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B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.

Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

Background: Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells., Methods: SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies., Results: We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA., Conclusion: The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.

Lymphomas associated with post-transplant lymphoproliferative disease (PTLD) represent a significant complication of immunosuppression in transplant recipients. In immunocompetent individuals, EBV-specific cytotoxic T lymphocytes (CTL) prevent the outgrowth of activated B lymphoblasts through apoptosis induction. Soluble versions of TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL) can induce apoptosis in numerous tumor cell types. Given the therapeutic potential of TRAIL, we examined the sensitivity of EBV+ spontaneous lymphoblastoid cell lines (SLCL) derived from patients with PTLD to treatment with soluble TRAIL. Despite abundant expression of TRAIL receptors (TRAIL-R), resistance to TRAIL-induced apoptosis was observed in all SLCL examined. This resistance could not be overcome by concomitant treatment with several pharmacological agents. Unlike BJAB positive control cells, for each SLCL tested, cleavage and activation of caspase 8 was inhibited due to failed recruitment of FADD and caspase 8 to TRAIL receptors upon stimulation. Further indicative of a proximal defect, TRAIL receptor aggregation could not be detected on the cell surface of SLCL following ligand engagement. These results suggest that the use of TRAIL for eliminating PTLD-associated tumors may be of limited clinical utility, and illustrate another mechanism by which EBV+ B lymphoma cells can evade tumor surveillance at the level of death receptor signaling.