The Novel Proteasome Inhibitor NPI-0052 Induces the Expression of Raf-1 Kinase Inhibitor Protein RKIP) in B-NHL via Inhibition of the Transcription Repressor Snail: Roles of Snail and RKIP in Sensitization to TRAIL Apoptosis

A subset of patients with B cell malignancies does not respond to conventional current cytotoxic therapies. Novel immunotherapeutic approaches have been considered such as TRAIL and agonist DR4/DR5 mAbs that are currently in phase I/II clinical trials in patients with a variety of tumor types. However, most tumors are not sensitive to TRAIL/agonist antibodies due to the development of a generalized resistance to apoptotic stimuli. Therefore, for these therapies to be effective it is a requisite that resistance is modified by the use of sensitizing agents that target cell survival anti-apoptotic pathways or, more selectively, specific gene products in the apoptotic pathways. Proteasome inhibitors such as bortezomib and the novel inhibitor NPI-0052 have shown both direct cytotoxicity and also sensitizing ability when used in combination with biologics and cytotoxic drugs. We have recently reported that treatment of TRAIL-resistance B-NHL cell lines with NPI-0052 (not toxic by itself) in combination with TRAIL resulted in significant potentiation of apoptosis and synergy was achieved. Sensitization was due, in part, to inhibition of NF-kB activity and induction of Raf-1 kinase inhibitor protein (RKIP) [Baritaki et al., J Immunol 80:6199, 2008]. The role of RKIP in TRAIL apoptosis by NPI-0052 in Ramos cells was examined as well as the mechanism of NPI-0052-induced expression of RKIP. Like NPI-0052, overexpression of RKIP in Ramos cells sensitized the cells to TRAIL apoptosis and, in contrast, treatment with siRNA RKIP reversed this sensitizing effect. A recent report by our collaborators [Beach et al., Oncogene 27:2243,2008] demonstrated that RKIP transcription is negatively regulated, in part, by the metastasis-inducer transcription factor Snail. Thus, we hypothesized that NPI-0052-induced upregulation of RKIP may be due to inhibition of Snail. Indeed, treatment of Ramos cells with NPI-0052 significantly downregulated Snail expression concomitantly with upregulation of RKIP. Treatment of Ramos cells with siRNA Snail resulted in upregulation of RKIP and sensitization to TRAIL apoptosis. In contrast, ectopic expression of Snail repressed RKIP expression. Further, ectopic expression of RKIP inhibited Snail expression while siRNA RKIP upregulated Snail. These findings revealed the existence of a regulatory RKIP-NF-kB-Snail-RKIP loop. NPI-0052-induced inhibition of Snail also resulted in inhibition of Snail-induced gene products involved in metastasis. These findings demonstrate that NPI-0052 regulates B-NHL sensitivity to TRAIL apoptosis via the inhibition of Snail and induction of RKIP. These findings support the potential therapeutic application of the combination of NPI-0052 and TRAIL in the treatment of patients with resistant B-NHL and other malignancies.