Arginine (R) at Position 595 in FLT3 Is Critical for Transforming Potential of FLT3-Length Mutations.

Activating mutations in the juxtamembrane region of FMS-like tyrosine kinase-3 (FLT3-length mutations, FLT3-LM)-are found in 20–25% of patients with acute myeloid leukemia (AML). FLT3-LM consists of duplications and additional insertions that show a variable length and site of insertion. Although these different length mutations lead to constitutive activation of FLT3 and subsequent downstream signalling pathways, several questions still remain unanswered. Here, we focussed on the question whether a common sequence motif in the duplicated region can be identified in patients carrying FLT3-LM. To address this topic we sequenced the juxtamembrane region of FLT3 from 274 patients with acute leukemias. We found that the length of mutation (tandem duplications) varied from 2–42 amino acids with a median of 17 AA. Furthermore, the duplicated amino acids centered around the motif DFREYEY of FLT3. Since the length of the motif DFREYEY varied from patient to patient, we focussed our study on the frequency of single amino acids within the duplicated region. We found that arginine (R) 595 in the motif DFREYEY is duplicated in 77% of patients. Further studies showed that a single duplication of arginine at position 595 in FLT3 is able to confer factor independent growth to Ba/F3 cells. In vitro, deletion or substitution of duplicated R 595 in two FLT3-LMs with different lengths showed a 50% reduction in the factor independent growth when compared to undeleted/non-substituted FLT3-LMs. The reduced proliferative capacity of Ba/F3 cells expressing FLT3-LM with deletions of R-595 was associated with a reduction of STAT5 activation.

Our data provide important insights into the molecular mechanisms of transformation by FLT3-LM and define duplicated R 595 as a critical mediator of the leukemic potential of these mutants.