Bortezomib Resistance Is Mediated by Increased Signaling through the Insulin-Like Growth Factor-1/Akt Axis.

Inhibition of the proteasome as an anti-cancer strategy was first validated with the approval of bortezomib (VELCADE®), which is now a standard of care in relapsed and/or refractory multiple myeloma, and is also being used in the front-line setting, where it has shown improved efficacy compared to previously available induction therapies. However, myeloma remains incurable, and the use of bortezomib up-front may limit its later utility in the relapsed setting. Indeed, response rates on retreatment of patients with previously bortezomib-sensitive disease have been as low as 23%, indicating the rapid development of resistance. The mechanisms by which plasma cells survive and become resistant to bortezomib therapy have not been fully characterized, in part due to a lack of appropriate model systems. Discovery of these resistance mechanisms will facilitate the development of strategies to circumvent them, and help to identify potential novel therapies for the treatment of patients with disease showing resistance to bortezomib.

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. One of these pooled lines derived from RPMI 8226 cells was then subjected to single-cell cloning, and all of the lines where characterized by gene expression profiling to identify potential mechanisms of resistance.

Cell lines resistant to bortezomib, such as RPMI 8226.BR, retained their viability, and did not demonstrate increased Annexin V staining compared to drug-naïve cells in the presence of 50 nM or more of this proteasome inhibitor. Calculations of the degree of resistance revealed values of up to 4.3 or more. Bortezomib resistance was a stable phenotype, since these cells remained unaffected by bortezomib treatment even after a one month period of drug withdrawal. Mutation of the binding site of the β5 proteasome subunit has been reported as one mechanism of bortezomib resistance, but the chymotrypsin-like proteasome activity in RPMI 8226.BR cells could still be suppressed by bortezomib even in the face of increased β5 expression in some subclones. Increased drug clearance has also been proposed as a resistance pathway, but studies by mass spectrometry indicated increased intracellular concentrations of bortezomib and its metabolites in RPMI 8226.BR cells, suggesting that neither of these mechanisms contributed to resistance in our models. Gene expression profiling revealed evidence of up-regulation of insulin-like growth factor (IGF)-1 transcripts by up to 5.2-fold, along with increased expression of phosphoinositide 3-kinase (by up to 4.4-fold) and protein kinase B/Akt (up to 3.2-fold), but suppression of phosphatase and tensin homolog (by up to 5.3-fold). RPMI 8226.BR clones secreted increased levels of IGF-1 into their media leading, in combination with the above gene expression changes, to increased levels of cellular activated, phosphorylated IGF-1 receptor (IGF-1R) and Akt. Addition of exogenous IGF-1 did reduce bortezomib sensitivity in drug-naïve myeloma cells, but induced a much greater increase in resistance in the BR clones even greater than that present in un-supplemented media. In contrast, inhibition of the IGF-1/IGF-1R/Akt axis using picropodophyllin, a pharmacologic inhibitor of IGF-1R; LY294002, a PI3K inhibitor; or NVP-BEZ-235, a novel Akt inhibitor, enhanced sensitivity to bortezomib, and this enhancement was greatest in the BR clones. Similarly, suppression of IGF-1R expression using Lentiviral-mediated delivery of specific shRNAs re-sensitized bortezomib-resistant cells to bortezomib-induced apoptosis, and indeed returned their sensitivity to that of their drug-naïve counterparts.

Our data support the hypothesis that increased signaling through the IGF-1/Akt axis mediated by enhanced plasma cell secretion of IGF-1, and enhanced expression of PI3K and Akt, is another mechanism that mediates stable bortezomib resistance in vitro. Moreover, if validated by ongoing studies of primary patient samples, they provide a rationale for the introduction of combination therapies with bortezomib and either Akt or IGF-1 inhibitors to overcome or prevent the emergence of bortezomib resistance in the clinic.

No relevant conflicts of interest to declare.