ZAP-70 Enhances Infiltration of Malignant B Lymphocytes Into the Bone Marrow by Increasing Migratory and Survival Responses to CXCR4 Stimulation

Abstract 1779

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by the accumulation and proliferation of monoclonal CD5+ B lymphocytes in peripheral blood, secondary lymphoid tissues, and bone marrow. In CLL, high expression of ZAP-70 is a strong prognostic factor that identifies patients with higher probability of progression and shorter survival. Increasing evidence shows that the microenvironment plays a relevant role in the natural history of this disease, providing CLL cells with proliferative and anti-apoptotic signals. In this sense, despite that the role of ZAP-70 in the biology of CLL has not been fully elucidated, it is well known that it is associated with enhanced response to several microenvironmental stimuli. Herein, we aimed to analyze the effect of ZAP-70 on the interaction of B-cells with the microenvironment in an in vivo xenograft model of disseminated B-cell leukemia. For this, we stably transfected Raji B-cells with a vector expressing a GFP-ZAP-70 fusion protein or GFP only as a control. Cells were then intravenously injected into 7- to 9-weeks old female C.B-17 SCID mice (Raji GFP n=12; Raji GFP-ZAP-70 n=9). This xenograft model develops hind legs paralysis at around 14 to 19 days after cell injection due to central nervous system (CNS) infiltration, which precedes death by 1 to 2 days; therefore, paralysis was considered the end point of the study. The infiltration of the CNS was not influenced by ZAP-70 expression; thus, median survival was of 16 days for both Raji GFP and Raji GFP-ZAP-70-injected mice. Analysis of the infiltration of Raji B-cells in the different organs by flow cytometry and immunohistochemistry disclosed a significantly increased infiltration in the bone marrow of Raji GFP-ZAP-70 mice compared to Raji GFP mice (67% ± 5.76% vs 2.9% ± 1.49%; P <.001). Based on that, we analyzed the in vitro migrative capacity of cells toward SDF-1α, the main chemokine regulating lymphocyte trafficking into the bone marrow. We observed that, despite that Raji GFP-ZAP-70 and Raji GFP transfectants had similar surface expression of CXCR4, GFP-ZAP-70 cells had higher migration toward SDF-1α compared to GFP cells (migration index: 34.6 ± 4.11 vs 9.9 ± 1.47; P =.0079). Of note, B-cells expressing ZAP-70 also migrated more toward bone marrow stromal cells than B-cells non-expressing ZAP-70 (migration index: 27.1 ± 1.6 and 9.43 ± 0.77, respectively; P =.028). This increased migrative capacity independent of CXCR4 expression could be explained by the observation that Raji GFP-ZAP-70 cells showed enhanced response to CXCR4 stimulation by SDF-1α, evidenced by increased phosphorylation of ZAP-70, Akt and ERK1/2 proteins. Accordingly, we found that primary CLL subclones with high expression ZAP-70 also had increased migration toward SDF-1α. The relevance of the CXCR4/SDF-1α axis in the migrative capacity of Raji B-cells was demonstrated by the blockage of CXCR4 with a mAb, which resulted in reductions of more than 85% in the in vitro migration to both SDF-1α and to bone marrow stromal cells in both cell lines. To evaluate this axis in the xenograft model, SCID mice were injected with malignant B-cells treated with anti-CXCR4 mAb (Raji GFP n=5; Raji GFP-ZAP-70 n= 8) or isotypic control antibody (Raji GFP n=5; Raji GFP-ZAP-70 n= 8). In mice injected with Raji-GFP cells, CXCR4 blockage resulted in a delayed onset of the disease (median survival: 15 days vs 69 days; p<0.01), having these cells restored their CXCR4 expression. Surprisingly, CXCR4 blockage in Raji GFP-ZAP-70 cells produced different effects, with 7 out of 8 mice remaining asymptomatic without detectable malignant B-cells during follow-up (> 100 days). This suggests that ZAP-70-positive malignant B-cells developed dependence on CXCR4-derived survival signals. In summary, we showed that ZAP-70 is responsible for an enhanced cellular infiltration into the bone marrow caused by an amplified response to CXCR4 as regards signaling and migration. Interestingly, ZAP-70-positive malignant B-cells apparently became addicted to survival signals derived from CXCR4 as a consequence of the enhanced signaling. Further elucidation of the role of ZAP-70 in signaling from the microenvironment, particularly from the CXCR4/SDF-1α axis, will contribute to enlighten the biology behind the adverse clinical impact of high ZAP-70 expression in CLL, and will also provide a rationale for the development of novel therapies.