UBX-382 potently degrades BTK via proteasome action in B-cell malignant cell lines. (A) BTK degradation in response to increasing UBX-382 and UBX-382-Me doses in TMD-8 cells over 24 hours. BTK levels were measured by immunoblotting using specific antibodies and values for the remaining BTK were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) intensity as a loading control. The results represent 2 independent experiments (n = 2). (B) Immunoblotting analysis of time-dependent degradation of BTK by treatment with UBX-382 in TMD-8 cells. The cells were treated with 100 nM UBX-382 for 0.5, 1, 4, 16, 24, and 48 hours and harvested for the analysis of BTK levels using BTK antibody. The remaining BTK values were normalized using GAPDH intensity as a loading control. The results represent 2 independent experiments (n = 2). (C) The degradation effect of BTK by UBX-382 as determined by immunofluorescence analysis in the TMD-8 cells. Variations in BTK levels and CRBN as a result of treatment with UBX-382 for 24 hours were observed by immunofluorescence using specific antibodies as indicated in “Materials and methods.” The TMD-8 cells were visualized using a confocal microscope (original magnification ×400) green, BTK; red, CRBN; and blue, nucleus. Scale bars, 20 μm. (D) BTK polyubiquitination was induced by treatment with 100 nM UBX-382 for 2 hours in Ramos cells. The polyubiquitin chains on BTK were observed using TUBE1 pull-down experiments as described in “Materials and methods.” β-actin was used to confirm an equal amount of protein loading. The results represent 2 independent experiments (n = 2). (E) BTK degradation is mediated by the ubiquitin-proteasome system in TMD-8 cells. The TMD-8 cells were pretreated with 0.1 μM bortezomib, MLN4924, or bafilomycin for 1 hours and then treated with 0.1 μM UBX-382 for 4 hours. Immunoblotting was performed to verify BTK levels using specific antibodies. The immunoblotting results represent 2 independent experiments (n = 2). Data are expressed as the mean ± standard error of the mean (SEM). (F) Quantitative proteomics analysis was performed to evaluate proteome changes in TMD-8 cells. The cells were treated with 100 nM UBX-382 or dimethyl sulfoxide (DMSO) for 4 hours. Lysates were treated with the TMT-6 plex kit, which was followed by liquid chromatography-tandem mass spectrometry–based proteomics analysis. The volcano plot indicates protein ranking per the abundance ratio (log2 fold change) for DMSO and UBX-382 and the statistical P value. The nonaxial vertical line represents a fold change of ± 1.5 and the nonaxial horizontal line indicates a P value of 0.05 significance threshold. This experiment was performed in triplicate. A total of 7418 proteins were identified in this experiment.
