CXCR7 Is Highly Expressed in Acute Lymphoblastic Leukemia and Potentiates the Chemotactic Response to SDF-1

Abstract 1482

The crosstalk between leukemic cells and their microenvironment provides pivotal signals for the localization and progression of leukemias. The stromal derived factor-1 (SDF-1)/CXCR4 pathway is deregulated in hematology malignances, such as acute lymphoblastic leukemia (ALL). SDF-1 represents the major chemokine for initiating stem cell chemotactic migration. The majority of cytokines that mediate stem cell chemotaxis does so via modulation either of SDF-1 or of its receptor, CXCR4. CXCR4 receptor is expressed in leukemic cells enabling cells to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection of cell death resulting in a worse prognosis. Recently, CXCR7 was identified as another SDF-1-binding receptor, but its contribution to SDF-1 – mediated effects in hematopoietic cells is still poorly explored, even though the CXCR7 relationship with tumor progression in non-hematopoietic malignancies is well established. Given that SDF-1/CXCR4 signaling is altered in patients with leukemia and that there is little information regarding CXCR7 in leukemia we investigated its expression in patients and leukemia cell lines. In addition, the relationship of CXCR4 and CXCR7 in potentiating the SDF-1 response was also investigated.

mRNA expression of CXCR7 was analyzed by Real-time PCR (normalized by GAPDH and HPRT) in bone marrow samples of 29 acute myeloid leukemia (AML), 11 ALL patients and 12 control subjects (healthy donors). CXCR7 protein expression in myeloid cell lines (U937, P39, K562 and KG -1) and lymphoblastic cell lines (Jurkat, Molt-4, Raji e Daudi) was analyzed by western blot. Localization of CXCR4 and CXCR7 proteins was investigated using flow cytometry and confocal microscopic analysis and CXCR7 knockdown cells were obtained by transduction with lentivirus-mediated shRNA. These cells were treated with AMD3100 (antagonist CXCR4) and their chemotactic capacity was analyzed by transwell chemotaxis assay.

CXCR7 was significantly higher expressed in ALL compared to AML and control subjects (p=0.0008, Mann-Whitney test). CXCR7 protein (western blot with ABCAM antibody) was also higher expressed in lymphoblastic cell lines (Molt-4 and Jurkat) compared with myeloid cells. The subcellular location of CXCR7 and CXCR4 by confocal microscopy and flow cytometry evidenced CXCR7 in the membrane of Molt-4 cells and in the cytoplasm of Jurkat cells whereas CXCR4 was in the membrane of both cell lines. Interestingly, we also noticed that, after SDF-1 induction, Molt-4 cells have higher chemotactic ability compared with Jurkat (median Molt 4=52.0 ± 5 vs Jurkat=24.1 ± 3, p=0.0079, Mann-Whitney test) which may be related with the membrane availability of CXCR7. In addition, the inhibition of CXCR7 or CXCR4 resulted in significant changes in Molt4 and Jurkat chemotactic response (0.01>p< 0.02, Mann-Whitney test), however, the inhibition of both CXCR7 and CXCR4 resulted in a more significant reduction in cell migration (p=0.0079/Molt-4; p=0.0043/Jurkat, Mann-Whitney test).

Increased expression of CXCR7, as here observed in lymphoblastic leukemia cells, is a phenomenon already described in a variety of solid tumor cell lines such as brain, prostate and lung. In solid tumors, CXCR7 mainly increases the proliferation of malignant cells. These results suggest that the biological function of CXCR7 depends on its tissue and organ localization and that, in acute lymphoblastic leukemia may have a role in cell chemotaxis, potentiating CXCR4 response to SDF-1 and thus, could contribute for leukemia initiating cell recruitment to niches once occupied by normal hematopoietic stem cells.