The D816V C-Kit Mutation Confers Higher Proliferation Activity By JAK-STAT and Src Family Kinase Pathways Compared to N822K C-Kit Mutation in Core-Binding Factor Acute Myeloid Leukemia

Backgrounds: Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is defined as core-binding factor AML (CBF-AML) and has been considered an AML group with favorable prognosis. CBF-AML produces chimeric protein inhibiting differentiation of normal hematopoiesis such as AML1-ETO and CBFB-MYHII. These chimeric protein (class II) and mutation of the class I with cell proliferation activity such as c-kit, Ras, and Flt3ITD are strongly associated with leukemogenesis of CBF-AML. In recent years, it has been reported that CBF-AML with c-kit mutations show poor prognosis. The c-kitmutation in CBF-AML has been reported in the extracellular domain of exon 8, the juxtamembrane domain of exons 10 and 11, and the A-loop domain with tyrosine kinase activity of exon 17. Among these mutations, D816V in exon 17 is the most frequent mutation in CBF-AML. Some previous in vitro studies report that the D816V mutation confers higher tumor growth and anti-apoptotic potential compared to mutations in the extracellular domain of exon 8 or in the juxtamembrane domains of exons 10 and 11. Interestingly N822K in exon 17 is reported at frequency similar to D816V mutation in Asia. Our previous report (Leukemia. 2011;25(9):1423) suggested the possibility that the prognosis of CBF-AML with the N822K mutation was different from D816V. This result suggests that D816V and N822K may differ functionally even if this mutation is also in the same A-loop. Therefore, the aim of this study is to clarify a difference of the molecular biologic function for leukemogenesis between D816V and N822K.

Method: To analyze the molecular function of D816V and N822K, we generated amphotropic retroviral vector expressing c-kit (wild type, D816V, and N822K). After transduction with human IL3 dependent TF-1 cells, we established c-kit expressed TF-1 cells (TF-1 WT, TF-1 D816V, and TF-1 N822K). In these three cells, we compared the proliferation rate by cell growth assay and analyzed the signal pathways by human microarray (Agilent SurePrint G3 Human Gene Expression 8x60K v2) and Western blotting.

Results: Expression and phosphorylation analysis showed that wild type, D816V, and N822K c-kit cDNA were individually transduced to the TF-1 cells. Cell growth assay showed IL3 independent proliferation was observed in TF-1 D816V and TF-1 N822K but not TF-1 WT. The proliferation rate of TF-1 D816V is significantly higher than that of TF-1 N822K (18610±6269 cells vs 4354±1109 cells in day 10, p=0.026). Similar results were obtained in the presence of SCF (p=0.057).

Subsequently, we investigated the difference of signal pathway for cell proliferation between the TF-1 D816V and TF-1 N822K by Western blotting. Under the condition were starved from IL3, TF-1 WT showed much higher expression of c-kit protein than those of the TF-1 D816V and TF-1 N822K, but the TF-1 WT hardly induced phosphorylation of it. Whereas the same level of constitutive phosphorylation of c-kit protein were observed in the TF-1 D816V and TF-1 N822K. We investigated about the downstream of phosphorylated c-kit protein such as JAK-STAT, MAP kinase, Src family kinase, and PI3kinase pathways in the TF-1 D816V and TF-1 N822K. We observed a strong induction of phosphorylated STAT3 and Src protein in the TF-1 D816V, but phosphorylated ERK protein of MAP kinase pathway in the TF-1 N822K. Finally we compared gene expression profiling of these cells using microarrays to discover the novel signaling pathways which are specific for each cell. We found Mras and Jak3gene expressions in the TF-1 D816V were five or more times greater than that in the TF-1 N822K. Also similar results were shown by Western blotting.

Conclusions: We conclude that the D816V c-kit mutation confers higher proliferation activity by JAK-STAT and Src family kinase compared to N822K c-kit mutation. These findings suggest that biological functions of c-kit mutations differ depending on the mutation site, which may affect responsiveness to treatments.