Inhibition of Interleukin-6 Signaling with CNTO328 Sensitizes Multiple Myeloma Cells to Bortezomib and Attenuates Proteasome Inhibitor-Mediated Induction of the Anti-Apoptotic Heat Shock Protein Response.

Abstract

The proteasome inhibitor bortezomib represents a significant advance in the treatment of multiple myeloma, but its efficacy is limited by a number of resistance mechanisms. One of the most important is the heat shock protein (HSP) and stress response pathways which, through members such as HSP-70 and mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1, oppose the pro-apoptotic activities of bortezomib. Because interleukin (IL)-6 signaling augments the heat shock response through signal transducer and activator of transcription (STAT)-1 and heat shock transcription factor (HSF)-1, we hypothesized that downregulation of IL-6 signaling would attenuate HSP induction by bortezomib, thereby enhancing its anti-myeloma activity. Treatment of the IL-6-dependent multiple myeloma cell lines KAS-6 and ANBL-6 with the combination of bortezomib and CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, resulted in greater reduction of cell viability than with either drug alone in a time- and concentration-dependent manner. This was associated with an enhanced induction of apoptosis which, under some conditions, was greater than the sum of the two individual agents alone, suggesting a synergistic interaction. Similar findings were not seen when using isotype control antibodies, and in studies of the IL-6-independent RPMI 8226 myeloma cell line. Increased activity was seen when cells were pre-treated with CNTO328 followed by bortezomib, or when they were treated with both agents concurrently, compared to treatment with bortezomib followed by CNTO328. Treatment with CNTO328 potently inhibited IL-6-mediated downstream signaling pathways, as demonstrated by marked blockade of STAT-3 and p44/42 MAPK phosphorylation. Enhanced activity of the combination regimen correlated with attenuated induction by bortezomib of the heat shock and stress response proteins HSP-70 and MKP-1 by up to 45% and 90%, respectively. Notably, CNTO328 markedly reduced levels of transcriptionally active phospho-STAT-1 and hyperphosphorylated HSF-1. Other strategies to suppress the heat shock response, including the use of the pharmacologic inhibitor KNK437, also yielded evidence for a synergistic anti-myeloma effect in combination with bortezomib. The synergistic activity of KNK437 and bortezomib was reproduced in normal mouse embryo fibroblasts (MEFs), but blunted in HSF-1 knockout MEFs. Taken together, the above data demonstrate that inhibition of IL-6 signaling enhances the anti-myeloma activity of bortezomib. They also support the hypothesis that this occurs, at least in part, by attenuating proteasome inhibitor-mediated induction of the heat shock response through downregulation of transcriptionally active STAT-1 and HSF-1. These findings provide a strong rationale for future translation of the CNTO328/bortezomib combination into the clinic.