Enhancing Activity of Bortezomib Against Multiple Myeloma (MM) through Inhibition of Interleukin (IL)-6 Signaling: A Role for Repression of Myeloid Cell Leukemia (Mcl)-1.

Levels of Mcl-1, an anti-apoptotic Bcl-2 family member that plays an important role in MM cell survival, are tightly regulated by the proteasome, and recent data has demonstrated that proteasome inhibition with bortezomib leads to Mcl-1 accumulation, thereby attenuating its activity. Previously, we reported that inhibition of IL-6 signaling with the chimeric antibody, CNTO328, potentiated the anti-myeloma activity of bortezomib in IL-6-dependent cell line models of MM, and that the enhanced activity was associated with inhibition of downstream IL-6 signaling pathways and repression of bortezomib-mediated induction of the anti-apoptotic heat shock response. Based on our promising results with this combination and the role that IL-6 plays in Mcl-1 regulation, we have extended our preclinical studies and investigated whether CNTO328 could inhibit bortezomib-mediated Mcl-1 induction and how the bone marrow microenvironment affects the activity of the combination. Pre-treatment of the IL-6-dependent MM cell lines KAS-6 and ANBL-6 with CNTO328, but not an isotype control antibody, blunted bortezomib-mediated induction of anti-apoptotic Mcl-1_L, and enhanced the cytotoxicity and pro-apoptotic activity of bortezomib. In contrast, CNTO328 did not attenuate bortezomib-mediated Mcl-1_L accumulation in the IL-6-independent MM cell line RPMI 8226, nor did it enhance the cytotoxicity of bortezomib in these cells. In the presence of patient-derived bone marrow stromal cells, CNTO328 and bortezomib resulted in a greater reduction of cell viability than with either agent alone in both ANBL-6 and KAS-6 cells. Furthermore, although CNTO328 alone did not lead to an increase in apoptosis of ANBL-6 cells in the presence of bone marrow stroma, it significantly potentiated the pro-apoptotic activity of bortezomib in a synergistic manner at clinically achievable concentrations. As opposed to our cell viability data using the co-culture system, CNTO328 was not able to increase levels of apoptosis in KAS-6 cells either as a single agent or in combination with bortezomib, suggesting that CNTO328 may be inducing cell cycle arrest of KAS-6 cells in the presence of bone marrow stroma but is not able to potentiate the apoptotic activity of bortezomib. Finally, given the important role of IL-6 in glucocorticoid resistance and the additive preclinical and clinical activity of bortezomib and dexamethasone in MM, we evaluated the activity of dexamethasone in combination with CNTO328 and bortezomib in ANBL-6 cells. Whereas the combination of bortezomib (2.5 nM) and CNTO328 reduced viability to 58%, the addition of dexamethasone (10 mM) reduced viability further to 26%. Taken together, the above data demonstrate that CNTO328 enhances the activity of bortezomib in part by attenuating bortezomib-mediated Mcl-1 accumulation, and provide the rationale for clinical evaluation of CNTO328 and bortezomib, with or without dexamethasone, in patients with MM.