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

Abstract

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies 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-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation 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 formation by 50%. Despite its weak constitutive activation by Flt3-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.

FMS-Like Tyrosine Kinase 3 in Normal Hematopoiesis and Acute Myeloid Leukemia

“…Aberrant stimulation of proteins that confer a proliferative advantage was first identified in mutant-FLT3 myeloid leukemia cells. In vitro, the 32D mouse myeloid cell line harboring the ITD-mutation showed factor-independent proliferation and resistance to radiation-induced apoptosis [73,74]. Both STAT5 and Ras pathways were shown to be activated.…”

“…Constitutively phosphorylated STAT5 has been observed in primary AML blasts and in immortalized cell lines Ba/F3 and 32D that express the mutated FLT3 receptor [64,73,74,78,79]. The phosphorylation of STAT5 contrasts most clearly with the wild-type signaling pathway because normal FL-activated of FLT3 does not cause significant STAT5 activation.…”

Mechanisms of pre‐B‐cell receptor checkpoint control and its oncogenic subversion in acute lymphoblastic leukemia

“…These mainly occur in the activation loop of FLT3, mostly representing point mutations in codon D835 or, less frequently deletions of codon I836 (84). Only approximately 1% carry the in AML dominating FLT3 ITD that is result of a duplication of a stretch of several amino acids in the juxtamembrane region of the receptor and facilitate leukemic transformation by inducing constitutive kinase activation and signaling through the Ras and JAK/STAT5 pathways (92).…”

Patient‐specific analysis of FLT3 internal tandem duplications for the prognostication and monitoring of acute myeloid leukemia

“…Raf/MAP kinase pathways are induced by the ligand-activated wild type, where STAT5 pathway is induced particularly by mutated FLT3. STAT5 activates proliferative and antiapoptotic pathways, which are considered to play an essential role in FLT3-ITD-positive AML pathogenesis (29,30). Vempati et al (31) suggested that tyrosine residues in positions 589 and 591 are important for STAT5 phosphorylation, whereas tyrosine residues in positions 597 and 599 are necessary for the structural maintenance of FLT3-ITD for STAT5 recruitment.…”