LMP1, LMP2A and CD40 Are All Essential for EBV-Associated Lymphoma Cell Survival.

LMP1, LMP2A and CD40 induce signals that lead to activation of the transcription factor NF-kB. Since inhibition of NF-kB leads to the apoptosis of EBV-infected lymphoblastoid

and lymphoma cell lines, we sought to determine whether elimination of one or more of these signals is sufficient to induce apoptosis of lymphoma cells expressing this proteins.

We developed and tested siRNA targeting LMP1 and LMP2A, which were transfected into two EBV positive (Type III)-lymphoma cell lines (IBL-1 and BCKN1) and two lymphoblastoid

cell lines (RPMI-8402 and LCL-9001). LMP1 or LMP2A suppression with siRNA resulted in inhibition of basal NF-kB by approximately 75% in the Type III cell

lines tested, indicating that a significant proportion of the constitutive NF-kB activity is being induced by both LMP1 and LMP2A. Simultaneous elimination of both LMP1 and

LMP2A showed no significant additive or synergistic effect. Elimination of LMP1 and or LMP2A was sufficient to induce apoptosis of EBV infected cells, as evaluated by flow

cytometry for Annexin V after suppression of these proteins. No effect was seen in LMP1 negative controls. Simultaneous suppression of both proteins resulted in similar levels of apoptosis, with no additive or synergistic effect. As EBV+ lymphoma cell lines express CD40, as well as CD154 (CD40 ligand) causing an autocrine loop, we hypothesized that inhibition of CD40 signaling may enhance the apoptotic effect induced by suppression of LMP1 and LMP2A. Treatment with blocking antibody to CD154 (CD40 ligand) in conjunction with LMP1 or LMP2A suppression increased apoptosis by approximately 20%. These data indicate that CD40, LMP-1 and LMP2A are critical for survival of EBV-infected lymphoma cells expressing these proteins. Thus CD40 represents a potential cellular target, and LMP1 and LMP2A may be useful viral targets for the treatment of EBV-associated lymphomas.