Effect of AZD1208 on downstream signaling in AML cell lines. (A) Pim signaling and interaction with PI3K/AKT/mTOR pathway showing activating (green) and inactivating (red) phosphorylation events. Rheb, Ras homolog enriched in brain. (B) Modulation of various biomarkers seen in MOLM-16, EOL-1, KG-1a, and OCI-M1 cells after 3 hours of treatment with control (“C”) dimethylsulfoxide (DMSO) or 0.01, 0.1, or 1 μM AZD1208 by immunoblot analysis of cell lysates. For p70S6K, the band shown represents the ∼60 kDa S6K1 isoform. (C) Effect of AZD1208 on cap-dependent translation complex formation after 3 hours of treatment with AZD1208 at the indicated doses. eIF4E was immunoprecipitated from treated cell lysates by using methyl-7-guanosine-5′-triphosphate cap sepharose beads (the eluted immunoprecipitated material is shown in the third row) and immunoblotted with eIF4G (first row) and 4EBP1 (second row) to assess the extent of association of each protein with eIF4E before and after treatment with AZD1208. The fourth row shows effects of drug treatment on p4EBP1 S65 prior to immunoprecipitation (IP) of cell lysates. (D) Effects of Pim kinase inhibition on polysomal assembly. OCI-M1 or EOL-1 cell lines were treated with solvent control (DMSO), or 1 μM AZD1208 for 9 hours, and the lysates were layered on a 10% to 50% sucrose gradient. The gradients were subjected to ultracentrifugation, and fractions were collected by continuous monitoring of optical density (OD) at 254 nm. The OD 254 nm was plotted as a function of gradient depth for each treatment. A representative profile from 1 of 2 studies is shown and the polysomal (PS) and monosomal (MS) peaks are indicated. The area under the polysome and monosome peaks was quantified for each treatment by using ImageJ software. The ratio of area under the polysomal and polysomal plus monosomal peaks was calculated for each treatment, and the results of 2 independent studies for each cell line are presented as percentage of respective DMSO control. Expt, experiment.