Primary WM cells express higher level of the immunoproteasome, and ONX0912 targets the CT-L activity in WM cells. Distribution of the caspaselike (C-L), trypsinlike (T-L), and chymotrypsin-like (CT-L) subunits of the constitutive proteasome (β1, β2, β5) and immunoproteasome (LMP2, MECL1, LMP7) was assessed by ELISA on protein lysates obtained from primary cells (average of 5 WM patients is shown; WM), and from peripheral blood–derived CD19⁺ cells of healthy subjects (average of 4 healthy donors is shown; HD) (A), BCWM.1 cells, and IgM-secreting low-grade lymphoma cells (RL, MEC.1; B). Anti-β1, -β2, -β5, -LMP2, -MECL1, and -LMP7 primary, and horseradish peroxidase–conjugated secondary antibodies were used. Each subunit was measured as nanograms per microgram of total protein, according to the SuperSignal ELISA Pico Kit manufacturer's instructions. (C-E) BCWM1 cells have been treated with ONX0912 (2.5-50nM) for 2 hours and ONX0912 effects on CT-L, T-L, and C-L activities of the proteasome have been evaluated as described earlier in the legend. (F) Primary CD19⁺ tumor cells from 3 patients with WM were incubated for 2 hours in the presence of diluent or ONX0912 (5-20nM). The chymotrypsin-like (CT-L) activity of the 20S proteasome of BCWM.1 was determined by measurement of fluorescence generated from the cleavage of the fluorogenic substrate suc-LLVY-amc. ONX0912-induced modulation of CT-L activity has been expressed as fold of untreated samples. In all panels, error bars represent SD.