Samples from each site and time point were also analyzed for CXCR4 occupancy by using flow cytometry with antibody 12G5

Samples from each site and time point were also analyzed for CXCR4 occupancy by using flow cytometry with antibody 12G5. consistent with loss of SDF-1/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and -catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications. Introduction The interaction between acute myeloid leukemia (AML) cells and the bone marrow (BM) microenvironment has been postulated to be important for resistance to chemotherapy and disease relapse in AML.1 The C-X-C chemokine receptor type 4 (CXCR4) and its ligand, stromal cellCderived factor 1 (SDF-1 [CXCL12]), are key mediators of this interaction. Stromal cellCderived factor 1 (SDF-1) is produced in the BM microenvironment, activates CXCR4 on leukemic cells, facilitates leukemia cell trafficking and YM90K hydrochloride homing in the BM microenvironment, and keeps leukemic cells in close contact YM90K hydrochloride with the stromal cells and extracellular matrix that constitutively generate growth-promoting and anti-apoptotic signals.2 Indeed, high CXCR4 expression on AML blasts is known to be associated with poor prognosis.3,4 Our group and others have tested small-molecule inhibitors against CXCR4: AMD3100 (Plerixafor), approved by the US Food and Drug Administration, and its analog AMD3465. These agents disrupted the SDF-1/CXCR4 axis and enhanced the anti-leukemic effects of chemotherapy, markedly reducing leukemic burden and prolonging overall survival in xenograft models.5,6 Disruption of the SDF-1/CXCR4 axis by CXCR4 antagonists is therefore an attractive investigational therapeutic approach for AML and is being tested in clinical trials. A phase 1/2 study recently reported that adding AMD3100 to cytotoxic chemotherapy increased response rates in patients with relapsed AML.1 However, the mobilization of leukemic blasts induced by AMD3100 is transient, and cell counts return to baseline levels within 12 hours,5 likely because of incomplete inhibition of the SDF-1/CXCR4 axis and the short in vivo half-life (3-5 hours) of AMD3100.7 Furthermore, AMD3100 and AMD3465 did not show antileukemic effects as single agents in vivo,5,6 although they did have inhibitory effects on multiple cancers of nonhematologic origin.8-16 LY2510924 is a novel and potent selective peptide antagonist of CXCR4.17 A recent phase 1 study in advanced cancers revealed good tolerability with mostly grade 1 to 2 2 adverse events, favorable pharmacokinetics, and target engagement as indicated by YM90K hydrochloride dose-dependent increases in CD34+ cell mobilization.18 Here, we report preclinical studies using LY2510924 to disrupt the SDF-1/CXCR4 axis in AML cells in vitro and in vivo. Il17a Materials and methods Please refer to supplemental Methods available on the Web site for detailed descriptions of the methods and reagents used. Cell lines, primary samples, and cultures Human AML cell lines OCI-AML3, U937, and MOLM-13 (supplemental Table 1) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA) and 1% penicillin-streptomycin (Gibco Laboratories, Grand Island, NY). Cells were harvested during the log phase of growth and seeded at a density of 0.2 106 cells per milliliter. Peripheral blood samples from patients with AML were collected during routine diagnostic procedures after informed consent was obtained in accordance with Institutional Review Board regulations of The University of Texas MD Anderson Cancer Center and the Declaration of Helsinki. Mononuclear cells were separated by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation. Flow cytometry The expression of surface CXCR4 protein was analyzed by using a Gallios flow cytometer (Beckman.