Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.     

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor β (TGF-β) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.     

Phosphatidylinositol 3-kinase is a determinant of responsiveness to B cell antigen receptor-mediated Epstein-Barr virus activation

B cell Ag receptor (BCR) cross-linking with anti-Ig Abs efficiently induces activation of latently infected EBV in some B cell lines, but not in others. The present study was aimed at defining the molecular mechanisms that determine the response to BCR-mediated EBV activation. Comparison of Burkitt's lymphoma-derived Akata, Mutu-I, and Daudi cells, which are representative responders and nonresponders to BCR-mediated EBV activation, respectively, indicated that three signaling pathways, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), were activated in anti-Ig-treated Akata and Mutu-I cells. However, in anti-Ig-treated Daudi cells PI3K was not activated, ERK was faintly activated, and p38 MAPK was constitutively phosphorylated irrespective of anti-Ig treatment. Restoration of PI3K activity with insulin-like growth factor 1 restored ERK and p38 MAPK pathways, and was accompanied by EBV activation in anti-Ig-treated Daudi cells. In contrast, a specific inhibitor for PI3K, wortmannin, inhibited EBV activation by anti-Ig Abs in Akata and Mutu-I cells. Transfection assays in EBV-negative Daudi cells revealed that PI3K activated a promoter for BZLF1, which is a switch of EBV activation from a latent infection, in the absence of other EBV products suggesting that the BZLF promoter was a target of BCR signaling, and that PI3K was important for BCR-mediated BZLF1 activation. These results indicate that the absence of PI3K impedes the progression of signals through the BCR and becomes a determinant of unresponsiveness to BCR-mediated EBV activation.