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.

Highlights d Direct comparison of proteasome inhibitors by activity-based probes and Ub -G76V-GFP d Short-term b5 inhibition alone is not cytotoxic for MM cells d b5/b2 co-inhibition is the most effective in PI-sensitive and PI-resistant MM d From the available PI, only high-dose carfilzomib provides b5/b2 co-inhibition

Proteasome inhibitor (PI) carfilzomib (CFZ) has activity superior to bortezomib (BTZ) and is increasingly incorporated in multiple myeloma (MM) frontline therapy and relapsed settings. Most MM patients ultimately experience PI-refractory disease, an unmet medical need with poorly understood biology and dismal outcome. Pharmacologic targeting of ABCB1 improved patient outcomes, including MM, but suffered from adverse drug effects and insufficient plasma concentrations. Proteomics analysis identified ABCB1 overexpression as the most significant change in CFZ-resistant MM cells. We addressed the functional role of ABCB1 overexpression in MM and observed significantly upregulated ABCB1 in peripheral blood malignant plasma cells (PCs) vs untreated patients’ bone marrow PC. ABCB1 overexpression reduces the proteasome-inhibiting activity of CFZ due to drug efflux, in contrast to BTZ. Likewise, the cytotoxicity of established anti-MM drugs was significantly reduced in ABCB1-expressing MM cells. In search for potential drugs targeting ABCB1 in clinical trials, we identified the HIV protease inhibitors nelfinavir (NFV) and lopinavir (LPV) as potent functional modulators of ABCB1-mediated drug export, most likely via modulation of mitochondria permeability transition pore. NFV and LPV restored CFZ activity at therapeutically relevant drug levels and thus represent ready-to-use drugs to be tested in clinical trials to target ABCB1 and to re-sensitize PC to established myeloma drugs, in particular CFZ.

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Up to now neuropeptide Y (NPY) receptors, which belong to the large family of G-protein-coupled receptors and are involved in a broad range of physiological processes, are believed to act as monomers. Studies with the Y 1 -receptor antagonist and Y 4 -receptor agonist GR231118, which binds with a 250-fold higher affinity than its monomer, led to the first speculation that NPY receptors can form homodimers. In the present work we used the fluorescence resonance energy transfer (FRET) to study homodimerization of the hY 1 -, hY 2 -, and hY 5 -receptors in living cells. For this purpose, we generated fusion proteins of NPY receptors and green fluorescent protein or spectral variants of green fluorescent protein (cyan, yellow, and red fluorescent protein), which can be used as FRET pairs. Two different FRET techniques, fluorescence microscopy and fluorescence spectroscopy, were applied. Both techniques clearly showed that the hY 1 -, hY 2 -, and hY 5 -NPY receptor subtypes are able to form homodimers. By using transiently transfected cells, as well as a stable cell line expressing the hY 2 -GFP fusion protein, we could demonstrate that the Y-GFP fusion proteins are still functional and that dimerization varies from 26 to 44% dependent on the receptor. However, homodimerization is influenced neither by NPY nor by G␣ protein binding. G-protein-coupled receptors (GPCRs)1 represent a superfamily of proteins characterized by seven transmembrane ␣-helices that interact with a family of heterotrimeric GTP-binding proteins, referred to as G-proteins (1). GPCRs are found in a wide range of organisms, and many kind of chemical messengers act through them, for example adrenalin, angiotensin, or neuropeptide Y (NPY). Ligands for GPCRs are involved in a broad range of physiological functions, and their malfunction is responsible for many diseases (2, 3).Until recently GPCRs were thought to function as monomers. However, a growing number of evidence suggests that they may exist as homodimers and heterodimers (4 -9). The existence of homodimers has been shown for several GPCRs including ␤ 2 -adrenergic receptor (10 -12), ␦-and -opioid receptors (6, 13), metabotropic glutamate receptor 5 (14), calcium-sensing receptor (15-17), m3 muscarinic receptor (18, 19), vasopressin V2-receptor (20), somatostatin (21, 22), and dopamine receptors (23)(24)(25). Whereas homodimerization of the somatostatin receptor 5 (21), the ␦-opioid receptor (13), and the ␤ 2 -adrenergic receptor (11) are agonist-mediated, dimerization of the -opioid receptor (26) is agonistindependent.So far photoaffinity labeling (27), cross-linking studies (15, 24), Western blot analysis (14), and immunoprecipitation (17,28,29) are the most frequently applied methods for the investigation of receptor homodimerization. Because of the development of new fluorescent dyes, novel fluorescent proteins, and new instrumentation, the fluorescence resonance energy transfer (FRET) obtained a renaissance (30) and could be applied recently for the investigation of receptor dimerization...