Erythropoietin Down-Regulates Stem Cell Factor Receptor (Kit) Expression in the Leukemic Proerythroblast: Role of Lyn Kinase

Overexpression of the transcription factor Spi-1/PU.1 by transgenesis in mice induces a maturation arrest at the proerythroblastic stage of differentiation. We have previously isolated a panel of spi-1 transgenic erythroleukemic cell lines that proliferated in the presence of either erythropoietin (Epo) or stem cell factor (SCF). Using these cell lines, we observed that EpoR stimulation by Epo down-regulated expression of the SCF receptor Kit and induced expression of the Src kinase Lyn. Furthermore, enforced expression of Lyn in the cell lines increased cell proliferation in response to Epo, but reduced cell growth in response to SCF in accordance with Lyn ability to down-regulate Kit expression. Together, the data suggest that Epo-R/Lyn signaling pathway is essential for extinction of SCF signaling leading the proerythroblast to strict Epo dependency. These results highlight a new role for Lyn as an effector of EpoR in controlling Kit expression. They suggest that Lyn may play a central role in during erythroid differentiation at the switch between proliferation and maturation

Disappearance of slan-positive non-classical monocytes for diagnosis of chronic myelomonocytic leukemia with an associated inflammatory state

International audienceNo abstract availabl

Régulation transcriptionnelle de l'hématopoïèse par l'activité tyrosine kinase de BCR-ABL (exemples des gènes Cxr4 et Fli-1)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Oncogenic kit triggers Shp2/Erk1/2 pathway to down-regulate the pro-apoptotic protein Bim and to promote apoptosis resistance in leukemic cells.

Oncogenic mutations leading to persistent kinase activities are implicated in various human malignancies. Thereby, signaling pathway-targeted therapies are powerful customized treatment to eradicate cancer cells. In murine and human leukemia cells harboring mutations in Kit, we previously showed that distinct and independent pathways controlled resistance to apoptosis or cell cycle. A treatment with PI3Kinase inhibitors to reduce cell proliferation combined with inhibitors of Erk1/2 activity to promote apoptosis had synergistic effects allowing eradication of leukemia cell growth. We reported here that Bim(EL), a pro-apoptotic member of the Bcl2 family proteins, is the target of Erk1/2 signaling and that its down-regulation is responsible for the apoptosis resistance of murine and human leukemic cells. Downstream of Kit mutant, the tyrosine phosphatase Shp2 maintains Bim(EL) expression at a low level, through Erk/2 activation and proteosomal Bim(EL) degradation. This process is controlled by Shp2 independently of other signaling pathways activated downstream of oncogenic Kit, demonstrating that Shp2 is a key regulator of Bim expression in the context of an oncogenic signaling. The increase in Bim(EL) expression is associated to an increased apoptosis. Moreover, the depletion of Bim overcomes apoptosis associated with Erk1/2 inactivation in UO126-treated leukemic cells, thereby establishing the contribution of Bim to drug-induced apoptosis. These data provide a molecular rationale for using BH3 mimetics in combination with PI3K inhibitors to treat leukemia, especially in the case of an oncogenic signaling refractory to Tyrosine Kinase inhibitors

Epo controls Lyn expression.

<p>A: Lyn and EpoR expression were studied in 633, 663 and 812 cells continuously grown in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). Whole cell lysates were subjected to Western blot analysis with antibodies directed against Lyn, EpoR and β-actin as a loading control. B: Switch from SCF to Epo induces the expression of Lyn. 663 and 812 cells cultured with SCF (100 ng/mL) were extensively washed with medium without cytokine and then expanded for 48 hrs with Epo at the indicated doses. Whole cell lysates were subjected to Western blot analysis with antibodies directed against Lyn, Stat5, phosphorylated-Stat5 and β-actin as loading control. C: AG490 inhibits the expression of Lyn. 663 and 812 cells were cultured for 48 hrs in a medium containing 10% serum in the presence or absence of AG490 (10 µM) and in the presence of Epo (1 U/ml). Whole cell lysates were subjected to Western blot analysis with antibodies to Lyn, EpoR and β-actin. Western blots are from a representative experiment. D: RT-PCR analysis of <i>Lyn</i> transcription in 633, 663 and 812 cells cultured in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). DNAs were amplified with specific primers for <i>Lyn</i> or <i>gapdh</i> as control.</p

Down-regulation of Kit expression by Epo.

<p>A: Kit and EpoR expression were studied in the 633, 663 and 812 cells grown continuously in the presence of either Epo (1 U/mL) or SCF (100 ng/mL) or a combination of Epo (1 U/mL)+SCF (100 ng/mL). Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. Western blots are from a representative experiment. The membrane was exposed in an Imager, and the resulting signal was quantified using the ImageGauge software package (Fuji, Paris, France). Values were normalized to β actin expression. The fold change in Kit expression between SCF-cultured cells and either Epo or Epo+SCF-cultured cells is indicated under Kit immunoblotting. B: Representative diagram of flow cytometry analysis showing Kit membrane expression in cells cultured with SCF (100 ng/mL; black line) or Epo+SCF (dotted line). Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of four independent experiments. C: AG490 inhibits the down-regulation of Kit by Epo. Cells were cultured for 48 hrs in a medium containing 10% FBS, 1 U/mL Epo and in the presence or absence (-) of AG490 (10 µM). Representative Western blot analysis of whole cell lysates with antibodies directed against Kit, Stat5 and phosphorylated Stat5. β-actin was used as loading control. The fold change in Kit expression between AG490-treated and untreated cells is indicated under Kit immunoblotting. Representative diagram of flow cytometry analysis showing Kit membrane expression in 663 and 812 cells cultured with Epo and treated (black line) or not (dotted line) with AG490 (10 µM) for 48 hrs. Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of positive cells in three independent experiments. D: Cells were cultured for 24, 48 and 72 hrs in the presence of a combination of Epo (1 U/mL)+SCF (100 ng/mL) or Epo (0.1 U/mL)+SCF (100 ng/mL) and viable cells were numbered. Data are mean±SD of five experiments, each performed in duplicate. E: The regulation of Kit expression is transcriptional: RT-PCR analysis of <i>Kit</i> and <i>EpoR</i> transcripts in 633, 663 and 812 cells cultured in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). cDNAs were amplified with specific primers for <i>Kit</i>, <i>EpoR</i> or <i>gapdh</i> as control.</p

Reversible down-regulation of Kit in response to Epo.

<p>Switch from Epo to SCF (A) and switch from SCF to Epo (B): 663 and 812 cells continuously cultured with Epo (1 U/mL) or SCF (100 ng/mL) were extensively washed with medium without cytokine and then expanded for 48 hrs with SCF (100 ng/mL) or Epo (1 U/mL), respectively. Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. The fold change in Kit expression following the switch in cytokines is indicated under Kit immunoblotting. Representative diagrams of flow cytometry analysis showing Kit expression on the surface of cells expanded with Epo (dotted line) or SCF (black line). Control IgG profile is shown in grey. The tables indicate the mean fluorescence intensity (MFI)±SD of positive cells in at least four independent experiments. C: Switch from SCF to various concentrations of Epo. 663 cells cultured with SCF (100 ng/mL) were extensively washed with medium without cytokine and then expanded for 48 hrs with Epo at doses indicated. Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. The fold decrease in Kit expression following the switch from SCF to various doses of Epo is indicated under Kit immunoblotting. Western blots are from a representative experiment repeated 3 times.</p

Proliferation of <i>spi-1</i> transgenic proerythroblasts and expression of EpoR, Kit and Stat5.

<p>A: Cells were continuously cultured in the presence of Epo (1 U/mL) or SCF (100 ng/mL). Number of living cells was monitored at 24, 48 and 72 hours using the Trypan blue exclusion staining and a Vi-Cell analyzer (Becton Coulter). Mean number of living cells and standard deviations were determined from 3 independent experiments performed in duplicate. B: Representative Western blot of lysates from cells grown with Epo (1 U/mL) or SCF (100 ng/mL). Antibodies raised against the proteins are indicated on the left of the panel. P-Stat5 and P-Kit antibodies detect Stat5 and Kit phosphorylated forms. The blot was probed with an anti-β-actin antibody to visualize the protein loading. The membrane was exposed in an Imager, and the resulting signal was quantified using the ImageGauge software package (Fuji, Paris, France). Values were normalized to β actin expression. The fold change in Kit expression between Epo or SCF-cultured cells is indicated at the bottom of each cell line. C: Representative diagram of flow cytometry analysis showing cell surface expression of Kit in 663 and 812 cells cultured with Epo (dotted line) or SCF (black line). Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of positive cells from four independent experiments.</p

The tyrosine phosphatase Shp2 controls the down-regulation of Bim.

<p>(A) Knocking-down of Shp2 in 606HS2 and 931HS2 cells. Whole cell extracts were immunoblotted using antibodies raised against Shp2, P-Erk1/2, Erk1/2, Bim and the cleaved form of caspase-3. (B) Overexpression of Shp2 in 606HS2 and 931HS2 cells. Whole cell extracts were immunoblotted using indicated antibodies. The expression of Shp2^WT-MT and Shp2^C459S-MT was detected as the band above the endogenous Shp2. (C) Whole cell extracts from 606HS2 and 931HS2 cells treated or not (–) for 4 h with PI3K inhibitor NVP-BEZ235 (120 nM) were immunoblotted using indicated antibodies. (D) Knocking-down of Stat5 in 606HS2 and 931HS2 cells. Whole cell extracts were immunoblotted using anti-Bim, anti-Stat5 antibodies and anti-β actin antibody as loading control.</p