The PP2A regulatory subunit B56α is the master regulator of the observed treatment synergy in vitro and in vivo. Schematic representation and western blot analysis of B56α (A) and B55α (B) in HL-60 parental cells, wild-type clones (WT, 1 and 4), and clones with CRISPR-Cas9 inactivation of PPP2R5A (2 and 3) and PPP2R2A (5 and 6) genes. (C) Viability percentages, CI values, and cell viability curves show that loss of B56α rescues the synergistic treatment effect. (D) NSG mice were injected with HL-60 clone 1 (WT) or clone 2 (B56α inactivation) cells. When tumors reached 100 to 150 mm3, FTY720 and venetoclax were administrated by oral gavage. (E) Time-dependent tumor volume growth upon treatment of injected NSG mice (n = 8 mice per group). Data were analyzed by two-way ANOVA and Tukey post hoc test. (F) Western blot analysis of B56α, Ser70p-BCL2, Thr202/Tyr204p-ERK, and MCL1 in tumors isolated from mice after 15 days of treatment. Two-tailed unpaired t test or Mann-Whitney U test was used for statistical analyses. (G) Boxplot graphs show PPP2R5A mRNA expression in AML cell lines (left, n = 14) and AML primary cells (right, n = 23; samples with good mRNA quality were assessed by quantitative reverse transcription polymerase chain reaction). Both cell lines and AML primary blasts sensitive to the combined therapy have significantly higher PPP2R5A mRNA levels. Data were analyzed by two-tailed Mann-Whitney U test (cell lines) or unpaired t test (AML primary blasts). (H) Schematic illustration of the molecular mechanism underlying the synergistic effect between PP2A-activating drugs and venetoclax. In panels E-G: ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. ANOVA, analysis of variance; ns, not statistically different.
