Antileukemic properties of atuveciclib in vitro. (A) The indicated AML cell lines were seeded in 96-well plates and treated with increasing concentrations of atuveciclib for 4 days. Viability was assessed using a WST-1 assay. Data are expressed as a percentage of DMSO control-treated cells. Shown are the means ± SE of at least 3 independent experiments. IC₅₀ values are shown to the right. (B) U937 cells were treated for 48 hours with control (DMSO) or atuveciclib (1 μM), and apoptosis was assessed using flow cytometry, for Annexin V and PI staining. The left panel shows a representative flow cytometry plot. The right panel shows quantitation of the Annexin V–positive and PI-negative and double-positive Annexin V and PI-stained cells. Shown are the means + SE of 4 independent experiments. *P < .05 using a paired Student t test. (C) U937 cells were incubated with vehicle or atuveciclib at the indicated doses for 48 hours. Proteins from whole cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) The effects of atuveciclib (0.5 µM and 1 µM) on U937-derived leukemic progenitors were assessed in clonogenic assays in methylcellulose. Effects on CFU-L CFU-L are shown. Data shown are the means + SE of 4 different experiments. ***P < .001 using a paired Student t test. (E) The effects of atuveciclib (0.5 µM and 1 µM) on CFU-L from different patients with AML were assessed in clonogenic assays in methylcellulose. Shown are the mean + SE of 5 independent experiments using cells from 5 different AML patients. **P < .01, ****P < .0001 using a paired Student t test.