Rituximab-Mediated Inhibition of the Constitutive NIK/IKK/IκB/NF-κB Signaling Pathway in Non-Hodgkin’s Lymphoma (NHL) B-Cell Lines: Role in Chemo-Sensitization.

The chimeric anti-CD20 antibody rituximab (Rituxan, IDEC-C2B8) is widely used in the clinical treatment of patients with non-Hodgkin’s lymphoma (NHL). Rituximab sensitizes NHL B-cell lines to drug-induced apoptosis via selective down-regulation of Bcl-_xL expression (Jazirehi et al., Mol. Cancer Therapeutics 2:1183, 2003). We hypothesized that rituximab-mediated down-regulation of Bcl-_xL expression may be due, in part, to inhibition of constitutive NF- κB activity that regulates Bcl- _xL expression. This hypothesis was tested following treatment with rituximab of CD20+ drug-resistant Ramos (Bcl-2⁻/ Bcl-_xL⁺) and Daudi (Bcl-2⁺/ Bcl-_xL⁺) cell lines. Rituximab decreased the phosphorylation of NIK, IKK and κB-Iα, diminished IKK kinase activity and decreased NF- κB DNA-binding activity. These events and down-regulation of Bcl-_xL expression occurred with similar kinetics and were observed 3–6 h post rituximab treatment. The role of NF- κB in the regulation of Bcl-_xL transcription in both Ramos and Daudi cells was demonstrated by using 1) promoter reporter assays in which deletion of the two tandem NF- κB binding sites in the upstream promoter region significantly reduced the luciferase activity 2) IκB super-repressor expressing cells and 3) by NF- κB specific inhibitors. The underlying mechanism of the inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to upregulation of Raf-1 kinase inhibitor protein (RKIP) expression, thus, interrupting the NF- κB signaling pathway through the physical association between NIK and RKIP, which was concomitant with Bcl-_xL down-regulation. The direct role of Bcl-_xL in drug-resistance was evaluated by using Bcl-_xL over-expressing Ramos cells, which exhibited higher resistance to drugs that was partially reversed by rituximab. These findings reveal a novel mechanism of action of rituximab-mediated signaling by inducing RKIP expression that negatively regulates the constitutive NF- κB pathway resulting in Bcl-_xL down-regulation and chemo-sensitization of the NHL B-cells. Furthermore, these findings identify several targets, namely RKIP, Bcl-_xL and the components of the NF- κB signaling pathway, for therapeutic intervention in combination with cytotoxic agents to reverse adaptive and acquired resistance of B-NHL.