FLT3/ITD Expression Increases Expansion, Survival and Entry into Cell Cycle of Human Hematopoietic Stem Cells.

FMS-like tyrosine kinase-3 (FLT3) is a Class III receptor tyrosine kinase that is important for normal hematopoiesis. Activating mutations of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region are the most common molecular aberrations found in acute myeloid leukemia (AML). The contributions of FLT3 activating mutations in the leukemic transformation of normal human hematopoietic stem/progenitor cells (HSCs) have not yet been fully elucidated. In this study, using a single lentiviral vector containing two promoters, we achieved consistent and efficient coexpression of FLT3/ITD and green fluorescent protein (GFP) in transduced human CD34+ HSCs. When cultured in medium containing SCF, TPO and FLT3 ligand (FL), FLT3/ITD-transduced cells survived with enhanced self-renewal and survival potential, which was not affected by the withdrawal of FL. These cells retained a surface immunophenotype typical of HSCs (CD34+CD38−). Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSCs had a higher fraction of cells in cell cycle. Clonogenic assays showed that FLT3/ITD-transduced HSCs produced fewer CFU-GM, implying that they were at least partially blocked in their ability to differentiate along the myeloid lineage. FLT3/ITD-transduced HSCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor. In the FLT3/ITD-transduced HSCs, we detected increased expression of Pim-1, a serine/threonine kinase with an important role in cell survival, proliferation and differentiation, c-myc, a transcription factor involved in cell proliferation and cell cycle regulation, and Cyclin D3, a key factor in cell cycle regulation, each of which may contribute to the altered genetic program instituted by FLT3/ITD signaling.

These results together indicate that FLT3/ITD mutations may contribute to leukemic transformation of normal HSCs by prolonging survival, promoting proliferation, and blocking differentiation. CEP-701 may act as a potent agent for AML stem cells harboring FLT3/ITD mutations.