Flt3 Internal Tandem Duplications Cooperate with Wnt Signaling in Leukemic Signal Transduction.

Flt3 internal tandem duplications cooperate with Wnt signaling in leukemic signal transduction

Lara Tickenbrock, Joachim Schwable, Markus Wiedehage, Bjoern Steffen, Chunaram Choudhary, Wolfgang E. Berdel, Carsten Muller-Tidow and Hubert Serve

Department of Medicine, Hematology and Oncology, University of Muenster, D-48149 Muenster, Germany

The type III receptor tyrosine kinase (RTK) Flt3 plays an important role in survival and proliferation of hematopoietic progenitor cells. Somatic mutations of Flt3 consisting of internal tandem duplications (ITD) occur in 25% of patients with acute myeloid leukemia (AML), and are associated with a poor prognosis. They result in Flt3-ligand (FL) independent kinase activation of Flt3. The Wnt signal transduction pathway has recently been implicated to be an important regulator of early hematopoietic stem cell fate decisions. Also, we previously showed that activation of Wnt-dependent Tcf transcriptional activity is induced by the leukemia-associated fusion proteins PML-RAR? and AML1-ETO. Here, we show that Flt3-ITD mutations enhance basal and Wnt3a-stimulated Tcf activity in myeloid cells. Microarray experiments analyzing Flt3-ITD target genes in 32D cells revealed up to 8-fold induction of Frizzled-4, a cell surface receptor for Wnt proteins, by Flt3-ITD mutations. This could be verified by real-time RT-PCR and Western Blot analyses. In functional analyses, we explored the synergism of Flt3-ITD and the activation of Wnt signaling. Flt3-ITD mutations induced the accumulation of ?-Catenin and TCF-dependent transcriptional activity. Also, the presence of Flt3-ITD highly sensitized cells for ?-Catenin-induction by the Wnt3a. Wnt3a incubation enhanced 32D cell proliferation in the presence of activated Flt3 receptors. More importantly, Flt3-ITD-mediated clonogenic growth highly depended on the activity of Tcf transcription factors, since transient transfection of Flt3-ITD cells with dominant negative TCF4 almost abolished 32D cell colony growth. Our results indicate that Flt3-ITD mutations mediate their leukemogenic effects in part through the activation of Wnt-dependent signaling pathways, possibly defining this signal system as a converging point of leukemogenic events elicited by Flt3-mutations and leukemia-associated fusion proteins.