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 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. (Blood. 2000;96:3907-3914)
Mizuki
M
M
Department of Medicine/Hematology and Oncology, University of Münster, Germany.
Fenski
R
R
Halfter
H
H
Matsumura
I
I
Schmidt
R
R
Müller
C
C
Grüning
W
W
Kratz-Albers
K
K
Serve
S
S
Steur
C
C
Büchner
T
T
Kienast
J
J
Kanakura
Y
Y
Berdel
W E
WE
Serve
H
H
D000208:Acute Disease; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002455:Cell Division / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects*; D002999:Clone Cells / Q000166:cytology; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D005260:Female; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics / Q000503:physiopathology*; D051379:Mice; D008809:Mice, Inbred C3H; D008894:Milk Proteins; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D022423:Myeloid Cells / Q000187:drug effects / Q000502:physiology; D009374:Neoplasms, Experimental / Q000401:mortality / Q000473:pathology; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D020080:Tandem Repeat Sequences / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D051941:fms-Like Tyrosine Kinase 3; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins