AMD3465 inhibits SDF-1α– or stroma-induced migration and suppresses prosurvival signaling pathways in FLT3-mutated cells. (A) Migration of either Ba/F3-ITD or Ba/F3-FLT3 cells in response to 4 hours SDF-1α was examined as described in “Chemotaxis studies” in the presence or absence of 1 μM AMD3465 and/or 10 nM sorafenib. (B) The effects of AMD3465 on the SDF-1α–induced or MS-5–induced up-regulation of pAKT, pERK, and pCXCR4 were analyzed by Western blot analysis in Ba/F3-ITD cells. (C) The combined effects of sorafenib and AMD3465 on AKT, ERK, and CXCR4 phosphorylation were examined in Ba/F3-ITD cells in the presence of MS-5 cells. (D) Primary AML cells with FLT3-ITD mutation grown alone or cocultured with MS-5 cells were exposed to the indicated concentration of AMD3465 alone, 1 μM sorafenib alone, or AMD3465 combined with sorafenib. Phosphorylation of AKT, ERK, and CXCR4 was analyzed by immunoblotting after 24 hours of treatment, and (E) the inhibitory effects of AMD3465 with or without sorafenib on cell migration was measured after 4 hours. The intensity of the phosphorylation bands was quantified by densitometry and displayed as ratios of phosphoproteins either to total proteins or to the loading control GAPDH.