Cell death signaling induced by combined targeting of antiapoptotic proteins. (A-B) The interactions of proapoptotic and antiapoptotic proteins were analyzed upon exposure of leukemia cells to apoptosis-inducing drugs by coimmunoprecipitation (IP). RCH-ACV cells (A) were exposed to AZD4320 (100 nM) and/or AZD5991 (1 μM) and NALM-6 cells (B) to AZD4320 (2 μM) and/or AZD5991 (2 μM) for 4 hours. The IP lanes show the interactions of BCL-2, BCL-XL, and MCL-1 with BIM, and the input lanes show whole protein lysates. Representative immunoblots of 2 independent experiments are shown. (C-E) Dynamic BH3 profiling was performed in RCH-ACV cells. The cells were treated with 10-nM AZD4320 (C) or 100-nM AZD5991 (D-E) for 2 hours. After permeabilization, the cells were exposed to 0.3 μM of the proapoptotic BH3-peptide BAD (binding to BCL-2, BCL-W, and BCL-XL), 30-μM HRK (BCL-XL), or 10-μM MS1 (MCL-1), followed by fixation and staining with an anticytochrome c antibody that selectively binds to mitochondrial cytochrome c. Delta priming was calculated as the percentage of drug-induced cytochrome c release minus the percentage of cytochrome c release induced by the control. The bar graphs show mean values ± standard deviations derived from 3 independent experiments in triplicates. (F) Schematic overview of ex vivo coculture of BCP-ALL PDX cells on MSCs. On day –1, MSCs were seeded in 96-well plates before the addition of ALL PDX cells. On day 0, samples were exposed to increasing concentrations (0.1, 1, 2.5, 5, 10, 100, and 500 nM) of AZD4320 and/or AZD5991 in a drug matrix for 72 hours. Cell death rates were determined by PI staining and flow cytometry measurement after drug exposure. Created with BioRender.com. (G-I) Heat maps showing cell death rates from dose-response matrix analyses of ALL PDX samples (G), PBMCs (H), and MSCs (I). Cell death rates were assessed by PI staining after 72 hours of drug exposure and coculture with MSCs. Efficacy scores were calculated as the mean of normalized cell death rates across the matrix. Synergy effects were visualized using SynergyFinder, and synergy scores were analyzed using the Bliss independence model. Dashed lines indicate the MSA. (J) Comparison of efficacy scores of the drug matrix analyses of AZD4320 and AZD5991 between ALL PDX samples, PBMCs, and MSCs. Bar graphs show mean ± standard deviation. Mann-Whitney U test; ∗P < .05. DMSO, dimethyl sulfoxide; MSA, most synergistic area.

Cell death signaling induced by combined targeting of antiapoptotic proteins. (A-B) The interactions of proapoptotic and antiapoptotic proteins were analyzed upon exposure of leukemia cells to apoptosis-inducing drugs by coimmunoprecipitation (IP). RCH-ACV cells (A) were exposed to AZD4320 (100 nM) and/or AZD5991 (1 μM) and NALM-6 cells (B) to AZD4320 (2 μM) and/or AZD5991 (2 μM) for 4 hours. The IP lanes show the interactions of BCL-2, BCL-XL, and MCL-1 with BIM, and the input lanes show whole protein lysates. Representative immunoblots of 2 independent experiments are shown. (C-E) Dynamic BH3 profiling was performed in RCH-ACV cells. The cells were treated with 10-nM AZD4320 (C) or 100-nM AZD5991 (D-E) for 2 hours. After permeabilization, the cells were exposed to 0.3 μM of the proapoptotic BH3-peptide BAD (binding to BCL-2, BCL-W, and BCL-XL), 30-μM HRK (BCL-XL), or 10-μM MS1 (MCL-1), followed by fixation and staining with an anticytochrome c antibody that selectively binds to mitochondrial cytochrome c. Delta priming was calculated as the percentage of drug-induced cytochrome c release minus the percentage of cytochrome c release induced by the control. The bar graphs show mean values ± standard deviations derived from 3 independent experiments in triplicates. (F) Schematic overview of ex vivo coculture of BCP-ALL PDX cells on MSCs. On day –1, MSCs were seeded in 96-well plates before the addition of ALL PDX cells. On day 0, samples were exposed to increasing concentrations (0.1, 1, 2.5, 5, 10, 100, and 500 nM) of AZD4320 and/or AZD5991 in a drug matrix for 72 hours. Cell death rates were determined by PI staining and flow cytometry measurement after drug exposure. Created with BioRender.com. (G-I) Heat maps showing cell death rates from dose-response matrix analyses of ALL PDX samples (G), PBMCs (H), and MSCs (I). Cell death rates were assessed by PI staining after 72 hours of drug exposure and coculture with MSCs. Efficacy scores were calculated as the mean of normalized cell death rates across the matrix. Synergy effects were visualized using SynergyFinder, and synergy scores were analyzed using the Bliss independence model. Dashed lines indicate the MSA. (J) Comparison of efficacy scores of the drug matrix analyses of AZD4320 and AZD5991 between ALL PDX samples, PBMCs, and MSCs. Bar graphs show mean ± standard deviation. Mann-Whitney U test; ∗P < .05. DMSO, dimethyl sulfoxide; MSA, most synergistic area.

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