IntroductionZAP-70 protein is a 70-kDa member of the Syk family of protein tyrosine kinases that was first identified as a crucial element for proximal signaling from the T-cell receptor. 1 Similar to Syk protein after the B-cell receptor (BCR) stimulation, ZAP-70 is recruited to the phosphorylated immunoreceptor tyrosine activation motifs of the -and CD3-chains present in the T-cell receptor, where it subsequently becomes phosphorylated and initiates several signaling cascades. 2 Expression of ZAP-70 protein was considered to be restricted to T lymphocytes and natural killer cells. However, it has also been found expressed in normal B-cell precursors and in some subsets of activated B cells. [3][4][5][6] Among B cell-derived malignancies, ZAP-70 is mainly expressed in chronic lymphocytic leukemia (CLL; 37%-57% of cases), 7-9 B-acute lymphoblastic leukemia (B-ALL; 56%-59% of cases) 4,10 and Burkitt lymphoma (8%-31% of cases). 11,12 An increased ZAP-70 expression in CLL has been associated with particular adverse biologic features, such as the presence of unmutated IgHV genes 7 or high CD38 expression, 13 and correlates with a poor clinical outcome. [7][8][9] In the same vein, ZAP-70 expression in B-ALL correlates with a short survival as well. 10 Although ZAP-70 expression in B-cell malignancies has an adverse prognostic influence, its role in the biology of the tumoral B cell is not fully defined. In this regard, the expression of ZAP-70 protein in CLL cells has been related to an enhanced BCR signaling. [13][14][15][16][17] In addition, increased ZAP-70 expression has been associated with increased migration capabilities of CLL cells toward different chemokines, such as CXCL12, CCL19, and CCL21, [18][19][20] and with increased signaling and survival on CXCL12 treatment. 18,21 However, whether these increased migrative capabilities are a direct effect of ZAP-70 expression or a mere reflection of the distinct biology features of ZAP-70-expressing cells needs to be further investigated.To ascertain the direct implication of ZAP-70 in B-cell signaling and migration, we analyzed the phenotypic effects of ectopic ZAP-70 expression in a B-cell system and studied the expression of adhesion molecules and chemokine receptors in CLL primary cells with high or low ZAP-70 expression within the same patient. Herein, we report that IgM, but not IgD, stimulation mobilizes and activates ZAP-70, which in turn enhances BCR-induced ERK1/2 and Akt phosphorylation and delays IgM and CD79b internalization. Moreover, we show that ZAP-70 induces the expression of the chemokine receptor CCR7 via ERK1/2 activation, thus directly enhancing the capacity of signaling and migration of the ZAP-70-expressing B cells toward CCL21. Finally, we show that CLL cells with higher ZAP-70 expression within the same patient have a different expression profile of adhesion molecules and chemokine receptors and enhanced migration capacity toward CCL21. Methods Cell lines and primary cellsThe Burkitt lymphoma B-cell lines Raji and Ramos were obtained from ...

Chronic lymphocytic leukemia (CLL) cells located in proliferation centers are constantly stimulated by accessory cells, which provide them with survival and proliferative signals and mediate chemotherapy resistance. Herein, we designed an experimental strategy with the aim of mimicking the microenvironment found in the proliferative centers to specifically target actively proliferating CLL cells. For this, we co-cultured CLL cells and bone marrow stromal cells with concomitant CD40 and Toll-like receptor 9 stimulation. This co-culture system induced proliferation, cell-cycle entry and marked resistance to treatment with fludarabine and bendamustine. Proliferating CLL cells clustered together showed a typical morphology of activated B cells and expressed survivin protein, a member of the inhibitor of apoptosis family that is mainly expressed by CLL cells in the proliferation centers. With the aim of specifically targeting actively proliferating and chemoresistant CLL cells, we investigated the effects of treatment with YM155, a small-molecule survivin inhibitor. YM155 treatment suppressed the co-culture-induced survivin expression and that was sufficient to inhibit proliferation and effectively induce apoptosis particularly in the proliferative subset of CLL cells. Interestingly, sensitivity to YM155 was independent from common prognostic markers, including 17p13.1 deletion. Altogether, these findings provide a rationale for clinical development of YM155 in CLL.

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironment, being the BCR pathway one key player in this crosstalk. Among proteins participating, ZAP-70 enhances response to microenvironmental stimuli. MicroRNA-21 (miR-21) is overexpressed in diverse neoplasias including CLL, where it has been associated to refractoriness to fludarabine and to shorter time to progression and survival. To further elucidate the role of ZAP-70 in the biology of CLL, we studied its involvement in miR-21 regulation. MiR-21 expression was higher in CLL cells with high ZAP-70. Ectopic expression of ZAP-70 induced transcription of miR-21 via MAPK and STAT3, which subsequently induced downregulation of tumor suppressors targeted by miR-21. The co-culture of primary CLL cells mimicking the microenvironment induced ZAP-70 and miR-21 expression, as well as downregulation of miR-21 targets. Interestingly, the increase in miR-21 after co-culture was significantly impaired by ibrutinib, indicating that the BCR signaling pathway is involved in its regulation. Finally, survival of CLL cells induced by the co-culture correlated with miR-21 upregulation. In conclusion, stimuli from the microenvironment regulate miR-21 and its targeted tumor suppressor genes via a signaling pathway involving ZAP-70, thus contributing to the cytoprotection offered by the microenvironment particularly observed in CLL cells expressing ZAP-70.

BackgroundThe aim of this study was to analyze whether in chronic lymphocytic leukemia the cytosolic release of histone H1.2, a new apoptogenic mechanism induced by DNA damage, was associated with the presence of genetic abnormalities and with the response to treatment.

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.