Activating FLT3 Mutations Display a Wide Range Of Transforming Potential and a Characteristic Pathway Profile Associated With Prognosis In Acute Myeloid Leukemia
Authors: Janke Hanna , Schneider Friederike , Schumacher Daniela , Herold Tobias , Karl-Peter Hopfner , Schneider Stephanie , Berdel Wolfgang E. , Büchner Thomas , Woermann Bernhard J. , Subklewe Marion , Bohlander Stefan K. , Hiddemann Wolfgang , Polzer Harald , Spiekermann Karsten 
Published: October 2019

Abstract

Background
Internal tandem duplication (ITD) and pointmutations in the tyrosine kinase domain (TKD) of the receptor tyrosine kinase FLT3 occur in about 30% of patients with acute myeloid leukemia (AML). In contrast to the negative prognostic impact of FLT3-ITD in normal karyotype AML, FLT3 pointmutations occurring in the TKD and juxtamembrane (JM) region are less frequent and of unclear clinical impact. Although TKD mutations can induce resistance to tyrosine kinase inhibitors the individual transforming potential of FLT3 pointmutations has not been analysed in detail. In this study we have performed a comprehensive analysis of various FLT3 mutants in a comparative setting in vitro and analyzed gene expression profiles, and clinical outcome with respect to FLT3mutation status.

Material and Methods
We analyzed relapse and survival in 672 cytogenetically normal AML patients and the FLT3 status at diagnosis and relapse in 156 patients. In the murine Ba/F3 cell model we analyzed the transforming potential, subcellular localization, phosphorylation status and signaling properties of eight different FLT3 mutants. The investigated FLT3 mutations include three ITD of different length and insertion site, V592A in the JM region, common FLT3-TKD mutations D835V and D835Y as well as D839G and I867S in the second TKD. FLT3-D839G and -I867S were recently found in AML patients by our group during routine diagnostics but have not been functionally characterized before. The corresponding remission samples did not express these mutations. Further a gene expression profile analysis with respect to FLT3-ITD and -TKD mutation status and evaluation of differences in activation of predefined STAT5 target gene set was performed.

Results
In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with an inferior relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%).
In vitro, FLT3-ITD induced a fully transformed phenotype in Ba/F3 cells, whereas FLT3 pointmutations showed a weaker but clearly transformed phenotype with gradual increase in proliferation and protection from apoptosis. The transforming capacity of the investigated mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD transformed cells, further gene expression profile analyses displayed differences in predefined STAT5 target genes between FLT3-ITD and FLT3-TKD mutations. In contrast, FLT3-non-ITD mutants had an enhanced signal of AKT and MAPK activation. Further differences were found on mRNA level presenting deregulation of SOCS2, ENPP2, PRUNE2 and ART3 expression between FLT3-ITD, FLT3-TKD and FLT3-WT.

Conclusion
Although apparently divergent in response to treatment all functionally characterized mutants showed a clear gain-of-function phenotype with a wide range of transforming activity associated with clinical prognosis and signaling.

Disclosures:
No relevant conflicts of interest to declare.