Drug modulators of B cell signaling pathways and Epstein-Barr virus lytic activation

Abstract

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. EBV is the causative agent for several epithelial and B cell cancers mostly in immunocompromised patients. In order to maintain a reservoir of EBV over an infected persons lifetime, EBV must periodically reactivate from latency and enter lytic replication to produce infectious virions, which go on to infect new cells. This can happen spontaneously or by stimulus, such as activating the B cell receptor (BCR) pathway. While the importance of BCR signaling in relation to EBV activation has not been described in-vivo, B cell receptor signaling may have an impact on EBV activation in patients. Some B cell malignancies are treated with drugs that inhibit the BCR pathway. Thus it is conceivable that these drugs might affect the viral reservoir maintained by basal B cell receptor signaling. Here, we show that a group of BCR signaling pathway kinase inihibitors used to treat hematologic malignancies, ibrutinib, idelalisib, and dasatinib, block BCR-mediated EBV lytic induction at clinically relevant doses. siRNA experiments where the targets of these drugs were depleted resulted in the inability of these drugs to block BCR-mediated EBV induction. We found that these drugs specifically block phosphorylation of their targets in the presence of BCR stimulation. Other lytic inducers were tested in the presence of these drugs, and none of the drugs were able to block EBV induction by the other inducers. Thus we believe that blocking BCR-mediated lytic induction by ibrutinib, idelalisib and dasatinib is specific and clinically relevant. In this work, we also confirmed that the immunosuppressive drugs cyclosporine and tacrolimus inhibit BCR-mediated lytic induction, while rapamycin does not. Rapamycin targets mTOR, which is downstream but still part of the BCR pathway. Treatment of cells with torin2, which inhibits both mTORC1 and mTORC2 prior to EBV induction via the BCR pathway shows that mTORC2 contributes to BCR-mediated lytic induction and that FKBP12 binding alone is not adequate to block activation. Additionally, we show that BCR signaling can activate EBV in freshly isolated B cells from peripheral blood mononuclear cells (PBMC) and that this activation can be inhibited by ibrutinib or idelalisib. Taken together, this work shows that ibrutinib, idelalisib and dasatinib block BCR-mediated lytic induction in a specific manner and at clinically relevant doses. While rapamycin does not block BCR-mediated lytic induction on its own, it was determined that mTORC2 plays a role in the BCR pathway and contributes to BCR-mediated lytic activation of EBV. We found that we were able to replicate this system in patient-derived PBMCs, thus suggesting that this system may be relevant in humans and could play a role in future studies on EBV maintenance in the context of long-term treatment by BCR pathway inhibitors.