Point Mutations Found in the Juxtamembrane Domain of FLT3 Define a New Class of Activating Mutations in AML.

In acute myeloid leukemia (AML) FMS-like tyrosine kinase-3 (FLT3) has been shown to be mutated in about one third of patients. Until now, two distinct activating mutations are known: FLT3-length mutations (FLT3-LM) in the juxtamembrane (JM) domain in 20–25% and FLT3-point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7–10% of patients. Here, we have characterized a new class of activating point mutations (PM) that cluster in a 16 amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PM).

Stable expression of four distinct FLT3-JM-PM in IL-3 dependent murine Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3-ligand and resistance to apoptotic cell death compared to FLT3-WT-expressing cells. As a molecular mechanism, we could show activation of STAT5 and upregulation of Bcl-x(L) by all FLT3-JM-PM. A selective FLT3-inhibitor, PKC412, was able to abrogate the factor-independent growth of FLT3-JM-PM. Mapping of the FLT3-JM-PM on the crystal structure of FLT3 showed that these mutations probably reduce the stability of the JM domain in the autoinhibitory conformation, and provide a structural basis for their transforming capacity.

Our results show that point mutations in the autoinhibitory JM domain represent a new class of gain-of-function mutations able to activate the transforming potential of FLT3.