Bortezomib (Velcade™) and the Bcl-2 Inhibitor HA14-1 Synergistically Stimulate Apoptosis in EBV-Transformed Lymphocytes: A Potential Approach to Treatment of EBV-Associated Lymphoproliferative Disorders.

Epstein-Barr virus (EBV) transforms B-cells into immortalized lymphoblastoid cell lines (LCLs) by triggering signaling pathways that lead to the activation of multiple transcription factors, including NF-kappaB. EBV appropriation of the NF-kappaB pathway via EBV-encoded latent membrane protein-1 plays a pivotal role in EBV-mediated up-regulation of cellular growth-promoting and anti-apoptotic genes, including Bcl-2. Proteosome inhibitors provide a possible approach to inhibit constitutive activation of NF-kappaB in EBV-transformed B-cells. To determine whether the proteosome inhibitor bortezomib (PS-341, Velcade™) may have potential as a therapeutic anti-tumor agent in EBV-driven B-cell neoplasms, we evaluated the effect of bortezomib in EBV-immortalized LCLs (Sweig) in the presence and absence of the small molecular inhibitor of Bcl-2, HA14-1. Sweig cells were treated with increasing concentrations of bortezomib (1–1000 nM) for 24 hrs in the absence and presence of HA14-1 (10 uM), and assayed for cell proliferation (by MTT assay), apoptosis (by quantitation of DNA fragmentation in a flow cytometric TUNEL assay), caspase activation (by Western blotting), and levels of Bcl-2 family proteins (by Western blotting). Bortezomib alone inhibited proliferation of LCLs in a dose-dependent manner with an IC₅₀ of 50 nM. At this concentration, bortezomib stimulated apoptosis in only 20% of LCLs, and we observed minimal caspase-3 cleavage. HA14-1 treatment alone had no significant effects on proliferation, apoptosis, or caspase-3 cleavage. When the Bcl-2 inhibitor HA14-1 was added to bortezomib, we observed marked enhancement of anti-proliferative and pro-apoptotic effects in LCLs. This synergistic interaction was observed with sequential exposure of LCLs to bortezomib followed by HA14-1 (8 hrs) or simultaneous exposure to both drugs for 24 hrs, but not when HA14-1 was added 8 hrs prior to bortezomib. In the presence of HA14-1, the IC₅₀ for bortezomib decreased to 10 nM. Bortezomib (25 nM)/HA14-1 stimulated apoptosis in 80% of LCLs compared to bortezomib alone (20%). Moreover, bortezomib/HA14-1 triggered pronounced cleavage of both caspase 9 and 8, as well as caspase 3, suggesting activation of apoptosis through both mitochondrial and extrinsic pathways. Interestingly, Bcl-2 protein levels were increased, with appearance of the pro-apoptotic Bcl-2 cleavage fragment, in LCLs treated with bortezomib/HA14-1. These studies have demonstrated for the first time that bortezomib mediates anti-proliferative and pro-apoptotic effects in EBV-transformed lymphocytes, and that these effects are dramatically enhanced in the presence of a Bcl-2 inhibitor. These findings support further investigation of bortezomib in EBV-associated lymphoproliferative diseases and suggest that combining bortezomib with Bcl-2 antagonists, such as rituximab or Bcl-2 antisense oligonucleotides, may enhance anti-tumor efficacy. Studies are in progress to further delineate the mechanism(s) of interaction between bortezomib and HA14-1 in LCLs, and to evaluate the anti-tumor efficacy of bortezomib in the pre-clinical SCID/human model of EBV-associated lymphoproliferative disease.