AML (Fig.5C), including clofarabine (44) and fludarabine (45). In partic-ular, we observed that the IC50 values decreased from2.57 to 0.66mmol/L (3.9-fold increased sensitivity) for cytarabine, from 0.82to 0.028 mmol/L (29-fold) for clofarabine, and from 34 to 0.056mmol/L (608-fold) for fludarabine, respectively. These findingsare consistent with the ability of HEK293 cells to activate nucleo-sides through phosphorylation (Fig. 3E), and confirm thatOCTN1 can sensitize cells to xenobiotic nucleoside analogues.Likewise, we observed that deficiency inOCTN1 expression couldbe overcome at intermediate (10 mmol/L) and high (100 mmol/L)

doses of cytarabine and related nucleoside analogues (Fig. 5D).Although the mechanism(s) of drug uptake under these condi-tions require further investigation, these findings are consistentwith the observed Km value of 1.95 mmol/L, where at higherconcentrations OCTN1 is saturated and not contributing tomechanisms of uptake.

Collectively, our findings indicate that OCTN1, a transporterthought to function primarily as a carrier of ergothioneine, canfacilitate the intracellular accumulation of cytarabine, a prereq-uisite to drug-induced antileukemic activity. These results iden-tified OCTN1 as an important, previously unrecognized, con-tributor to the cellular uptake and efficacy of cytarabine, andthis process may be relevant for other related nucleoside ana-logues used in the treatment of AML, such as clofarabine andfludarabine. Our results also suggest a role of OCTN1 in cellularsensitivity to a range of nucleoside analogues with broadimplications to additional diseases.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerNone of the funding bodies had a role in the preparation of the

manuscript.

Authors' ContributionsConception and design: C.D. Drenberg, A.A. Gibson, S.D. Baker, A. SparreboomDevelopment of methodology: C.D. Drenberg, A.A. Gibson, S. Hu, G. Du,A.T. Nies, M. SchwabAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.D. Drenberg, A.A. Gibson, D.P. Rhinehart, S. Hu,N. Pabla, S.D. BakerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.D. Drenberg, A.A. Gibson, S.B. Pounds, L. Shi,D.P. Rhinehart, S. Hu, A.T. Nies, M. Schwab, N. Pabla, T.A. Gruber, S.D. Baker,A. SparreboomWriting, review, and/or revision of the manuscript: C.D. Drenberg, S.B.Pounds, D.P. Rhinehart, S. Hu, S. Hu, W. Blum, S.D. Baker, A. SparreboomAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): C.D. Drenberg, A.A. Gibson, L. Li, S. Hu,S.D. BakerStudy supervision: S.D. Baker, A. Sparreboom

AcknowledgmentsWe thank Lara Sucheston-Campbell (The Ohio State University, Colum-

bus, OH) for critical review of the manuscript, Dario Vignali (St. JudeChildren's Research Hospital, Memphis, TN) for providing the MSCV-IRES-GFP vector, and Chung-Ming Tse (Johns Hopkins University, Balti-more, MD) for providing the PK15 and PK15NTD cell lines. The resultsshown are in part based upon data generated by the TCGA Research Network(http://cancergenome.nih.gov/).

Grant SupportThe project was supported in part by NIH grants F32CA180513,

P30CA021765, R01CA138744, R01CA151633, and R25CA023944, the Amer-ican Lebanese Syrian Associated Charities (ALSAC), the Robert-Bosch Founda-tion, and the Interfaculty Center for Pharmacogenomics and Drug Research(ICEPHA).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 19, 2016; revised January 6, 2017; accepted January 24,2017; published OnlineFirst February 16, 2017.

Drenberg et al.




http://cancergenome.nih.gov/


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2017;77:2102-2111. Published OnlineFirst February 16, 2017.Cancer Res Christina D. Drenberg, Alice A. Gibson, Stanley B. Pounds, et al. OCTN1 Is a High-Affinity Carrier of Nucleoside Analogues

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