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

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

Knocking-down of Bim expression leads to attenuation of apoptosis induced by UO126 in HS2 cells.

<p>(<b>A</b>) Knocking-down of Bim in 606HS2 and 931HS2 cells. Whole cell extracts were immunoblotted using indicated antibodies. <b>(B)</b> Erk1/2 activation and Bim_EL expression in UO126 treated cells. Whole cell extracts from Bim-shRNA-94-HS2 cells or control ns-shRNA-HS2 cells treated or not (–) during 48hours with UO126 (10 or 20 µM) were immunoblotted using indicated antibodies. <b>(C)</b> For mortality analysis, cells were plated at 2×10⁵ cells/mL. Bim-shRNA-94-HS2 cells or control ns-shRNA-HS2 cells were treated or not (–) during 48hours with UO126 (10 or 20 µM) 24 h after lentiviral infection. The percentage of dead cells was evaluated by trypan blue exclusion. The percentage of apoptotic cells was determined by labeling the cells with cleaved caspase3-antibody and flow cytometry analysis. For both cell death and cell apoptosis, data are mean ±SEM (n = 3). Statistical differences from the value of the control (–) are indicated as follows: * <i>P</i><0.05;** <i>P</i><0.01; *** <i>P</i><0.001 (Student <i>t</i> test). <b>(D)</b> Proteosomal degradation of Bim_EL. Cells were treated or not (–) for 2 h or 4 h with MG132 (10 µM). Whole cell extracts were immunoblotted using indicated antibodies. Data are a representative experiment out of three.</p

High sensitivity of the Hematoflow™ solution for chronic myelomonocytic leukemia screening

International audienceBackground: Accumulation of classical monocytes CD14++CD16– (also called MO1) ≥ 94% can accurately distinguish chronic myelomonocytic leukemia (CMML) from reactive monocytosis. The HematoFlow™ solution, able to quantify CD16 negative monocytes, could be a useful tool to manage monocytosis which remains a common issue in routine laboratories.Methods: Classical monocytes were quantified from 153 whole blood samples collected on EDTA using both flow cytometry methods, either MO1 percentage determination by the multiparameter assay previously published and regarded here as the reference method, or CD16 negative monocyte percentage determination by the means of HematoFlow™.Results: Both methods of classical monocyte percentage determination were highly and significantly correlated (r = 0.87, P < 0.0001). The HematoFlow™ solution leant toward an overestimation of the genuine classical monocyte percentages obtained by the reference method. Percentages of CD16 negative monocytes provided by HematoFlow were higher than 94% for all the 73 patients displaying classical monocytes MO1 found ≥94% by the reference method, indicating a sensitivity of 100%. Furthermore, the calculation of CD16 negative monocyte percentage can be easily computerized and integrated to the middleware.Conclusions: We propose a new application of the Hematoflow™ solution that can be used as a flag system for monocytosis management and CMML detection

CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia

International audienceAbstract Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α ( SIRPα ), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ. In mouse models of human T-cell acute lymphoblastic leukemia (T-ALL), infusion of human monocytes (Mos) engineered to overexpress p21 (p21TD-Mos) leads to Mo differentiation into phagocytosis-proficient TAMs that, after leukemia cell engulfment, undergo pro-inflammatory activation and trigger the reprogramming of bystander TAMs, reducing the leukemic burden and substantially prolonging survival in mice. These results reveal p21 as a trigger of phagocytosis-guided pro-inflammatory TAM reprogramming and highlight the potential for p21TD-Mo-based cellular therapy as a cancer immunotherapy

<i>TET2</i> mutational status affects myelodysplastic syndrome evolution to chronic myelomonocytic leukemia

International audienc