Figure 2.

The S63845 + venetoclax combination impairs the interactions of MCL-1 and BCL-2 with the pro-apoptotic protein BIM. (A) BIM shows three major isoforms: BIM_EL, BIM_L and BIM_S. MM.1S clones KO for BIM_EL and BIM_L isoforms were generated by electroporation of a Cas9 ribonucleoprotein complex [containing a guide RNA and a Cas9 enzyme (Integrated DNA Technologies)], using the Neon Transfection System (Thermo Fisher Scientific) and subsequent single cell sorting. Clones KO for BIM_EL and BIM_L isoforms and control clones (electroporated with the Cas9 enzyme only) were exposed to increasing doses of S63845+venetoclax for 24 hours, keeping a constant 1:50 S63845:venetoclax ratio. Cell viability was analyzed by MTT assay. (B and C) MM.1S and KMS12-BM cell lines (least sensitive and most sensitive to S63845 and venetoclax) were respectively treated with S63845 (12.5 and 2 nM) and venetoclax (625 and 4 nM), in monotherapy or in combination for 24 hours (S63845 and venetoclax doses were adjusted for each cell line so that the combination would induce 13-25% apoptosis as measured by Annexin-V and PI staining). Protein lysates were subjected to immunoprecipitation with an anti-BIM antibody, and MCL-1, BCL-2 and BCL-X_L bound to BIM were then analyzed by immunoblotting. Their levels were quantified by densitometry analysis of bands using ImageJ software, normalized to those of BIM, and depicted as bar diagrams. Whole cell lysates of each cell line are also shown.