Combination of histone deacetylase and proteasome inhibitors counteracts the anti-apoptotic and cell cycle regulatory function of EBNA-3C protein in Epstein-Barr virus-positive burkitt and lymphoblastoid cells

Abstract

BACKGROUND AND AIMS: Epstein-Barr virus (EBV) drives the development of post-transplant lymphoproliferative disorder (PTLD) via the concerted action of Epstein-Barr nuclear antigen (EBNA) proteins. We previously reported that combination of histone deacetylase inhibitor (HDACi) and proteasome inhibitor (Pi) could synergistically induce apoptosis of Burkitt lymphoma (BL) and lymphoblastoid cells (LCL; an in-vitro cell model of PTLD) which express the EBNA-3A, -3B and -3C proteins. In this study, we aim to investigate the mechanism by which combined HDACi/Pi counteracts the survival function of EBNA-3A, -3B or -3C proteins. METHODS: Spontaneous LCLs (sLCLs) and a panel of BL cell lines (BL31) harboring EBNA-3A, -3B or -3C knockout (KO) EBV genome and corresponding cell lines harboring the knockout virus together with the individual gene revertant (Rev) were treated with combination of HDACi (vorinostat) and Pi (bortezomib), followed by analyses of apoptosis and cell cycle. In-vivo testing of B cell xenografts in SCID mice was also performed. RESULTS: Isobologram analysis showed that combined HDACi/Pi induced significantly greater synergistic killing in 3C Rev than 3C KO cells. Such differential response was not observed in either 3A or 3B Rev and KO cells. Furthermore, higher percentage of sub-G1 population and stronger proteolytic cleavage of apoptotic markers including PARP and caspase-3 as well as up-regulation of p21 were found in EBNA-3C expressing cells when compared to 3C KO cells. Combined HDACi/Pi also mediated G2/M arrest in 3C KO cells but not in EBNA-3C expressing cells and produced enhanced suppression of the growth of EBNA-3C expressing but not 3C KO BL31 xenografts in SCID mice, supporting that EBNA-3C might be the major counteracting target of the drug combination. Furthermore, significant increase in percentage of sub-G1 population without G2/M arrest and up-regulation of cleaved PARP and caspase-3 as well as p21 were also observed in paediatric PTLD patient-derived sLCLs upon treatment with combined HDACi/Pi. CONCLUSIONS: Combined HDACi/Pi induces potent apoptosis in EBV-positive BL and LCL through counteracting the anti-apoptotic and cell cycle regulatory function of EBNA-3C. This drug regimen may be tested as treatment for EBV driven lymphoproliferative diseases. The work is funded by CRCG grant (#104003676) of KFH and CRCG (#104002068, #104002845 and #104003674) and EBV research (#200004525) grants of AKSC.