Point mutations in the juxtamembrane domain of FLT3 define a new class of activating mutations in AML.
In acute myeloid leukemia (AML), two clusters of activating mutations are known in the FMS-like tyrosine kinase-3 (FLT3) gene: FLT3-internal tandem duplications (FLT3-ITDs) in the juxtamembrane (JM) domain in 20% to 25% of patients, and FLT3 point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7% to 10% of patients, respectively. Here, we have characterized a new class of activating point mutations (PMs) that cluster in a 16-amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PMs). Expression of 4 FLT3-JM-PMs in interleukin-3 (IL-3)-dependent Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3 ligand, and resistance to apoptotic cell death. FLT3-JM-PM receptors were autophosphorylated and showed a higher constitutive dimerization rate compared with the FLT3-wild-type (WT) receptor. As a molecular mechanism, we could show activation of STAT5 and up-regulation of Bcl-x(L) by all FLT3-JM-PMs. The FLT3 inhibitor PKC412 abrogated the factor-independent growth of FLT3-JM-PM-expressing cells. Compared with FLT3-ITD and FLT3-TKD mutants, the FLT3-JM-PMs showed a weaker transforming potential related to lower autophosphorylation of the receptor and its downstream target STAT5. Mapping of the FLT3-JM-PMs on the crystal structure of FLT3 showed that these mutations reduce the stability of the autoinhibitory JM domain, and provides a structural basis for the transforming capacity of this new class of gain-of-function mutations of FLT3.
Reindl
Carola
C
Department of Medicine III, University Hospital Grosshadern, CCG Leukemia, GSF-National Research Center for Environment and Health, Marchioninistr 25, 81377 Munich, Germany.
Bagrintseva
Ksenia
K
Vempati
Sridhar
S
Schnittger
Susanne
S
Ellwart
Joachim W
JW
Wenig
Katja
K
Hopfner
Karl-Peter
KP
Hiddemann
Wolfgang
W
Spiekermann
Karsten
K
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry; D051941:fms-Like Tyrosine Kinase 3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004273:DNA, Neoplasm; D007377:Interleukin-3; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D014443:Tyrosine; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3; D019311:Staurosporine; C059539:midostaurin