Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of intern al tandem duplications (ITD) occur in 20% of patients with acute myeloid le ukemia. They are associated with a poor prognosis of the disease. In this s tudy, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the mur ine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence, Flt3 isofo rms containing ITD mutations (Flt3-ITD) induced factor-independent growth a nd resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed coloni es in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-I TD only slightly activated the MAP kinases Erk1 and 2 and the protein kinas e B (Akt) in the absence of ligand and retained ligand-induced activation o f these enzymes, However, Flt3-ITD led to strong factor-independent activat ion of STAT5. The relative importance of the STAT5 and Ras pathways for ITD -induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony for mation by 50%, Despite its weak constitutive activation by Flt3D-ITD, dnRas also strongly inhibited Flt3-ITD-mediated colony formation. Taken together , Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways. (C) 2000 by The American Society of Hematology.