Expression and Functional Analysis of Autotaxin, a Relevant Motility and Survival Factor, in FLT3-ITD Positive Acute Myeloid Leukemia and Primary Hematopoietic Stem Cells.

FLT3-mutations are among the most common abnormalities in adult acute myeloid leukemia. These mutations induce constitutive activation of the receptor and downstream signaling pathways, including RAS/ERK, PI3K and STAT5-signalling. The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with inferior prognosis. We have recently demonstrated by microarray analysis that leukemic samples of patients with FLT3-ITD mutations have significantly upregulated expression levels of Autotaxin (ATX). The ATX protein acts as a secreted lysophospholipase D (lysoPLD) through generating lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). LPA has several important functions in cell migration and proliferation and acts via G-protein coupled receptors. It has been shown that ATX represents an aberrantly expressed motility and growth factor in a variety of cancer cells. However data on ATX in myeloid leukemias and especially in AML are missing. To study more deeply the role of ATX in leukemogenesis, we screened a series of human leukemic model cell lines for ATX expression. Using retroviral transduction, two alternatively spliced transcripts of ATX were expressed in several human leukemic cell lines without detectable levels of endogenous ATX. To investigate the ATX function and expression in normal haematopoiesis we used CD34+ human progenitor cells. The expression of ATX transcripts was confirmed at both mRNA and protein levels by RT-PCR and Western blotting, respectively. Transwell migration assays in the presence of LPC or LPA were performed to study effects of ATX on cell motility. Proliferation and clonogenic potential were investigated using MTT and colony forming assays. Moreover, we examined the effect of the FLT3 inhibitor N-benzoylstaurosporine (PKC412) on ATX expression and ATX mediated migration in MV4-11 cells. High ATX expression was found primarily in malignant cells. Western blot analysis showed that detectable levels of ATX are secreted into medium within 2 hours. In normal cells, highest ATX mRNA expression was found in purified CD34 cells, but could also be found at lower levels in T-cells. Stable overexpression of FLT3-ITD in OCI-AML3 cells induced an increased ATX expression (up to 6 fold). Vice versa, inhibition of FLT3-ITD by sublethal doses of PKC412 in MV4-11 cells resulted in a significant reduction of ATX expression down to 10% of the initial expression level. Furthermore, PKC412 treatment resulted in a complete loss of LPC or LPA induced specific migratory capacity in MV4-11 cells. The transduction of ATX increased the colony-forming capacity by 75% and significantly increased the short term proliferation. LPA increased chemotaxis in human leukemic cell lines and human CD34+ progenitors in a dose dependent manner and induced significantly higher migratory rates by at least 50%. LPC induced chemotaxis by 80–200% only in cells with high expression of endogenous or exogenous ATX, demonstrating the autocrine activity of ATX. Ongoing studies on the mechanism of ATX expression showed that ionomycin, an activator of the Ca2+–calcineurin–NFAT signalling pathway, upregulated ATX mRNA in several leukemic cell lines as well as in human primary progenitors up to 5-fold, which could be completely blocked by cyclosporine A treatment, indicating an involvement of NFAT in the regulation of ATX. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration of hematopoietic stem cells and its deregulation may contribute to the pathogenesis of AML, especially in patients with FLT3-ITD mutations.