LY2510924 rapidly and durably blocks surface CXCR4 and inhibits SDF-1α–induced chemotaxis and prosurvival signals of leukemic cells. Both (A) OCI-AML3 and (B) U937 cells were cultured with different concentrations of LY2510924 or AMD3100 for 150 minutes, and surface CXCR4 was measured by flow cytometry with antibody 12G5, which is blocked by receptor occupancy with either agent. Results are expressed as percentage change in the mean fluorescent intensity (MFI) compared with control (untreated) cells. (C) OCI-AML3 cells were cultured with 1 nM LY2510924, and surface CXCR4 12G5 binding was measured by flow cytometry at different time points. (D) OCI-AML3 cells were cultured with different concentrations of LY2510924 or AMD3100 for 72 hours, and surface CXCR4 12G5 binding was measured by flow cytometry. (E) OCI-AML3 (0.5 × 10⁶) or (F) primary AML (1.0 × 10⁶; n = 3) cells were plated onto the upper chamber of Transwell plates and exposed to 50 ng/mL SDF-1α in the lower chamber with or without 1 nM LY2510924 or AMD3100 for (E) 2.5 hours or (F) 12 hours. The results are expressed as percentage of migrating cells relative to the number of input cells. After overnight serum starvation, (G) 1 × 10⁶ OCI-AML3 or (H) U937 cells in RPMI medium containing 0.5% bovine serum albumin were or were not pretreated with 1 µM LY2510924 for 1 hour and were exposed to 100 ng/mL SDF-1α for 10 minutes. Phosphorylation of AKT (pAKT) and ERK (pERK) was detected by western blot analysis, and the intensity of the bands was quantified by densitometry and displayed as the ratio of phosphorylated protein to control phospho-protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. All results are expressed as the mean ± SD, with the exception of (F), expressed as the mean ± SEM.