An orthogonal approach to target MCL1 confirms that most myeloma cell lines are rapidly killed when MCL1 is targeted. (A) Selectivity of wild-type BIM or its variants for the prosurvival BCL2 proteins. The BH3-only protein BIM binds to all the prosurvival BCL2 proteins with high affinity.⁴⁰  BAD binds selectively to BCL2, BCLXL, and BCLW,⁴⁰  whereas BIM2A, in which 2 of the key residues in the BIM BH3 domain are replaced with alanines, retains high binding affinity only for MCL1.⁴¹  BIM4E is an inert variant that fails to bind any of the prosurvival proteins.⁴⁰  (B) Table summarizing the sensitivity of 25 human myeloma cell lines to expression of wild-type (WT) BIM or its variants. The myeloma cell lines were infected with lentiviruses expressing different BIM variants (A). The viability of the infected cells was determined using the CellTiter-Glo assay 24 hours after addition of DOX to induce expression of WT BIM or its variants, and normalized to that observed when the inert BIM variant (BIM4E) was expressed. A discrete heat map representation of the data are shown: red represents very strong killing (<15% viable cells), whereas blue (>75% viable cells) indicates the cells tolerated expression of the BIM variants. Notably, 17/25 lines (68% of those tested) are sensitive (viability reduced to <25%) to selective MCL1 inhibition with BIM2A. The data represent the mean values of 3 triplicate wells from a single experiment; the raw data are shown in supplemental Table 6. (C) Correlation between the sensitivity of myeloma cell lines to killing when MCL1 is genetically targeted by CRISPR/Cas9 (Figure 4) or by introduction of a MCL1-selective peptidyl antagonist, BIM2A (Figure 5B). The loss of viability of 19 myeloma cell lines after MCL1 is targeted by a sgRNA (y-axis) is correlated with the killing observed with a selective MCL1 ligand BIM2A (x-axis) (r=0.48, P = .036; the r and P values were determined with Pearson correlation analysis). (D) Most myeloma cell lines are sensitive to MCL1 inhibition. Venn diagram summarizing the sensitivity of a panel of 24 myeloma cell lines to inhibition of MCL1 (viability ≤ 25%; Figure 5B) with the selective peptidyl antagonist BIM2A, BCL2 (by ABT-199; Figure 1B) or BCLXL (by A5463; Figure 1B). Note that the WL-2 cell line, which is highly sensitive to both ABT-199 and A5463 (Figure 1B), but not to BIM2A (Figure 5B), cannot be accurately depicted, and therefore is excluded on this Venn diagram. (E) Myeloma cells lines that have deregulated TP53 function remain highly sensitive to MCL1 inhibition. Pie charts depict the degree of killing of the myeloma cell lines (left: 25 cell lines in total; right: 17 lines with mutated TP53) by the MCL1-selective peptidyl ligand BIM2A. Note that the fraction of cell lines harboring mutant TP53 that were killed by BIM2A (right) is indistinguishable from the cell line panel as a whole (left).