Proteasome stress predicts sensitivity of primary MMCs to bortezomib. Primary, patient-derived MMCs were selected by immunomagnetic purification from bone marrow biopsies and divided in 3 pools. The first pool was seeded onto polylysinated slides and assayed for accumulation of polyubiquitinated proteins and Ig light chain (Ig-L) expression by immunofluorescence. The second pool was plated in multiwell plates, challenged with increasing doses of bortezomib for 24 hours, and apoptotic responses assessed by highly sensitive FACS analysis (LSRII) on labeling with conjugated annexin V and propidium iodide. The third pool was assayed in vitro for proteasome-specific chymotryptic activity by means of a specific fluorogenic peptide, as in Figure 3. (A) Spontaneous accumulation of polyubiquitinated proteins in primary MMCs. While in the CD138⁻ fraction polyubiquitinated proteins accumulate only upon treatment with PIs (16 hours with 100 nM bortezomib [Btz] in the bottom left panel), CD138⁺ MMCs (hallmarked by Ig-L expression) show Fk2⁺ fluorescence in basal conditions (2 representative cases shown). Images were deconvoluted with DeltaVision and SoftWorx (see “Methods”); single z sections are shown. Size bars equal 5 μm. (B) Polyubiquitinated proteins are highly specific of Ig-L⁺ MMCs. Automated quantification of fluorescence in Ig-L⁺ and Ig-L⁻ nucleated cells was performed using the IN Cell Investigator software. The box plot shows the average fluorescence intensity (FI), with 2.5 top and bottom percentiles; *P < .001. (C) Intrapatient positive correlation between polyubiquitinated accumulation and Ig-L content. Each dot corresponds to a single MM cell, and each plot corresponds to 1 patient. Scatters from 3 representative patients are shown. r > 0.6; P < .001. (D) Positive correlation of polyubiquitinated protein accumulation and Ig-L content among different patients. MFI indicates mean fluorescence intensity. r = 0.95; P < .05. (E) Primary MM endowed with high proteasome capacity is intrinsically less responsive to Btz. In 4 primary samples we measured overall proteasome activity in cell lysates by means of a fluorogenic peptide specifically probing the 26S chymotryptic activity (LLVY-amc).^13,14  Proteasome-specific chymotryptic activity is expressed as nanomoles probe cleaved per minute per milligram of protein. Dose-response curves were generated by 24-hour treatments with Btz. To determine the intrinsic sensitivity of each MM clone, EC₅₀ values were calculated using nonlinear regression with Prism version 5.0 software (GraphPad Software). (F) A model of proteasome-related determinants of apoptotic sensitivity to PI. Relatively PI-resistant MMCs are equipped with high proteasome pools, both in absolute terms, and relative to degradative demands. As a result, few proteins await degradation, and high levels of free ubiquitin are available to target new proteasome substrates. In contrast, PI-sensitive MMCs are equipped with poor proteasome levels, despite high metabolic demands. As a result, huge amounts of polyubiquitinated proteins accumulate upstream of overloaded proteasomes, and little ubiquitin is available for proteins to be degraded. Although cells are healthy, stresses that further challenge the ubiquitin-proteasome system will unveil a lower apoptotic threshold.