Sina Koch

Abnormal Localization and Accumulation of FLT3-ITD, a Mutant Receptor Tyrosine Kinase Involved in Leukemogenesis

Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus.

Autotaxin is expressed in FLT3-ITD positive acute myeloid leukemia and hematopoietic stem cells and promotes cell migration and proliferation

Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.

Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells

Summary FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3 ITD) is an important oncoprotein in acute myeloid leukemia (AML). Owing to its constitutive kinase activity FLT3 ITD partially accumulates at endomembranes, a feature shared with other disease-associated, mutated receptor tyrosine kinases. Because Ras proteins also transit through endomembranes we have investigated the possible existence of an intracellular FLT3-ITD/Ras signaling pathway by comparing Ras signaling of FLT3 ITD with that of wild-type FLT3. Ligand stimulation activated both K- and N-Ras in cells expressing wild-type FLT3. Live-cell Ras–GTP imaging revealed ligand-induced Ras activation at the plasma membrane (PM). FLT3-ITD-dependent constitutive activation of K-Ras and N-Ras was also observed primarily at the PM, supporting the view that the PM-resident pool of FLT3 ITD engaged the Ras/Erk pathway in AML cells. Accordingly, specific interference with FLT3-ITD/Ras signaling at the PM using PM-restricted dominant negative K-RasS17N potently inhibited cell proliferation and promoted apoptosis. In conclusion, Ras signaling is crucial for FLT3-ITD-dependent cell transformation and FLT3 ITD addresses PM-bound Ras despite its pronounced mislocalization to endomembranes.