Cancer Therapy: Preclinical

Lenalidomide, Thalidomide, and PomalidomideReactivate the Epstein–Barr Virus Lytic Cyclethrough Phosphoinositide 3-Kinase Signaling andIkaros ExpressionRichard J. Jones1, Tawin Iempridee2, Xiaobin Wang3, Hans C. Lee1, Janet E. Mertz4,Shannon C. Kenney4, Heather C. Lin5, Veerabhadran Baladandayuthapani5,Christopher W. Dawson6, Jatin J. Shah1, Donna M.Weber1, and Robert Z. Orlowski1,7

Abstract

Purpose: Lenalidomide, thalidomide, and pomalidomide(LTP) are immunomodulatory agents approved for use in mul-tiple myeloma, but in some settings, especially with alkylatingagents, an increase in Hodgkin lymphoma and other secondaryprimary malignancies (SPM) has been noted. Some of thesemalignancies have been linked to Epstein–Barr virus (EBV),raising the possibility that immunomodulatory drugs disruptlatent EBV infection.

Experimental Design: We studied the ability of LTP to reacti-vate latently infected EBV-positive cell lines in vitro and in vivo, andevaluated the EBV viral load in archived serum samples frompatients who received a lenalidomide, thalidomide, and dexa-methasone (LTD) combination.

Results: Treatment of EBV-infected B-cell lines with LTP atphysiologically relevant concentrations induced the immediate

early geneBZLF1, the early geneBMRF1, and the late proteins VCAand BCFR1. This occurred in the potency order pomalidomide >lenalidomide > thalidomide, and the nucleoside analogue gan-ciclovir enhanced the cytotoxic effects of lenalidomide andpoma-lidomide in Burkitt lymphoma cells in vitro and in vivo. EBVreactivation was related to PI3K stimulation and Ikaros suppres-sion, and blocked by the PI3Kd inhibitor idelalisib. Combina-tions of lenalidomide with dexamethasone or rituximabincreased EBV reactivation compared with lenalidomide aloneand, importantly, lenalidomide with melphalan produced evengreater reactivation.

Conclusions: We conclude LTP may reactivate EBV-positiveresting memory B cells thereby enhancing EBV lytic cycle andhost immune suppression. Clin Cancer Res; 22(19); 4901–12.�2016AACR.

IntroductionThe immunomodulatory drugs lenalidomide, thalidomide,

and pomalidomide (LTP) have contributed to an improvementin survival in patients withmultiple myeloma (1). Consequently,they have become part of the backbone of many therapeuticregimens in the upfront, relapsed, and relapsed/refractory set-

tings. Moreover, lenalidomide is now included in maintenancetherapy (MT) in both transplant-eligible and transplant-ineligiblepopulations (2–4).

Although the efficacy of LTP is without question, concern hasarisen about a possible increase in secondary primary malignan-cies (SPM). This has been seenwithmaintenance aftermelphalan-based high-dose chemotherapy and autologous stem cell trans-plant (SCT; refs. 2, 3), or following melphalan-based inductiontherapy (4). Interestingly, lenalidomide maintenance increasedthe incidence of Hodgkin lymphoma in two studies, an uncom-mon SPM seen in patients with multiple myeloma. Indeed, Attaland colleagues reported four Hodgkin lymphoma cases inpatients with multiple myeloma who had undergone SCT andnone in the placebo group (2), whereas McCarthy reported onecase (3). Moreover, a retrospective analysis of three phase III trialsin relapsed patients with multiple myeloma demonstrated anSPM incidence of 4% in patients who received lenalidomideversus 1.4% in those who received placebo (5).

One possible reason for the development of Hodgkin lympho-ma as an SPM is reactivation of Epstein–Barr virus (EBV). Thisoncogenic gammaherpes virus is associatedwith, and contributesto the development and maintenance of a variety of humanmalignancies, includingHodgkin lymphoma, Burkitt lymphoma,and posttransplant lymphoproliferative disease (PTLD; ref. 6).Primary infection occurs during childhood, and lifelong infectionis maintained latently in resting memory B cells, with occasional

1Department of Lymphoma and Myeloma,The University of Texas MDAnderson Cancer Center, Houston, Texas. 2National NanotechnologyCenter (NANOTEC), National Science and Technology DevelopmentAgency (NSTDA), Pathum Thani, Thailand. 3Urology Department,ShengJing Hospital, China Medical University, ShenYang, China.4McArdle Laboratory for Cancer Research, University of WisconsinSchool of Medicine and Public Health, Madison, Wisconsin. 5Depart-ment of Biostatistics, The University of Texas MD Anderson CancerCenter, Houston, Texas. 6Birmingham Cancer Research UK CancerCentre, School of Cancer Sciences, University of Birmingham, Bir-mingham, United Kingdom. 7Department of Experimental Therapeu-tics, The University of Texas MD Anderson Cancer Center, Houston,Texas.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Richard J. Jones, University of Texas MD AndersonCancer Center, 7455 Fannin ST, Unit 403, SCR2.3206, Houston, TX 77054.Phone: 713-745-5654; Fax: 713-792-6887; E-mail: rjones@mdanderson.org

doi: 10.1158/1078-0432.CCR-15-2242

�2016 American Association for Cancer Research.

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viral reactivation. In the absence of immune control, such as inpatients who undergo SCT, loss of EBV control can give rise toPTLD (7). The use of HYPER-CVAD regimens in leukemia andlymphoma patients, and methotrexate use in patients with auto-immune disease, increases the incidence of EBV-positive lympho-mas (8, 9). It is also interesting to note that lenalidomide-MT inrelapsed/refractory multiple myeloma patients following allo-graft SCT induced herpes virus reactivation (EBV and herpessimplex virus) requiring acyclovir prophylaxis (10). In contrast,chemotherapeutics such as doxorubicin or gemcitabine have beenshown to reactivate EBV from a latent infection in EBV-positivemalignancies to a lytic infection, which has led to clinicallysignificant regression of EBV-positive tumors (11). Hence, theability of chemotherapy agents to induce lytic EBV infection canbe a therapeutic modality for EBV-positive malignancies in com-binationwith the nucleoside analogue ganciclovir, or it could giverise to secondary EBV-positive malignancies in immunocompro-mised hosts. These possibilities drove us to determine whetherlenalidomide reactivates the EBV lytic cycle, which could sensitizetumor cells to the effects of ganciclovir and/or contribute to thedevelopment of Hodgkin lymphoma as an SPM.

Herewe show that LTP reactivated lytic EBV infection in latentlyinfected Burkitt lymphoma cell lines. This reactivation wasresponsible for sensitization of the cells to ganciclovir andenhanced the efficacy of lenalidomide in a Burkitt lymphomaxenograft. LTP reactivated EBV through the stimulation of PI3Kand suppression of Ikaros, whereas the combination of lenalido-mide with dexamethasone and rituximab, as well as with mel-phalan, enhanced EBV reactivation. Taken together, these dataprovide a potential mechanism through which lenalidomide candrive reactivationof EBV to a lytic form, and thismay contribute tothe increased incidence of Hodgkin lymphoma in the post-SCT/lenalidomide maintenance setting.

Materials and MethodsReagents

Lenalidomide was from Celgene, whereas thalidomide, poma-lidomide, idelalisib, LY294002, SB202190, PD98059, and dexa-methasonewere purchased fromSelleckChemicals. Doxorubicin,melphalan, and ganciclovir were purchased from Sigma-Aldrichas were methotrexate and bortezomib. Rituximab was from TheMD Anderson Cancer Center Pharmacy. All drugs were dissolved

in DMSO, except for melphalan, which was dissolved in ethanol,and rituximab, which was in 0.9% sodium chloride.

Cell culture and patient samplesMUTU-I and KEM-I (gifts from Alan Rickinson [Birmingham

Cancer Research UK Cancer Centre, School of Cancer Sciences,University of Birmingham, Birmingham,United Kingdom] and JeffSample [Department ofMicrobiology and Immunology, The Penn-sylvania State University College of Medicine, Pennsylvania]), areEBVþBurkitt lymphoma cell lines. The EBVþBurkitt lymphomacellline DAUDI and the lymphoblastoid cell line (LCL) B95.8 werefrom the ATCC, whereas the donor 4 (D4-WT) LCL and BZLF-1deleted LCL (D4-ZKO) were described previously (12). All lineswere validated through The MD Anderson Cancer Center Charac-terized Cell Line Core Facility. Cells were grown in RPMI1640 (LifeTechnologies) with 10% FBS, 100 U/mL penicillin, and 100 mg/mLstreptomycin (Sigma-Aldrich). Archived serum samples weresourced frompatientswithmyelomaenrolled inaphase I/II clinicaltrial (13),whichwas in compliancewith theDeclarationofHelsinkiaccording to an Institutional Review Board–approved protocol.

ImmunoblottingProtein expression was measured by immunoblot analysis as

described previously (14). The antibody toBZLF-1was fromSantaCruz Biotechnology, whereas the anti-AKT, phospho (p)-GSK3a/bSer21/9, p-AKTSer473, FoxO1, and Ikaros antibodies were fromCell Signaling Technology. Antibody to BMRF1 was obtainedfrom Millipore, anti-b-actin from Sigma-Aldrich, and anti-VCAp18 antibodies were from Thermo Scientific. Total AKT andpAKTSER473 levels were also measured using sandwich ELISA Kitas recommended by themanufacturer Cell Signaling Technology.

Cell proliferation assayThe WST-1 tetrazolium reagent from Roche was used to deter-

mine the effects on cell proliferation. Viable cell numbers(Annexin-V- and TO-PRO-3-negative) were measured usingAnnexin-V-Pacific Blue and TO-PRO-3 (Life Technologies) incombinationwithCountBright Beads ona Fortessaflowcytometer(Becton Dickson) using FlowJo, version 10 (Tree Star, Inc.).

Blockade of PI3K signalingDAUDI cells were pretreated for 1 hour with DMSO alone or

with LY294002, PD98059, SB202190, idelalisib, and in combi-nation with LTP for 48 hours.

Lentiviral overexpression of IkarosMUTU-I cells were infected with a lentivirus encoding Ikaros or

a control virus as described previously (15).

In vivo xenograftExperiments were performed in accordance with protocols

approved by the institutional Animal Care and Use Facility. CB-17SCIDmice (HarlanLaboratories, Inc.)were inoculated in the rightflank subcutaneously with 5 � 106 MUTU-I cells. When tumorburdens reached approximately 100 mm3 in volume, mice wererandomized into groups of five mice to receive intraperitonealvehicle (PBS) daily, lenalidomide daily, ganciclovir thrice weekly,or both agents.

Quantitative real-time PCR and semiquantitative reverse-transcription PCR analysis

Total RNA harvested from cells was reverse transcribed asdescribed previously (16). cDNA was subjected to PCR using

Translational Relevance

The expanding use of immunomodulatory drugs in multi-ple myeloma, other hematologic malignancies, and diseasesother than cancer demonstrates the potential effectiveness ofthis class of agents. Their long-term use, such as in the main-tenance therapy setting, however, may reveal previouslyunidentified downstream effects and toxicities. Our findingsshow the reactivation of the Epstein–Barr virus (EBV) into itslytic life cycle from a previously latent state occurs primarilythrough the stimulation of PI3K signaling. This phenomenonhas thepotential to be anovel therapeutic strategy against EBV-positive malignancies in combination with ganciclovir. How-ever, these findings also raise concern about the immunosup-pressive effects of these drugs, and provides a possible link toEBV-related second primary malignancies.

Jones et al.

Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research4902

primers and conditions for BZLF1, BCRF1, and b2M as publishedpreviously (16, 17).Quantitative real-time PCR (qPCR) for BGLF-4 was performed on a StepOne PCR analyzer (Applied Biosys-tems) using SYBR Green Master Mix (Life Technologies) withsense primer 50-TGACGGAGCTGTATCACGAG-30 and antisenseprimer 50-CCAGGGGCTCAATACTACCA-30 based on the Gen-Bank EBV sequence: AJ507799.2. EBV viral load in serum sampleswasmeasured using the EBV R-gene Kit (Argene), according to themanufacturer's instructions.

Drug synergy assaysTo detect the presence of synergistic interactions, the methods

of Chou and Talalay were used (18). Data were analyzed usingCalcuSyn Version 2 software (Biosoft), and combination indices(CI) calculated.

Statistical analysesData were subjected to statistical analyses using the SEM. The

significance of drug–effect relationships was determined by one-tailed unpaired t tests using Excel software (Microsoft Corpora-tion), and results were considered significantwhen P <0.05. For invivo studies, an analysis of cooperative effects of lenalidomide andganciclovir on tumor growth was performed using a Bayesianbootstrapping approach (19). Linear mixed-effect models wereused to study the change of EBV viral load over time in patientserum samples and the effects of valacyclovir treatment. Anunstructured covariance model was used to account for interpa-tient variability and the longitudinal nature of the data. Thetransformation of logarithm to the base 10 of the EBV viral loadwas used in the analyses to satisfy the normality assumption of themodels. SAS version 9.2 and S-Plus version 8.04 (SAS Institute)were used to carry out the computations for all analyses.

ResultsImmunomodulatory agents reactivate latent EBV infection

We sought to determine whether LTP-induced EBV reactivationin latently infected LCL and Burkitt lymphoma cell lines. Clini-cally relevant concentrations (20–22) of LTP weakly enhancedexpression of the immediate early gene product BZLF1, and theearly viral gene product BMRF1 in B95.8 and D4 LCL cells(Fig. 1A). Reverse-transcription PCR (RT-PCR) analysis alsoshowed a dose-dependent increase in BZLF1 transcription andinduction ofBCRF1 (viral IL-10), amarker of the late stages of EBVreplicationwith lenalidomide treatment (Fig. 1B). In contrast, theBurkitt lymphoma cell linesDAUDI, KEM-I, andMUTU-1 showedrobust BMRF1 and BZLF1 induction, along with the expression ofthe late protein, VCA (Fig. 1C). BMRF1 inductionwas similar with1 and 5mmol/L lenalidomide and pomalidomide, and equivalentto methotrexate, a known EBV reactivation inducer (9). Pomali-domide was particularly effective in DAUDI and KEM-I, followedby lenalidomide and thalidomide (Fig. 1C). This potency inreactivating EBV parallels the known clinical efficacy of theseagents in multiple myeloma (23).

EBV lytic cycle induction by lenalidomide and pomalidomideenhances their activity in Burkitt lymphoma and LCL cells

To determine the contribution of the EBV lytic cycle to thecytotoxic effects of immunomodulatory drugs, we evaluated D4cells bearing wild-type (WT) EBV or D4 cells transformed using aBZLF1 gene–deleted (D4-ZKO) EBV, rendering them incapable ofentering lytic cycle. Treatment of WT LCLs with LTP reduced theviable cell number to 60% with lenalidomide, 90% with thalid-omide, and 50% with pomalidomide (Fig. 2A), but the ZKO LCLcells displayed little to no change in viability with lenalidomideand thalidomide, whereas pomalidomide only reduced viability

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Figure 1.

Immunomodulatory agents reactivate lytic EBV infection. A, B95.8 and D4 LCL cell lines were treated for 48 hours with vehicle, LTP, or methotrexate as apositive control, and extracts were immunoblotted with the indicated antibodies. B, RT-PCR on D4 LCLs following treatment with lenalidomide (LEN) for 48 hourswith primers for BZLF1, BCRF1, and a loading control using b2M or a 1:10 dilution of the cDNA. C, the EBVþ Burkitt lymphoma cell lines DAUDI, KEM-I, andMUTU-I were treated as above. Protein levels of BZLF1, BMRF1, and VCA, along with b-actin as a loading control, were determined. Representative imagesare shown from one of three independent experiments.

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10% (Fig. 2A). We next evaluated the effect of LTP on DAUDI,MUTU-I, and KEM-I. Pomalidomide was the most effective insuppressing DAUDI and MUTU-I proliferation, with IC50s of0.3 and 0.25 mmol/L, respectively, whereas the KEM-I IC50

plateaued at approximately 1 to 3 mmol/L (Fig. 2B). Lenalido-mide in the three lines did not achieve an IC50, but did reduceproliferation to 75% in DAUDI at 0.6 mmol/L and 53% inMUTU-I cells at 1 mmol/L (Fig. 2B). Lenalidomide had nosignificant effect in KEM-I, whereas thalidomide had no sig-nificant effect in any of them (Fig. 2B).

As we cannot introduce a BZLF1-deleted EBV into Burkittlymphoma cells because these cells harbor an endogenous WTEBV,wenext evaluatedwhether lenalidomide andpomalidomide

stimulated the lytic cycle enough to result in conversion ofganciclovir to its active triphosphate form. This can then competewith deoxyguanosine triphosphate (dGTP) and be inserted intothe cellular DNA, leading to apoptosis of the infected cells (24).DAUDI and MUTU-I cells were treated with lenalidomide (Fig.2C, left) or pomalidomide (Fig. 2C, right) alone or in combina-tion with 50 mmol/L ganciclovir, and the live cell number deter-mined using Annexin-V/TO-PRO-3 exclusion FACS. Lenalido-mide alone reduced the DAUDI live cell population to 80%, butlenalidomide with ganciclovir reduced the live cell number to49%. InMUTU-I cells, lenalidomide reduced the live cell numberto 62% alone, and to 45% with ganciclovir (Fig. 2C, left).Similarly, the combination of pomalidomide and ganciclovir in

Figure 2.

EBV lytic cycle enhances growth inhibition in response to lenalidomide (LEN) and pomalidomide (POM) and synergize with ganciclovir (GCV) in SCID mice. A, LCLcells bearing a wild-type EBV (D4 WT) or a BZLF1-deleted EBV (D4 ZKO) were treated with LTP (1 mmol/L) or vehicle for 1 week. Flow cytometric analysiswas then performed after staining with Annexin-V/TO-PRO-3 and Count Bright beads, from which the viable cell number was calculated and normalized to thevehicle control group. B, Burkitt lymphoma (BL) cell lines were treated for 4 days with lenalidomide, thalidomide (THAL), pomalidomide, or vehicle, cellviability was determined using the WST-1 reagent, and results were expressed as the percentage viability relative to the vehicle control, which was arbitrarily set at100%. C, DAUDI and MUTU-I cells were treated for 1 week with either lenalidomide (1 mmol/L) or pomalidomide (0.25 mmol/L) alone, or in combination withganciclovir (50 mmol/L). Annexin-V/TO-PRO-3 and Count Bright bead flow cytometry were used to determine the viable cell numbers. Values represent the mean� SEM from three independent experiments. An unpaired t-test was performed to evaluate for significance and "�" denotes P values of <0.01. D, SCID mice wereinoculated with MUTU-I cells subcutaneously and monitored until tumors were established. Five mice per group were injected intraperitoneally with vehicle,lenalidomide (50 mg/kg) daily, ganciclovir (50 mg/kg) three times per week, or the combination. Tumor volumes were measured and are plotted as afunction of time for each group. Statistically significant differences comparing the combination to the single agents were determined using an unpaired t-test, and aP value of <0.02 is indicated by "�". MUTU-I was treated with DMSO, LTP at 1 or 5 or 1 mmol/L methotrexate as a positive control for 48 hours. RNA washarvested and cDNA synthesized and qPCR performed for BGLF-4with RQ values normalized to the DMSO control. An unpaired t test was performed to evaluate forsignificance and "�" denotes P values of <0.05 relative to the DMSO control.

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DAUDI cells reduced the live cell fraction to 12% versus 30%withpomalidomide alone. MUTU-I cells were even more sensitive tothe combination, with a reduction of the live cells to 8% versus21% for pomalidomide alone (Fig. 2C, right).

To determine the presence of any synergistic interactions withvarying LTP and ganciclovir concentrations, we performed syn-ergy assays with DAUDI, MUTU-I, and KEM-I treated with LTP organciclovir alone, or in combination with ganciclovir. DAUDIcells demonstrated an enhanced suppression of cell growth withLTP in combination with ganciclovir with concentrations as lowas 0.4 mmol/L lenalidomide or pomalidomide and 4 mmol/Lganciclovir. In comparison, 10 mmol/L thalidomide with 100mmol/L ganciclovir actually overcame the stimulation of growthobservedwith thalidomide (Supplementary Fig. S1A). KEM-I cellsdemonstrated less growth suppression with lenalidomide andpomalidomide both requiring 10 mmol/L in combination with100 mmol/L ganciclovir, whereas no activity was present withthalidomide and ganciclovir (Supplementary Fig. S1B). MUTU-Icells, in contrast, showed an enhanced suppression of cell growthwith LTP and ganciclovir at a range of concentrations, withpomalidomide and ganciclovir being particularly effective,whereas thalidomide and ganciclovir overcame the growth stim-ulation of thalidomide (Supplementary Fig. S1C). Isobologramanalysis was performed to determine whether these interactionswere synergistic as defined by Chou and Talalay (18). MUTU-Itreated with lenalidomide and ganciclovir demonstrated synergyat all the concentrations used, with CI values ranging from 0.031to 0.314. Similarly, pomalidomide and ganciclovir were alsohighly synergistic across the concentrations, with CIs of 0.02 to0.55. In comparison, only 0.4 mmol/L thalidomide and 4 mmol/Lganciclovir showed synergy, with a CI of 0.141 (SupplementaryTable S1). Pomalidomide and ganciclovir were also synergisticagainst KEM-I across the concentration ranges used, with CIs of0.03 to 0.2 (Supplementary Table S1). In DAUDI cells, pomali-domide with ganciclovir at the lowest concentrations were syn-ergistic, with CIs of 0.027 to 0.670, whereas lenalidomide andganciclovir were synergistic to additive, withCIs of 0.417 to 1.154.Note that no CIs are shown for lenalidomide and thalidomidewith KEM-I, or thalidomide with DAUDI, as the single agents hadno change in viability or stimulated growth (i.e., cell viability over100%), which precludes isobologram analysis (SupplementaryTable S1). These data show that EBV lytic cycle induction is in partresponsible for the anti-proliferative effects of lenalidomide andpomalidomide in LCLs, and to a greater extent in Burkitt lym-phoma cell lines.

Lenalidomide in combination with ganciclovir enhances thetherapeutic effect in vivo

To determine whether EBV lytic cycle induction is sufficient toresult in effects in vivo, we established MUTU-I xenografts andtreated them with vehicle, lenalidomide daily, ganciclovir thriceweekly, or the combination. Lenalidomide and ganciclovir aloneeach had a slight effect on suppressing MUTU-I tumor growth(Fig. 2D), but statistical analysis showed this effect was notsignificant. In contrast, the lenalidomide and ganciclovir combi-nation inhibited tumor growth, with tumor volumes from day 3of treatment onwards being significantly smaller compared withthe vehicle, or lenalidomide or ganciclovir alone (Fig. 2D). Foreach time point comparing the lenalidomide and ganciclovircombination to single agents, P values were <0.02 (Supplemen-tary Table S2), with no significant differences between lenalido-

mide or ganciclovir alone. An analysis of the cooperative effects oflenalidomide and ganciclovir found, from day 3 onward, theposterior probability of cooperative effect was equal to 1, mean-ing there was a 0 in 10,000 chance that the combination did nothave a cooperative effect. To confirm that LTP stimulate the BGLF-4 kinase responsible for phosphorylating ganciclovir to its activeform in EBV-positive cells (25), we treated MUTU-I for 48 hourswith LTP or methotrexate and performed qPCR for BGLF-4.Lenalidomide and pomalidomide at concentrations as low as1 mmol/L stimulated a four- and fivefold increase in BGLF-4,respectively. Interestingly, the 5 mmol/L concentration actuallyinduced less BGLF-4 with both lenalidomide and pomalidomide,whereas thalidomide induced minimal BGLF-4 at the two con-centrations used (Fig. 2D, right).

Blockade of PI3K signaling suppresses lenalidomide-inducedEBV reactivation

EBV reactivation by chemotherapy agents is mediated throughstimulation of PI3K, MAPK kinase (MEK), and p38-MAPK sig-naling (26–28). We therefore assessed the effects of MEK, PI3K,and p38-MAPK inhibitors in combination with LTP on EBVreactivation. LTP were unable to consistently induce BMRF1 orBZLF1 in the presence of LY294002 or SB202190, whereas theMEK inhibitor had a limited effect on reactivation (Fig. 3A). Theinduction of the EBV lytic cycle by LTP also coincided withstimulation of PI3K signaling, as evidenced by enhanced expres-sion of phospho-glycogen synthase kinase (GSK) 3 a/bSer21/9 andprotein kinase B/AKTSer473 (Fig. 3B).

Suppression of EBV reactivation using SB202190 or LY294002maybe anoff-target effect as these inhibitors have a broad range ofmechanisms, including suppression of casein kinase 1 andNF-kB,which may influence EBV reactivation. We therefore evaluatedidelalisib, a highly specific PI3K-p110d inhibitor (29). Treatmentof DAUDI cells with lenalidomide and pomalidomide inducedsignificant BMRF1 expression, and enhanced pGSK3 a/bSer21/9

and pAKTSer473 levels (Fig. 3C). In contrast, concomitant additionof a clinically relevant concentration of idelalisib completelysuppressed stimulation of BMRF1, pGSK3a/bSer21/9, andpAKTSer427 (Fig. 3C). LTP may also regulate EBV reactivationthrough PI3K-mediated suppression of the transcription factorForkhead-box-O1 (FoxO1), which, if suppressed, leads to loss ofIkaros (30), an EBV latency regulator (15). Treatment of DAUDIcells with lenalidomide slightly decreased FoxO1 and depletedIkaros, leading to the enhancement of BZLF1 and BMRF1 and theinduction of pAKTSer473 (Fig. 3D). The addition of idelalisib withlenalidomide led to a further depletion of Ikaros and greatersuppression of FoxO1. This was in contrast to treatment withidelalisib alone,which actually enhanced FoxO1anddid not haveany effect on Ikaros. These data confirm the role of PI3K stimu-lation by LTP in EBV reactivation.

LTP display differential abilities to stimulate PI3KTo further understand the varying ability of LTP to stimulate

EBV reactivation, we evaluated the ability of different LTP con-centrations to increase PI3K signaling as measured by apAKTSER473 ELISA and the effect of idelalisib on blocking thelytic cycle as it relates to cell viability. Lenalidomide and poma-lidomide concentrations as low as 0.1 mmol/L increasedpAKTSER473 three- to 3.5-fold, respectively, comparedwith vehiclecontrols. At 0.5 mmol/L, this effect increased to fivefold withlenalidomide and sevenfold with pomalidomide, remaining

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stable at concentrations of up to 5 mmol/L (Fig. 4A). In contrast,thalidomide did not increase pAKTSER473 significantly at lowconcentrations. At 1 mmol/L, thalidomide resulted in a twofoldincrease, which increased to threefold at 5 mmol/L. This pattern ofinduction of pAKTSER473 followed by a plateau directly reflectedthe induction of BMRF1 observed in Fig. 1B.

As shown in Fig. 3, idelalisib suppressed pAKTSER473 at theclinically relevant concentration of 1 mmol/L.Wenext determinedthe optimal concentration in vitro at which idelalisib could sup-press pAKTSER473. Treatment of DAUDI cells with concentrationsof idelalisib as low as 50 nmol/L led to a 95%decrease in the basallevels of pAKTSER473,with complete suppression at concentrationsup to 1mmol/L (Fig. 4B).We also determined that a concentrationof idelalisib as low as 25 nmol/L was in the effective range tosuppress basal pAKTSER473 levels andhadno effect on cell viability(Fig. 4B, right). We therefore used idelalisib (25 nmol/L) incombination with varying LTP concentrations to determinewhether it could reverse the LTP-induceddecrease inproliferation.

Single-agent lenalidomide suppressed the growth of DAUDI cellsto 70% compared with the vehicle control, and this remainedconstant across the concentration range used; the addition ofidelalisib completely reversed this growth suppression at allconcentrations used (P < 0.05 compared with the control; Fig.4C). Pomalidomide as a single agent decreased the cell viability to90% at 0.08 mmol/L and to 80% at 0.4 mmol/L, and this growthsuppression was reversed by idelalisib (P < 0.05). At pomalido-mide, concentrations of 2 and 10 mmol/L, however, idelalisib wasnot able to reverse the cell growth suppression (Fig. 4C, right).Thalidomide did not induce any decrease in cell viability, aspreviously observed, and the addition of Idelalisib resulted inno significant change to the cell viability (Fig. 4C, middle).

Overexpression of Ikaros attenuates lenalidomide-inducedEBV reactivation

Lenalidomide induces proteasome-mediated degradation ofthe Ikaros family of proteins (31), and Ikaros suppresses EBV

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Inhibition of PI3K suppresses lenalidomide (LEN)-induced EBV reactivation. A, DAUDI cells were incubated with vehicle, LTP (5 mmol/L), or in combination withinhibitors of MEK (PD98059; 50 mmol/L), PI3K (LY294002; 15 mmol/L), or p38 (SB202190; 20 mmol/L) for 48 hours, and immunoblotted with the indicatedsera. B, immunoblotting of DAUDI cells treated with LTP (5 mmol/L) was performed for markers of PI3K activation and EBV reactivation. C,DAUDI cells were treatedwith LTP (5 mmol/L) alone or in combination with the PI3Kd subunit inhibitor idelalisib (1 mmol/L) for 48 hours, and lysates probed for PI3K activationmarkers and EBV reactivation markers. D, DAUDI cells were treated with lenalidomide (5 mmol/L), idelalisib (1 mmol/L), or the combination for 24 hours andimmunoblotted for the markers of PI3K activation, EBV reactivation, FoxO1, and Ikaros. Representative images are shown from one of three independentexperiments.

Jones et al.

Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research4906

reactivation in Burkitt lymphoma cells (15). We therefore eval-uated whether LTP-mediated Ikaros degradation is associatedwith EBV reactivation. Although exposure of DAUDI and KEM-Icells to 1 mmol/L of lenalidomide or pomalidomide stronglyinduced Ikaros degradation, coinciding with strong BMRF1and BZLF1 induction, no change was seen with thalidomide(Fig. 5A). Overexpression of Ikaros in MUTU-I cells attenuatedboth BZLF1 and BMRF1 induction by lenalidomide at lowconcentrations (0.1–0.5 mmol/L), compared with the controlvirus (Fig. 5B). However, this effect was lost at higher concen-trations (1–5 mmol/L) of lenalidomide, with complete loss ofthe overexpressed Ikaros at 5 mmol/L (Fig. 5B). We also eval-uated the ability of the proteasome inhibitor bortezomib toblock Ikaros-mediated degradation by lenalidomide. Although

lenalidomide alone induced loss of Ikaros, with accompanyingincreases in BMRF1, BZLF1, and pAKTSER473 (Fig. 5C), additionof a low concentration of bortezomib (5 nmol/L) with lena-lidomide suppressed the induction of BMRF1, BZLF1, andpAKTSER473 but failed to rescue Ikaros expression (Fig. 5C).

Combinations of lenalidomide with chemotherapy agentsenhance EBV reactivation

A combination of lenalidomide and thalidomide with dexa-methasone in lenalidomide resistant/refractorymyelomapatientsmay to some extent overcome lenalidomide resistance (13). Thiscombination could also result in enhanced EBV reactivation, ascorticosteroid use with methotrexate enhanced EBV reactivation,raising the risk of development of EBV lymphomas (9). We

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Idelalisib blocks LTP-mediated stimulation of PI3K at nanomolar concentrations and reverses LTP-mediated inhibition of proliferation.A,DAUDI cellswere incubatedwith vehicle or LTP (0.1–5 mmol/L) for 24 hours, and PI3K activity evaluated in cell lysates using a pAKTSer473 ELISA. pAKTSer473 values were normalized tothose of the total AKT (also determined by ELISA), and the fold change was compared to that with the vehicle control. B, idelalisib (0.01–1 mmol/L) was added toDAUDI cells for 24 hours, the suppression of PI3K was measured using a pAKTSer473 ELISA (left), and normalization was as described above. DAUDI cellswere treated with either vehicle or increasing concentrations of idelalisib (25–1,000 nmol/L) for 72 hours, cell viability was determined using theWST-1 reagent, andresults were expressed as the percentage viability relative to the vehicle control, which was arbitrarily set at 100% (right). "�" denotes P values of <0.05compared to the vehicle control inA and B. C,DAUDI cells were treated with LTP (0.016–10 mmol/L) alone or in combination with idelalisib (25 nmol/L) for 72 hours,cell viability was determined using the WST-1 reagent, and results were expressed as the percentage viability relative to the vehicle control, which wasarbitrarily set at 100%. "�" denotes P values of <0.05 comparing the single-agent LTP to the idelalisib combination.

Immunomodulatory Drugs Reactivate Lytic EBV

www.aacrjournals.org Clin Cancer Res; 22(19) October 1, 2016 4907

therefore examined the EBV viral load in 18 archived serumsamples frompatientswho received a lenalidomide, thalidomide,and dexamethasone combination (Supplementary Fig. S2Aand Fig. 6A; ref. 13) and for whom a serum sample was availableat baseline and after the first and last cycles of therapy (number ofcycles, 3–15). qPCR evaluation of the baseline pretreatmentserum indicated that most patients had no or low levels of virus,with amedian load of 3.5� 10 copies/mL (note: two patients hada high titer of virus at baseline) and a range of 0 to 3.7 � 108

copies/mL (Fig. 6A; Supplementary Table S3). After one cycle ofthe treatment combination, themedian viral load increased to 1.0� 102 copies/mLwith a range of 0 to 3.9� 109 copies/mL, but thisincrease was not significant when compared with the baselineserum value (P¼ 0.08). However, analysis after the final cycle forthe 18 patients demonstrated an increase in themedian viral loadto 2.3� 105 copies/mL, with a range of 0 to 4.0� 108, which washighly significant compared with the baseline (P ¼ 0.0016; Fig.6A; Supplementary Table S3). Six patients received valacyclovirfor varicella zoster infection at intermittent times (and not con-tinuously) throughout the trial. To account for any differences intreatment outcome due to valacyclovir, we compared the sixpatients receiving valacyclovir to the 12 who did not and did notobserve any statistical difference in the EBV viral load (P¼0.3628;Supplementary Fig. S2B).

To see if we could model this effect in vitro, we treated DAUDIcells with the agents alone or in dual or triplicate combinations.Lenalidomide alone stimulated strong BMRF1 expression alongwith BZLF1 and VCA, whereas a similar thalidomide concentra-tion stimulated BMRF1 weakly (Fig. 6B). As expected, a clinicallyrelevant concentration of dexamethasone also stimulated BMRF1,BZLF1, and VCA expression (Fig. 6B). The lenalidomide andthalidomide combination did not increase BMRF1 expression

over either agent alone, whereas lenalidomide and dexametha-sone or thalidomide and dexamethasone combinations inducedsignificantly higher BZLF1, BMRF1, and VCA expression. A com-bination of all three agents was no more efficacious at EBVreactivation than the lenalidomide and dexamethasone combi-nation (Fig. 6B).

Finally, we sought to investigate the interplay of lenalidomidewith other drugs used to treat myeloma and lymphoma patients.In DAUDI cells, lenalidomide, methotrexate, doxorubicin, andmelphalan all induced significant amounts of BMRF1, BZLF1, andVCA, whereas rituximab induced BZLF1, VCA, and BMRF1 weak-ly, and a low concentration of the proteasome inhibitor, borte-zomib, had no effect (Fig. 6C, left). When lenalidomide wascombinedwith doxorubicin ormelphalan, enhanced reactivationwas observed, whereas bortezomib suppressed the lenalidomideinduction of BMRF1 and VCA. In comparison, EBV reactivationwas induced in MUTU-I cells by a low dose (0.5 mmol/L) oflenalidomide, and strongly induced by single-agentmethotrexate,doxorubicin,melphalan, and, to a lesser extent, by rituximab (Fig.6C, right), whereas bortezomib again had no effect. However,when lenalidomide was combined with methotrexate, doxoru-bicin, melphalan, or rituximab, enhanced BMRF1, BZLF1, andVCA induction was observed beyond the single agents alone.

DiscussionIncorporation of thalidomide, lenalidomide, and pomalido-

mide into the myeloma therapeutic armamentarium has contrib-uted substantially to patient survival. Given the contributory role,EBV plays in approximately 50% of Hodgkin lymphoma, andreports of Hodgkin lymphoma in patients on lenalidomide-MT,we hypothesized that lenalidomide, and its analogues thalido-mide and pomalidomide, may induce EBV reactivation. This

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Ikaros overexpression attenuates EBVreactivation by lenalidomide (LEN).A,KEM-I and DAUDI were treated witheither vehicle, various concentrationsof LTP (1–5 mmol/L) or methotrexate(1 mmol/L) for 48 hours, and celllysates were immunoblotted forIkaros, BZLF1, BMRF1, and b-actin.B, MUTU-I cells were infected with acontrol Lentivirus or a Lentivirusinducing expression of Ikarosfor 48 hours, and then treatedwith lenalidomide (0.1–5 mmol/L)for 48 hours. Cell lysates wereimmunoblotted for Ikaros, BZLF1,BMRF1, and b-actin as a loadingcontrol. C, DAUDI cells were treatedwith lenalidomide (1 mmol/L),bortezomib (BZB; 5 nmol/L), orboth for 24 hours, and cell lysateswere immunoblotted with theindicated sera.

Jones et al.

Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research4908

hypothesis was further supported by the fact that the immuno-suppressant methotrexate in nonmalignant diseases such as rheu-matoid arthritis enhanced the risk of EBV-positive lymphomas(9). In this study, we found that LTP reactivated EBV in latentlyinfected B-cell lines in the order of efficacy pomalidomide >lenalidomide > thalidomide. LCL cells were weakly inducibleinto lytic cycle, whereas Burkitt lymphoma cells were readilyinduced (Fig. 1). Combinations of ganciclovir with lenalidomideor pomalidomide enhanced the growth inhibitory effect in Burkittlymphoma cells (Fig. 2C and Supplementary Fig. S1), and thiswasfurther borne out in amousemodel (Fig. 2D). The ability of LTP toreactivate EBV and sensitize cells to the bystander effect of gan-ciclovir indicates a potential use in EBV reactivation therapies forEBV-positive B-cell malignancies. Notably, similar approacheswere shown to be effective using in vitro and in vivo models(11, 32, 33), and clinically with combinations of gemcitabine,valproic acid, and ganciclovir in nasopharyngeal carcinomapatients, which produced disease stabilization (34).

One mechanism of action of LTP against multiple myeloma isthrough induction of proteasomal degradation of Ikaros (31, 35,

36). We observed this effect with lenalidomide and pomalido-mide at concentrations as low as 1 mmol/L, which correlated withEBV reactivation (Fig. 5A). Interestingly, the addition of a lowconcentration of bortezomib (5 nmol/L) failed to rescue Ikarosbut did suppress the lytic cycle along with PI3K signaling, as seenby the decrease in pAKTSer473 (Fig. 5C). The inability of borte-zomib to prevent proteasomal suppression of Ikraos mediated bylenalidomide has been shown to be both dose and time depen-dent, requiring pretreatment for 1 hour with 100 nmol/L borte-zomib to prevent Ikaros loss (37). The low concentration range of3 to 5 nmol/Lwas shown to be less effective in that study, as in ourexperiments. The ability of low bortezomib concentrations toblock induction of the lytic cycle is most likely due to its reportedability to suppress pAKTSer473 by decreasing upstream kinases inthe PI3K signaling cascade (38).

Ikaros is known to suppress the EBV lytic cycle through regu-lation of Octamer transcription factor-2 and Paired Box-5,B-cell transcription factors which promote latency (15, 39, 40),leading to the assumption that LTP-mediated EBV reactivationoccurs through this mechanism. Indeed, we found that Ikaros

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Lenalidomide (LEN) and commonly used chemotherapy agents enhance EBV reactivation. A, archived serum samples from a trial with patients with myelomareceiving thalidomide (THAL) and lenalidomide continuously with weekly dexamethasone in a 28-day cycle were evaluated for their EBV viral load by qPCR. EBVviral load copies per milliliters was calculated for the baseline and first and last cycles of therapy for each patient. "�" denotes P values of <0.01 comparing thefinal cycle to the baseline viral load. B, DAUDI cells were treated with lenalidomide, THAL (1mmol/L), or dexamethasone (DEX; 100 nmol/L), or in two- orthree-drug combinations for 48 hours, and cell lysates were immunoblotted with the indicated sera. C, MUTU-I and DAUDI cells were treated with lenalidomide,methotrexate, doxorubicin, bortezomib (BZB), melphalan, or rituximab alone or in combination with lenalidomide at the indicated concentrations. Proteinlysates were immunoblotted with the indicated sera. Representative images are shown from one of three independent experiments.

Immunomodulatory Drugs Reactivate Lytic EBV

www.aacrjournals.org Clin Cancer Res; 22(19) October 1, 2016 4909

overexpression attenuated lenalidomide induction of the lyticcycle, but did not result in complete blockade (Fig. 5B). This islikely due to our additional finding that PI3K played a role in lyticEBV induction. Lenalidomide is known to stimulate PI3K activity(41), and we also showed that LTP stimulated PI3K activity,coinciding with EBV reactivation (Figs. 3 and 4). Importantly,this stimulation was reversed by PI3K inhibition with idelalisib,which led to complete suppression of EBV reactivation. Of note isthe fact that lenalidomide and pomalidomide at low concentra-tions (0.1 mmol/L) can eliminate Ikaros and also stimulate sig-nificant PI3K signaling, as shown by significant pAKTSer473 upre-gulation (Figs. 4A and 5B). This effect plateaued at 0.5mmol/L anddid not increase further for both lenalidomide and pomalido-mide, thereby explaining why no significant changes in BMRF1occurred between 1 and 5 mmol/L in the Burkitt lymphoma andLCL cells (Fig. 1). However, thalidomide did show a dose-depen-dent increase in pAKTSer473 at 5 mmol/L but does not suppressIkaros, which suggests that the ability of LTP to induce EBVreactivation is primarily due to the stimulation of PI3K signaling,which is enhanced by lenalidomide and pomalidomide's simul-taneous suppression of Ikaros.

EBV reactivation by LTP, therefore, is likely due to severalmechanisms. First, stimulation of PI3K suppresses FoxO1, whosefunction is required for proper Ikaros mRNA splicing and whoseloss results in Ikaros suppression (30). This effect was apparentwith lenalidomide treatment (Fig. 3D). Second, LTP binding toCereblon induces Ikaros degradation, further suppressing Ikarosexpression. Finally, direct PI3K stimulation by LTP results in lyticcycle activation and appears to be a primary mechanism. Theweaker induction of lytic cycle in LCLs compared with Burkittlymphoma cells is likely due to the fact that LCLs have EBV latencystate III, with lower Ikaros levels compared with the Burkittlymphoma cells, which are in latency state I (15).

Finally, we evaluated EBV reactivation with lenalidomide incombination with commonly used chemotherapeutics for myelo-ma and lymphoma in patient serum samples and cell lines. In asmall number of samples from patients with myeloma whoreceived a lenalidomide/thalidomide/dexamethasone combina-tion, we could see that multiple cycles of the combination resultedin a significant increase in the EBV load in the serum (Fig. 6A andSupplementary Table S3), and this was reproduced in vitro using celllines (Fig. 6B). This was in line with previously reported datashowing glucocorticoids stimulate EBV reactivation (42), whichwe found was enhanced by addition of lenalidomide or thalido-mide. Furthermore, we demonstrated expression of the late proteinVCA in lenalidomide- and pomalidomide-exposed Burkitt lym-phoma cells (Fig. 6B) and by RT-PCR we detected the late proteinBCRF1 in LCLs (Fig. 1), indicating full activation of the EBV lyticcycle, which will result in viral release. Melphalan, doxorubicin andmethotrexate also reactivated EBV, which was enhanced with thelenalidomide addition (Fig. 6C). Rituximab did not induce EBVreactivation, but as reported for rituximab/dexamethasone combi-nations (43), rituximab/lenalidomide increased EBV reactivation.

Our findings that LTP reactivate EBV in latently infected B cellsraises several interesting questions, particularly in light of lena-lidomide being linked to induction of SPMs in patients withmyeloma on lenalidomide-MT (44, 45). Although the mostcommon hematologic SPMs were acute myeloid leukemia andmyelodysplastic syndromes, two lenalidomide maintenancephase III trials reported Hodgkin lymphoma in the lenalidomidearms, with four cases in Attal and colleagues (2) and one in

McCarthy and colleagues (3), with no cases in the placebo arms.In the context of thalidomide, two case reports on patients withmyeloma found EBV-positive Hodgkin lymphoma as an SPMafter thalidomide-containing regimens. One patient receivedthalidomide maintenance for 2 years with prior melphalan ther-apy (46), and another received three cycles of vincristine, doxo-rubicin and dexamethasone, and four cycles of bortezomib,thalidomide, and dexamethasone salvage with development ofHodgkin lymphoma 5 years later (47). A study examining SPMincidence in patients with myeloma treated in the pre-lenalido-mide era (1997–2008) reported only one case of Hodgkin lym-phoma out of 589 patients (48). In contrast, four patients devel-oped Hodgkin lymphoma as an SPM on lenalidomide-MT out of306 patients treated by Attal and colleagues.

Patients with myeloma frequently display varying degrees ofimmunosuppression due to secondary hypogammaglobuline-mia, high-dose chemotherapy, use of LTP, proteasome inhibitors,or corticosteroids. In the face of varying degrees of immunosup-pression, lenalidomide could reactivate dormant EBV-positive Bcells, whichwouldnormally attract bothhumoral and cytotoxic T-cell responses, resulting in killing of the EBV-positive B cells andelimination of infectious virions. Lack of immune surveillance inthe face of continual EBV stimulation may eventually exhaustremaining protective immunity. This would increase the smallpool of latently infected EBV-positive B cells, estimated to bearound 1 in 106 cells, which would then have the potential tobecome transformed and, potentially, malignant. Combinationsof dexamethasone with lenalidomide could result in enhancedEBV reactivation and immune suppression, and the "R2" regimencomprising lenalidomide and rituximab used in lymphomapatients (49) could have a similar effect. It has been reportedthat lenalidomide-treated patients with myeloma have an in-creased incidence of varicella zoster and herpes simplex virusinfections (50), suggesting LTP may reactivate the herpes virusfamily per se. The potential relationship between lenalidomideand EBV reactivation should be a consideration for patientstreated with lenalidomide long term. Further studies are neededto determine if reactivation contributes to lenalidomide-mediat-ed clinical toxicities, if there is a possible contribution to SPMs,and if the risk of this could be reduced by the addition of anti-herpes virus agents such as acyclovir or valacyclovir in high-riskpatient groups.

Disclosure of Potential Conflicts of InterestR.Z. Orlowski reports receiving commercial research grants from and is a

consultant/advisory board member for Celgene Corporation. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R.J. Jones, T. Iempridee, R.Z. OrlowskiDevelopment of methodology: R.J. Jones, T. Iempridee, J.E. MertzAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.J. Jones, T. Iempridee, X. Wang, S.C. Kenney,C.W. Dawson, J.J. ShahAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.J. Jones, T. Iempridee, J.E. Mertz, H.C. Lin,V. Baladandayuthapani, R.Z. OrlowskiWriting, review, and/or revision of the manuscript: R.J. Jones, T. Iempridee,H.C. Lee, J.E. Mertz, S.C. Kenney, C.W. Dawson, J.J. Shah, R.Z. OrlowskiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.J. Jones, X. Wang, J.E. MertzStudy supervision: R.J. Jones, R.Z. OrlowskiOther (patient samples): D. Weber

Jones et al.

Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research4910

AcknowledgmentsFlow cytometry services were provided by theMDAnderson FlowCytometry

Core Facility, which is supported by the MD Anderson Cancer Center SupportGrant (P30CA016672). R.Z.Orlowski would like to acknowledge support fromthe National Cancer Institute (P50 CA142509, R01s CA184464 and 194264,and U10 CA032102).

Grant SupportThis work was supported in part by funding from the US NIH by

grants P50 CA142509 (to R.Z. Orlowski), R01 CA184464 and 194264

(to R.Z. Orlowski), P30 CA016672, and P01 CA22443 (to J.E. Mertz andS.C. Kenney).

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 14, 2015; revised April 29, 2016; acceptedMay 22, 2016;published OnlineFirst June 13, 2016.

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