Mechanism of action of drug combination regimens in the treatment of Epstein-Barr virus-associated lymphoproliferative diseases

Abstract

Epstein-Barr virus (EBV) is strongly associated with lymphoproliferative diseases (LPDs) in both immunocompetent and immunocompromised hosts, through establishing different latency patterns. Post-transplant lymphoproliferative disorder (PTLD) expresses the full spectrum of latent proteins in the type III latency, including six EBV nuclear antigens (EBNA-1, -2, -3A, -3B, -3C and -LP) and three latent membrane proteins (LMP-1, -2A and -2B) whilst Burkitt’s lymphoma (BL) with Wp-restricted latency expresses EBNA-1, -3A, -3B, -3C, -LP and BHRF-1. EBNA-3C binds to Bcl-6 resulting in ubiquitination and proteasomal degradation of Bcl-6 with subsequent increase in the expression of cyclin D1 and Bcl-2, thereby promoting G1-S cell cycle progression and inhibition of apoptosis. EBNA-3C also recruits histone deacetylases (HDACs) to repress the transcription of the cyclin-dependent kinase inhibitors, p16INK4A and p21WAF1. LMP-1 is a CD40 receptor homolog which constitutively activates NF-κB via the proteasomal degradation of IκBα, thereby up-regulating the transcription of pro-survival proteins, such as Bcl-2 and Mcl-1, to inhibit apoptosis. We postulate that these pro-survival pathways associated with EBNA-3C and LMP-1 are possible candidates of targeted therapy of EBV-driven lymphoproliferative diseases (EBV-LPDs). We first tested whether combining bortezomib (proteasome inhibitor) with suberoylanilide hydroxamic acid (HDAC inhibitor) [bortezomib/SAHA] could mediate the synergistic killing of EBV-LPDs through counteracting the functions of EBNA-3C. Bortezomib/SAHA preferentially killed BL cells expressing EBNA-3C through the upregulation of p16INK4A and p21WAF1 and reduction of EBNA-3C’s phosphorylation of the cell cycle-dependent CDC25C phosphatase, thereby abrogating the pro-survival functions of EBNA-3C. However, bortezomib/SAHA synergistically killed lymphoblastoid cell line (LCL), which is an in vitro model of PTLD, irrespective of EBNA-3C. We proceeded to test whether combining bortezomib with venetoclax (Bcl-2 inhibitor) [bortezomib/venetoclax] could counteract pro-survival functions of viral proteins and induce synergistic killing of LCLs. Isobologram analysis showed that bortezomib/venetoclax induced cell death synergism in spontaneous LCLs (sLCLs) derived from patients with PTLD and EBV-associated haemophagocytic lymphohistiocytosis, respectively. The mechanism of killing was related to the suppression of NF-κB signaling pathway induced by LMP-1 and inhibition of proteasomal degradation of Bcl-6 mediated by EBNA-3C. Upon treatment with bortezomib/venetoclax, the levels of cyclin D1 and phosphorylated Bcl-2 (at serine 70) were significantly decreased in the sLCLs. Production of reactive oxygen species and activation of DNA damage response were detected in the sLCLs. Noxa, a pro-apoptotic BH3-only protein, was induced by bortezomib to enhance the susceptibility of the sLCLs to apoptosis upon treatment with venetoclax whilst the knockdown of Noxa in the sLCLs led to the resistance of the cells to apoptosis. In vivo study demonstrated that bortezomib/venetoclax significantly inhibited the growth of xenografts of the sLCL in SCID mice. Taken together, bortezomib/venetoclax synergistically induced potent apoptosis in sLCLs both in vitro and in vivo. The induction of apoptosis was mediated by the suppression of NF-κB signaling by LMP-1 and the reduction of phosphorylated Bcl-2 and cyclin D1 associated with EBNA-3C. Noxa induced by bortezomib sensitized the sLCLs to killing by venetoclax. We conclude that the combination regimen of bortezomib and venetoclax specifically targets the cell survival dependencies of EBV-driven LPDs on EBNA-3C and LMP-1.