Adaptive resistance of myeloma to proteasome inhibition represents a clinical challenge, whose biology is poorly understood. Proteasome mutations were implicated as underlying mechanism, while an alternative hypothesis based on low activation status of the unfolded protein response was recently suggested (IRE1/XBP1-low model). We generated bortezomib- and carfilzomib-adapted, highly resistant multiple myeloma cell clones (AMO-BTZ, AMO-CFZ), which we analyzed in a combined quantitative and functional proteomic approach. We demonstrate that proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition, irrespective of a proteasome mutation, and uniformly show an 'IRE1/XBP1-low' signature. Adaptation of myeloma cells to proteasome inhibitors involved quantitative changes in >600 protein species with similar patterns in AMO-BTZ and AMO-CFZ cells: proteins involved in metabolic regulation, redox homeostasis, and protein folding and destruction were upregulated, while apoptosis and transcription/translation were downregulated. The quantitatively most upregulated protein in AMO-CFZ cells was the multidrug resistance protein (MDR1) protein ABCB1, and carfilzomib resistance could be overcome by MDR1 inhibition. We propose a model where proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition owing to metabolic adaptations that favor the generation of reducing equivalents, such as NADPH, which is supported by oxidative glycolysis. Proteasome inhibitor resistance may thus be targeted by manipulating the energy and redox metabolism.

Adaptive resistance of myeloma cells to proteasome inhibition is poorly understood. It is suggested to base on point mutations in PSMB5 and/or downmodulation of the activation state of the unfolded protein response (UPR) via reduced activity of its major regulatory axis IRE-1/XBP-1. We have generated subclones of the AMO-1 myeloma cell line resistant to bortezomib > 1000 nM (AMO-BTZ), or carfilzomib > 1000 nM (AMO-CFZ), that do or do not carry the PSMB5 A310G mutation in the β5 substrate pocket. We combine this model with a global quantitative proteomics approach, the analysis of the activation status of the IRE-1/XBP-1 pathway, and with an advanced set of proteasome activity-specific fluorescent affinity probes that allow direct, selective, simultaneous visualization of the activity of all six active β-subunits of the constitutive and the immunoproteasome. Our results demonstrate that the A310G mutation has a modest impact on β5c proteasome inhibition by bortezomib (increasing the IC50 from 25nM to 80 nM), and likewise by carfilzomib (IC50 increase from 10 nM to 50 nM). Strikingly, when AMO-CFZ or AMO-BTZ were exposed to the same functional level of proteasome inhibition (> 90% inhibition of β5c/5i, 20% inhibition of β1/1i, β2/2i) that resulted in 70-90% cytotoxicity in AMO-1 cells, no cytotoxicity was observed in AMO-BTZ and AMO-CFZ cells. Likewise, AMO-BTZ and AMO-CFZ cells were resistant to the next generation proteasome inhibitors ixazomib, oprozomib and dalanzomib, irrespective of the presence or absence of the PSMB5 mutation. Analysis of the UPR and its major regulators on protein and mRNA levels revealed that all clones of AMO-BTZ and AMO-CFZ showed significantly lower expression of IRE-1 and its product, spliced XBP-1, compared to AMO-1 cells, in contrast to all other major regulators of the UPR (ATF6, PERK, elF2a). Proteasome inhibitor treatment induced phosphorylation of IRE-1 and the induction of sXBP1 similarly in AMO-1, AMO-BTZ and AMO-CFZ cells, however, the induction of downstream proteins of the UPR (ATF4, PDI) was exclusively found in AMO-1 cells. Mass spectrometry-based quantitative global proteomic analysis was performed to compare AMO-1 cells with AMO-BTZ and AMO-CFZ with a cut off of at least a 50% change in abundance of differentially expressed proteins in at least 2 out of triplicate experiments. This yielded > 3500 identified individual proteins in proteasome inhibitor adapted cells, of which > 600 were differentially expressed and subsequently subjected to a protein-protein interaction (PPI) search and a Gene Ontology (GO) analysis, resulting in an average of 30 GO terms for the overexpressed proteins and 10 for downregulated species in AMO-BTZ and AMO-CFZ. Manual grouping of GO into functionally related clusters resulted in 5-6 groups that were largely concordant between AMO-BTZ and AMO-CFZ. The clusters found overexpressed in AMO-BTZ and AMO-CFZ were proteins involved in protein catabolism, redox control and protein folding. Uniform downregulation was observed for protein clusters involved in transcription/translation, differentiation, apoptosis and structural/cytoskeletal functions. The quantitatively largest group of proteins with significantly altered expression levels in AMO-BTZ/AMO-CFZ vs. AMO-1 control cells consisted of proteins involved in metabolic regulation. This big cluster comprised close to 50 % of all polypeptides with significant quantitative changes, suggesting a key role for metabolic homeostasis. The quantitatively most significantly upregulated protein in both AMO-CFZ and AMO-BTZ was NADPH dehydrogenase, the most important reducing enzyme in eukaryotic cells (4-6 x upregulated). The top individual upregulated protein in AMO-CFZ was the p-glycoprotein 1 (Pgp, 12 x upregulated), while the transcription factor IKZF3 was among the top downregulated proteins in AMO-BTZ cells (0.2 x). Our data indicate that proteasome gene mutations are not required for proteasome inhibitor resistance of myeloma cells, that proteasome inhibitor adapted myeloma cells can compensate subtotal proteasome inhibition irrespective of the type of inhibitor used, and that they have undergone complex adaptive changes in particular in proteins that regulate metabolic functions. Thus we suggest that the metabolic machinery rather than the proteasome should be explored for drug targets in myeloma cells with acquired proteasome inhibitor resistance. Disclosures No relevant conflicts of interest to declare.

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