MAP Kinase Activation Is Essential for the Development of Acute Leukemia by MLL Fusion Protein and FLT3 Tyrosine Kinase Mutation.

MLL (mixed lineage leukemia)-fusion-mediated acute leukemia in infants has frequently been found to have FLT3 overexpression or tyrosine kinase domain (TKD) mutation. We have recently developed both in vitro and in vivo leukemogenesis models where MLL fusion proteins cooperate with another FLT3 mutant, internal tandem duplication (ITD). However, little has been clear about molecular mechanism of the cooperativity between MLL fusion protein and FLT3 mutants, not only FLT3-ITD but also FLT3-TKD. The present study demonstrates that MLL-SEPT6 fusion protein associated with infantile acute leukemia cooperates in vitro with FLT3-TKD mainly through activated MAP kinase pathway, while MLL-SEPT6 cooperates with FLT3-ITD mainly through activated STAT5 pathway. We first found that the interleukin (IL) -3 dependent murine hematopoietic cell line immortalized by MLL-SEPT6, named HF6, was transformed to grow without IL-3 by forced expression of FLT3 mutants which activated MAP kinase and STAT5, as shown in IL-3 dependent murine pro-B Ba/F3 cells. A dominant negative mutant of STAT5A suppressed the proliferation of the HF6 cells transformed by FLT3-ITD more effectively than that by FLT3-TKD, similarly to the transformed Ba/F3 cells. However, unlike the transformed Ba/F3 cells, the proliferation of transformed HF6 cells was suppressed with an MEK inhibitor more effectively in the HF6 cells transformed by FLT3-TKD than by FLT3-ITD. These results suggested that, in the transformation of HF6 cells, MAP kinase activation is more critical for FLT3-TKD than STAT5, while STAT5 activation is more critical for FLT3-ITD than MAP kinase. Furthermore, HF6 cells became IL-3 independent by direct activation of Raf-MAP kinase, while Ba/F3 cells did not. In contrast, a constitutively active mutant of STAT5 enabled, not HF6, but Ba/F3 cells to grow without IL-3, thus suggesting the essential role of activation of the Raf-MAP kinase cascade in the growth of the cells expressing MLL fusion protein. We next examined the oncogenic potential of MLL-SEPT6 and either of the FLT3 mutants by leukemogenesis assays in vivo using bone marrow transplantation. Interestingly, FLT3-TKD cooperated with MLL-SEPT6 in vivo to induce acute leukemia in mice rapidly (26±5.5 days), similarly to FLT3-ITD (27±5.1 days), although the individual oncogenic potential of FLT3-TKD leading to T-cell lymphoma (119±11 days), was much weaker than that of FLT3-ITD leading to myeloproliferative disease (56±16 days). Taken together, these results suggest that MLL fusion protein can induce human acute leukemia in concert with MAP kinase activation through secondary genetic events including FLT3-TKD mutation or other mechanisms which activate MAP kinase.