AZD1208 prevents in vivo tumor growth in an ATL mouse model. (A) Dose response of ED-40515(–) with AZD1208, demonstrates complete loss of proliferation in treated cells. Cell counts (left) and XTT assays (right) were repeated at least twice. Results represent the percentage of cells alive after 5 days of AZD1208 (0, 1, 3, 5, 10, 20, or 50 μM) compared with 5 days treated with DMSO. (B) Carboxyfluorescein diacetate succinimidyl ester staining of ED-40515(–) cells treated with or without AZD1208. DMSO and AZD1208 cells were stained and analyzed with carboxyfluorescein diacetate succinimidyl ester on day 0, and then re-analyzed on Day 5. (C) AZD1208 induces apoptosis in ATL cells with minimal cell death in normal PBMCs. Annexin V/PI staining was performed 5 days after treatment (percentage of cells PI⁺/Annexin V⁺ is indicated). (D) Loss of Pim1 activity is associated with deregulation of prosurvival and apoptotic proteins (caspase3, Bcl2, survivin, and Mcl-1, along with Pim1-regulated proteins) in ED-40515(–) cells after 5 days of AZD1208 (10 μM) or DMSO treatment. (E-G) In vivo growth of NOG mice engrafted with ED-40515(–) cells treated with AZD1208. Mice were treated every day with either vehicle (n = 7) or AZD1208 (n = 7) at a dose of 30 mg/kg. (E) Representative images of tumors extracted from mice. (F) Growth curves of in vivo tumor growth, plotted as the average tumor volume (mm³) (calculated as the [width²  × length]/2) per every 2 days. P values are calculated using a 2-sided Student t test between the tumor volumes for vehicle vs AZD1208-treated. (G) Tumor weight (in grams) of ED-40515(–) tumors taken at the time of sacrifice. P values are calculated using a 2-sided Student t test between the tumor weight for vehicle vs AZD1208-treated. The mean tumor weight is indicated with a bar and green square, with the standard error of the mean indicated.