Bad Phosphorylation Is Reduced in Response to Inhibition of Constitutively Activated FLT3.

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Previously we showed that human leukemia-derived myeloid cell lines having constitutive activation of FLT3 undergo apoptotic cell death when treated with FLT3 inhibitors. Examining the anti- and pro-apoptotic proteins in constitutively activated FLT3 signaling in BaF3/ITD, MOLM-14 and MV4-11 cell lines, we found that the level of Bad phosphorylation was greatly decreased by FLT3 inhibitor treatment. Bad is a proapoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-x_L, resulting in cell death. Survival factors such as IL-3 inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136. Both Ser112 and Ser136 are rapidly dephosphorylated after treatment with FLT3 inhibitors in BaF3/ITD, MOLM-14, and MV4-11 cells. We also found Bad to be highly phosphorylated in both wild-type and mutant FLT3 primary AML samples. In confirmation of the cell line data, Bad was rapidly dephosphorylated after treatment of the primary samples with lestaurtinib (CEP-701). Upstream molecules responsible for phosphorylation of Bad include Akt, Erk/MAPK, Pim-1, and Pim-2. We previously reported Pim-1 as one of the genes whose expression is induced by constitutively activated FLT3. We and other groups have also shown that constitutively activated FLT3 induces multiple signaling pathways, including PI3/Akt, Erk/MAPK, and Jak/STAT. The dephosphorylation of Bad in response to FLT3 inhibition also correlates with deactivation of Stat5, MEK1/2 and Akt kinase activities and decreased expression of Pim-1. Thus, Bad may be one of the points that these multiple signaling pathways converge in FLT3-mediated cell survival. Thus, it appears that activated FLT3 phosphorylates Bad, resulting in the inability of Bad to bind to Bcl-x_L, therefore blocking apoptosis. This is the first report of deactivation of a pro-apoptotic Bcl-2 family member induced by constitutively activated FLT3.