Effect of ONX-0914 on MM cells. (A) Expression of immunoproteasomes in MM cell lines as determined by the activity-based probes. Immortalized B-cell line LG2, which expresses high levels of immunoproteasomes, and HeLa cells, which express little or no immunoproteasomes were used as references (n = 2). (B) Cleavage rates of β5i-specific substrate Ac-ANW-amc and of β5c and β5i substrate Suc-LLVY-amc were measured in extracts of primary MM cells isolated from relapsed/refractory patients using EasySep Human CD138 Positive Selection Kit II (STEMCELL Technologies, Catalog #18357), and ANW:LLVY ratio was used as readout of relative β5i expression. (C) ONX-0914 is cytotoxic to MM cells. Top: cells were treated with ONX-0914 for 1 hour followed by Alamar Blue assay 47 hours later (n = 2-3; see supplemental Table 1 for exact number of cells for each cell line). In a parallel experiment, proteasome inhibition was measured by site-specific fluorogenic substrates (middle graph) or activity-based probes (bottom graph) immediately after 1-hour treatment with ONX-0914 (n = 4). Dashed line indicates in vivo relevant inhibition (eg, concentration that causes ≥95% of β5i and ∼50% of β5c inhibition similar to maximal tolerated dose in vivo¹⁸ ). (D) Clinically relevant concentrations of bortezomib or carfilzomib (n = 2-3) have cytotoxicity similar to that of ONX-0914. Dashed lines indicate clinically relevant doses. Except for KMS-11 (n = 5), bortezomib data are from Figure 1 of Shabaneh et al.²⁵  (E) IFN-γ treatment increases the sensitivity of MM cells to ONX-0914, but not to carfilzomib. Cells were treated with IFN-γ for 5 days and then treated with ONX-0914 (upper left and bottom right) or carfilzomib (bottom left) as in panel C. Upper right panel shows increase in β5i activity after 4 days as measured by BODIPY-NC-005-VS (n = 2-3). Sp., specific.