After 8 and 24 hours, cells were analyzed for DNA content by PI staining and flow cytometry. protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition. == Introduction == FLT3 (FMS-like tyrosine kinase 3) is a type III receptor tyrosine kinase (RTK) closely related to the platelet-derived growth factor (PDGF) receptor and c-Kit with important functions in the regulation of early hematopoietic cells. FLT3 is frequently mutated in patients YH249 with acute myeloid leukemia (AML), which correlates with poor prognosis and decreased patient survival.1-4The most common mutations are internal tandem duplications (ITD) of YH249 the juxtamembrane domain, which cause ligand-independent dimerization and autophosphorylation. FLT3-ITD expression causes malignant transformation and factor-independent growth when expressed in factor-dependent cell lines.5,6 Development of RTK inhibitors selective for FLT3 has emerged as attractive drugs for treatment of AML patients. Several inhibitors have been described, such as AG1295, CEP701, PKC412, YH249 and SU-11 248, with cytotoxic effects to cell lines and primary AML cells in vitro expressing mutant FLT3. PKC412 is one of several FLT3 inhibitors that is currently evaluated in late-stage clinical trials in AML patients carrying FLT3 mutations.7However, as single agents, these inhibitors are able to sustain only limited cytotoxic responses in AML patients, and relapse occurs after the initial response.8-11Therefore combination therapy has emerged as a therapeutic strategy. Clarifying the downstream signaling components of FLT3-ITD could identify attractive targets for such intervention and enhance the long-term therapeutic benefits. FLT3-ITD activates intracellular effector proteins mediating proliferation and survival. The Ras pathway and constitutive phosphorylation of mitogen-activated protein (MAP) kinase has been demonstrated in cells expressing FLT3-ITD.5,6In primary AML blasts and in cell lines expressing FLT3-ITD, increased activation of the survival AKT kinase has been observed.12-14AKT is transiently activated by normal FLT3 signaling, which leads to inhibition of apoptosis by phosphorylating FOXO3a, a Forkhead family member involved in apoptosis and cell-cycle control.15Studies have confirmed that AKT inhibits FOXO3a in FLT3-ITDexpressing cells,13,14suggesting that the pathway could be efficient for FLT3 intervention. Phosphorylated AKT is also found in AML samples expressing wild-type FLT3,16indicating that overexpression of FLT3 is sufficient to trigger AKT activation. Increasing evidence suggest that the mechanisms by which chemotherapeutic drugs and novel inhibitors eliminate malignant cells is mainly by apoptotic induction. The BH3-only proteins Bim (Bcl-2interacting modulator of cell death) and Puma (p53 up-regulated modulator of apoptosis), proapoptotic members of the Bcl-2 family, can be activated in response to cytotoxic stimuli, including chemotherapeutic drugs.17They have a prominent role among the BH3-only proteins, because they bind with high affinity to all antiapoptotic Bcl-2 family proteins,18thereby promoting mitochondrial release of cytochrome c, which subsequently activates caspase 9 and caspase 3 and death effector molecules. Recent results suggest that Bim and Puma have overlapping effects as well as distinct roles YH249 in vivo. This was Srebf1 demonstrated in genetically modified mice in which glucocorticoid-induced apoptosis varied with cell type and was dependent on lack of Bim or Puma.19Hence, exploring the regulation of Bim and Puma upon chemotherapeutic treatment may prompt new developments in treatment of AML. Studies indicate that BCR-ABL protein in chronic myelogenous leukemia (CML) and oncogenic FLT3-ITD support cell survival through down-regulation of Bim expression by a FOXO3a-dependent mechanism.13,14,20-22Recently, it was demonstrated that FOXO3a increases Puma expression in response to growth factor deprivation in lymphoid cells and mouse embryonic fibroblasts, suggesting that Puma together with Bim may have overlapping functions as FOXO3a downstream targets,23and that they may be involved in the effector mechanisms by which RTK inhibitors affect AML cells expressing FLT3-ITD. Here we show that the FOXO3a pathway is essential in mediating apoptosis of FLT3-ITDexpressing cells upon treatment with AG1295 and PKC412. We found that FLT3-ITD activated AKT and ERK when introduced into the factor-dependent progenitor cell line FDC-P1. Inhibition of the PI3-kinase pathway and downstream FOXO3a was crucial for survival, and a phosphorylation-deficient, constitutively active variant of FOXO3a up-regulated both Bim and Puma during FLT3-ITD signaling and induced cell death. Importantly, Bim and Puma were up-regulated in human leukemic cell.