Abstract 3982

Poster Board III-918

FMS-like Tyrosine kinase 3 (FLT3), also known as CD135, is one of the key proteins driving normal haematopoiesis. Multiple studies along the last decade have demonstrated that activation of FLT3 also plays a role in the leukaemic counterpart of haematopoiesis and leads to an increased proliferation, a resistance to apoptosis and the loss of cytokine dependence in blast cells. These findings have been confirmed in clinical settings by various groups that showed the association of activating mutations with a worse outcome in normal karyotype acute myeloid leukaemia (AML) and lead to the development in clinical trial of FLT3 inhibitors such as staurosporine derivatives (PKC 412) or multi hit tyrosine kinase inhibitors like sorafenib. Most of the works on these activating mutations were focused on Internal Tandem Duplication (ITD) found in exon 12 or 14 and on point mutation (D835) in the kinase domain (exon 20). It represents up to 30 to 35% of the molecular abnormalities found in normal karyotype AML. Of note, other membrane TK receptors, like c-kit, could also be activated during leukaemogenesis and cancer. Some of the molecular events responsible of the oncogenic potential are shared between the two receptors and notably TK domain point mutations (namely c-kit D816). Besides these similarities, other mutations could lead to c-kit activation and especially extracellular juxtamembrane domain mutation as described in mastocytosis. Interestingly, the mirror image in FLT3 has not been explored and we decided to address this issue by performing a wide range sequencing (exon 8 to 14 and exon 20) of FLT3 in a series of normal karyotype AML patients treated in our centre (n=100). Our screen found 25% of FLT3 ITD and 9% of TK domain point mutation. No relevant point mutations were found on exon 8 to 14. Of note, 7 patients harboured a deletion of exon 10, confirmed by another set of primers, 4 of them have also a FLT3 ITD. Considering the TK domain point mutation, we found 7 mutation interesting residue D835, one mutation D841V and one mutation A848P. Interestingly the patient with that last mutation was suffering from a very aggressive clinical presentation and this mutation (which corresponds to A829 in c-kit) was not documented in the literature. After site-directed mutagenesis of the wild type human FLT3 receptor, we introduced FLT3-A848P cDNA into Ba/F3 cells. This resulted in an interleukin-3 (IL-3)-independent cell proliferation, receptor phosphorylation, and constitutive activation of extracellular regulatory kinase (ERK), suggesting that the receptor is constitutively activated. Non specific FLT3 inhibitors have been tested: both sunitinib and sorafenib inhibited the growth of Ba/F3-FLT3-A848P cells (IC50 10 nM and 50 nM respectively) but to a lesser extend as compared to Ba/F3-FLT3-ITD cells (IC50 0.5 nM and 1 nM respectively) or Ba/F3-FLT3-D835V cells (IC50 0.1 nM and 2 nM respectively). These inhibitors also reduced tyrosine phosphorylation of the mutant receptor, and inhibited ERK phosphorylation. 3D-models show that A848P mutation dramatically modifies the activation loop conformation, thus promoting constitutive activation of the receptor and either penalizing the docking of inhibitors or increasing the affinity to ATP. These results suggest that mutations on A848 should be considered as a significant new activating mutation in patients with AML, and that patients with such mutations may less respond to tyrosine kinase inhibitors.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.