Abstract B66: Resistance to bortezomib in myeloma cell lines is mediated by increased IGF-1/Akt signaling to activate Cot kinase and NF-κB

Abstract

Abstract Background: Targeting the proteasome as an anti-cancer therapy was first realized with bortezomib, now one of the standards used in relapsed/refractory multiple myeloma and mantle cell lymphoma. Despite this advance, response rates have been as low as 23%, indicating the importance of innate or acquired resistance to bortezomib. Methods: To improve our understanding of the mechanisms responsible for bortezomib resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and -independent (RPMI 8226 and OPM-2) bortezomib-resistant (BR) myeloma by continuously exposing drug-naïve cells to increasing bortezomib concentrations. All of the lines where characterized by gene expression profiling to identify potential mechanisms of resistance. Results: Cell lines resistant to bortezomib retained their viability in the presence of 50 nM or more of bortezomib with degrees of resistance up to 4.3. Gene expression profiling revealed up-regulation of insulin-like growth factor (IGF)-1 transcripts (5.2-fold), Akt (3.2-fold), and cancer Osaka thyroid (Cot) kinase (2.3-fold). Consistent with our profiling data, BR clones had increased soluble IGF-1 levels, increased IGF-1 protein, and phospho-IGF-1R activation. Next we examined the effect of overexpression of Cot on BR clones. Cot functions to regulate nuclear factor kappa B (NF-κB)-dependent transcription in response to Akt by promoting post-translational processing of inactive NF-κB p105 to active p50. Basel levels of nuclear activated p50 were increased up to 4.7-fold in BR clones. Activated NF-κB dimers were also found to be significantly increased in ANBL-6.BR and 8226.BR cells as evidenced by phosphorylation of p65 (RelB). Our examination into Cot expression in drug-naive and BR cells showed a large increase of Cot protein and phospho-Cot (Thr290) activation in 8226.BR cells that was enhanced with 24-hour bortezomib treatment. A transcriptional target of NF-κB is Bcl-2 was also found to be increased in profiling studies (5.5-fold) and correlates with increased expression in BR cell lines tested. Treatment with 10 nM bortezomib for 6 hours led to a decreased in Bcl-2 protein both drug-naïve and drug-resistant cells. A decrease in Bcl-2 protein was also observed in ANBL-6.BR, RPMI 8226.BR, and OPM-2.BR cells in response to 24-hour treatment with picropodophyllin, a pharmacologic inhibitor of IGF-1R activation, consistent with the hypothesis that the IGF-1/Akt signaling axis in important in Bcl-2 protein expression in bortezomib-resistant clones. Conclusions: Our data support the hypothesis that increased signaling through the IGF-1/Akt axis and enhanced expression of Cot kinase is another mechanism that mediates stable bortezomib resistance in vitro through the activation of NF-κB and its downstream transcriptional targets, including Bcl-2. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B66.