Interplay of CXCR4 and FLT3-ITD in the Regulation of Stem Cell Migration and Proliferation.

Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. CXCR4 and its ligand SDF-1 are of central importance for stem cell homing to the bone marrow. In FLT3-ITD positive AML, CXCR4 expression on stem cells is negatively correlated to overall survival. Using retroviral vector transduction we achieved overexpression of the human FLT-ITD transgene, the FLT3-wt or the GFP control gene in greater than 70% of cord blood hematopoietic progenitors (HP). In contrast to previously published data in murine cell lines, we found that FLT3-ITD positive human cells display strongly reduced migration towards the CXCR4 ligand SDF-1. Analysis of SOCS3 (suppressor of cytokine signaling 3) expression revealed strongly increased mRNA expression in FLT3-ITD overexpressing CD34+HP and FLT3-ITD positive AML blasts. Since SOCS3 is a known antagonist of CXCR4 signaling, increased SOCS3 levels might cause the observed inhibition of SDF-1 dependent migration. For individually tested CD34+HP from 6 cord blood donors, co-cultivation of FLT3-ITD overexpressing HP on the irradiated mouse stromal cell line M210-B4 or on primary mesenchymal stromal cells resulted in an increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, FLT3-wt or GFP-transduced HP. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced cord blood HP. Similar results were obtained using FLT3-ITD positive AML cells from 3 patients. Using CFSE to track cell proliferation and PI/Annexin staining to analyze apoptosis, we demonstrate that the addition of AMD3100 had no effect on apoptosis but decreased the proliferation rate of FLT3-ITD positive blast cells. Using primary AML blasts and FLT3-ITD transgene overexpressing cord blood HP, we demonstrate that the robust FLT3-ITD mediated proliferation advantage can be partly inhibited by the CXCR4 antagonist AMD3100.