STAT3 Silencing as a Novel Therapeutic Strategy for Activated B Cell-Type Diffuse Large B-Cell Lymphoma.

Abstract 926

Among the non-Hodgkin's lymphomas, the diffuse large B cell lymphoma (DLBCL) represents the most frequent (30%) of the aggressive lymphomas. Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. IL-6 and IL-10 are major activators of STAT3 signaling and are important in the pathophysiology of DLBCL. STAT3 has been found to be persistently active in activated B cells (ABC), which are non-germinal center-derived DLBCL cells. We studied the consequences of STAT3 inhibition on multiple biological functions in two representative human cell lines of this group, Ly3 and Ly10 cells.

For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells, control lentivirus Ly3 cells, STAT3 shRNA-expressing lentivirus Ly10 cells and control lentivirus Ly10 cells. The stable expression of STAT3 shRNA results in 40-50% reduction of total STAT3 protein levels in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus cells. STAT3 down-regulation induced inhibition of cell proliferation (approximately 40%). Ly3 cells respond to IL-10 more than to IL-6 in terms of proliferation; both cytokines induced less proliferation in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus Ly3 cells. Similar results were obtained in Ly10 cells, which respond more to IL-6 than to IL-10 in terms of proliferation.

We analyzed by quantitative real-time PCR the mRNA levels of different STAT3 target genes and observed significant reduction in mRNA levels of Mcl-1, Bcl-xL and Survivin in STAT3 shRNA lentivirus Ly3 cells, as well as significant reduction of Cyclin D2 and up-regulation of STAT1 in shRNA lentivirus Ly10 cells. Comparison of these gene expression profiles with data obtained from other B-cell lymphoma cell lines revealed that silencing of STAT3 resulted in down-regulation of different STAT3 target genes in a cell-dependent manner. We also observed that both STAT3 and control lentivirus Ly3 cells have the same protein levels of c-Myc; nevertheless STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc.

We next investigated the effect of STAT3 inhibition on adhesion to bone marrow stroma and chemotaxis. STAT3 shRNA lentivirus Ly3 cells adhered less to the stroma layer than control cells, and the longer they were cocultured with the stroma cells in the presence of serum-free media the more they lost the ability to adhere. Moreover, STAT3 shRNA lentivirus Ly3 cells had decreased capacity to migrate toward SDF-1 alpha, an important factor that mediates proliferation, survival, chemotaxis, migration and adhesion into bone marrow stroma.

Radiation, in combination with chemotherapy, is one of the therapies used for DLBCL patients. We therefore investigated whether STAT3 down-regulation sensitized Ly3 cells to radiation. Radiation induced a higher accumulation of phospho-H2A.X (first sentinel event following DNA damage such as DSBs) and apoptosis in STAT3 shRNA lentivirus cells compared to control cells. Moreover, IL-6 and IL-10 protected the STAT3 shRNA lentivirus Ly3 cells less than the control cells from the induction of phospho-H2A.X following radiation.

We further investigated the effect of STAT3 silencing in animal models of Ly3 lymphoma (Nude or NOD-SCID mice). Tumors in control lentivirus Ly3-bearing mice grew robustly, whereas tumors in STAT3 shRNA lentivirus Ly3-bearing mice regressed 5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis, significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin (approximately 40% to 60% inhibition), and reduction of cytokine production such as IL-10, IL-15, Leptin and Thrombopoietin. Taken together, these results suggest that inhibition of STAT3 is a potential promising approach in the therapy of ABC-type DLBCL.