LY2510924 induces gene expression changes in leukemic cells in vivo that are consistent with loss of SDF-1α/CXCR4 signaling. In the OCI-AML3/Luc/GFP xenograft model, engraftment was confirmed in 8 mice on day 40 by proportion of leukemic cells in blood (3.3% ± 1.1%). Groups of these mice were then given daily LY2520924 treatment and euthanized after 24 hours (n = 3 on day 41) or 72 hours (n = 2 on day 43). Control mice receiving no treatment (n = 3) were euthanized on day 42. Leukemic cells were sorted and separated from BM, blood, and spleen by fluorescence-activated cell sorting (FACS) using the specific markers human CD45 and GFP. (A) Samples from each site and treatment time point were also analyzed by FACS with CXCR4 antibody 12G5 as an inverse indicator of CXCR4 occupancy by LY2510924. (B) From genome-wide GEP of 24 samples (3 groups, 3 tissue sites, 2 or 3 mice per group), log₂ values of each sample’s genes from the treated mice were subtracted by the average for the corresponding gene in the control BM samples. The heat map shows subtracted values converted to fold-change for genes with an absolute subtracted log₂ value of at least 1.5 for at least 6 samples, with samples and genes hierarchically clustered for similarity in variation. The color bar indicates fold-change values, and the dendrogram indicates similarity between samples, which are labeled according to duration of LY2510924 treatment. (C) Selected genes whose expression levels are highly correlated with a score of the presumed relative degree of loss of SDF-1α/CXCR4 signaling, based on non-BM localization and/or LY2510924 treatment and its duration. Score values are shown above sample names, and gene expression values are shown by fold-change from the mean, according to the color bar. The upper positively correlated group of genes is related to myelomonocytic differentiation. The lower negatively correlated genes are involved in differentiation, proliferation, or apoptosis. Values for genes shown twice were detected by different probes. (D) Western blot analysis was performed with leukemic cells sorted and separated from spleen and/or BM by FACS in 2 mice given daily LY2520924 treatment and euthanized after 72 hours compared with 2 control mice. The intensity of the bands was quantified by densitometry and displayed as the ratio of phosphorylated protein to control phospho-protein. GAPDH and β-tubulin was used as a loading control. Results of (A) are expressed as the mean ± SEM. BM, bone marrow; Con, control; PB, peripheral blood; SP, spleen.