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Home , Lenalidomide, Pomalidomide

Cancer Therapy: Preclinical Clinical Cancer Research , , and Reactivate the Epstein–Barr Virus Lytic Cycle through Phosphoinositide 3-Kinase Signaling and Ikaros Expression Richard 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 early gene BZLF1, the early gene BMRF1, and the late proteins VCA (LTP) are immunomodulatory agents approved for use in mul- and BCFR1. This occurred in the potency order pomalidomide > tiple myeloma, but in some settings, especially with alkylating lenalidomide > thalidomide, and the nucleoside analogue gan- agents, an increase in and other secondary ciclovir enhanced the cytotoxic effects of lenalidomide and poma- primary (SPM) has been noted. Some of these lidomide in Burkitt lymphoma cells in vitro and in vivo. EBV malignancies have been linked to Epstein–Barr virus (EBV), reactivation was related to PI3K stimulation and Ikaros suppres- raising the possibility that immunomodulatory drugs disrupt sion, and blocked by the PI3Kd inhibitor idelalisib. Combina- latent EBV . tions of lenalidomide with or Experimental Design: We studied the ability of LTP to reacti- increased EBV reactivation compared with lenalidomide alone vate latently infected EBV-positive cell lines in vitro and in vivo, and and, importantly, lenalidomide with produced even evaluated the EBV viral load in archived serum samples from greater reactivation. patients who received a lenalidomide, thalidomide, and dexa- Conclusions: We conclude LTP may reactivate EBV-positive methasone (LTD) combination. resting memory B cells thereby enhancing EBV lytic cycle and Results: Treatment of EBV-infected B-cell lines with LTP at host immune suppression. Clin Cancer Res; 22(19); 4901–12. 2016 physiologically relevant concentrations induced the immediate AACR.

Introduction tings. Moreover, lenalidomide is now included in maintenance therapy (MT) in both transplant-eligible and transplant-ineligible The immunomodulatory drugs lenalidomide, thalidomide, populations (2–4). and pomalidomide (LTP) have contributed to an improvement Although the efficacy of LTP is without question, concern has in survival in patients with (1). Consequently, arisen about a possible increase in secondary primary malignan- they have become part of the backbone of many therapeutic cies (SPM). This has been seen with maintenance after melphalan- regimens in the upfront, relapsed, and relapsed/refractory set- based high-dose and autologous trans- plant (SCT; refs. 2, 3), or following melphalan-based induction 1Department of Lymphoma and Myeloma, The University of Texas MD therapy (4). Interestingly, lenalidomide maintenance increased Anderson Cancer Center, Houston, Texas. 2National Nanotechnology the incidence of Hodgkin lymphoma in two studies, an uncom- Center (NANOTEC), National Science and Technology Development mon SPM seen in patients with multiple myeloma. Indeed, Attal Agency (NSTDA), Pathum Thani, Thailand. 3Urology Department, ShengJing Hospital, China Medical University, ShenYang, China. and colleagues reported four Hodgkin lymphoma cases in 4McArdle Laboratory for Cancer Research, University of Wisconsin patients with multiple myeloma who had undergone SCT and School of Medicine and Public Health, Madison, Wisconsin. 5Depart- none in the placebo group (2), whereas McCarthy reported one ment of Biostatistics, The University of Texas MD Anderson Cancer case (3). Moreover, a retrospective analysis of three phase III trials Center, Houston, Texas. 6Birmingham Cancer Research UK Cancer Centre, School of Cancer Sciences, University of Birmingham, Bir- in relapsed patients with multiple myeloma demonstrated an mingham, United Kingdom. 7Department of Experimental Therapeu- SPM incidence of 4% in patients who received lenalidomide tics, The University of Texas MD Anderson Cancer Center, Houston, versus 1.4% in those who received placebo (5). Texas. One possible reason for the development of Hodgkin lympho- Note: Supplementary data for this article are available at Clinical Cancer ma as an SPM is reactivation of Epstein–Barr virus (EBV). This Research Online (http://clincancerres.aacrjournals.org/). oncogenic gamma herpes virus is associated with, and contributes Corresponding Author: Richard J. Jones, University of Texas MD Anderson to the development and maintenance of a variety of human Cancer Center, 7455 Fannin ST, Unit 403, SCR2.3206, Houston, TX 77054. malignancies, including Hodgkin lymphoma, Burkitt lymphoma, Phone: 713-745-5654; Fax: 713-792-6887; E-mail: [email protected] and posttransplant lymphoproliferative disease (PTLD; ref. 6). doi: 10.1158/1078-0432.CCR-15-2242 Primary infection occurs during childhood, and lifelong infection 2016 American Association for Cancer Research. is maintained latently in resting memory B cells, with occasional

www.aacrjournals.org 4901 Jones et al.

in DMSO, except for melphalan, which was dissolved in ethanol, Translational Relevance and rituximab, which was in 0.9% sodium chloride. The expanding use of immunomodulatory drugs in multi- ple myeloma, other hematologic malignancies, and diseases Cell culture and patient samples other than cancer demonstrates the potential effectiveness of MUTU-I and KEM-I (gifts from Alan Rickinson [Birmingham this class of agents. Their long-term use, such as in the main- Cancer Research UK Cancer Centre, School of Cancer Sciences, tenance therapy setting, however, may reveal previously University of Birmingham, Birmingham, United Kingdom] and Jeff unidentified downstream effects and toxicities. Our findings Sample [Department of Microbiology and Immunology, The Penn- sylvania State University College of Medicine, Pennsylvania]), are show the reactivation of the Epstein–Barr virus (EBV) into its þ þ lytic life cycle from a previously latent state occurs primarily EBV Burkitt lymphoma cell lines. The EBV Burkitt lymphoma cell through the stimulation of PI3K signaling. This phenomenon line DAUDI and the lymphoblastoid cell line (LCL) B95.8 were has the potential to be a novel therapeutic strategy against EBV- from the ATCC, whereas the donor 4 (D4-WT) LCL and BZLF-1 positive malignancies in combination with ganciclovir. How- deleted LCL (D4-ZKO) were described previously (12). All lines ever, these findings also raise concern about the immunosup- were validated through The MD Anderson Cancer Center Charac- pressive effects of these drugs, and provides a possible link to terized Cell Line Core Facility. Cells were grown in RPMI1640 (Life m EBV-related second primary malignancies. Technologies) with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin (Sigma-Aldrich). Archived serum samples were sourced from patients with myeloma enrolled in a phase I/II clinical trial (13), which was in compliance with the Declaration of Helsinki viral reactivation. In the absence of immune control, such as in according to an Institutional Review Board–approved protocol. patients who undergo SCT, loss of EBV control can give rise to PTLD (7). The use of HYPER-CVAD regimens in leukemia and Immunoblotting lymphoma patients, and use in patients with auto- Protein expression was measured by immunoblot analysis as immune disease, increases the incidence of EBV-positive lympho- described previously (14). The to BZLF-1 was from Santa a mas (8, 9). It is also interesting to note that lenalidomide-MT in Cruz Biotechnology, whereas the anti-AKT, phospho (p)-GSK3 / bSer21/9 473 relapsed/refractory multiple myeloma patients following allo- , p-AKTSer , FoxO1, and Ikaros were from graft SCT induced herpes virus reactivation (EBV and herpes Cell Signaling Technology. Antibody to BMRF1 was obtained b simplex virus) requiring acyclovir prophylaxis (10). In contrast, from Millipore, anti- -actin from Sigma-Aldrich, and anti-VCA fi chemotherapeutics such as or gemcitabine have been p18 antibodies were from Thermo Scienti c. Total AKT and SER473 shown to reactivate EBV from a latent infection in EBV-positive pAKT levels were also measured using sandwich ELISA Kit malignancies to a lytic infection, which has led to clinically as recommended by the manufacturer Cell Signaling Technology. significant regression of EBV-positive tumors (11). Hence, the ability of chemotherapy agents to induce lytic EBV infection can Cell proliferation assay be a therapeutic modality for EBV-positive malignancies in com- The WST-1 tetrazolium reagent from Roche was used to deter- bination with the nucleoside analogue ganciclovir, or it could give mine the effects on cell proliferation. Viable cell numbers rise to secondary EBV-positive malignancies in immunocompro- (Annexin-V- and TO-PRO-3-negative) were measured using fi mised hosts. These possibilities drove us to determine whether Annexin-V-Paci c Blue and TO-PRO-3 (Life Technologies) in fl lenalidomide reactivates the EBV lytic cycle, which could sensitize combination with Count Bright Beads on a Fortessa ow cytometer tumor cells to the effects of ganciclovir and/or contribute to the (Becton Dickson) using FlowJo, version 10 (Tree Star, Inc.). development of Hodgkin lymphoma as an SPM. Blockade of PI3K signaling Here we show that LTP reactivated lytic EBV infection in latently DAUDI cells were pretreated for 1 hour with DMSO alone or infected Burkitt lymphoma cell lines. This reactivation was with LY294002, PD98059, SB202190, idelalisib, and in combi- responsible for sensitization of the cells to ganciclovir and nation with LTP for 48 hours. enhanced the efficacy of lenalidomide in a Burkitt lymphoma xenograft. LTP reactivated EBV through the stimulation of PI3K Lentiviral overexpression of Ikaros and suppression of Ikaros, whereas the combination of lenalido- MUTU-I cells were infected with a lentivirus encoding Ikaros or mide with dexamethasone and rituximab, as well as with mel- a control virus as described previously (15). phalan, enhanced EBV reactivation. Taken together, these data provide a potential mechanism through which lenalidomide can In vivo xenograft drive reactivation of EBV to a lytic form, and this may contribute to Experiments were performed in accordance with protocols the increased incidence of Hodgkin lymphoma in the post-SCT/ approved by the institutional Animal Care and Use Facility. CB- lenalidomide maintenance setting. 17 SCID mice (Harlan Laboratories, Inc.) were inoculated in the right flank subcutaneously with 5 106 MUTU-I cells. When tumor 3 Materials and Methods burdens reached approximately 100 mm in volume, mice were randomized into groups of five mice to receive intraperitoneal Reagents vehicle (PBS) daily, lenalidomide daily, ganciclovir thrice weekly, Lenalidomide was from , whereas thalidomide, poma- or both agents. lidomide, idelalisib, LY294002, SB202190, PD98059, and dexa- methasone were purchased from Selleck Chemicals. Doxorubicin, Quantitative real-time PCR and semiquantitative reverse- melphalan, and ganciclovir were purchased from Sigma-Aldrich transcription PCR analysis as were methotrexate and . Rituximab was from The Total RNA harvested from cells was reverse transcribed as MD Anderson Cancer Center Pharmacy. All drugs were dissolved described previously (16). cDNA was subjected to PCR using

4902 Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research Immunomodulatory Drugs Reactivate Lytic EBV

BZLF1, BCRF1 b M primers and conditions for , and 2 as published Results previously (16, 17). Quantitative real-time PCR (qPCR) for BGLF- Immunomodulatory agents reactivate latent EBV infection 4 was performed on a StepOne PCR analyzer (Applied Biosys- We sought to determine whether LTP-induced EBV reactivation tems) using SYBR Green Master Mix (Life Technologies) with 0 0 in latently infected LCL and Burkitt lymphoma cell lines. Clini- sense primer 5 -TGACGGAGCTGTATCACGAG-3 and antisense 0 0 cally relevant concentrations (20–22) of LTP weakly enhanced primer 5 -CCAGGGGCTCAATACTACCA-3 based on the Gen- expression of the immediate early gene product BZLF1, and the Bank EBV sequence: AJ507799.2. EBV viral load in serum samples early viral gene product BMRF1 in B95.8 and D4 LCL cells was measured using the EBV R-gene Kit (Argene), according to the (Fig. 1A). Reverse-transcription PCR (RT-PCR) analysis also manufacturer's instructions. showed a dose-dependent increase in BZLF1 transcription and induction of BCRF1 (viral IL-10), a marker of the late stages of EBV Drug synergy assays replication with lenalidomide treatment (Fig. 1B). In contrast, the To detect the presence of synergistic interactions, the methods Burkitt lymphoma cell lines DAUDI, KEM-I, and MUTU-1 showed of Chou and Talalay were used (18). Data were analyzed using robust BMRF1 and BZLF1 induction, along with the expression of CalcuSyn Version 2 software (Biosoft), and combination indices the late protein, VCA (Fig. 1C). BMRF1 induction was similar with (CI) calculated. 1 and 5 mmol/L lenalidomide and pomalidomide, and equivalent to methotrexate, a known EBV reactivation inducer (9). Pomali- Statistical analyses domide was particularly effective in DAUDI and KEM-I, followed Data were subjected to statistical analyses using the SEM. The by lenalidomide and thalidomide (Fig. 1C). This potency in fi – signi cance of drug effect relationships was determined by one- reactivating EBV parallels the known clinical efficacy of these t tailed unpaired tests using Excel software (Microsoft Corpora- agents in multiple myeloma (23). tion), and results were considered significant when P < 0.05. For in vivo studies, an analysis of cooperative effects of lenalidomide and EBV lytic cycle induction by lenalidomide and pomalidomide ganciclovir on tumor growth was performed using a Bayesian enhances their activity in Burkitt lymphoma and LCL cells bootstrapping approach (19). Linear mixed-effect models were To determine the contribution of the EBV lytic cycle to the used to study the change of EBV viral load over time in patient cytotoxic effects of immunomodulatory drugs, we evaluated D4 serum samples and the effects of valacyclovir treatment. An cells bearing wild-type (WT) EBV or D4 cells transformed using a unstructured covariance model was used to account for interpa- BZLF1 gene–deleted (D4-ZKO) EBV, rendering them incapable of tient variability and the longitudinal nature of the data. The entering lytic cycle. Treatment of WT LCLs with LTP reduced the transformation of logarithm to the base 10 of the EBV viral load viable cell number to 60% with lenalidomide, 90% with thalid- was used in the analyses to satisfy the normality assumption of the omide, and 50% with pomalidomide (Fig. 2A), but the ZKO LCL models. SAS version 9.2 and S-Plus version 8.04 (SAS Institute) cells displayed little to no change in viability with lenalidomide were used to carry out the computations for all analyses. and thalidomide, whereas pomalidomide only reduced viability

A B B95.8 D4 LCL D4 WT LCL DMSO 1 μmol/L 5 μmol/L LEN THAL POM MTX LEN THAL POM MTX μmol/L 5 1 5 1 1 1 5 Vehicle 5 1 Vehicle 5 1 1 1 5 RT RT RT - - +RT +RT 1:10 1:10 - +RT BZLF1 1:10 BZLF1 BMRF1 BCRF1 β M β-Actin 2

C DAUDI KEM-I MUTU-I LEN THAL POM MTX LEN THAL POM MTX LEN THAL POM MTX

Vehicle μmol/L Vehicle Vehicle 1 1 1 5 5 1 1 5 5 5 1 5 5 1 1 1 1 5 1 1 5 BZLF1

BMRF1

VCA p18 β-Actin

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 a positive control, and extracts were immunoblotted with the indicated antibodies. B, RT-PCR on D4 LCLs following treatment with lenalidomide (LEN) for 48 hours with 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, and MUTU-I were treated as above. Protein levels of BZLF1, BMRF1, and VCA, along with b-actin as a loading control, were determined. Representative images are shown from one of three independent experiments.

www.aacrjournals.org Clin Cancer Res; 22(19) October 1, 2016 4903 Jones et al.

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, LCL cells 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 analysis was 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 the vehicle control group. B, Burkitt lymphoma (BL) cell lines were treated for 4 days with lenalidomide, thalidomide (THAL), pomalidomide, or vehicle, cell viability was determined using the WST-1 reagent, and results were expressed as the percentage viability relative to the vehicle control, which was arbitrarily set at 100%. 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 with ganciclovir (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 were inoculated 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 a function of time for each group. Statistically significant differences comparing the combination to the single agents were determined using an unpaired t-test, and a P 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 was harvested and cDNA synthesized and qPCR performed for BGLF-4 with RQ values normalized to the DMSO control. An unpaired t test was performed to evaluate for significance and "" denotes P values of <0.05 relative to the DMSO control.

10% (Fig. 2A). We next evaluated the effect of LTP on DAUDI, stimulated the lytic cycle enough to result in conversion of MUTU-I, and KEM-I. Pomalidomide was the most effective in ganciclovir to its active triphosphate form. This can then compete suppressing DAUDI and MUTU-I proliferation, with IC50sof with deoxyguanosine triphosphate (dGTP) and be inserted into 0.3 and 0.25 mmol/L, respectively, whereas the KEM-I IC50 the cellular DNA, leading to of the infected cells (24). plateaued at approximately 1 to 3 mmol/L (Fig. 2B). Lenalido- DAUDI and MUTU-I cells were treated with lenalidomide (Fig. mide in the three lines did not achieve an IC50, but did reduce 2C, left) or pomalidomide (Fig. 2C, right) alone or in combina- proliferation to 75% in DAUDI at 0.6 mmol/L and 53% in tion with 50 mmol/L ganciclovir, and the live cell number deter- MUTU-I cells at 1 mmol/L (Fig. 2B). Lenalidomide had no mined using Annexin-V/TO-PRO-3 exclusion FACS. Lenalido- significant effect in KEM-I, whereas thalidomide had no sig- mide alone reduced the DAUDI live cell population to 80%, but nificant effect in any of them (Fig. 2B). lenalidomide with ganciclovir reduced the live cell number to As we cannot introduce a BZLF1-deleted EBV into Burkitt 49%. In MUTU-I cells, lenalidomide reduced the live cell number lymphoma cells because these cells harbor an endogenous WT to 62% alone, and to 45% with ganciclovir (Fig. 2C, left). EBV, we next evaluated whether lenalidomide and pomalidomide Similarly, the combination of pomalidomide and ganciclovir in

4904 Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research Immunomodulatory Drugs Reactivate Lytic EBV

DAUDI cells reduced the live cell fraction to 12% versus 30% with mide or ganciclovir alone. An analysis of the cooperative effects of pomalidomide alone. MUTU-I cells were even more sensitive to lenalidomide and ganciclovir found, from day 3 onward, the the combination, with a reduction of the live cells to 8% versus posterior probability of cooperative effect was equal to 1, mean- 21% for pomalidomide alone (Fig. 2C, right). ing there was a 0 in 10,000 chance that the combination did not To determine the presence of any synergistic interactions with have a cooperative effect. To confirm that LTP stimulate the BGLF- varying LTP and ganciclovir concentrations, we performed syn- 4 kinase responsible for phosphorylating ganciclovir to its active ergy assays with DAUDI, MUTU-I, and KEM-I treated with LTP or form in EBV-positive cells (25), we treated MUTU-I for 48 hours ganciclovir alone, or in combination with ganciclovir. DAUDI with LTP or methotrexate and performed qPCR for BGLF-4. cells demonstrated an enhanced suppression of cell growth with Lenalidomide and pomalidomide at concentrations as low as LTP in combination with ganciclovir with concentrations as low 1 mmol/L stimulated a four- and fivefold increase in BGLF-4, as 0.4 mmol/L lenalidomide or pomalidomide and 4 mmol/L respectively. Interestingly, the 5 mmol/L concentration actually ganciclovir. In comparison, 10 mmol/L thalidomide with 100 induced less BGLF-4 with both lenalidomide and pomalidomide, mmol/L ganciclovir actually overcame the stimulation of growth whereas thalidomide induced minimal BGLF-4 at the two con- observed with thalidomide (Supplementary Fig. S1A). KEM-I cells centrations used (Fig. 2D, right). demonstrated less growth suppression with lenalidomide and pomalidomide both requiring 10 mmol/L in combination with Blockade of PI3K signaling suppresses lenalidomide-induced 100 mmol/L ganciclovir, whereas no activity was present with EBV reactivation thalidomide and ganciclovir (Supplementary Fig. S1B). MUTU-I EBV reactivation by chemotherapy agents is mediated through cells, in contrast, showed an enhanced suppression of cell growth stimulation of PI3K, MAPK kinase (MEK), and p38-MAPK sig- with LTP and ganciclovir at a range of concentrations, with naling (26–28). We therefore assessed the effects of MEK, PI3K, pomalidomide and ganciclovir being particularly effective, and p38-MAPK inhibitors in combination with LTP on EBV whereas thalidomide and ganciclovir overcame the growth stim- reactivation. LTP were unable to consistently induce BMRF1 or ulation of thalidomide (Supplementary Fig. S1C). Isobologram BZLF1 in the presence of LY294002 or SB202190, whereas the analysis was performed to determine whether these interactions MEK inhibitor had a limited effect on reactivation (Fig. 3A). The were synergistic as defined by Chou and Talalay (18). MUTU-I induction of the EBV lytic cycle by LTP also coincided with treated with lenalidomide and ganciclovir demonstrated synergy stimulation of PI3K signaling, as evidenced by enhanced expres- at all the concentrations used, with CI values ranging from 0.031 sion of phospho-glycogen synthase kinase (GSK) 3 a/bSer21/9 and to 0.314. Similarly, pomalidomide and ganciclovir were also protein kinase B/AKTSer473 (Fig. 3B). highly synergistic across the concentrations, with CIs of 0.02 to Suppression of EBV reactivation using SB202190 or LY294002 0.55. In comparison, only 0.4 mmol/L thalidomide and 4 mmol/L may be an off-target effect as these inhibitors have a broad range of ganciclovir showed synergy, with a CI of 0.141 (Supplementary mechanisms, including suppression of casein kinase 1 and NF-kB, Table S1). Pomalidomide and ganciclovir were also synergistic which may influence EBV reactivation. We therefore evaluated against KEM-I across the concentration ranges used, with CIs of idelalisib, a highly specific PI3K-p110d inhibitor (29). Treatment 0.03 to 0.2 (Supplementary Table S1). In DAUDI cells, pomali- of DAUDI cells with lenalidomide and pomalidomide induced domide with ganciclovir at the lowest concentrations were syn- significant BMRF1 expression, and enhanced pGSK3 a/bSer21/9 ergistic, with CIs of 0.027 to 0.670, whereas lenalidomide and and pAKTSer473 levels (Fig. 3C). In contrast, concomitant addition ganciclovir were synergistic to additive, with CIs of 0.417 to 1.154. of a clinically relevant concentration of idelalisib completely Note that no CIs are shown for lenalidomide and thalidomide suppressed stimulation of BMRF1, pGSK3a/bSer21/9, and with KEM-I, or thalidomide with DAUDI, as the single agents had pAKTSer427 (Fig. 3C). LTP may also regulate EBV reactivation no change in viability or stimulated growth (i.e., cell viability over through PI3K-mediated suppression of the transcription factor 100%), which precludes isobologram analysis (Supplementary Forkhead-box-O1 (FoxO1), which, if suppressed, leads to loss of Table S1). These data show that EBV lytic cycle induction is in part Ikaros (30), an EBV latency regulator (15). Treatment of DAUDI responsible for the anti-proliferative effects of lenalidomide and cells with lenalidomide slightly decreased FoxO1 and depleted pomalidomide in LCLs, and to a greater extent in Burkitt lym- Ikaros, leading to the enhancement of BZLF1 and BMRF1 and the phoma cell lines. induction of pAKTSer473 (Fig. 3D). The addition of idelalisib with lenalidomide led to a further depletion of Ikaros and greater Lenalidomide in combination with ganciclovir enhances the suppression of FoxO1. This was in contrast to treatment with therapeutic effect in vivo idelalisib alone, which actually enhanced FoxO1 and did not have To determine whether EBV lytic cycle induction is sufficient to any effect on Ikaros. These data confirm the role of PI3K stimu- result in effects in vivo, we established MUTU-I xenografts and lation by LTP in EBV reactivation. treated them with vehicle, lenalidomide daily, ganciclovir thrice weekly, or the combination. Lenalidomide and ganciclovir alone LTP display differential abilities to stimulate PI3K each had a slight effect on suppressing MUTU-I tumor growth To further understand the varying ability of LTP to stimulate (Fig. 2D), but statistical analysis showed this effect was not EBV reactivation, we evaluated the ability of different LTP con- significant. In contrast, the lenalidomide and ganciclovir combi- centrations to increase PI3K signaling as measured by a nation inhibited tumor growth, with tumor volumes from day 3 pAKTSER473 ELISA and the effect of idelalisib on blocking the of treatment onwards being significantly smaller compared with lytic cycle as it relates to cell viability. Lenalidomide and poma- the vehicle, or lenalidomide or ganciclovir alone (Fig. 2D). For lidomide concentrations as low as 0.1 mmol/L increased each time point comparing the lenalidomide and ganciclovir pAKTSER473 three- to 3.5-fold, respectively, compared with vehicle combination to single agents, P values were <0.02 (Supplemen- controls. At 0.5 mmol/L, this effect increased to fivefold with tary Table S2), with no significant differences between lenalido- lenalidomide and sevenfold with pomalidomide, remaining

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A + LEN + THAL + POM B LEN THAL POM Vehicle BMRF1 pGSK3α/βSer21/9 THAL PD98059 (MEK) LY294002 (PI3K) SB202190 (P38) Vehicle LEN PD98059 (MEK) LY294002 (PI3K) SB202190 (P38) POM PD98059 (MEK) LY294002 (PI3K) SB202190 (P38) Vehicle

Vehicle Total GSK3 BZLF1 pAKTSer473 BMRF1 Total AKT β-Actin β-Actin

Idelalisib C D Idelalisib POM THAL LEN LEN THAL POM Idelalisib Vehicle Idelalisib BMRF1 Vehicle LEN LEN & FoxO1 pGSK3α/βSer21/9

Total GSK3 Ikaros pAKT Ser473 BMRF1 Total AKT BZLF1

β-Actin pAKTSer473

Total AKT β-Actin

Figure 3. Inhibition of PI3K suppresses lenalidomide (LEN)-induced EBV reactivation. A, DAUDI cells were incubated with vehicle, LTP (5 mmol/L), or in combination with inhibitors of MEK (PD98059; 50 mmol/L), PI3K (LY294002; 15 mmol/L), or p38 (SB202190; 20 mmol/L) for 48 hours, and immunoblotted with the indicated sera. 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 treated with 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 activation markers and EBV reactivation markers. D, DAUDI cells were treated with lenalidomide (5 mmol/L), idelalisib (1 mmol/L), or the combination for 24 hours and immunoblotted for the markers of PI3K activation, EBV reactivation, FoxO1, and Ikaros. Representative images are shown from one of three independent experiments.

stable at concentrations of up to 5 mmol/L (Fig. 4A). In contrast, Single-agent lenalidomide suppressed the growth of DAUDI cells thalidomide did not increase pAKTSER473 significantly at low to 70% compared with the vehicle control, and this remained concentrations. At 1 mmol/L, thalidomide resulted in a twofold constant across the concentration range used; the addition of increase, which increased to threefold at 5 mmol/L. This pattern of idelalisib completely reversed this growth suppression at all induction of pAKTSER473 followed by a plateau directly reflected concentrations used (P < 0.05 compared with the control; Fig. the induction of BMRF1 observed in Fig. 1B. 4C). Pomalidomide as a single agent decreased the cell viability to As shown in Fig. 3, idelalisib suppressed pAKTSER473 at the 90% at 0.08 mmol/L and to 80% at 0.4 mmol/L, and this growth clinically relevant concentration of 1 mmol/L. We next determined suppression was reversed by idelalisib (P < 0.05). At pomalido- the optimal concentration in vitro at which idelalisib could sup- mide, concentrations of 2 and 10 mmol/L, however, idelalisib was press pAKTSER473. Treatment of DAUDI cells with concentrations not able to reverse the cell growth suppression (Fig. 4C, right). of idelalisib as low as 50 nmol/L led to a 95% decrease in the basal Thalidomide did not induce any decrease in cell viability, as levels of pAKTSER473, with complete suppression at concentrations previously observed, and the addition of Idelalisib resulted in up to 1 mmol/L (Fig. 4B). We also determined that a concentration no significant change to the cell viability (Fig. 4C, middle). of idelalisib as low as 25 nmol/L was in the effective range to suppress basal pAKTSER473 levels and had no effect on cell viability Overexpression of Ikaros attenuates lenalidomide-induced (Fig. 4B, right). We therefore used idelalisib (25 nmol/L) in EBV reactivation combination with varying LTP concentrations to determine Lenalidomide induces proteasome-mediated degradation of whether it could reverse the LTP-induced decrease in proliferation. the Ikaros family of proteins (31), and Ikaros suppresses EBV

4906 Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research Immunomodulatory Drugs Reactivate Lytic EBV

A * **

473 * ** * * * pAKT Ser Fold change * (normalized to control) 0 1 5 0 1 5 0 1 5 0.1 0.5 0.1 0.5 0.1 0.5 Drug (mmol/L) B

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0 5 1 0 0 0 01 . 25 50 0.1 0 10 500 00 0. 0.05 1, Idelalisib (mmol/L) Idelalisib (nmol/L)

C 150 150

100 100 * * * **** Cell viability Cell

50 viability Cell viability Cell 50 Lenalidomide Thalidomide Pomalidomide relative to vehicle (%) Lenalidomide & idelalisib (25 nmol/L) (%) to vehicle relative Thalidomide & idelalisib (25 nmol/L) (%) to vehicle relative Pomalidomide & idelalisib (25 nmol/L) 0 0 0.01 0.1 1 10 0.01 0.1 1 10 Lenalidomide (mmol/L) Thalidomide (mmol/L) Pomalidomide (mmol/L)

Figure 4. Idelalisib blocks LTP-mediated stimulation of PI3K at nanomolar concentrations and reverses LTP-mediated inhibition of proliferation. A, DAUDI cells were incubated with 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 to those 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 to DAUDI cells for 24 hours, the suppression of PI3K was measured using a pAKTSer473 ELISA (left), and normalization was as described above. DAUDI cells were treated with either vehicle or increasing concentrations of idelalisib (25–1,000 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 was arbitrarily set at 100% (right). "" denotes P values of <0.05 compared to the vehicle control in A 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 was arbitrarily set at 100%. "" denotes P values of <0.05 comparing the single-agent LTP to the idelalisib combination.

reactivation in Burkitt lymphoma cells (15). We therefore eval- lenalidomide alone induced loss of Ikaros, with accompanying uated whether LTP-mediated Ikaros degradation is associated increases in BMRF1, BZLF1, and pAKTSER473 (Fig.5C),addition with EBV reactivation. Although exposure of DAUDI and KEM-I of a low concentration of bortezomib (5 nmol/L) with lena- cells to 1 mmol/L of lenalidomide or pomalidomide strongly lidomide suppressed the induction of BMRF1, BZLF1, and induced Ikaros degradation, coinciding with strong BMRF1 pAKTSER473 but failed to rescue Ikaros expression (Fig. 5C). and BZLF1 induction, no change was seen with thalidomide (Fig. 5A). Overexpression of Ikaros in MUTU-I cells attenuated Combinations of lenalidomide with chemotherapy agents both BZLF1 and BMRF1 induction by lenalidomide at low enhance EBV reactivation concentrations (0.1–0.5 mmol/L), compared with the control A combination of lenalidomide and thalidomide with dexa- virus (Fig. 5B). However, this effect was lost at higher concen- methasone in lenalidomide resistant/refractory myeloma patients trations (1–5 mmol/L) of lenalidomide, with complete loss of may to some extent overcome lenalidomide resistance (13). This the overexpressed Ikaros at 5 mmol/L (Fig. 5B). We also eval- combination could also result in enhanced EBV reactivation, as uated the ability of the bortezomib to corticosteroid use with methotrexate enhanced EBV reactivation, block Ikaros-mediated degradation by lenalidomide. Although raising the risk of development of EBV lymphomas (9). We

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A KEM-I DAUDI LEN THAL POM MTX LEN THAL POM MTX

μmol/L 5 1 1 5 1 1 5 5 1 1 Vehicle 1 5 Vehicle 1 5 Figure 5. Ikaros overexpression attenuates EBV IKAROS reactivation by lenalidomide (LEN). A, KEM-I and DAUDI were treated with BZLF1 either vehicle, various concentrations of LTP (1–5 mmol/L) or methotrexate BMRF1 (1 mmol/L) for 48 hours, and cell lysates were immunoblotted for β-Actin Ikaros, BZLF1, BMRF1, and b-actin. B, MUTU-I cells were infected with a control Lentivirus or a Lentivirus B MUTU-I C inducing expression of Ikaros for 48 hours, and then treated Control IKAROS with lenalidomide (0.1–5 mmol/L) for 48 hours. Cell lysates were LEN Vehicle BZB BZB LEN & immunoblotted for Ikaros, BZLF1, BMRF1, and b-actin as a loading Ikaros 0.1 0.5 0.1 Vehicle Vehicle 0.5 μmol/L LEN 5.0 5.0 1.0 1.0 control. C, DAUDI cells were treated Ikaros with lenalidomide (1 mmol/L), BMRF1 bortezomib (BZB; 5 nmol/L), or BZLF1 BZLF1 both for 24 hours, and cell lysates pAKT Ser473 were immunoblotted with the indicated sera. BMRF1 Total AKT β-Actin β-Actin

therefore examined the EBV viral load in 18 archived serum over either agent alone, whereas lenalidomide and dexametha- samples from patients who received a lenalidomide, thalidomide, sone or thalidomide and dexamethasone combinations induced and dexamethasone combination (Supplementary Fig. S2A significantly higher BZLF1, BMRF1, and VCA expression. A com- and Fig. 6A; ref. 13) and for whom a serum sample was available bination of all three agents was no more efficacious at EBV at baseline and after the first and last cycles of therapy (number of reactivation than the lenalidomide and dexamethasone combi- cycles, 3–15). qPCR evaluation of the baseline pretreatment nation (Fig. 6B). serum indicated that most patients had no or low levels of virus, Finally, we sought to investigate the interplay of lenalidomide with a median load of 3.5 10 copies/mL (note: two patients had with other drugs used to treat myeloma and lymphoma patients. a high titer of virus at baseline) and a range of 0 to 3.7 108 In DAUDI cells, lenalidomide, methotrexate, doxorubicin, and copies/mL (Fig. 6A; Supplementary Table S3). After one cycle of melphalan all induced significant amounts of BMRF1, BZLF1, and the treatment combination, the median viral load increased to 1.0 VCA, whereas rituximab induced BZLF1, VCA, and BMRF1 weak- 102 copies/mL with a range of 0 to 3.9 109 copies/mL, but this ly, and a low concentration of the proteasome inhibitor, borte- increase was not significant when compared with the baseline zomib, had no effect (Fig. 6C, left). When lenalidomide was serum value (P ¼ 0.08). However, analysis after the final cycle for combined with doxorubicin or melphalan, enhanced reactivation the 18 patients demonstrated an increase in the median viral load was observed, whereas bortezomib suppressed the lenalidomide to 2.3 105 copies/mL, with a range of 0 to 4.0 108, which was induction of BMRF1 and VCA. In comparison, EBV reactivation highly significant compared with the baseline (P ¼ 0.0016; Fig. was induced in MUTU-I cells by a low dose (0.5 mmol/L) of 6A; Supplementary Table S3). Six patients received valacyclovir lenalidomide, and strongly induced by single-agent methotrexate, for varicella zoster infection at intermittent times (and not con- doxorubicin, melphalan, and, to a lesser extent, by rituximab (Fig. tinuously) throughout the trial. To account for any differences in 6C, right), whereas bortezomib again had no effect. However, treatment outcome due to valacyclovir, we compared the six when lenalidomide was combined with methotrexate, doxoru- patients receiving valacyclovir to the 12 who did not and did not bicin, melphalan, or rituximab, enhanced BMRF1, BZLF1, and observe any statistical difference in the EBV viral load (P ¼ 0.3628; VCA induction was observed beyond the single agents alone. Supplementary Fig. S2B). To see if we could model this effect in vitro, we treated DAUDI Discussion cells with the agents alone or in dual or triplicate combinations. Incorporation of thalidomide, lenalidomide, and pomalido- Lenalidomide alone stimulated strong BMRF1 expression along mide into the myeloma therapeutic armamentarium has contrib- with BZLF1 and VCA, whereas a similar thalidomide concentra- uted substantially to patient survival. Given the contributory role, tion stimulated BMRF1 weakly (Fig. 6B). As expected, a clinically EBV plays in approximately 50% of Hodgkin lymphoma, and relevant concentration of dexamethasone also stimulated BMRF1, reports of Hodgkin lymphoma in patients on lenalidomide-MT, BZLF1, and VCA expression (Fig. 6B). The lenalidomide and we hypothesized that lenalidomide, and its analogues thalido- thalidomide combination did not increase BMRF1 expression mide and pomalidomide, may induce EBV reactivation. This

4908 Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research Immunomodulatory Drugs Reactivate Lytic EBV

ABDAUDI mol/L) m mol/L) m THAL& DEX LEN,THAL,DEX Vehicle DEX (100 nmol/L) LEN & THAL LEN & DEX LEN (1 THAL (1 BZLF1

BMRF1 VCA p18 β-Actin

C DAUDI MUTU-I

+ LEN + LEN mol/L) mol/L) mol/L) g/mL) g/mL) m m m mol/L) mol/L) mol/L) m m m m m Vehicle MTX DOX BZB BZB (5 nmol/L) DOX (50 nmol/L) MTX (1 MLPH (5 MLPH (5 MTX DOX BZB MTX (1 DOX (50 nmol/L) BZB (5 nmol/L) LEN (0.5 LEN (1 Vehicle MLPH RTX RTX (50 MLPH RTX (50 RTX BZLF1

BMRF1 VCA p18 β-Actin

Figure 6. Lenalidomide (LEN) and commonly used chemotherapy agents enhance EBV reactivation. A, archived serum samples from a trial with patients with myeloma receiving thalidomide (THAL) and lenalidomide continuously with weekly dexamethasone in a 28-day cycle were evaluated for their EBV viral load by qPCR. EBV viral 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 the final 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- or three-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. Protein lysates were immunoblotted with the indicated sera. Representative images are shown from one of three independent experiments. hypothesis was further supported by the fact that the immuno- 36). We observed this effect with lenalidomide and pomalido- suppressant methotrexate in nonmalignant diseases such as rheu- mide at concentrations as low as 1 mmol/L, which correlated with matoid arthritis enhanced the risk of EBV-positive lymphomas EBV reactivation (Fig. 5A). Interestingly, the addition of a low (9). In this study, we found that LTP reactivated EBV in latently concentration of bortezomib (5 nmol/L) failed to rescue Ikaros infected B-cell lines in the order of efficacy pomalidomide > but did suppress the lytic cycle along with PI3K signaling, as seen lenalidomide > thalidomide. LCL cells were weakly inducible by the decrease in pAKTSer473 (Fig. 5C). The inability of borte- into lytic cycle, whereas Burkitt lymphoma cells were readily zomib to prevent proteasomal suppression of Ikraos mediated by induced (Fig. 1). Combinations of ganciclovir with lenalidomide lenalidomide has been shown to be both dose and time depen- or pomalidomide enhanced the growth inhibitory effect in Burkitt dent, requiring pretreatment for 1 hour with 100 nmol/L borte- lymphoma cells (Fig. 2C and Supplementary Fig. S1), and this was zomib to prevent Ikaros loss (37). The low concentration range of further borne out in a mouse model (Fig. 2D). The ability of LTP to 3 to 5 nmol/L was shown to be less effective in that study, as in our reactivate EBV and sensitize cells to the bystander effect of gan- experiments. The ability of low bortezomib concentrations to ciclovir indicates a potential use in EBV reactivation therapies for block induction of the lytic cycle is most likely due to its reported EBV-positive B-cell malignancies. Notably, similar approaches ability to suppress pAKTSer473 by decreasing upstream kinases in were shown to be effective using in vitro and in vivo models the PI3K signaling cascade (38). (11, 32, 33), and clinically with combinations of gemcitabine, Ikaros is known to suppress the EBV lytic cycle through regu- valproic acid, and ganciclovir in nasopharyngeal lation of Octamer transcription factor-2 and Paired Box-5, patients, which produced disease stabilization (34). B-cell transcription factors which promote latency (15, 39, 40), One of LTP against multiple myeloma is leading to the assumption that LTP-mediated EBV reactivation through induction of proteasomal degradation of Ikaros (31, 35, occurs through this mechanism. Indeed, we found that Ikaros

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overexpression attenuated lenalidomide induction of the lytic McCarthy and colleagues (3), with no cases in the placebo arms. cycle, but did not result in complete blockade (Fig. 5B). This is In the context of thalidomide, two case reports on patients with likely due to our additional finding that PI3K played a role in lytic myeloma found EBV-positive Hodgkin lymphoma as an SPM EBV induction. Lenalidomide is known to stimulate PI3K activity after thalidomide-containing regimens. One patient received (41), and we also showed that LTP stimulated PI3K activity, thalidomide maintenance for 2 years with prior melphalan ther- coinciding with EBV reactivation (Figs. 3 and 4). Importantly, apy (46), and another received three cycles of vincristine, doxo- this stimulation was reversed by PI3K inhibition with idelalisib, rubicin and dexamethasone, and four cycles of bortezomib, which led to complete suppression of EBV reactivation. Of note is thalidomide, and dexamethasone salvage with development of the fact that lenalidomide and pomalidomide at low concentra- Hodgkin lymphoma 5 years later (47). A study examining SPM tions (0.1 mmol/L) can eliminate Ikaros and also stimulate sig- incidence in patients with myeloma treated in the pre-lenalido- nificant PI3K signaling, as shown by significant pAKTSer473 upre- mide era (1997–2008) reported only one case of Hodgkin lym- gulation (Figs. 4A and 5B). This effect plateaued at 0.5 mmol/L and phoma out of 589 patients (48). In contrast, four patients devel- did not increase further for both lenalidomide and pomalido- oped Hodgkin lymphoma as an SPM on lenalidomide-MT out of mide, thereby explaining why no significant changes in BMRF1 306 patients treated by Attal and colleagues. occurred between 1 and 5 mmol/L in the Burkitt lymphoma and Patients with myeloma frequently display varying degrees of LCL cells (Fig. 1). However, thalidomide did show a dose-depen- due to secondary hypogammaglobuline- dent increase in pAKTSer473 at 5 mmol/L but does not suppress mia, high-dose chemotherapy, use of LTP, proteasome inhibitors, Ikaros, which suggests that the ability of LTP to induce EBV or corticosteroids. In the face of varying degrees of immunosup- reactivation is primarily due to the stimulation of PI3K signaling, pression, lenalidomide could reactivate dormant EBV-positive B which is enhanced by lenalidomide and pomalidomide's simul- cells, which would normally attract both humoral and cytotoxic T- taneous suppression of Ikaros. cell responses, resulting in killing of the EBV-positive B cells and EBV reactivation by LTP, therefore, is likely due to several elimination of infectious virions. Lack of immune surveillance in mechanisms. First, stimulation of PI3K suppresses FoxO1, whose the face of continual EBV stimulation may eventually exhaust function is required for proper Ikaros mRNA splicing and whose remaining protective immunity. This would increase the small loss results in Ikaros suppression (30). This effect was apparent pool of latently infected EBV-positive B cells, estimated to be with lenalidomide treatment (Fig. 3D). Second, LTP binding to around 1 in 106 cells, which would then have the potential to Cereblon induces Ikaros degradation, further suppressing Ikaros become transformed and, potentially, malignant. Combinations expression. Finally, direct PI3K stimulation by LTP results in lytic of dexamethasone with lenalidomide could result in enhanced cycle activation and appears to be a primary mechanism. The EBV reactivation and immune suppression, and the "R2" regimen weaker induction of lytic cycle in LCLs compared with Burkitt comprising lenalidomide and rituximab used in lymphoma lymphoma cells is likely due to the fact that LCLs have EBV latency patients (49) could have a similar effect. It has been reported state III, with lower Ikaros levels compared with the Burkitt that lenalidomide-treated patients with myeloma have an in- lymphoma cells, which are in latency state I (15). creased incidence of varicella zoster and herpes simplex virus Finally, we evaluated EBV reactivation with lenalidomide in (50), suggesting LTP may reactivate the herpes virus combination with commonly used chemotherapeutics for myelo- family per se. The potential relationship between lenalidomide ma and lymphoma in patient serum samples and cell lines. In a and EBV reactivation should be a consideration for patients small number of samples from patients with myeloma who treated with lenalidomide long term. Further studies are needed received a lenalidomide/thalidomide/dexamethasone combina- to determine if reactivation contributes to lenalidomide-mediat- tion, we could see that multiple cycles of the combination resulted ed clinical toxicities, if there is a possible contribution to SPMs, in a significant increase in the EBV load in the serum (Fig. 6A and and if the risk of this could be reduced by the addition of anti- Supplementary Table S3), and this was reproduced in vitro using cell herpes virus agents such as acyclovir or valacyclovir in high-risk lines (Fig. 6B). This was in line with previously reported data patient groups. showing glucocorticoids stimulate EBV reactivation (42), which we found was enhanced by addition of lenalidomide or thalido- Disclosure of Potential Conflicts of Interest mide. Furthermore, we demonstrated expression of the late protein R.Z. Orlowski reports receiving commercial research grants from and is a VCA in lenalidomide- and pomalidomide-exposed Burkitt lym- consultant/advisory board member for Celgene Corporation. No potential phoma cells (Fig. 6B) and by RT-PCR we detected the late protein conflicts of interest were disclosed by the other authors. BCRF1 in LCLs (Fig. 1), indicating full activation of the EBV lytic cycle, which will result in viral release. Melphalan, doxorubicin and Authors' Contributions methotrexate also reactivated EBV, which was enhanced with the Conception and design: R.J. Jones, T. Iempridee, R.Z. Orlowski lenalidomide addition (Fig. 6C). Rituximab did not induce EBV Development of methodology: R.J. Jones, T. Iempridee, J.E. Mertz Acquisition of data (provided animals, acquired and managed patients, reactivation, but as reported for rituximab/dexamethasone combi- provided facilities, etc.): R.J. Jones, T. Iempridee, X. Wang, S.C. Kenney, nations (43), rituximab/lenalidomide increased EBV reactivation. C.W. Dawson, J.J. Shah Our findings that LTP reactivate EBV in latently infected B cells Analysis and interpretation of data (e.g., statistical analysis, biostatistics, raises several interesting questions, particularly in light of lena- computational analysis): R.J. Jones, T. Iempridee, J.E. Mertz, H.C. Lin, lidomide being linked to induction of SPMs in patients with V. Baladandayuthapani, R.Z. Orlowski myeloma on lenalidomide-MT (44, 45). Although the most Writing, 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. Orlowski common hematologic SPMs were and Administrative, technical, or material support (i.e., reporting or organizing myelodysplastic syndromes, two lenalidomide maintenance data, constructing databases): R.J. Jones, X. Wang, J.E. Mertz phase III trials reported Hodgkin lymphoma in the lenalidomide Study supervision: R.J. Jones, R.Z. Orlowski arms, with four cases in Attal and colleagues (2) and one in Other (patient samples): D. Weber

4910 Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research Immunomodulatory Drugs Reactivate Lytic EBV

Acknowledgments (to R.Z. Orlowski), P30 CA016672, and P01 CA22443 (to J.E. Mertz and Flow cytometry services were provided by the MD Anderson Flow Cytometry S.C. Kenney). Core Facility, which is supported by the MD Anderson Cancer Center Support The costs of publication of this article were defrayed in part by the Grant (P30 CA016672). R.Z. Orlowski would like to acknowledge support from payment of page charges. This article must therefore be hereby marked advertisement the National Cancer Institute (P50 CA142509, R01s CA184464 and 194264, in accordance with 18 U.S.C. Section 1734 solely to indicate and U10 CA032102). this fact. Grant Support This work was supported in part by funding from the US NIH by Received September 14, 2015; revised April 29, 2016; accepted May 22, 2016; grants P50 CA142509 (to R.Z. Orlowski), R01 CA184464 and 194264 published OnlineFirst June 13, 2016.

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