Abstract 1243: Inside of multiple mechanism of adaption towards the proteasome inhibitors in multiple myeloma
Authors: Besse Andrej , Besse Lenka , Kraus Marianne , Bader Jurgen , Novkovic Mario , Kryukov Fedor , Nemec Pavel , Silzle Tobias , Driessen Christoph 
Published: July 2016

Abstract
Malignant plasma cells drive massive overproduction of monoclonal immunoglobulin in multiple myeloma (MM). Hence MM cells critically rely on a proper balance between protein biosynthesis and folding, and degradation of unfolded/ misfolded protein orchestrated by the unfolded protein response (UPR). Inhibitors of the proteasome, the terminal protease along the UPR pathway, are highly effective against MM. Adaptive resistance to current proteasome inhibitors (PI) (Bortezomib-Btz, Carfilzomib-Cfz) is a major cause of treatment failure. Btz is a peptide boronate-type reversible inhibitor, whereas Cfz is of epoxyketone-type and binds irreversibly to the same proteasomal B5 active site target. Next generation PI are likewise B5-directed and either of the peptide boronate (ixazomib, dalanzomib), or the epoxyketone type (oprozomib). PI resistance is currently understood via two alternative concepts, the load/capacity concept characterized by proteasome upregulation in conjunction with downregulation of the UPR via reduced IRE/XBP-1 signaling, and the mutation concept basing on B5 active site mutations that prevent PI binding. Only IRE1/XBP1 downregulation have been confirmed in patient-derived material. We generated an in vitro model where AMO-1 myeloma cells (IC50: 3.1nM for Btz, 4.9nM Cfz) were adapted to Btz (AMOaBtz; IC50: 298.7nM Btz, 43.2nM Cfz) or to Cfz, (AMOaCfz; IC50: 27.9nM Btz, 411.5nM Cfz). AMOaBtz cells showed preferential resistance against ixazomib and dalanzomib, while AMOaCfz cells were preferentially resistant to oprozomib and other epoxyketone-type experimental inhibitors. AMOaBtz and AMOaCfz likewise downregulated IRE/XBP-1, but only AMOaBtz contained an acquired proteasome mutation (A310G in PSMB5), however this had only a modest influence on Btz binding and proteasome inhibition. Global gene expression analysis revealed 1430 differentially expressed genes in AMOaBtz/ AMOaCfz (FC&gt;2, p&lt;0.05). Analysis of uniquely upregulated genes in AMOaCfz identified the multi-drug resistance gene ABCB1 (MDR1) that was found 120-fold upregulated by qPCR. Inhibition of ABCB1 with Verapamil or Reserpin strongly increased Cfz sensitivity, confirming functional significance. Genes responsible for immunoglobulin production were selectively downregulated in AMOaBtz, and paraprotein production (IgA) was abolished as measured by ELISA. In conclusion, AMOaCfz is the first model combining the key features found in PI-resistant patient cells (lack of PSMB5 mutation and low IRE1/XBP1 activity). The comparison of AMOaCfz and AMOaBtz suggests the presence of class-preferential adaptation mechanisms, which may be overcome by using alternative type proteasome inhibitors or mMDR1 inhibitors.
Citation Format: Andrej Besse, Lenka Besse, Marianne Kraus, Jurgen Bader, Mario Novkovic, Fedor Kryukov, Pavel Nemec, Tobias Silzle, Christoph Driessen. Inside of multiple mechanism of adaption towards the proteasome inhibitors in multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1243.