Inhibition of the Akt Pathway in MM Cell Lines by the Anti-TfR-IgG3-Avidin Fusion Protein (Anti-TfR-IgG3-Av): Role in Chemosensitization to CDDP-Induced Apoptosis

Multiple Myeloma (MM) is an incurable disease of monoclonal malignant plasma cells. Treatment of MM with conventional chemotherapeutic drugs has resulted in improved response rates, however, with no sufficient improvement in overall survival. Bortezomib has been recently used and results in significant clinical responses in refractory MM. However, many patients relapse and become refractory to cytotoxic therapies and, hence, the need for new therapies. We have generated an Anti-TfR-IgG3-Avidin Fusion Protein (Anti-TfR-IgG3-Av) that can bind MM which express high levels of transferrin receptor and can deliver biotinylated molecules into cancer cells (Ng et al PNAS2002; 79:10706). We have reported that treatment of MM with Anti-TfR-IgG3-Av results in inhibition of cell proliferation and direct cytotoxicity in few cell lines. Further, we have also found that Anti-TfR-IgG3-Av can sensitize resistant MM cells to drugs (eg. CDDP)-induced apoptosis. Sensitization by Anti-TfR-IgG3-Av resulted in the inhibition of several anti-apoptotic gene products like XIAP, Bid, Bcl-2 and BclXL. Since these gene products are regulated by the NF-κB pathway, we hypothesized that Anti-TfR-IgG3-Av may inhibit the AKT pathway in MM cell lines. The AKT signaling inactivates several pro-apoptotic factors, such as Bad, which is phosphorylated and inhibits its binding and inactivation of BclXL. AKT also activates IκB kinase (IKK) to phosphorylate IκB (inhibitor of NF-κB ) and leading to its proteasomal degradation and NF-κB nuclear localization. The AKT and NF-κB pathways result in the transcription of many anti-apoptotic gene products like XIAP, Bcl-2, survivin and BclXL. Treatment of MM cell lines with Anti-TfR-IgG3-Av resulted in inhibition of phospho-AKT and inhibition of NF-κB activity and downstream inhibition of above anti-apoptotic gene products. We then examined the roles of AKT and NF-κB in Anti-TfR-IgG3-Av-induced sensitization of MM to CDDP-apoptosis. Treatment of IM-9 cells with siRNA AKT, not control siRNA, resulted in inhibition of AKT concomitantly with inhibition of Bcl-2 and survive in. The cells treated with si-RNA AKT were sensitized to CDDP-induced apoptosis. These findings suggested that Anti-Anti-TfR-IgG3-Av-induced sensitization to CDDP may be due, in part, to inhibition of AKT. Likewise, the role of NF-κB inhibition by Anti-TfR-IgG3-Av in the sensitization to CDDP was demonstrated by the use of the specific NF-κB inhibitor, DHMEQ. Thus, both inhibition of AKT and NF-κB pathways by Anti-TfR-IgG3-Av play a major role in Anti-TfR-IgG3-Av-induced sensitization to CDDP. The apoptosis achieved by the combination of Anti-TfR-IgG3-Av and CDDP resulted from the complementation of several gene products modified by each agent alone and resulting in the activation of caspases 9, 8 and 3 and apoptosis. The above findings provide an underlying mechanism of Anti-TfR-IgG3-Av-induced cell signaling modification that renders drug-resistant MM cells sensitive to apoptosis by drugs.