LY2510924 durably blocks surface CXCR4, induces mobilization of leukemia cells, and inhibits AKT and ERK intracellular signaling in vivo, retarding progression of primary AML xenografts. Primary AML cells (0.4 × 106 per mouse) were intravenously injected into NSG mice. Mice were divided into two groups, control (n = 13) and LY2510924 (n = 15), after engraftment was documented in peripheral blood on day 25 and began to receive daily treatment. (A) In 5 representative mice of each group, percentages of circulating primary AML cells before and 3 or 24 hours after the first LY2510924 injection on days 25 and 26 were compared with those of untreated mice. (B) Identification of circulating leukemic cells by flow cytometry and staining for CXCR4 with antibody 12G5 (inversely reflective of receptor occupancy) at points during the initial 2 days of daily LY2510924 administration are shown for a representative mouse. (C) CXCR4 staining with antibody 12G5 after 5 days of LY2510924 treatment was compared in 5 representative mice of each group. (D-E) Three representative mice per group were euthanized on day 45, and cells from BM, spleen, and blood in all mice (control, n = 6; LY2510924, n = 11) on day 45 were analyzed by flow cytometry; dual-positive cells (human CD34 and CD45) were compared between each group in terms of (D) expression of CXCR4 12G5 and (E) proportion of leukemic cells. (F) AKT and ERK phosphorylation in dual-positive cells (human CD34 and CD45) recovered from BM and spleen were measured by multiparametric phospho-flow cytometry in a representative mouse from each group on day 48. The results showed that receptor occupancy by LY2510924 correlates with reduced AKT and ERK phosphorylation. (G) Overall survival rate in each group was estimated by the Kaplan-Meier method. Results of (A), (C-E), and (G) are expressed as the mean ± SEM. *P < .05; **P < .01.
