PP2A activation decreases stemness in vivo and ex vivo. (A) Proportion of lineage negative (Lin⁻) cells in the CD45.2 AML cell population on Veh (n = 3) or OSU-2S (n = 5), in BM of treated mice or BMT only control mice. OSU-2S–treated mice have significant lower proportion of CD45.2 (AML) Lin⁻ cells as detected in the BM (P = .0011), indicating lower leukemic stem and progenitors. (B) Number of colonies (colony forming units [CFU]) formed by cells isolated from BM of Veh- or OSU-2S–treated mice (n = 7). OSU-2S–treated mice BM have significantly lower colony forming units vs that of Veh-treated mice (P = .0022). *P < .05; **P < .01; ***P < .001; ****P < .0001. (C) Mass cytometric analysis of primary AML cells treated with Veh or OSU-2S (3.5 μM, 48 hours) reveals decreased surface progenitor marker CD34, decreased CD34⁺/CD38⁻ LSCs and increased more mature CD34⁻/CD38⁺ or CD34⁻/CD38⁻ cells. (D) Representative SPADE analysis run on primary AML cells (n = 3) treated with Veh or OSU-2S, clustering on 23 CD markers revealed a decrease in CD34⁺/CD38⁻ LSCs and CD34⁺/CD38⁺ and progenitor population. The T-cell population was unchanged. The patient-derived samples had minimum to no B cells. The number of cells is denoted by node size. Color scheme denotes intensity of pRb which also decreases with OSU-2S. (E) Percentage of relative colony forming units (CFUs) in primary AML cells (IC₅₀, 0.147 µM) vs nonleukemic CD34⁺ cells (IC₅₀, 1.107 µM) treated with OSU-2S, plated on methylcellulose and counted 2 weeks after. Primary AMLs have sevenfold higher sensitivity to OSU-2S. (F) Schematic of the limiting dilution assay (LDA) and bulk LTC-IC assay performed on primary AML samples and normal donor cord blood and BM cells. (G) Fold LTC-IC frequency of Veh- and OSU-2S (0.5 µM)–treated primary AML and normal donor samples (n = 6, AML 1, 15, 22, 26, 35, and 36). OSU-2S treatment significantly decreased the frequency of LTC-IC in primary AML samples. No significant difference was observed in nonleukemic samples (n = 4). (H) The PP2A-mediated model of growth arrest and differentiation in AML. OSU-2S disrupts SET-PP2A interaction, reactivating PP2A in leukemic cells. PP2A dephosphorylates Myc, leading to Myc degradation and relieving Myc-mediated suppression of p21. Induction of p21 results in hypophosphorylation of Rb, arresting cells at S phase entry, and arrested cells undergo differentiation, possibly via CEBP factors activated by Rb or other differentiation factors induced by p21 or Rb, resulting in upregulation of differentiation markers and downregulation of stemness markers. *P < .05; **P < .01; ***P < .001.