Abstract
Epstein-Barr Virus (EBV)+ lymphomas are an important subgroup of aggressive malignant lymphomas which include lymphomas in the post-transplantation setting, Burkitt’s lymphomas (BL), AIDS-related lymphomas (ARL), and some forms of Hodgkin, T-cell, and natural killer (NK) cell lymphomas. EBV is a member of the herpes virus family, characterized by their ability to support two different life cycles: the productive “lytic” cycle leading to the production and release of new virions and the non-productive “latent” cycle. Most lymphoma cells are infected with latent EBV, and only few viral genes are expressed at low levels. Several groups of broad-acting chemical agents are able to reactivate EBV and induce herpes thymidine kinase (TK) expression in vitro and in vivo. NF-kB has been described to play a critical role in regulating the balance between latency and lytic replication of EBV. Therefore, we hypothesized that the proteasome inhibitor Bortezomib can be used to initiate EBV lytic antigen expression in EBV-related malignancies enabling the antiviral drug Ganciclovir to kill EBV+ lymphoma cells. The human cell line HR-1, derived from a Burkitt’s lymphoma and latently infected with EBV, was cultured in the presence of 50nM bortezomib for 24 hrs. The immediate early lytic phase EBV antigens ZEBRA and RTA were induced and expressed as measured by flow cytometry. The EBV-VCA and EBV-R antigens were not expressed in untreated controls but were induced as demonstrated by western blot analysis, indicating the switch to the lytic life-cycle of EBV. These results were successfully repeated using the EBV+ Akata cell line. Induction of viral thymidine kinase (vTK) was shown by QRT-PCR as well. Histone deacetylase inhibitors are a well known group of broad-acting chemical agents able to reactivate EBV. In combination experiments, we found that Bortezomib plus sodium butyrate or SAHA act at least additive in inducing the EBV lytic life cycle in HR-1 or RAJI cells. Bortezomib sensitizes the EBV+ Akata cell line 2A8-1 to growth inhibitory effects of ganciclovir as shown by MTT assays. The cells were treated with two different non-toxic drug concentrations which were chosen low enough not to induce apoptosis (bortezomib: 1nM and 2nM; ganciclovir:15 and 30μM). Bortezomib induces the lytic EBV life cycle in vivo. In murine xenograft models growing the Akata A.15 line subcutaneously bortezomib induces the immediate-early protein ZEBRA as shown by immunohistochemistry and vTK as shown by QRT-PCR. Experiments to induce lytic phase EBV in murine xenograft models using the Akata cell line and to combine EBV induction with the nucleoside analogue ganciclovir are in progress. Murine studies with EBV-transformed lymphoblastoid cell line (LCL) xenograft models, combination bortezomib plus ganciclovir, and molecular imaging with FHBG specific PET probes are in progress. Reactivating EBV with proteasome inhibitors alone or in combinations with low concentrations of histone deacetylase inhibitors may be a less toxic therapeutic strategy for EBV-associated lymphomas.
Understanding Epstein-Barr Virus Life Cycle with Proteomics: A Temporal Analysis of Ubiquitination During Virus Reactivation