Aberrant Intracellular Retention of Mutated Receptor Tyrosine Kinase FLT3 in Human Leukemic Cells as a Potential Mechanism Supporting the Pathogenesis of Acute Myeloid Leukaemia.

The exact regulation of FLT3 expression, a type III receptor tyrosine kinase (RTK), contributes to controlled proliferation and differentiation of blood cells at different stages during hematopoiesis. Activating mutations of FLT3 are found in AML, ALL and MDS and contribute to their pathogenesis, but it is unknown how mutant FLT3 deregulates hematopoiesis. It has been reported that murine mutant FLT3 signal transduction, namely with the most frequent Internal Tandem Duplication (ITD), differs from FLT3 wild type signalling. One possible mechanism for a different signalling quality leading to pathogenic effects observed in leukaemia, might be an aberrant intracellular trafficking of mutant FLT3, like shown for the other mutated type III RTKs PDGFR and KIT, which are aberrantly localized in human cancer. The localization of mutant FLT3-ITD as well as wild type FLT3 was investigated in retrovirally transduced COS7 cells, human leukemic cells and cell lines and primary CD34+ hematopoietic stem cells, enriched by MACS technique with an anti-CD34 antibody. Subcellular distribution of FLT3 was determined by confocal laser scanning microscopy. Internalization kinetics was studied by fluorescently labelling the ligand (FL) and time lapse imaging. The effects on signal transduction were investigated by Western blot analysis of protein phosphorylation for FLT3 and downstream targets (ERK, STAT5, PIM1). FLT3 localized to the plasma membrane, whereas mutant FLT3- ITD accumulated in the trans Golgi network (TGN), and not as previously speculated in the Endoplasmic Reticulum. FL was not internalized in FLT3-ITD expressing cells, while internalization in FLT3 expressing cells started at around 4 min. This observation is consistent with the non-surface localization of FLT3-ITD. Inhibition of Golgi specific glycosylation by Swainsonine led to TGN retention of FLT3. The STAT5 pathway was FL-independently activated only by FLT3-ITD. TGN accumulation of human FLT3- ITD, possibly caused by immature glycosylation, and the lack of FL internalization might support FL-independent autonomous signalling. The TGN environment may provide interactions with additional/different substrate(s), altering subsequent signalling quality (as shown here for STAT5) and thereby favouring uncontrolled proliferation of undifferentiated cells.