The mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by proteintyrosine phosphatases (PTPs). IntroductionAcute myeloid leukemia (AML) is the most frequent leukemia in adults with improving but still limited treatment possibilities, notably in elderly patients. 1,2 It arises by malignant transformation of myeloid progenitor cells. Among the contributing genetic lesions, mutations in the class III receptor tyrosine kinase (RTK) FMS-like tyrosine kinase 3 (FLT3) occur in approximately 30% of patients. 3 The prevalent type of FLT3 mutations are internal tandem duplications (ITD) of amino acid stretches in the juxtamembrane domain or in the tyrosine kinase domain, 4,5 which confer cytokineindependent proliferation and resistance to apoptosis, and causally contribute to AML in combination with additional genetic lesions. 1 Compared with the ligand-activated wild-type (WT) FLT3, FLT3 ITD mutants exhibit not only elevated but also altered signaling quality, with very pronounced activation of signal transducer and activator of transcription (STAT)5 as one characteristic feature. 6,7 FLT3 ITD also causes the production of high levels of reactive oxygen species (ROS). 8,9 Signal transduction of RTKs is regulated by protein-tyrosine phosphatases (PTPs). PTPs prevent ligand-independent RTK activation, and contribute to modulation and termination of ligand-induced signaling. 10 The activity of PTPs is regulated at several different levels. 11 One regulatory principle is the reversible oxidation of the PTP active-site cysteine, which leads to reversible inactivation. 12,13 Temporary inactivation of negatively regulating PTPs by this mechanism is believed to be important for efficient RTK signal propagation in the cell. 14 A major ROS causing cellular PTP oxidation is hydrogen peroxide (H 2 O 2 ), which can be generated by a dismutase reaction from superoxide anions, the reaction products of NADPH-oxidases. Activation of the NADPH oxidase isoform 1 (NOX1) occurs downstream of RTK activation, and involves activation and membrane translocation of the small guanosine triphosphate (GTP)ase Rac1. 15 ROS generation in the cell is counteracted by efficient ROS decomposing systems. 16 These include peroxiredoxins (Prx), which have a very low K m for H 2 O 2 and can eliminate it even at low concentrations. 17 Relatively little is known about PTPs regulating FLT3 signal transduction. We have previously shown that the nontransmembrane PTPs PTP1B and SHP-1 can potently dephosphorylate FLT3 on overexpression. Further, PTP1B appears important for suppressing signaling of newly synthesized FLT3. 7,18 SHP-2 acts as a positive regulator, because it is important for Erk activation and proliferation induced by ligand-activated WT FLT3. However, it is dispensable for FLT3 ITD-mediated transformation. 19 We previously performed a shRNA-based screen to identify PTPs regulating WT FLT3. The initial screen assessed the effects of shRNAs for 20 PTPs on FL-induced Erk1/2 activation in WT FLT3-expressing 32D cells. Among several potentia...

Phosphorylation of wild type FLT3 and AML-associated mutant FLT3 was recently analyzed using site-specific phosphotyrosine antibodies (15). Interestingly, the phosphorylation pattern of the different FLT3 variants showed quantitative and also qualitative differences. Although FLT3-ITD or mutations in the kinase domain resulted in ligand-independent FLT3 autophosphorylation and signaling activity, the wild type receptor is only autophosphorylated in response to stimulation with its cytokine FL.Signaling of receptor tyrosine kinases is modulated by protein-tyrosine phosphatases (PTP) (16), and aberrations in PTP function play a role in carcinogenesis (17). Some PTP, notably SHP-2, have been found to positively influence growth-stimulatory signaling pathways, and mutations leading to gain-offunction of these PTP can potentially be oncogenic. It has been demonstrated that SHP-2 directly interacts with FLT3 in a phosphorylation-dependent manner via phosphotyrosine 599. Table S1. 1 To whom correspondence should be addressed. Tel.: 49-3641-9395634; Fax:49-3641-9395602; E-mail: joerg.mueller2@med.uni-jena.de.2 The abbreviations used are: AML, acute myeloid leukemia; PTP, proteintyrosine phosphatase; FL, FLT3 ligand; PLC␥, phospholipase C␥; ITD, internal tandem duplication; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4(-/-)) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.

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