The combination of FTY720 and venetoclax promotes caspase-dependent apoptosis through modulation of p-BCL2 and ERK-dependent MCL1 degradation. (A) Time-dependent caspase-3 and -7 activation plus apoptotic cells in AML cell lines after cell treatment. Statistical significance was determined by one-way ANOVA and Tukey post hoc test analysis (data at 24 hours); ∗∗P < .01, ∗∗∗P < .001. (B) Western blot analysis shows decreased Ser70p-BCL2 and MCL1 protein expression levels after 24 hours of treatment with FTY720 and venetoclax in combination in AML cell lines. (C) Half-life of normalized MCL1 protein levels as a function of time after adding CHX or CHX plus FTY720 and venetoclax. β-actin was used as loading control for normalization of MCL1 protein levels, which were expressed as fold change of the control (assigned a value of 1). Analyses were performed by using 1-phase decay equations. (D) Levels of Thr202/Tyr204p-ERK and ERK protein expression were significantly decreased in treated AML cell lines (24 hours of treatment). (E) Western blot analyses after MCL1 protein immunoprecipitation show that Thr163p-MCL1 was significantly reduced in AML cell lines upon treatment (24 hours). (F) Schematic representation showing that FTY720 plus venetoclax negatively regulates ERK-dependent stabilization of MCL1. (G) Bar graphs show reduction of venetoclax IC50 values when combined with trametinib in AML cell lines (48 hours of treatment). (H) Western blot analyses show significantly decreased MCL1 and Thr202/Tyr204p-ERK after 24 hours of treatment with trametinib and venetoclax combination. ANOVA, analysis of variance; CHX, cycloheximide.