Rare flt3 deletion mutants may provide additional treatment options to patients with aml: an approach to individualized medicine

Access through your institution Buy or subscribe The receptor tyrosine kinase (RTK) FLT3 (Fms-like tyrosine kinase-3) is essential for the proliferation, differentiation and survival of

hematopoietic cells.1, 2 Mutations in the gene for _FLT3_ have been described in acute myeloid leukemia (AML) as internal tandem duplications (ITD) localized in the juxtamembrane (JM) domain

and _FLT3_ tyrosine kinase domain (TKD) mutations amounting to 2–30% and 7–10% of patients, respectively. In patients with AML, _FLT3_-ITD confers an unfavorable prognosis.3 In AML,

recently discovered _FLT3_ mutations involve point mutations as well as short deletions in the JM domain, highlighting the importance for a deeper understanding and further studies of these

aberrations.4, 5 The FLT3 JM domain (Supplementary Figure 1a) is indeed the critical regulator region of receptor autoinhibition.6 Kiyoi _et al._7 had shown constitutive activation of the

FLT3 receptor by deletion of a stretch of amino acids in the JM domain, generally duplicated in FLT3-ITD. Transient transfection into HEK293T cells confirmed activation of associated

signaling proteins by several deletion mutants. Even deletion of just one amino acid led to the activation of downstream signaling (Supplementary Figures 1b and c). When retrovirally

expressed in the IL3-dependent pro-B cell line Ba/F3, the _FLT3_ Stop and delLeu (Supplementary Table 1, Id 3 and Id 2, respectively) mutants were not further studied in this work, as we

focused on activating mutations in the JM domain. The delEY and the delIns mutants led to constitutive activation of STAT3, ERK1/2, SFK (Src family kinases), SHP2 and AKT (Figure 1a).

Similar to _FLT3_-ITD, they also activated STAT5. As seen in HEK293T cells, only FLT3-ITD induced upregulated phosphorylation of Y589/Y591. These tyrosines were previously thought to be

essential for STAT5 activation and transforming capacity of the RTK.9 Our data show that STAT5 was partly activated by SFKs in deletion mutant expressing cells (Figure 1b), whereas JAK2

inhibition had no effect on STAT5 phosphorylation (Supplementary Figure 2a). A similarity between FLT3 deletion mutants and the TKD mutant _FLT3_ D835Y (p.Asp835Tyr) is conceivable, as both

do not show an increase in Y589/Y591 phosphorylation.9 Nevertheless, the missing phosphorylation of Y589/591 was associated with low STAT5 activation in D835Y expressing cells but not for

_FLT3_ deletion mutants (Figure 1a). Immunoprecipitation experiments confirmed the phosphorylation of additional tyrosines independent from Y598/Y591 in the deletion mutants, as was already

shown for _FLT3_-ITD (Supplementary Figure 2b).9 FLT3 ligand stimulation of the WT receptor led to the phosphorylation of ERK1/2 and AKT but not of STAT5. Interestingly, STAT3 was not

activated by _FLT3_-ITD, suggesting that the deletion mutants and ITD induced different patterns of downstream signaling. These results were in line with those of Frohling _et al._10 of

STAT3 phosphorylation driven by FLT3 JM point mutations. We found that endoplasmic reticulum targeting or hypoglycosylation of FLT3 delEY mutant by brefeldin A and tunicamycin treatment,

respectively, clearly diminished STAT5 and STAT3 phosphorylation, whereas there was no change with FLT3-ITD (Supplementary Figure 2c), as published.11 The same was seen for the delIns mutant

only with tunicamycin treatment. All mutants exhibited decreased phosphorylation of AKT after brefeldin A and tunicamycin treatment, confirming receptor surface expression as a prerequisite

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3076–3081. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Kristina Feldberg for expert technical assistance. We acknowledge Frank D Böhmer for providing the

pcDNA3-FLT3 (WT)-HA vector and M-D Hagel for initial cloning of vectors. This work was in part supported by the Novartis Foundation. AUTHOR CONTRIBUTION NC, RCP, GR, JR, PC, MS, TH, THB, SS

and SK designed research. NC, GR, RCP and JR performed research. NC, GR, RCP, PC, TH, SS and SK analyzed data. NC, RCP, GR, SS and SK wrote the paper. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany N Chatain, J Rossa, M

Schemionek, T H Brümmendorf & S Koschmieder * Computational Biophysics, German Research School for Simulation Sciences and Computational Biomedicine, Institute for Advanced Simulation

IAS-5 and Institute of Neuroscience and Medicine INM-9, Forschungszentrum Jülich, Jülich, Germany R C Perera, G Rossetti & P Carloni * Jülich Supercomputing Centre, Forschungszentrum

Jülich, Jülich, Germany G Rossetti * MLL Munich Leukemia Laboratory, Munich, Germany T Haferlach & S Schnittger Authors * N Chatain View author publications You can also search for this

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AUTHOR Correspondence to S Koschmieder. ETHICS DECLARATIONS COMPETING INTERESTS SK has received honoraria for consultancy from Novartis, Pfizer, Brystol-Meyers Squibb, and Ariad. TH and SS

have equity ownership of MLL. THB has received honoraria for consultancy from Novartis, Pfizer, Brystol-Meyers Squibb. The remaining authors declare no conflict of interest. ADDITIONAL

INFORMATION Supplementary Information accompanies this paper on the Leukemia website SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION 1 (DOC 5166 KB) SUPPLEMENTARY INFORMATION 2 (DOC 146

KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chatain, N., Perera, R., Rossetti, G. _et al._ Rare FLT3 deletion mutants may provide additional

treatment options to patients with AML: an approach to individualized medicine. _Leukemia_ 29, 2434–2438 (2015). https://doi.org/10.1038/leu.2015.131 Download citation * Published: 27 May

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