Pharmacological Pim kinase inhibitors inhibit ATL cell proliferation. (A) Pim1 is overexpressed in ATL-derived cell lines and primary ATL patient samples. Normal PBMCs (n = 3) and HL60, an AML line that does not express Pim1, were used as controls. Primary acute/lymphoma ATL patient samples (n = 14) were used. (B) Overexpression of Pim1 target genes, p-4EBP1 (Thr37/46), and p-Bcl2 (Ser70) in ATL-derived cell lines and ATL patient samples (n = 7). (C-D) Loss of proliferation in ATL-derived cell lines by the Pim-kinase inhibitors, Smi-4a (C), and AZD1208 (D). Cell counts were repeated at least twice. Results represent the percentage of cells alive after 5 days of Pim inhibitor treatment, compared with 5 days treated with DMSO. For Smi-4a treatment, cells were treated with 0, 10, or 20 μM Smi-4a, and for AZD1208, cells were treated with 0, 5, or 10 μM AZD1208. Normal PBMCs (n = 2) were used as a control. Western blots indicate loss of Pim1 targets, p-4EBP1 (Thr37/46), p-p70S6K (Thr389), and loss of pBad (Ser20) (negligible for Smi-4a) after 24 hours with 0, 10, 20, or 40 μM Smi-4a; 0, 5, or 10 μM AZD1208; or DMSO control. (E-F). Loss of cell viability in ATL-derived cell lines treated with the Pim-kinase inhibitors, Smi-4a (E) and AZD1208 (F). XTT cellular metabolic assays were performed on ATL lines treated with Pim inhibitors. Results represent the percentage of XTT-positive cells after 5 days of Pim inhibitor treatment, compared with 5 days treated with DMSO. Bars represent the average of at least 2 independent experiments.