Expression de Zap-70 dans les lymphocytes périphériques de sang normal et de Leucémie Lymphoïde Chronique dans un modèle unicellulaire

Zap-70 est une protéine majeure de la signalisation intracellulaire dans les lymphocytes T et NK. Zap-70 est exprimée à tous les stades de maturation dans les lymphocytes B murins, tandis que chez l homme Zap-70 est détectée dans certaines hémopathies lymphoïdes B, dans les précurseurs B normaux, et les lymphocytes B activés.L expression de Zap-70 dans les lymphocytes B normaux, matures, périphériques chez l homme est un sujet de controverse.Pour répondre à cette question, nous avons mis au point une RT-PCR qualitative pour détecter Zap-70 dans les lymphocytes B périphériques à l échelon unicellulaire. Nos résultats sont en faveur d une absence d expression de Zap-70 dans les lymphocytes B périphériques matures. Nous mettons en évidence une hétérogénéité d expression de Zap-70 dans les lymphocytes B de LLC, ainsi que dans les lymphocytes T et NK périphériques à l échelon unicellulaire.PARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocSudocFranceF

Etude de l'incidence de la signalisation B dans la physiopathologie de la leucémie lymphoïde chronique

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Analyse des altérations structurales du génome dans la Leucémie Lymphoïde Chronique (apport de l hybridation génomique comparative sur puces à ADN (CGH array))

La leucémie Lymphoïde Chronique (LLC) est la plus fréquente des hémopathies occidentales. Les anomalies génétiques détectées par la technique d hybridation in situ par fluorescence (FISH) ont une valeur pronostique bien établie. Dans ce travail, j ai étudié les anomalies génétiques de la LLC à l aide de plusieurs techniques. La FISH a été la méthode la plus sensible pour détecter la présence d une délétion dans la région 13q14, ciblée sur le marqueur D13S319, dans 54% des cas d une cohorte de 314 patients. La représentativité du clone variait de 7 à 90% ; le plus souvent monoallélique, la délétion portait sur les 2 allèles dans 9% des cas et des délétions mono- et bi-alléliques coexistaient dans 15% des cas. Nous avons réalisé une étude en Hybridation Génomique Comparative sur micro-puces recouvertes d oligonucléotides (CGH-a) qui permet de quantifier les copies d un gène sur l ADN de patients par rapport à un témoin. Nous avons ainsi observé une grande variation de la taille de la délétion allant d une région minimale, restreinte à une zone appelée BCMS comprenant les MicroARN 15 et 16, à une grande région de 60 M bases couvrant une zone allant du gène du Rétinoblastome à celui de la protocadherine. La CGH nous a également permis dans 3 cas, de mettre en évidence une délétion sur le chromosome 14, impliquant notamment le gène codant les immunoglobulines. Nous avons identifié un gène de fusion impliquant un nouveau partenaire : ZFP36L1 membre de la famille des tristétraprolines facteurs régulateurs de la transcription. Le rôle fonctionnel de ce nouveau transcrit dans la leucémogenèse est en cours d étude.B-cell chronic lymphocytic leukemia (CLL) is the most frequent leukemia in western countries. Genomic abnormalities detected by Fluorescence in situ hybridization (FISH) are emerging prognostic indicators important in CLL. These ones are observed in about 80% of CLL patients and have been associated to defined subgroups with different clinical outcomes. In this project, different techniques have been performed aiming at the identification of new genetic abnormalities in CLL. FISH was the most sensitive one, identifying deletion del13q14 in 54% of 314 patients with a restriction to the D13S319 marker. We showed that the del13q deletion was heterogeneous and the clonality of the deletion was highly variable, ranging from 7 to 90 %, monoallelic in most cases but biallelic in 9.5% and concomitantly mono and bi-allelic in 15% of the cases.We also used Comparative Genomic Hybridization performed on DNA array (CGH-a) that measured differences in DNA copy number between patients and reference genomes. We observed variable sizes of deletion ranging from a Minimal deleted region (BCMS) including microRNAs 15 /16 to a very large region of 60 Mb, including RB1 or protocadherin in the opposite sides. CGH-array allowed to detect a deletion del14q in 3 cases, with a fusion between Immunoglobulin gene and a new partner that we identified as ZFP36L1 a member of the tris tetrapolin family of proteins involved in mRNA decay. The functional role of this new fusion gene in leukemogenesis is currently under study.PARIS13-BU Sciences (930792102) / SudocSudocFranceF

Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

Abstract Background We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL. Methods Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry. Results We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed. Conclusions This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis

Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma.

International audienceBACKGROUND: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL. METHODS: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry. RESULTS: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed. CONCLUSIONS: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis

Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Abstract Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases. [Cancer Res 2009;69(16):6387–95

Survival Response to B-Cell Receptor Ligation Is Restricted to Progressive Chronic Lymphocytic Leukemia Cells Irrespective of Zap70 Expression

International audienceDespite very similar gene expression profiles, the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is heterogeneous. Immunoglobulin VH (IgVH) mutational status and expression of B-cell receptor (BCR) signaling mediators have been associated with disease progression. However, the consequences of BCR engagement on cell survival and evolution of the disease remain unclear. We show here that B-CLL cell survival is dependent on the threshold of BCR stimulation induced by immobilized antibody, in contrast to soluble anti-mu F(ab)'2 antibody, which leads to apoptosis. Measurement of metabolic activity and apoptotic response discriminated two subgroups. "Nonresponders" showed low metabolic activity and unmodified apoptotic response upon BCR stimulation. In contrast, "responders" exhibited increased metabolic activity and inhibition of spontaneous apoptosis. This survival advantage was associated to a BCR-dependent activation profile leading to induction of cyclin D2/cyclin-dependent kinase 4 (cdk4) expression and G1 cell cycle progression. The ability to respond to BCR ligation correlated with an unfavorable clinical course and allowed to define an additional group of patients among IgVH-mutated cases exhibiting a risk of progression. Remarkably, we show that Zap70 expression was neither mandatory nor sufficient to generate downstream survival signals and cyclin D2/cdk4 up-regulation. In conclusion, BCR engagement has a significant effect on B-CLL cell survival, activation, and G1 progression. Furthermore, our results provide new insights in the physiopathology of progressive IgVH-mutated cases

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia

International audienceAbstract B-cell antigen receptor (BCR)–mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium–calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets