Multistep pathogenesis of leukemia via the MLL-AF4 chimeric gene/Flt3 gene tyrosine kinase domain (TKD) mutation-related enhancement of S100A6 expression

Objective Concerning MLL-AF4 leukemogenesis, previous mouse models suggest that the tumorigenesis capacity of MLL-AF4 alone is insufficient for causing leukemia. Based on the finding that an Fms-like tyrosine kinase 3 (Flt3) gene mutation in the tyrosine kinase domain (TKD) was observed in approximately 15% of mixed lineage leukemia (MLL), we investigated synergistic leukemogenesis effects of the two genes in vitro. Materials and Methods In a mouse interleukin-3 (IL-3)−dependent cell line, 32Dc, expression of MLL-AF4 and mutant Flt3 was induced using a lentiviral vector. We analyzed apoptosis induction in the absence of IL-3 and the granulocyte colony-stimulating factor−related induction of differentiation, gene expression profiling, and the mechanism involved in the synergistic effects of MLL-AF4 and Flt3-TKD. Results Neither Flt3-expressing 32Dc (32Dc Flt3-TKD ) nor MLL-AF4−expressing 32Dc (32Dc MLL-AF4 ) acquired IL-3−independent proliferative capacity in semisolid/liquid media. However, Flt3-TKD+MLL-AF4−expressing 32Dc (32Dc Flt3-TKD+MLL-AF4 ) acquired a non−IL-3−dependent proliferative capacity by inhibiting apoptosis in the two media. The 32Dc Flt3-TKD and 32Dc MLL-AF4 cells differentiated into granulocytes in the presence of granulocyte colony-stimulating factor. However, in the 32Dc Flt3-TKD+MLL-AF4 cells, there was no differentiation. Subsequently, we performed gene expression profiling. The enhancement of Hox genes expression was not identified. However, expression of S100A6 was synergistically enhanced in the presence of both MLL-AF4 and Flt3-TKD genes. Moreover, anti-S100A6 small interfering RNA downregulated leukemic proliferation. Conclusion We conclude that their synergistic enhancement of S100A6 expression plays an important role in MLL-AF4−associated leukemogenesis.