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. 2016 Oct 1;22(19):4901-4912.
doi: 10.1158/1078-0432.CCR-15-2242. Epub 2016 Jun 13.

Lenalidomide, Thalidomide, and Pomalidomide Reactivate the Epstein-Barr Virus Lytic Cycle through Phosphoinositide 3-Kinase Signaling and Ikaros Expression

Richard J Jones  1 , Tawin Iempridee  2 , Xiaobin Wang  3 , Hans C Lee  4 , Janet E Mertz  5 , Shannon C Kenney  5 , Heather C Lin  6 , Veerabhadran Baladandayuthapani  6 , Christopher W Dawson  7 , Jatin J Shah  4 , Donna M Weber  4 , Robert Z Orlowski  8
Affiliations

Lenalidomide, Thalidomide, and Pomalidomide Reactivate the Epstein-Barr Virus Lytic Cycle through Phosphoinositide 3-Kinase Signaling and Ikaros Expression

Richard J Jones et al. Clin Cancer Res. .

Abstract

Purpose: Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin lymphoma and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein-Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection.

Experimental design: We studied the ability of LTP to reactivate latently infected EBV-positive cell lines in vitro and in vivo, and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide, and dexamethasone (LTD) combination.

Results: Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene BZLF1, the early gene BMRF1, and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide > lenalidomide > thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt lymphoma cells in vitro and in vivo EBV reactivation was related to PI3K stimulation and Ikaros suppression, and blocked by the PI3Kδ inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared with lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation.

Conclusions: We conclude LTP may reactivate EBV-positive resting memory B cells thereby enhancing EBV lytic cycle and host immune suppression. Clin Cancer Res; 22(19); 4901-12. ©2016 AACR.

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Conflict of interest statement

The remaining authors declare no financial conflicts of interest.

Figures

Figure 1
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 MTX as a positive control, and extracts were immunoblotted with the indicated antibodies. (B) Reverse-transcriptase (RT) PCR on D4 LCLs following treatment with LEN for 48 hours with primers for BZLF1, BCRF1, and a loading control using β2M or a 1:10 dilution of the cDNA. (C) The EBV+ BL cell lines DAUDI, KEM-I and MUTU-I were treated as above. Protein levels of BZLF1, BMRF1 and VCA, along with β-actin as a loading control, were determined. Representative images are shown from 1 of 3 independent experiments.
Figure 2
Figure 2. EBV lytic cycle enhances growth inhibition in response to LEN and POM and synergize with 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 µM) 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) BL cell lines were treated for 4 days with LEN, THAL, POM 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 LEN (1 µM) or POM (0.25 µM) alone, or in combination with GCV (50 µM). Annexin-V/TO-PRO-3 and Count Bright bead flow cytometry were used to determine the viable cell numbers. Values represent the mean +/− the standard error of the mean from 3 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, LEN (50 mg/kg) daily, GCV (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 µM or 1 µM MTX 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.
Figure 3
Figure 3. Inhibition of PI3 kinase suppresses LEN induced EBV reactivation
(A) DAUDI cells were incubated with vehicle, LTP (5 µM), or in combination with inhibitors of MEK (PD98059; 50 µM), PI3K (LY294002; 15 µM), or p38 (SB202190; 20 µM) for 48 hours, and immunoblotted with the indicated sera. (B) Immunoblotting of DAUDI cells treated with LTP (5 µM) was performed for markers of PI3K activation and EBV reactivation. (C) DAUDI cells were treated with LTP (5 µM) alone or in combination with the PI3Kδ subunit inhibitor idelalisib (1 µM) for 48 hours, and lysates probed for PI3K activation markers and EBV reactivation markers. (D) DAUDI cells were treated with LEN (5 µM), idelalisib (1 µM) or the combination for 24 hours and immunoblotted for the markers of PI3K activation, EBV reactivation, FoxO1 and Ikaros. Representative images are shown from 1 of 3 independent experiments.
Figure 4
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 µM) 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 µM) was added to DAUDI cells for 24 hours, the suppression of PI3K was measured using a pAKTSer473 ELISA (left panel), and normalization was as described above. DAUDI cells were treated with either vehicle or increasing concentrations of idelalisib (25–1000 nM) 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 panel). “*” 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 µM) alone or in combination with idelalisib (25 nM) 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.
Figure 5
Figure 5. Ikaros overexpression attenuates EBV reactivation by LEN
(A) KEM-I and DAUDI were treated with either vehicle, various concentrations of LTP (1–5 µM) or MTX (1 µM) for 48 hours, and cell lysates were immunoblotted for Ikaros, BZLF1, BMRF1, and β-actin. (B) MUTU-I cells were infected with a control Lentivirus or a Lentivirus inducing expression of Ikaros for 48 hours, and then treated with LEN (0.1–5 µM) for 48 hours. Cell lysates were immunoblotted for Ikaros, BZLF1, BMRF1, and β-Actin as a loading control. (C) DAUDI cells were treated with LEN (1 µM), BZB (5 nM), or both for 24 hours, and cell lysates were immunoblotted with the indicated sera.
Figure 6
Figure 6. LEN and commonly used chemotherapy agents enhance EBV reactivation
(A) Archived serum samples from a trial with myeloma patients receiving THAL and LEN continuously with weekly DEX in a 28-day cycle were evaluated for their EBV viral load by qPCR. EBV viral load copies per ml 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 LEN, THAL (1µM), or DEX (100 nM), 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 LEN, MTX, DOX, BZB, MLPH, or RTX alone or in combination with LEN at the indicated concentrations. Protein lysates were immunoblotted with the indicated sera. Representative images are shown from 1 of 3 independent experiments.
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