Synergy in Apoptosis Is Achieved in B-NHL by the Combination of Rituximab and the Novel Proteasome Inhibitor NPI-0052: Pivotal Role of Induction of the Immune Surveillance Cancer Gene Product RKIP
Authors: Vega Mario I. , Paniagua Melisa Martinez- , Huerta-Yepez Sara , Suzuki Eriko , Umezawa Kazuo , Yeung Kam , Libra Massimo , Baritaki Stavroula , Palladino Michael A. , Bonavida Benjamin 
Published: April 2020

Abstract
Treatment of patients with B-NHL with a combination of rituximab and CHOP resulted in significant clinical response in greater than 90% of patients. The underlying mechanism of synergy achieved in-vivo is not clear; however, our recent studies with B-NHL cell lines revealed rituximab-induced inhibition of intracellular survival pathways that were responsible for reversal of resistance. The combination of rituximab and CHOP is associated with drug-induced toxicity, and thus, it is desirable to have a nontoxic agent that can replace CHOP with similar and improved clinical responses. The proteasome inhibitor, NPI-0052, has been shown to exert minimal toxicity, and induce cytotoxic activity against certain tumor cell lines and is currently in Phase I/II clinical trials as single agent and in combination with Zolinza against various cancers. We have reported that rituximab inhibits the NF-κB pathway concomitantly with the induction of Raf-1 kinase inhibitor protein (RKIP) and inhibition downstream of anti-apoptotic gene products (e.g. Bcl-2, Bc-lXL, Mcl-1, etc.). Likewise, NPI-0052 has also recently been shown to induce the expression of RKIP and inhibits downstream anti-apoptotic gene products. Based on the above findings, we hypothesized that treatment of resistant B-NHL cells with the combination of rituximab and NPI-0052 may result in the complementary induction of apoptosis through additive and/or synergistic effects as a result of inhibiting several survival and anti-apoptotic gene products regulated by NF-κB and induction of RKIP. This study was designed to test this hypothesis. Treatment of Ramos B-NHL cells with rituximab (20 μg/ml for 24 h) or NPI-0052 (20–40 nM) did not yield any significant apoptosis; however, the combination treatment resulted in significant potentiation of apoptosis and synergy was achieved. Treatment with rituximab or NPI-0052 alone resulted in inhibition of the NF-κB pathway, namely, IκBα and downstream BclXL and Mcl-1 and there was no activation of caspases. There was, however, significant induction of RKIP expression by each agent alone. The combination treatment resulted in additive effects with the activation of caspases 8, 9 and 3 and induction of apoptosis. The role of NF-κB inhibition by rituximab in synergy was corroborated with the use of the NF-κB inhibitor, DHMEQ, which sensitized the cells to apoptosis by NPI-0052. The role of RKIP induction in the regulation of apoptosis by NPI-0052 was demonstrated in cells over-expressing RKIP, which were sensitized to NPI-0052-induced apoptosis. In contrast, treatment with si-RNA RKIP reversed rituximab-induced sensitization to NPI-0052-induced apoptosis. Altogether, these findings reveal one mechanism by which rituximab sensitizes B-NHL cells to NPI-0052 apoptosis as the result of the concomitant induction of RKIP and inhibition of the NF-κB survival pathway. The findings also suggest the potential clinical application of rituximab and NPI-0052 in the treatment of patients with B-NHL with minimal toxicity. Furthermore, the findings suggest that agents that can induce RKIP may mimic rituximab in the sensitization to NPI-0052-induced apoptosis and their therapeutic application in patients who are not responsive to rituximab.