Regulation of Drug Resistance Via the Overexpression of Phopho-RKIP (P-RKIP) in Multiple Myeloma: Dephosphorylation of P-RKIP Reverses Drug Resistance

Background: Multiple myeloma (MM) remains an incurable disease. We have reported that MM cell lines and patient-derived MM cells overexpress the gene product Raf-Kinase Inhibitor Protein (RKIP) in its inactivated phosphorylated (p-RKIP) form.¹ Active RKIP inhibits both the Raf/MEK/ERK and NF-κB pathways and resulting in the inhibition of proliferation, metastasis, and sensitization to chemo and immune drugs.²

Objective: Drug resistance is a common feature of patients with MM. The delineation of factors that regulate resistance and their targeting may reverse resistance and leads to prolongation of survival.

Hypothesis: We hypothesized that dephosphorylation of RKIP in MM may restore the activity of RKIP and resulting in the sensitization of drug-resistant MM cells to drug-induced cytotoxicity.

Experimental Design: The expression of RKIP and p-RKIP (specific anti-p-RKIP antibody) was analyzed in both MM cell lines and fresh tumor cells derived from bone marrow aspirates of MM patients using Western, flow cytometry and IHC and a specific anti-pRKIP monoclonal antibody. We also examined the effect of a PKC inhibitor, bisindolymalemide-1 (BIM) treatment in MM cell lines and their response to treatment with anti-MM drugs such as bortezomib, melphalan, and bendamustine.

Findings: The analyses of MM cell lines and patient-derived MM cells revealed the overexpression of RKIP, the majority of which was in its inactive phosphorylated form, p-RKIP. Treatment of MM cell lines with the PKC inhibitor BIM resulted in the dephosphorylation of p-RKIP and the treated drug resistant MM cells were sensitized to bortezomib, melphalan, and bendamustine-induced cell death. We have also found a correlation between the RKIP/p-RKIP ratios in MM and clinical response; Kaplan-Meyer analysis demonstrated that there was a correlation between the high expression of RKIP and poor survival.

Conclusions and Clinical Implication: The findings demonstrated that the overexpression of p-RKIP in MM was involved, in part, on the regulation of drug resistance since dephosphorylation of p-RKIP reversed the drug resistance. The role of p-RKIP in the regulation of drug resistance in MM is corroborated in our prior reported studies in other cancer types that demonstrated that knockout of RKIP rendered the sensitive tumor cells drug resistant, whereas in contrast, overexpression of RKIP sensitized the drug-resistant cells to drug-induced apoptosis. We conclude that the combination treatment of a PKC inhibitor and anti-MM drugs can be clinically relevant in the treatment of patients who are resistant to drug treatment. Also, we propose that the examination of the levels of p-RKIP in MM may reveal a novel prognostic biomarker and the determination of proper treatment regimens.

Future studies: We propose to investigate the molecular and biochemical means by which p-RKIP is overexpressed in MM and characterizing the specific PKC that is involved in the phosphorylation of RKIP in MM.

References

¹ Baritaki, S, Huerta-Yepez, S, Cabrava-Haimandez, M, Sensi, M, Canevari, S, Libra, M, Penichet, M, Chen, H, Berenson, JR, Bonavida, B. Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma. Forum on Immunopathological Diseases and Therapeutics, 2: 179-188, 2011.

² Bonavida B. RKIP-mediated chemo-immunosensitization of resistant cancer cells via disruption of the NF-κB/Snail/YY1/RKIP resistance-driver loop. Crit Rev Oncog. 19:431-45, 2014.