IntroductionAcute myeloid leukemia (AML) is an aggressive form of a malignant disorder of the hematopoietic system that shows increasing incidence with age and is characterized by highly proliferative blast cells. [1][2][3] Aberrant activity of tyrosine kinases has been shown to be present in various malignant diseases and AML is no exception. The FMS-like tyrosine kinase 3 (FLT3) gene in chromosome band 13q12 encodes a receptor tyrosine kinase that belongs to the same family as FMS, KIT, and the 2 genes encoding PDGFR␣ and ␤. [4][5][6][7] It is normally expressed by hematopoietic stem/progenitor cells (HSPCs) and as hematopoietic cells differentiate FLT3 expression is lost. [8][9][10][11] A large body of work has shown that FLT3 plays roles in survival, proliferation, and differentiation. Aside from its role in regulating normal hematopoiesis, FLT3 is also highly expressed in several hematologic malignancies. 7 Mutations in the receptor, in the form of internal tandem duplication (ITD) of the juxtamembrane domain and point mutations of the kinase domain, both result in constitutive activation. 7 These mutations occur in one third of AML patients making it one of the most commonly mutated genes in AML. 12,13 Patients with FLT3/ITD mutations have been demonstrated to have very poor prognosis. 5,14,15 However, the molecular basis by which FLT3/ITD mutations lead to aggressive disease and poor prognosis in AML is not yet clearly understood.The process of activation, internalization, and degradation of FLT3 occurs in a similar fashion to other members of the class III receptor-type tyrosine kinases (RTKs) family. 16 Binding of FLT3 ligand (FL) causes homodimerization, tyrosine kinase activation, receptor autophosphorylation, and initiation of downstream signaling cascades. 7 The FLT3 receptor kinase shares structural homology with other type III receptor kinases, such as KIT and FMS, with all 3 playing an important role in survival, proliferation, and differentiation of hematopoietic cells. 17 We and other investigators have shown that STAT5 is one of the principal pathways involved in mediating gene expression in response to constitutive receptor activation through mutation. FLT3 signaling results in activation of pathways through phosphorylation of STAT5, MAPK, AKT, VAV, CBL, and BAD. [18][19][20] Phosphorylation and activation of STAT5 by FLT3/ITD mutants are particularly strong compared with its phosphorylation in the wild-type FLT3 allele. 21 STAT5 tyrosine phosphorylation mediates STAT5 protein dimerization through a mechanism involving SH2 domains and N-terminal regions and results in translocation to the nucleus where it activates transcription of a number of genes. 22 However, recent data suggest that STAT5 may have other interacting partners. 23,24 For example, signal-transducing adapter proteins (STAPs) have been shown to constitutively interact with inactive STAT5 in the cytoplasm but dissociate when STAT5 is phosphorylated. 24 STAT5 is also phosphorylated on serine residues, possibly by the ERK1/2 prote...

IntroductionFLT3 (Fms-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which includes KIT (c-kit receptor tyrosine kinase), FMS (macrophage-stimulating factor receptor), and 2 genes for the platelet-derived growth factor (PDGF) receptors. Both KIT and FMS play important roles in hematopoiesis. 1 FLT3 is expressed by a number of human and murine cell lines of both myeloid and B-lymphoid lineage. 2 FLT3 is also highly expressed on the malignant cells in most cases of acute myeloid leukemia (AML) and acute B-lineage leukemia (ALL). 3,4 Internal tandem duplication (ITD) mutations of the juxtamembrane domaincoding sequence of the FLT3 gene have been identified in 17% to 34% of patients with AML and 5% of patients with myelodysplastic syndrome (MDS). 5,6 In vitro studies have shown that FLT3/ITD receptors dimerize in a ligand-independent manner, leading to autophosphorylation of the receptor through constitutive activation of the tyrosine kinase domain. 7 Recently, constitutive activation of FLT3 has also been shown to occur through autocrine mechanisms. 8 Constitutive activation of FLT3 leads to autonomous, cytokineindependent growth with subsequent transformation of cells. 9,10 The signaling through constitutively activated FLT3 is mediated at least partially by the Ras and signal transducer and activator of transcription 5 (STAT5) pathways. 10-12 STAT5 appears to be an important target molecule in constitutively activated FLT3-mediated leukemogenesis, and its phosphorylation is inhibited by selective FLT3 tyrosine kinase inhibitors. [13][14][15][16][17] CEP-701 is a potent and selective inhibitor for autophosphorylation of both wild-type and constitutively activated mutant FLT3 forms. 13 CEP-701 improves survival in a murine leukemia model of FLT3/ITD-transformed cells and induces cytotoxicity in primary AML cells containing FLT3/ITD mutants. 13 The proto-oncogene Pim-1 was first identified by its induction by proviral insertion in murine leukemia virus-induced T-cell lymphomas. 18 It is a serine threonine kinase with increased expression in a variety of murine and human acute leukemias. 19,20 Expression of Pim-1 increases cell mitogenesis and survival independent of growth factors. 21 Pim-1 also synergizes with c-Myc in leukemogenesis and enhances transcriptional activity of c-Myb. 22,23 Pim-1 may also protect hematopoietic cells from apoptosis induced by genotoxic stress or growth factor withdrawal, perhaps by directly targeting nuclear effectors. 24 Expression of Pim-1 RNA and protein are normally induced by a number of cytokines, including granulocyte-macrophage colonystimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), IL-6, and interferon-␣ (IFN-␣). This suggests that Pim-1 may partially mediate signal transduction through these receptors. 25,26 Pim-1 is induced through Janus kinase For personal use only. on May 8, 2018. by guest www.bloodjournal.org From 2 (Jak2)/STAT5-mediated growth factor signaling, implying it may play a ro...

IntroductionConstitutive activation of the class III receptor tyrosine kinase, FLT3, plays important roles in leukemogenesis. [1][2][3][4] Internal tandem duplications in the juxtamembrane region (FLT3-ITD) or point mutations in the kinase domain (FLT3-PM) lead to constitutively activated FLT3. [5][6][7] FLT3 is also activated by coexpression of FLT3 ligand (FL) through intracrine, paracrine, and/or autocrine pathways. [8][9][10] The presence of FLT3-ITD mutations is associated with a poor prognosis in acute myelogenous leukemia (AML). 11-14 Activated FLT3 mediates signaling through at least 3 major downstream signaling pathways: signal transducers and activators of transcription (STAT5), PI3K/Akt, and Ras/mitogen-activated protein (MAP) kinase. [15][16][17][18][19][20][21][22][23][24][25] These signaling pathways have overlapping roles in cell differentiation, proliferation, and survival. FLT3 is expressed in most acute leukemias, including 94% of B-lineage acute lymphoblastic leukemia (ALL), 34% of T-lineage ALL, and 89% of AML cases. [26][27][28] These observations strongly suggest FLT3 as a candidate for molecularly targeted therapy.In fact, a number of FLT3 tyrosine kinase inhibitors (TKIs) have been developed. Some of the best studied to date include CEP-701 (lestaurtinib), PKC412, MLN518, SU11248 (sunitinib malate), and AG1295. 23,[29][30][31][32][33] Although these inhibitors vary in their potency and selectivity for FLT3, all are able to induce cytotoxicity in FLT3-expressing cells in vitro and/or in vivo. Furthermore, clinical trials with some of these inhibitors have demonstrated their ability to decrease peripheral blood and bone marrow blast counts in some patients. [34][35][36] CEP-701 is currently being tested on relapsed patients with FLT3 mutant AML in a randomized phase 2 clinical trial in combination with chemotherapy.Although FLT3 inhibitors demonstrate preclinical and clinical activity, they possess a number of limitations. Clinical trials have revealed that FLT3 TKIs used as single agents are able to significantly reduce peripheral blood and bone marrow blasts only in a minority of patients, and the effect is transitory. [34][35][36] This may be due to achieving insufficient levels of FLT3 inhibition in these patients, a lack of dependence of these cells on FLT3 signaling for proliferation and survival, and/or selection of resistant cell populations. Furthermore, most cases of AML and ALL do not express mutant FLT3, and it is unclear to what degree these cells depend on FLT3 signaling for sustaining the leukemic phenotype. At drug concentrations necessary to inhibit FLT3 phosphorylation past a critical threshold required to induce cytotoxicity, a varying spectrum of other kinases are frequently also inhibited, which can lead to toxicities.Even when cells are dependent on FLT3 signaling for survival and proliferation, prolonged exposure to TKIs are likely to select for resistant clones, as has been seen with imatinib mesylate (Gleevec), a TKI targeting BCR-ABL in chronic myelogenous leukemia...