PP2A-mediated cell death in response to OSU-2S. (A) Fold change in PP2A activity of AML cells lines treated with OSU-2S (5 µM, 4 hours), normalized to dimethyl sulfoxide (DMSO) vehicle control (Veh) (n = 3). OSU-2S significantly increased PP2A activity in HL-60 (P = .0227), MV4-11 (P = .0206), and Kasumi-1 (P = .0206) cells. (B) Fold change in PP2A activity of patient-derived AML (n = 7, AML 2-, 6-, 8-, 11-, 15-, 16-, and 2-fold1) treated with OSU-2S (5 µM, 4 hours), normalized to Veh. OSU-2S treatment significantly increased PP2A activity (P = .0037), comparable to the phosphatase activator control FTY720. (C) Percentage cell viability (measured by annexin V/PI staining) with OSU-2S treatment normalized to DMSO Veh control in AML cell lines. OSU-2S demonstrated potent dose-dependent cytotoxicity against AML cell lines HL-60 (n = 4, IC₅₀ [24 hours] ∼3.47 µM; IC₅₀ [48 hours] ∼2.76 µM), MV4-11 (n = 3, IC₅₀ [24 hours ] ∼3.58 µM; IC₅₀ [48 hours] ∼3.24 µM), U937 (n = 4, IC₅₀ [24 hours] ∼4.6 µM; IC₅₀ [48 hours] ∼4.56 µM), Kasumi-1 (n = 4, IC₅₀ [24 hours] ∼5.062 µM; IC₅₀ [48 hours] ∼4.22 µM) and MOLM-13 (n = 3, IC₅₀ [24 hours] ∼4.1 µM; IC₅₀ [48 hours] ∼2.3 µM). (D) Percentage of viable cells (measured by annexin V/PI staining) normalized to Veh in patient-derived AML cells (n = 13) treated with increasing concentrations of OSU-2S. OSU-2S treatment showed significant dose-dependent cytotoxicity (mean IC₅₀ [24 hours] ∼3.83 µM; IC₅₀ [48 hours] ∼3.13 µM, P [dose trend] < 0.0001). (E) Relative PP2Ac expression in PP2Ac (50 nM) siRNA transfected cells, normalized to nonspecific negative control (nc) siRNA transfected cells. PP2Ac protein levels were measured by densitometric quantification of immunoblots and normalized to actin loading control. Representative western blot images are shown. PP2Ac siRNA significantly knocked down PP2Ac protein expression. (F) Percentage of viable cells in HL-60 (n = 10) and MV4-11 (n = 6) cells transfected with nc siRNA or PP2Ac siRNA, normalized to Veh-treated, nc siRNA-transfected cells. Cells were treated 24 hours after transfection with Veh and 1.75 µM OSU-2S (HL-60) or 2.5 µM OSU-2S (MV4-11) for an additional 24 hours. OSU-2S showed significant cytotoxicity in nc siRNA-transfected cells; however, PP2Ac siRNA significantly reversed OSU-2S–mediated cytotoxicity (HL-60: P = .0001, 53.15% rescue in cell death; MV4-11: P = .0013, 46.36% rescue). (G) Relative PP2A-B56α (also denoted as B56α) expression in B56α siRNA (60 nM) transfected cells, normalized to nonspecific nc siRNA-transfected cells. B56α protein levels were measured by densitometric quantification of immunoblots and normalized to actin loading control. Representative western blot images are shown. B56α siRNA significantly knocked down PP2A B56α protein expression. (H) Percentage of viable cells in HL-60 (n = 10) and MV4-11 (n = 8) cells transfected with nc siRNA or B56α siRNA, normalized to Veh-treated, nc siRNA-transfected cells. Cells were treated 24 hours after transfection with Veh and 1.75 µM OSU-2S (HL-60) or 2.5 µM OSU-2S (MV4-11) for an additional 24 hours. B56α siRNA significantly reversed OSU-2S–mediated cytotoxicity (HL-60: P = .032, 58.23% rescue in cell death; MV4-11: P = .0004, 82% rescue). *P < .05; **P < .01; ***P < .001; ****P < .0001.