Selective FLT3 Inhibition Uncovers the Role of FL and FLT3 in Normal CD34+ Hematopoietic Stem Cells.

Several cytokines in the bone marrow act through stem cell or progenitor cell specific receptors to regulate the capacity of immature hematopoietic cells to potentiate downstream multilineage expansion. Ligand-mediated activation of the FMS-like tyrosine kinase-3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. FLT3 expression in the bone marrow is restricted to CD34+ cells and a subset of dendritic precursors. FLT3 as a member of the type III RTK subfamily is closely related to c-KIT, c-FMS and PDGFRα/β. Activating mutations of FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is a promising therapeutic option in treatment of AML patients. CEP-701, a potent FLT3 tyrosine kinase inhibitor is known to be cytotoxic to cell lines and primary AML cells harbouring FLT3 mutations and shows biological and clinical activity in patients with relapsed or refractory AML. In this study, we investigated the effect of FLT3 kinase inhibition in normal hematopoietic stem and progenitor cells in-vitro. Peripheral blood mobilized CD34+ cells were cultured in serum-free conditions supplemented with various cytokine combinations. Flt-3 inhibition was performed with addition of CEP-701 in different concentrations. FLT3 inhibition resulted in dose-dependent growth inhibition of CD34+ cells in in-vitro culture. This effect was independent of cytokine combinations chosen. A tetrazolium-based MTT assay was used to quantify 50% growth inhibition after 48h of exposure to CEP-701. The mean IC50 (± SD) for CEP-701 on normal CD34+ progenitor cells was 56 ± 19 nM. Immunophenotypic analysis of cell cultures showed a markedly decrease of CD34+ expressing cells under FLT3 inhibiting conditions. Surprisingly, inhibition of cell growth was even present, when cell culture was performed in absence of FL. In addition, the effect of FLT3 inhibition could be restored by addition of a neutralizing FL-antibody to cell culture even in conditions without FL substitution. Expression of FLT3 and FL under cytokine-supplemented culture conditions and FLT-3 inhibition were monitored by Western Blot analysis. In order to evaluate the effect of FLT-3 inhibition on the progenitor cell function, colony formation was analysed. Addition of CEP-701 into cell culture resulted in decrease of absolute colony production, however, relative colony formation per input cells was not significantly decreased showing that progenitor cell function of surviving cells was not markedly affected. These data demonstrate a significant inhibitory effect of CEP-701 on normal CD34+ cells. We could demonstrate for the first time, that FL might act as an autocrine mediator activating FLT3 in CD34+ cells as CD34+ cells similarily express both, FLT3 and FL. Finally, these findings might also explain hematotoxicity of various tyrosine kinase inhibitors, e.g. imatinib, which also show unspecific targeting of FLT3.