Synergistic effects of the chimeric anti-CD20 antibody rituximab with glucocorticoid on induction of lytic form of Epstein-Barr virus (EBV) infection, leading to susceptibility to ganciclovir in B-cell lymphoma cells with latent EBV infection.
Authors: Mariko Aoyagi, Fumihiro Higashino, Akiko Takahashi, Yasunori Totsuka, Takao Kohgo, Masanobu Kobayashi, Masanobu Shindoh
Journal: Cancer Research (2004)

3220  Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a number of human malignancies including Burkitt’s lymphoma, Hodgkin’s disease, post-transplantation lymphoproliferative disease, nasopharyngeal carcinoma, and gastric cancer. The presence of EBV genome in all of tumor cells suggests that novel therapies to specifically kill EBV-infected cells could be used to treat these malignancies. Since the majority of EBV-infected tumor cells carry the EBV genome in a latent form, antiviral therapy is less useful for treatment of the diseases. One approach would be to induce lytic EBV infection in tumor cells, which may make the cells susceptible to antiviral drugs such as ganciclovir (GCV), a cytotoxic prodrug that is converted into an active, cytotoxic form by the EBV-encoded lytic proteins. In this study, we first evaluated the effects of a glucocorticoid, dexamethasone, on induction of lytic form in EBV-latently infected Akata B-cell lymphoma cells. Cells were treated with dexamethasone at concentration of 10 nM, and 3 days later, FACS analysis with a monoclonal antibdody against the EBV-encoded lytic protein (BMRF1) was performed to quantitate the number of cells with lytic infection. Dexamethasone-treated cells had 5-8% of cells expressing the lytic protein. We then evaluated the effects of rituximab on induction of the EBV lytic infection. Rituximab, a chimeric anti-CD20 monoclonal antibody, has been used for treatment of B-cell non-Hodgkin lymphoma. Rituximab alone, up to the concentration of 100 μg/ml, did not significantly induce the EBV lytic infection. However, the combination of dexamathasone (10 nM) with rituximab (100 μg/ml) resulted in synergistic induction. Addition of rituximab enhanced the number of cells expressing the lytic proteins approximately 5 times in comparison with that of dexamethasone treatment alone. These results were confirmed by immunoblot analysis with monoclonal antibodies against EBV-lytic proteins (BZLF1 and BMRF1). We next examined whether GCV is effective for killing EBV-positive lymphoma cells treated with dexamethasone and rituximab. The isogenic EBV-positive and EBV-negative Akata cells were treated with dexamethasone and rituximab in the presence of GCV (30 μM) for 5 days, and the viable cells were determined by trypan blue exclusion. GCV alone was not toxic to the cells. Addition of GCV showed enhanced cell killing in EBV-positive cells treated with dexamethasone/rituximab. Importantly, the number of cells surviving the combination of GCV and dexamethasone/rituximab was reduced significantly in the EBV-positive cells but not in the EBV-negative cells. These data suggest that GCV in combination with rituximab and dexamethasone could be a virally targeted therapy for EBV-associated lymphoma.