NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells.

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.

Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3.

Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias.

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1.

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

Perinuclear localization of the protein-tyrosine phosphatase SHP-1 and inhibition of epidermal growth factor-stimulated STAT1/3 activation in A431 cells.

The SH2 domain protein-tyrosine phosphatase SHP-1 has been shown earlier to bind to the epidermal growth factor receptor and to have the capacity for receptor dephosphorylation. New bi- and tricistronic expression vectors (pNRTIS-21 and pNRTIS-33, respectively) based on the tetracycline system were constructed and employed to generate stable cell lines with inducible expression of SHP-1. Inducible overexpression of SHP-1 in A431 cells led to attenuation of epidermal growth factor (EGF) receptor autophosphorylation and of EGF-induced DNA binding of 'signal transducers and activators of transcription' (STAT) 1 and 3. SHP-1 was localized in the cytoplasm with an enrichment in the perinuclear compartment. Association of SHP-1 with perinuclear structures may form the basis for a partial cofractionation with nuclei observed in different types of transfected cells and also with endogenous SHP-1 in U-937 cells. Treatment of SHP-1-overexpressing A431 cells or of HaCaT human keratinocytes expressing SHP-1 endogenously with the Ca2+-ionophore A23187 resulted in partial nuclear accumulation of SHP-1. Thus, SHP-1 may interact with substrates or regulatory proteins in perinuclear or nuclear structures.

Activation of platelet-derived growth factor (PDGF) receptor dephosphorylation in intact Swiss 3T3 cells by elevators of intracellular Ca2+ and cAMP.

We investigated the modulation of platelet-derived growth factor (PDGF) receptor dephosphorylation in Swiss 3T3 cells using a novel assay permitting monitoring of receptor dephosphorylation in intact cells. PDGF treatment of the cells reduced the receptor dephosphorylation rate to 41%, the elevators of intracellular Ca2+, A23187 and thapsigargin increasing it to 227 and 138%, respectively. The cAMP elevators forskolin and isobutylmethylxanthine also accelerated PDGF receptor dephosphorylation. The involvement of Ca(2+)- and cAMP-dependent protein kinases in the regulation of PDGF receptor dephosphorylation is suggested.

The dephosphorylation characteristics of the receptors for epidermal growth factor and platelet-derived growth factor in Swiss 3T3 cell membranes suggest differential regulation of receptor signalling by endogenous protein-tyrosine phosphatases.

Comparison of the phosphotyrosine-specific dephosphorylation of the autophosphorylated receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in Swiss 3T3 cell membranes by the endogenous phosphatases revealed striking differences. EGF receptor dephosphorylation was clearly faster than PDGF receptor dephosphorylation and strongly inhibited by Triton X-100 and octylglucoside, whereas PDGF receptor dephosphorylation was to a lesser extent detergent-susceptible. PDGF receptor dephosphorylation was effectively inhibited by phenylarsineoxide, protamine and poly-lysine and partially by N-ethylmaleinimide, whereas EGF receptor dephosphorylation was not affected by these agents. We suggest that these differences in dephosphorylation of EGF and PDGF receptors are due to their differential interaction with membrane-associated protein-tyrosine phosphatases and important for differential regulation of receptor signalling.

[Differences in proline metabolism of a peptide alkaloid producing and a nonproducing strain of Claviceps purpurea]. / Unterschiede im Prolinstoffwechsel eines Peptidalkaloide-produzierenden und eines nichtproduzierenden Stammes von Claviceps purpurea.

Two strains (pepty 695 and pur 221) of Claviceps purpurea have been used to study the dependence of proline turnover on incubation time (1, 3, 6, and 12 hours) under different culture conditions (synthetic saccharose-citrate medium NL 720 and complex wort medium M 107) by means of tracer technique. In the saprophytically ergotoxine producing strain pepty 695 the proline is utilized in the protein and alkaloid biosyntheses. On the other hand, under non-producing conditions (M 10) the radioactivity of proline-14C is incorporated into many fractions and finally into CO2. In submerged culture (NL 720) the Claviceps strain pur 221 which does not produce ergolines uses proline only to a small extent in the protein biosynthesis, while most of this amino acid is not metabolized. The extent of uptake of proline is not correlated with the alkaloid synthesis. For comparison the turnover of L-glutamate-14C and D,L-ornithine-1-14C by strain pepty 695 cultivated in the NL 720 medium has been studied.