Cross-Talk Between CLL Cells and Endothelial Cells in the Bone Marrow Microenvironment: Role of Signal Transducer and Activator of Transcription (Stat)-3.

Abstract 1242

Poster Board I-264

We have recently found that Stat3, constitutively phosphorylated on serine 727 residues, activates genes that are known to be activated by tyrosine phospho-Stat3 in CLL cells (Hazan-Halevi et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 1058). Because Stat3 modulates VEGF expression in other cellular systems, we asked whether VEGF is a Stat3 target gene in CLL. Using chromatin immunoprecipitation (ChIP) we found that anti-Stat3 antibodies immunoprecipitated DNA of VEGF as well as of Stat3-regulated genes (such as Stat3 and p21). To further delineate this finding, we infected CLL cells with Stat3-shRNA and determined changes in RNA levels by qRT-PCR. In four out of four CLL samples, Stat3-shRNA induced a significant reduction in the RNA levels of VEGF, confirming that serine phosphorylated STAT3 upregulates VEGF gene transcription in CLL. Furthermore, anti-VEGF immunohistochemical staining detected high levels of VEGF in CLL bone marrow cells, and enzyme-linked immunosorbent assay (ELISA) detected significantly higher VEGF levels in the serum of CLL patients (N = 47) compared to serum of normal individuals (N = 14). Serum VEGF levels correlated with the patients' absolute lymphocyte count. Because VEGF is known to induce vasculogenesis, we studied bone marrow biopsies of 10 patients with CLL and demonstrated increased microvascular density in all specimens. We noted also attachment of CLL cells to endothelial and vascular endothelial cells in all studied marrow sections. To clarify the interaction between CLL and endothelial cells we used human umbilical vein endothelial cells (HUVEC) and studied their effect on CLL cell apoptosis by measuring Annexin V using flow cytometry. In co-culture studies, direct contact with HUVEC protected CLL cells from spontaneous apoptosis. This effect was abrogated by insertion of micropore filters between HUVEC and CLL cells, suggesting that the protective mechanisms required direct interaction between CLL and endothelial cells. Similarly, anti-VEGF receptor (VEGFR) antibodies partially reversed the protective effect of HUVECs on CLL cells, consistent with an antiapoptotic effect dependent on VEGF-VEGFR interaction. Taken together, our data suggest that constitutively activated Stat3 mediates the interaction between CLL cells and the bone marrow microenvironment via VEGF, thereby providing a survival advantage to the neoplastic cells.