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Apoptosis induced by selective BH3-mimetics
tion of pro- and anti-apoptotic proteins changed upon exposure to BH3-mimetics (Figure 4). In the BCL-2- dependent cell lines RIVA and U2932, the recently described displacement of BIM from BCL-231 was difficult to detect but some reduction in binding of BIM to BCL-2 was found in U2932 cells. In RIVA cells, a minor amount of BIM appeared bound to BCL-XL following treatment with ABT-199, which may indicate a low level of BIM displacement from BCL-2. In both cell lines, less BAX was bound to BCL-2 following treatment with ABT-199, indi- cating a direct displacement of BAX from BCL-2 (Figure 4A). Similarly, in the BCL-XL-dependent cell lines, BIM binding to BCL-XL was reduced upon treatment with A1331852. Strikingly, both BAX and BAK were less bound by BCL-XL upon A1331852 treatment, supporting the hypothesis that BH3-mimetics can directly displace BAX and BAK (Figure 4B). Treatment with S63845 in the
MCL-1-dependent cell lines resulted in less binding of BIM and BAK to MCL-1 (Figure 4C). In summary, these studies demonstrate that treatment with BH3-mimetics resulted in reduced binding of pro-apoptotic BCL-2 pro- teins.
The displacement of BIM could be functionally impor- tant for apoptosis induction, as released BIM could initi- ate apoptosis by binding directly to BAX and BAK and activating them. To investigate whether BIM is necessary, we performed siRNA-mediated knockdown of BIM fol- lowed by treatment with BH3-mimetics. Combined use of two distinct siRNA partially inhibited BH3-mimetic induced cell death in a treatment- and cell-line-dependent manner, as BIM knockdown reduced cell death in RIVA, SUDHL10 and to a lesser extent in U2946 cells (Figure 5A-C, Online Supplementary Figure S6), although efficient knockdown was achieved in all cell lines (Figure 5D). As
AB
C
Figure 4. On-target binding of BH3-mimetics displaces pro-apoptotic BCL-2 pro- teins. (A-C) The interaction of anti- and pro-apoptotic BCL-2 proteins (BIM, BAX and BAK) was studied upon treatment with the BH3-mimetics (A) ABT-199 (RIVA, 3 nM and U2932, 10 nM), (B) A1331852 (RCK8, 3 nM and SUDHL8, 10 nM) or (C) S63845 (SUDHL10, 100 nM and U2946, 300 nM) for 4 h. In order to exclude downstream caspase-mediated effects on protein expression, the broad range caspase inhibitor zVAD.fmk was added to the cells. Input lanes show the pres- ence of overall protein in the lysate, and immunoprecipitation (IP) lanes show interaction with BCL-2, BCL-XL or MCL-1. Protein G beads without primary anti- body were used to control for unspecific binding. Staining with BCL-2, BCL-XL, MCL-1 and GAPDH was performed to demonstrate efficient immunoprecipitation and equal protein loading, respectively. Representative western blots of two to five independent experiments are shown.
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