ATO/ATRA exert antileukemic activity in human NPM1-mutated AML cell lines. (A) Flow cytometric apoptosis assay (Annexin V/7AAD) on human AML cell lines harboring (OCI/AML3, IMS-M2) or not (U937, OCI/AML2) NPM1 mutation upon treatment with 3 μM ATO, 1 μM ATRA, or combination of both for 24 and 48 hours. Percentage of apoptotic cells at each time point is shown. Mean values ± SD from different experiments (OCI/AML3, n = 6; IMS-M2, n = 4; OCI/AML2, n = 3; U937, n = 3). Statistical significance for each treatment condition vs untreated control is indicated by asterisk(s). Two-tailed paired Student t test P values indicate statistical significance for ATO/ATRA cooperative effect vs ATO alone. (B) Graph expressing ATO-induced apoptosis at 24 hours from experiments in panel A as fold of induction, relative to control (untreated cells) (mean ± SD), with statistics between NPM1-mutated and NPM1 wild-type cells. Two-tailed paired Student t test P values are indicated. (C) Cell growth curves upon ATRA treatment (1 μM) in OCI/AML3 (mutated NPM1) vs OCI/AML2 and U937 (wild-type NPM1) human AML cell lines. Mean values of 3 independent experiments ± SD with statistics (2-tailed paired Student t test P values) are reported. (A,C) Significance levels are indicated by *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.