IntroductionReceptor tyrosine kinases (RTKs) bind extracellular growth factors and activate intracellular signaling networks. The magnitude and kinetics of RTKs activation are tightly regulated, since they determine the quality and extent of the biologic response. 1 Attenuation of RTK signaling occurs by endocytosis and subsequent protein degradation. [2][3][4] Cbl proteins have been shown to be central players in these processes. [5][6][7] The members of the Cbl family, c-Cbl, Cbl-b, and Cbl-3, contain an N-terminal phosphotyrosine-binding (PTB) domain that allows direct interaction with activated RTKs, and a RING finger domain that classifies Cbl proteins as E3 ubiquitin ligases. 5,[8][9][10] Ubiquitylation of RTKs is important for their internalization, endocytic sorting, and targeting for degradation. 2 In addition to their ubiquitin E3 ligase activity, Cbl proteins also associate with the endocytic machinery via their C-terminus by recruiting proteins such as CIN85 and endophilins. 11,12 However, not only are Cbl proteins important for RTK signal termination, but they also mediate positive RTK signaling events to downstream effectors. Upon phosphorylation, Cbl molecules bind signaling molecules including SHP-2, Gab2, and PI3-kinase. 13 In animal models, but not in human cancers, oncogenic forms of Cbl have been described that are characterized by loss of the E3 ubiquitin ligase activity. 14,15 It has been reported that these oncogenic mutants of Cbl interact with activated RTKs and function in a dominant-negative fashion. 16,17 Aberrant signaling by the type III RTK Flt3 is an important event in the pathogenesis of acute myeloid leukemia (AML). Flt3 strongly influences hematopoietic progenitor cell homeostasis and is highly expressed in AML. [18][19][20][21] Also, about one third of AML cases harbor somatic, activating Flt3 mutations that cause myeloid transformation. [21][22][23][24] In contrast to activating mutations, little is known about the potential functions of Flt3 in AML cases lacking Flt3 mutations. Also, the mechanisms of Flt3 signal regulation and the role of Cbl proteins in these processes remain undetermined, although it has been shown that Flt3 activation is followed by Cbl phosphorylation. 25 Here, we analyzed the role of c-Cbl in the internalization, ubiquitylation, and biologic functions of wild-type Flt3 (Flt3-WT) and the most commonly described Flt3 mutations in AML, internal tandem duplication (Flt3-ITD). We found that the inhibition of Cbl function severely disturbed Flt3 signal transduction kinetics by blocking Flt3 internalization and ubiquitylation. As a consequence, interference with Cbl function induced ligand-independent, autoactive biologic effects of Flt3. Also, we describe a novel E3-ligase inactivating c-Cbl mutation isolated from the blasts of one AML patient. This mutant Cbl protein interfered with the function of endogenous c-Cbl and displayed in vitro transforming activity in myeloid cells that was dependent on the presence of Flt3. To our knowledge, this represents the ...

No abstract

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 receptor (FLT3). Patients with FLT3-ITD mutations tend to have a poor prognosis. MicroRNAs (miRNAs) have a pivotal role in myeloid differentiation and leukemia. MiRNA-155 (MiR-155) was found to be upregulated in FLT3-ITD-associated AMLs. In this study, we discovered that FLT3-ITD signaling induces the oncogenic miR-155. We show in vitro and in vivo that miR-155 expression is regulated by FLT3-ITD downstream targets nuclear factor-κB (p65) and signal transducer and activator of transcription 5 (STAT5). Further, we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Knockdown of miR-155 or overexpression of PU.1 blocks proliferation and induces apoptosis of FLT3-ITD-associated leukemic cells. Our data demonstrate a novel network in which FLT3-ITD signaling induces oncogenic miR-155 by p65 and STAT5 in AML, thereby targeting transcription factor PU.1.

IntroductionFms-like tyrosine kinase 3 (FLT3) belongs to the class III receptor tyrosine kinase (RTK) family that includes FMS, platelet-derived growth factor receptor (PDGFR), and c-KIT. 1 FLT3 plays an important physiologic role in self-renewal and differentiation of hematopoietic stem and progenitor cells. [2][3][4] In acute myeloid leukemia (AML), activating mutations in the FLT3 gene occur in 30% to 40% of adult patients and have been demonstrated to play a crucial role in driving proliferation and survival of the leukemic clone. Twenty percent to 30% of AML patients harbour an internal tandem duplication (ITD) in the juxtamembrane (JM) region of FLT3. 5,6 ITDs found in FLT3 are always in frame and range from 3 base pairs (bp) to more than 400 bp. 7 Ten percent of AML patients have mutations within the activation loop of the second kinase domain, predominantly substitutions of aspartate at residue 835 (D835). However, additional activating mutations in this region have also been described. [8][9][10] Expression of FLT3_ITD receptors results in autophosphorylation of FLT3 and subsequent activation of downstream signaling. 11-13 Consequently, FLT3_ITD mutations render hematopoietic cells growth factor-independent by promoting cell proliferation and inhibition of apoptosis and lead to myeloproliferative disease in a murine transplantation model. 11,[13][14][15] Generally, it is believed that the ITD insertions within FLT3 occur in the zipper or linker peptide segment of JM (JM-Z) near the JM hinge region, thereby disrupting the autoinhibitory function of the JM-domain. 16 However, a detailed analysis of FLT3_ITD insertion sites has not been performed so far. Therefore, we have addressed this issue and have determined ITD insertion sites of 753 unselected FLT3_ITD-positive AML cases by direct cDNA sequencing. Methods Sequence analysis of FLT3_ITD from 753 unselected FLT3_ITD-positive AML casesMononucleated bone marrow cells from patients participating in standard diagnostic procedures and treated according to protocols of the AML Cooperative Group (AMLCG) study group or according to other intensive AML therapy protocols were obtained by Ficoll density gradient centrifugation. Some of the patients have already been described previously. 7 mRNA was extracted with the MagnaPureLC mRNA Kit I (Roche Diagnostics, Mannheim, Germany). cDNA synthesis of mRNA of an equivalent of 5 to 10 ϫ 10 6 cells was performed using SuperscriptII (Gibco BRL/Invitrogen, Karlsruhe, Germany) and random hexamer primers (Pharmacia, Freiburg, Germany). PCR and evaluation was performed as described previously. 7 Mutation detection was done by standard agarose gel electrophoresis and in parallel by fragment analysis on a capillary sequencer as described previously. 17 Direct sequencing of PCR products was performed as described 7 and primers were the same as used for PCR or RT-PCR amplification, respectively.Blood samples from AML patients were collected after informed consent was obtained in accordance with the Declaration of Helsinki. Labor...

IntroductionThe receptor tyrosine kinase (RTK) FLT3 belongs to the class III RTK subfamily that also includes KIT, FMS, and platelet-derived growth factor receptor (PDGF-R). 1,2 These class III RTK members are characterized by an extracellular domain consisting of 5 immunoglobulin-like domains, a juxtamembrane domain, and 2 kinase domains (KDs) interrupted by a kinase insert. 3 Ligand binding to the extracellular domain results in dimerization of the receptor followed by autophosphorylation on specific intracellular tyrosine residues. Subsequently, multiple downstream signaling pathways are activated. [4][5][6] Activation of the FLT3 receptor with its ligand (FL) plays an important role in proliferation and differentiation of early hematopoietic progenitors. [7][8][9] The FLT3 receptor tyrosine kinase is expressed on blast cells in most patients with acute myelogenous leukemia (AML), and activating mutations of FLT3 have been detected in approximately 30% of these patients. 10 Two distinct groups of FLT3 mutations are most common: internal tandem duplications (ITDs) of the juxtamembrane coding sequence in 20% to 27% of patients with AML 11-13 and point mutations at codon 835 (Asp835) within the second kinase domain in about 7% of patients with AML. 12,14 Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the effect of point mutations on the prognosis of patients with AML has not yet been defined. 11,12 Both types of mutations constitutively activate the FLT3 receptor, leading to activation of downstream signaling proteins, including STAT5 (signal transducer and activator of transcription 5) and MAP (mitogen-activated protein) kinase, and result in factorindependent proliferation of growth factor-dependent murine lymphoid and myeloid cells. [14][15][16] Additional evidence in support of an oncogenic role of FLT3 mutations stems from studies demonstrating that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. 17 Recently, a series of novel mutations within the activation loop were identified in patients with AML. In that study, deletion of isoleucine 836 (Ile836del) occurred in 13 of 87 patient samples carrying point mutations in the kinase domain, and one sample was found to contain a novel point mutation, changing isoleucine 836 to methionine with an additional arginine inserted after codon 836 (Ile836MetϩArg). 12 The effect of these mutations on FLT3 receptor signaling activity has not yet been investigated.Because the FLT3 receptor is expressed in blast cells of most patients with AML and a significant fraction of these patients carry an activating mutation conferring a worse prognosis, targeting the molecule by specific inhibitors may establish new treatment PKC412 is a Novartis compound. [18][19][20] In this study, we investigated the role of newly described FLT3 receptor mutations on receptor activation and cell growth. Our results indicate that both deletion and insertion mutants are sufficient to ...

FLT3-ITD–mediated leukemogenesis is associated with increased expression of oncogenic PIM serine/threonine kinases. To dissect their role in FLT3-ITD–mediated transformation, we performed bone marrow reconstitution assays. Unexpectedly, FLT3-ITD cells deficient for PIM1 failed to reconstitute lethally irradiated recipients, whereas lack of PIM2 induction did not interfere with FLT3-ITD–induced disease. PIM1-deficient bone marrow showed defects in homing and migration and displayed decreased surface CXCR4 expression and impaired CXCL12–CXCR4 signaling. Through small interfering RNA–mediated knockdown, chemical inhibition, expression of a dominant-negative mutant, and/or reexpression in knockout cells, we found PIM1 activity to be essential for proper CXCR4 surface expression and migration of cells toward a CXCL12 gradient. Purified PIM1 led to the phosphorylation of serine 339 in the CXCR4 intracellular domain in vitro, a site known to be essential for normal receptor recycling. In primary leukemic blasts, high levels of surface CXCR4 were associated with increased PIM1 expression, and this could be significantly reduced by a small molecule PIM inhibitor in some patients. Our data suggest that PIM1 activity is important for homing and migration of hematopoietic cells through modification of CXCR4. Because CXCR4 also regulates homing and maintenance of cancer stem cells, PIM1 inhibitors may exert their antitumor effects in part by interfering with interactions with the microenvironment.

Epidermal growth factor receptor (EGFR) overexpression and activation are hallmarks of non-small cell lung carcinoma (NSCLC). Although EGFR-targeted therapies are used, the prognosis of NSCLC remains poor. ADAM17 induces activation of the EGFR through ligand cleavage. However, we show that inhibition or knockdown of ADAM17 markedly reduces tumorigenesis and survival to a large part independently from EGFR ligand shedding in NSCLC cells. These findings strongly indicate additional oncogenic mechanisms regulated by ADAM17. We identified Notch1 signaling as an

CORRESPONDENCE