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Combination of suberoylanilide hydroxamic acid (SAHA) and bortezomib (SAHA/bortezomib) was shown to synergistically induce killing of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) of type III or Wp-restricted latency, both of which express EBNA3A, -3B and -3C proteins. We hypothesize that SAHA/bortezomib can counteract the survival functions conferred by the EBNA3 proteins. We tested the effect of SAHA/bortezomib on the survival of BL cell lines containing EBNA3A, -3B or -3C knockout EBV with or without the respective revertant EBNA3 genes. Isobologram analysis showed that SAHA/bortezomib induced significantly greater synergistic killing of EBNA3C-revertant cells when compared with EBNA3C-knockout cells. Such differential response was not observed in either EBNA3A or -3B revertant versus their knockout pairs. Interestingly, EBNA3C-knockout cells showed significant G2/M arrest whilst EBNA3C-revertant cells and LCLs escaped G2/M arrest induced by SAHA/bortezomib and became more susceptible to the induction of apoptosis. In parallel, SAHA/bortezomib induced stronger expression of p21WAF1 but weaker expression of p-cdc25c, an M-phase inducer phosphatase, in EBNA3C-expressing cells when compared with EBNA3C-knockout cells. SAHA/bortezomib also induced greater growth suppression of EBNA3C-expressing xenografts (EBNA3C-revertant and LCL) than that of EBNA3C-knockout xenografts in SCID mice. In conclusion, our data showed that SAHA/bortezomib could synergistically induce killing of BL and LCL through counteracting the survival functions of EBNA3C, providing a strong basis for clinical testing of this drug combination in patients with EBV-associated lymphoproliferative diseases.

Epstein-Barr virus (EBV) is strongly associated with lymphoproliferative diseases (LPDs), in particular B cell, in both immunocompetent and immunocompromised hosts. The virus is able to manipulate host cell machineries such as the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and the survival of the host cells through expression of various viral proteins in distinct latency patterns. Latent membrane protein-1 (LMP-1) is a constitutively active CD40 receptor homolog and activates NF-κB signaling pathways to induce Bcl-2 expression whilst Epstein-Barr nuclear antigen-3C (EBNA-3C) interacts with and mediates proteasomal degradation of Bcl-6 protein which, in turn, increases Bcl-2 expression. We hypothesize that combining a proteasome inhibitor, bortezomib, with a Bcl-2 inhibitor, venetoclax, will induce synergistic killing of EBV-driven LPDs expressing both LMP-1 and EBNA-3C in the latency III pattern. Isobologram showed that combination of bortezomib and venetoclax could synergistically induce potent apoptosis of spontaneous lymphoblastoid cell lines (sLCL), derived from patients with post-transplant lymphoproliferative disorder (PTLD), expressing LMP-1 and EBNA-3C proteins. The mechanism of killing was related to the suppression of NF-κB signaling pathway induced by LMP-1. The phosphorylation of serine 70 of Bcl-2, which enhances the anti-apoptotic activity of Bcl-2 through stabilization of its interaction with other pro-apoptotic proteins such as Bak and Bim, was decreased in the sLCL, but not in the LMP-1 or EBNA-3C knockdown LCL, upon treatment with the drug combination. Activation of DNA damage response and production of reactive oxygen species were observed in the sLCL, contributing to the synergistic cell death. Bortezomib induced the expression of pro-apoptotic initiator, NOXA, to enhance the susceptibility of the sLCL to apoptosis upon treatment with venetoclax whilst knockdown of NOXA in the sLCL led to the resistance of the cells to apoptosis induced by the drug combination. In-vivo study demonstrated that the drug combination significantly inhibited the growth of xenograft of sLCL in SCID mice (p Disclosures No relevant conflicts of interest to declare.