Figure 3.

Addition of LU-102 sensitizes cells to in vivo relevant concentrations of ONX-0914. (A) Effect of 48-hour treatment with LU-102 on viability MM cells as determined by Alamar Blue (n = 2-4). (B) Inhibition of proteasome in MM1.S cells after 2-hour treatment as determined by the Proteasome-Glo assay (n = 3) (Promega). (C) MM1.S cells were treated with ONX-0914 for 1 hour, and then treated with 1 μM LU-102 for 47 hours (n = 3). (D) MM1.S cells were cotreated for 4 hours with ONX-0914 and 3 μM LU-102 (n = 2). (E) Cells were treated by ONX-0914, followed by LU-102 (250 nM in H929 and MM1.R, 1 μM in all others) for 47 hours (n = 2-6), and half maximal inhibitory concentrations of MM cells was determined form dose-response curves (supplemental Figure 1). (F) Viability of cells treated with 900 nM ONX-0914 in experiment shown in panel E. Numbers are combination indexes (CIs). (G) Primary cells isolated from plural effusions of bortezomib-refractory patients were treated with 0.9 µM ONX-0914 for 4 hours with or without 2 μM LU-102, and viability of CD138⁺ cells was determined by flow cytometry at 48 hours using PE Mouse Anti-Human CD138 and Zombie Aqua Fixable Viability antibodies. (H) LU-102 blocks the recovery of β5i/β5c activity over time after 1-hour treatment. Total β5 activity was measured with Proteasome-Glo in RPMI-8226 cells, either immediately after 1-hour treatment with 1 µM ONX-0914 or 100 nM carfilzomib or after a 17-hour recovery in the presence or absence of 1 µM LU-102 (n = 2). Pt., patient.