Noelia Purroy

ZAP-70 enhances migration of malignant B lymphocytes toward CCL21 by inducing CCR7 expression via IgM-ERK1/2 activation

ZAP-70 in chronic lymphocytic leukemia (CLL) has been associated with enhanced B-cell receptor (BCR) signaling, survival, and migration. We investigated whether ZAP-70 can directly govern migration and the underlying mechanisms. In the ZAP-70 stably transfected Ramos cell line, IgM stimulation, but no IgD, enhanced phosphorylation of ERK1/2, Akt and Syk, and delayed IgM and CD79b internalization. In contrast, in the Raji cell line, where ZAP-70 was constitutively phosphorylated, ERK1/2, but not Akt, was phosphorylated, suggesting that MAPK pathway mediates ZAP-70 effects. BCR stimulation modulated the expression of CCR7, CXCR4, CXCR5, CD44, CD49d, and CD62L, which were up-regulated in ZAP-70-positive CLL primary subclones. The most dramatic change after BCR engagement in ZAP-70-transfected cells was CCR7 up-regulation, this being impaired by ERK1/2 inhibition and translating into both increased signaling and migration toward CCL21. Primary CLL subclones with high ZAP-70 expression showed increased migration toward CCL21. In conclusion, ZAP-70 ectopic expression led to enhanced BCR signaling after IgM stimulation and increased the expression of CCR7 predominantly via ERK1/2, increasing the response and migration toward CCL21. In primary CLL samples, cellular subsets with high ZAP-70 expression had increased expression of adhesion molecules and chemokine receptors in addition to an enhanced ability to migrate toward CCL21.

Long-term follow-up of dose-adjusted EPOCH plus rituximab (DA-EPOCH-R) in untreated patients with poor prognosis large B-cell lymphoma. A phase II study conducted by the Spanish PETHEMA Group

This prospective multi‐institutional phase II study was designed to assess the efficacy and safety of dose‐adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin) plus rituximab (DA‐EPOCH‐R) in untreated patients with poor prognosis large B‐cell lymphomas. Eighty‐one patients diagnosed with diffuse large B‐cell lymphoma (DLBCL, n = 68), primary mediastinal DLBCL (n = 6) and follicular lymphoma Grade 3b (n = 7), with an age‐adjusted International Prognostic Index >1, were eligible for analysis. Median age was 60 years (range: 21–77). Sixty‐five patients (80·2%) achieved complete response. After a median follow‐up time of 64 months, 10‐year event‐free survival and overall survival (OS) were 47·8% and 63·6%, respectively. None of the studied clinical and biological characteristics were associated with poorer outcome. Interestingly, patients with BCL6 rearrangement achieved a 10‐year OS of 100%, while patients with BCL2 rearrangement exhibited a poorer outcome compared to activated B‐cell tumours and germinal centre B‐cell without BCL2 rearranged tumours. Results achieved with DA‐EPOCH‐R showed a good long‐term outcome and a tolerable toxicity profile in high‐risk large B cell lymphoma patients. Outcome was not affected by tumour cell proliferation or by cell of origin, highlighting the requirement of new biological markers for patient subclassification of high‐risk DLBCL patients.

Targeting the proliferative and chemoresistant compartment in chronic lymphocytic leukemia by inhibiting survivin protein

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.

ZAP-70 Promotes the Infiltration of Malignant B-Lymphocytes into the Bone Marrow by Enhancing Signaling and Migration after CXCR4 Stimulation

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.

Inhibition of BCR signaling using the Syk inhibitor TAK-659 prevents stroma-mediated signaling in chronic lymphocytic leukemia cells.

Proliferation and survival of chronic lymphocytic leukemia (CLL) cells depend on microenvironmental signals coming from lymphoid organs. One of the key players involved in the crosstalk between CLL cells and the microenvironment is the B-cell receptor (BCR). Syk protein, a tyrosine kinase essential for BCR signaling, is therefore a rational candidate for targeted therapy in CLL. Against this background, we tested the efficacy of the highly specific Syk inhibitor TAK-659 in suppressing the favorable signaling derived from the microenvironment. To ex vivo mimic the microenvironment found in the proliferation centers, we co-cultured primary CLL cells with BM stromal cells (BMSC), CD40L and CpG ODN along with BCR stimulation. In this setting, TAK-659 inhibited the microenvironment-induced activation of Syk and downstream signaling molecules, without inhibiting the protein homologue ZAP-70 in T cells. Importantly, the pro-survival, proliferative, chemoresistant and activation effects promoted by the microenvironment were abrogated by TAK-659, which furthermore blocked CLL cell migration toward BMSC, CXCL12, and CXCL13. Combination of TAK-659 with other BCR inhibitors showed synergistic effect in inducing apoptosis, and the sequential addition of TAK-659 in ibrutinib-treated CLL cells induced significantly higher cytotoxicity. These findings provide a strong rationale for the clinical development of TAK-659 in CLL.

Microenvironment regulates the expression of miR-21 and tumor suppressor genes PTEN, PIAS3 and PDCD4 through ZAP-70 in chronic lymphocytic leukemia

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.

Risk adapted high-dose and dose-dense therapies modulate the impact of biological classification in diffuse large B-cell lymphoma prognosis

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous disease entity.[1][1] Young patients with high-intermediate and high aa-IPI score seem to be good candidates to receive alternative treatments to standard RCHOP-21 including EPOCH-R,[2][2] R-ACVBP+HDT-ASCT[3][3] and upfront autologous

Abstract 1344: ZAP-70 enhances migration of malignant B lymphocytes toward lymphoid organs in a Burkitt lymphoma xenograft model

Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation and proliferation of mature B-lymphocytes in the blood, bone marrow and secondary lymphoid organs. CLL patients with adverse outcome prognosis can be identified by the presence of a high ZAP-70 expression. ZAP-70, a protein tyrosine kinase that plays a crucial role in cellular activation in T and NK cells, has been related to aggressive features of the CLL cells, such as higher migrative capacity in vitro. In order to analyze the consequences of ZAP-70 ectopic expression in an in vivo model, we stably transfected a Burkitt cell line (Raji) with a vector expressing a ZAP-70-GFP fusion protein. Raji transfectants showed constitutively active ZAP-70 protein. Subsequently, thirty-four 8-week old SCID mice were inoculated intravenously with 5 x10 6 cells from each cell line (control Raji-GFP, 12 mice; Raji-ZAP-70-GFP, 9 mice). Mice were euthanized when hind legs paralysis, dyspnea, or tumor growth was observed. Organs were obtained to quantify the percentage of GFP-positive cells present in each organ by flow cytometry. Median survival of mice injected with the ZAP-70 cell line did not differ from that observed in the control mice (16 days, p=0.658). Percentage of GFP positive cells was analyzed by flow cytometry in different tissue compartments (Table 1). We observed a significantly higher percentage of infiltrating GFP-positive cells in the bone marrow from mice injected with ZAP-70 expressing cell line (58.8%±6.08 vs 4.2%±1.4, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1344. doi:1538-7445.AM2012-1344