Cancer Therapy: Preclinical Clinical Cancer Research Lenalidomide, Thalidomide, and Pomalidomide 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 Hodgkin lymphoma and other secondary ciclovir enhanced the cytotoxic effects of lenalidomide and poma- primary malignancies (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 infection. tions of lenalidomide with dexamethasone or rituximab 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 melphalan 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. Ó2016 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 multiple myeloma (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 chemotherapy and autologous stem cell 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 Ó2016 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 methotrexate 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 antibody 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 antibodies 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 doxorubicin 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