Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation

Abstract

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of leukemia-related deaths. Mutations in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent findings in this heterogeneous disease. The major types of mutations in FLT3 can be categorized as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs not only occur in the juxtamembrane region as originally described, but also in the kinase domain. Although the juxtamembrane ITDs have been well characterized, the tyrosine kinase domain ITDs have not yet been thoroughly studied due to their recent discovery. For this reason, we compared ITD mutations in the juxtamembrane domain with those in the tyrosine kinase domain, as well as with the most common activating point mutation in the tyrosine kinase domain, D835Y. The purpose of this study was to understand whether it is the nature of the mutation or the location of the mutation that plays the main role in leukemogenesis. The various FLT3 mutants were expressed in the murine pro-B cell line Ba/F3 and examined for their capacity to form colonies in semisolid medium. The size and number of colonies formed by Ba/F3 cells expressing either the internal tandem duplication within juxtamembrane domain of the receptor (JMD-ITD) or the tyrosine kinase domain (TKD)-ITD were indistinguishable, while Ba/F3 cells expressing D835Y/FLT3 failed to form colonies. Cell proliferation and cell survival was also significantly higher in TKD-ITD expressing cells, compared to cells expressing D835Y/FLT3. Furthermore, TKD-ITD is capable of inducing phosphorylation of STAT5, while D835Y/FLT3 fails to induce tyrosine phosphorylation of STAT5. Other signal transduction pathways such as the RAS/ERK and the PI3K/AKT pathways were activated to the same level in TKD-ITD cells as compared to D835Y/FLT3 expressing cells. Taken together, our data suggest that TKD-ITD displays similar oncogenic potential to the JMD-ITD but a higher oncogenic potential than the D835Y point mutation.

Keywords: Acute myeloid leukemia; FLT3; ITD; Internal tandem duplication; Receptor tyrosine kinase.

Fig. 1

Higher proliferation and cell survival in TKD-ITD mutants compared to D835Y. a Cells were cultured in the absence or presence of FL (100 ng/ml) and normalized against cell viability in the presence of IL-3. Forty-eight hours post-seeding, cell viability was assessed using the AlamarBlue cell viability assay. Each bar represents the mean ± SD of a representative triplicate experiment. b Apoptosis was measured using annexin V and 7-AAD kit after 48 h of cytokine depletion

Fig. 2

Activation of downstream signal transduction pathways by the different oncogenic mutants of FLT3 Ba/F3 cells were starved in serum- and IL-3-free media for 4 h before stimulating with 100 ng/ml of FL for 5 min. Cells were then lysed and lysates were used for SDS-PAGE and Western blotting analysis. The membrane was probed for different downstream signaling proteins and their phosphorylated forms

Fig. 3

Both JM-ITD and TKD-ITD induce clonogenic growth in semisolid media in the absence of IL-3, while D835Y fails to induce colonies. The Ba/F3 cells stably expressing the indicated FLT3 mutants were plated in triplicate at a concentration of 500 cells per well. The plates were cultured for 7 days. a Photographs of the representative areas of the wells demonstrating the number of colonies. WT FLT3-expressing Ba/F3 cells failed to induce colonies (data not shown) b Quantification of the number of colonies per plate. Each bar represents the mean ± SD of a representative triplicate experiment. c The total area of each colony was analyzed with help of ImageJ. The data presented indicate the relative size of the colonies (*p value = p < 0.05)

Fig. 4

Stability of the various FLT3 mutants. a Cells were treated with cycloheximide for 30 min and lysed or stimulated with FL (100 ng/ml) for additional 30 min. Western blotting analysis was used to measure the degree of degradation. b The graph presents a summary of quantified degradation of the FLT3 receptor. Each bar represents the mean ± SD of a representative triplicate experiment