Mechanism of action of A1210477 in sensitive myeloma cells. (A) Cytochrome c release and (B) immunoblots of caspase 3 and 9 activation in OPM2 cell line under A1210477 treatment. Results are representative of at least 2 independent experiments. (C) Correlation between A1210477 sensitivity and BH3 profiling. BH3 profiling of HMCLs was performed using MS1 peptide (10 μM), and loss of cytochrome c was analyzed by flow cytometry, as previously described.20 Values of cytochrome c negative cells corresponding to BH3 profiling are indicated in supplemental Table 3. Sensitivity to A1210477 (5 μM) was plotted vs cytochrome c negative cells. The Spearman rank correlation is indicated. (D) A1210477-induced cell death is impaired by BAK silencing, but neither BAX nor BIM silencing. OPM2 and KMM1 were transfected with the different siRNA, protein expression was determined 48 hours after transfection, and cells were treated with A1210477 for 24 hours before assessing cell death by Annexin V staining. The induction of apoptosis was compared with the nontreated controls. Results represent the mean of 4 independent experiments. Statistical analysis was performed by Kruskal-Wallis test. (E) A1210477 disrupts the complexes of MCL1 with its proapoptotic counterparts. Immunoprecipitation of MCL1 was performed after short A1210477 treatment (2 μM) of OPM2 cells, followed by western blotting of indicated proteins. Quantification of proteins bound to MCL1 was performed for each condition relative to endogenous complexes without treatment. Quantification of bound proteins was performed using ImageJ software. (F) Myeloma cells from patient #27 were treated with A1210477 (2 μM) for 1 hour. Lysates were obtained and MCL1 immunoprecipitates were analyzed by western blot, as in E. *Ab light chain (G) A1210477 binding to MCL1 impaired MCL1 ubiquitination. OPM2 cell line was preincubated during 3 hours with MG-132 (1 μM). Then, A1210477 (1.5 μM) was added for 30 minutes. Cell lysates were used for the detection of ubiquitinylated MCL1 captured by TUBEs, followed by western blotting analysis.