Abstract :
[en] ZAP-70 is a 70kDa protein tyrosine kinase (PTK) of the Syk-kinase family. It was identified in T lymphocytes where it plays an important role in T cell receptor (TCR) signaling (1). Normal B lymphocytes lack ZAP-70 but use another PTK for BCR signaling, namely Syk. However ZAP-70 was found in some B-cell malignancies such as B-cell chronic lymphocytic leukemia (CLL), mantle cell lymphomas (MCL) and diffuse large B-cell lymphomas (DLBCL) (1, 50-53, 89).CLL is characterized by an accumulation of CD5+ mature B-cells in the blood, lymph nodes and spleen. This accumulation is due to a resistance to apoptosis. The clinical course of the disease is highly variable and can be divided into two subgroups. About half of the patients show more aggressive disease with fast progression rates whereas the other half have a milder, slow progressing variant. These two subgroups can be distinguished based on ZAP-70 expression. ZAP-70 expression indicates a poor clinical outcome and thus the aggressive form of CLL (89, 91-93). While major progresses have been made in characterizing the expression of ZAP-70, little is known about its function and the consequences of its expression in these hematological malignancies. ZAP-70 has been reported however to correlate with an increased B cell signaling (13, 97, 98) higher migratory potential (103, 105-107) and increased resistance in apoptosis (97,108,110,112) but no mechanisms for these processes have been described so far.The specific aim of this project is to identify pathways that are involved in ZAP-70 expressing malignant B cells that can explain the poor prognosis associated with ZAP-70 expression.Because the lack of available CLL cell lines, and since this kinase has also been reported as expressed in other B cells malignancies such as DLBCL and MCL, we screened several B lymphoma cell lines for ZAP-70 expression and identified one DLBCL cell line (OCI-LY19) expressing this PTK. We silenced ZAP-70 using an siRNA approach in this cell line and compared on the one hand the tyrsosine phosphorylated proteins and on the other hand the proteome of the OCI-LY19 cells transfected with non-targeting (siCTL) or ZAP-70 targeting siRNA (siZAP70) using respectively a signal transduction array and 2D-DIGE and LC-ESI-MSMS. By 2D-DIGE, we identified several proteins differentially expressed between the two samples. Among them, the ERM (Ezrin/Radixin/Moesin) Ezrin and Moesin were the most represented. Ezrin and Moesin were present in the siCTL conditions, together with ZAP-70, and absent in the siZAP70 conditions. A correlation between ZAP-70 and ezrin expression was confirmed in CLL patient samples. We found that this correlation was only true at the protein level and not at the mRNA level suggesting that the decrease/absence of Ezrin observed is not due to decreased transcription of EZR locus or stabilization of the Ezrin mRNA, but more likely caused by decreased translation or increased degradation of Ezrin protein in the absence of ZAP-70 in OCI-LY19 cell line. There are indications that proteasomal degradation of Ezrin is a possible mechanism by which ZAP-70 might stabilize the ERM in CLL. We next evaluated the role of ZAP-70 and ERM proteins in cellular processes known to be important in CLL, such as apoptosis and migration. OCI-LY19 cells expressing ZAP-70 and Ezrin migrate better in response to SDF1alpha compared to OCI-LY19 cells depleted from ZAP-70 and/or Ezrin. Depletion of Ezrin by siRNA alone was sufficient to decrease migration and depleting Ezrin does not affect ZAP-70 expression levels. ZAP-70+/Ezrin+ CLL B cells also showed higher migratory potential toward SDF1alpha than ZAP-70-/Ezrin- CLL B cells. We observed that the presence of ZAP-70 in the OCI-LY19 cells protects them from spontaneous and drug-induced apoptosis. Specific depletion of Ezrin and Moesin by siRNA in OCI-LY19 induces spontaneous apoptosis without affecting ZAP-70 expression levels. To check for spontaneous apoptosis in primary CLL B cells, co-culture with HK feeder cells was compared with a mixed culture of PBMC. No difference in spontaneous apoptosis could be observed between ZAP-70+/Ezrin+ and ZAP-70-/Ezrin- CLL B cells. Furthermore cells that were kept in a mixed PBMC population were rapidly undergoing apoptosis, but viable cell rates remained stable over four days, indicating new possible culture conditions for primary CLL B cells without co-culture of feeder cells.Taken together since a correlation between ZAP-70 and Ezrin in B cell malignancies has been found and since Ezrin depletion affects cellular processes without affecting ZAP-70 expression levels, we can conclude that Ezrin is at least partially responsible for the poor prognosis of ZAP-70 expressing B cell malignancies through resistance in apoptosis and increased migration capacities.
