The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor

Abstract

In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor.

Figure 1. Identification of the mutation in the patient sample and expression of the truncated FLT3 p.Q569Vfs*2 receptor.

(a) Blast cells were isolated from the bone marrow of a relapsed AML patient. mRNA was isolated and reverse transcribed. The FLT3 cDNA was amplified and fragment analysis was performed. Arrows indicate fragments for FLT3 WT and FLT3 p.Q569Vfs*2. The peaks differ by eight base pairs in their fragment size. (b) Sanger sequencing revealed a deletion of eight nucleotides, leading to a frameshift and a premature stop codon within the FLT3 gene. The chromatogram is shown for the wild-type FLT3, nucleotide triplets and the corresponding amino acids are shown for the FLT3 WT and the frameshift mutation FLT3 p.Q569Vfs*2 sequence. (c) Phoenix eco cells were transfected with FLAG-tagged FLT3 WT and FLAG-tagged FLT3 p.Q569Vfs*2. After cell lysis the FLT3 protein was immunoprecipitated from whole cell lysates with an N-terminal FLT3 antibody (SF1.340). After blotting the FLAG-tagged FLT3 was detected with an FLAG M2 antibody. One representative experiment is shown. The blot was cropped to improve the clarity of the image. (d) Ba/F3 cells stably expressing the indicated constructs. After cell lysis the FLT3 protein was detected with an N-terminal FLT3 antibody (4B12). One representative experiment is shown. FLT3 bands are indicated by asterisks. The blot was cropped to improve the clarity of the image (MT = FLT3 p.Q569Vfs*2). (e) Immunofluorescence of FLT3 (red), glycoconjugates (green) and counterstaining of DNA (blue) in transiently transfected U2OS cells. One representative image of each construct is shown.

Figure 2. FL binding by the truncated FLT3 p.Q569Vfs*2 receptor, downstream signaling pathways, and proliferation of Ba/F3 cells.

(a) Ba/F3 cells stably expressing the indicated constructs were incubated with biotinylated human FL. Receptor bound biotinylated FL was detected with streptavidin-APC using flow cytometry. As a negative control biotinylated soybean trypsin inhibitor was used. One out of at least three independent experiments is shown. (b) Ba/F3 cells stably expressing the indicated constructs were seeded at a density of 4 × 10⁴/mL in the presence or absence of 50 ng FL. Viable cells were counted by trypan blue exclusion after 72 hours. Shown are mean values ± SEM of at least three independent experiments; *p < 0.05 (MT = FLT3 p.Q569Vfs*2). (c) Ba/F3 cells stably expressing the empty vector, FLT3 WT, FLT3 p.Q569Vfs*2 alone or both FLT3 WT and FLT3 p.Q569Vfs*2 were starved for 24 hours in cell culture media containing 0.3% fetal calf serum. Cells were left untreated (−) or stimulated (+) with 100 ng/mL FL for 60 minutes prior to cell lysis. Phosphorylation of ERK was analyzed by western blot. One representative experiment is shown. The blot was first incubated with phospho ERK antibody, stripped and reblotted with ERK and α-Tubulin antibody. The blot was cropped to improve the clarity of the image (MT = FLT3 p.Q569Vfs*2).