Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle Induction with Bortezomib Is a Response to ER Stress

Abstract 1736

EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta.