The role of BCL6 mediated genomic instability in germinal B cells and in lymphomagenesis.

RANUNCOLO, SM; MELNICK, AM

Saxton River, Vermont.

Congreso; Hematological Malignancies.; 2005

Federation of American Societies for Experimental Biology (FASEB)

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor plays a critical role in B-cell physiology, as it is required for establishment of germinal centers and immunoglobulin affinity maturation. BCL6 is upregulated when activated B-cells migrate to lymphoid follicles to form germinal centers, while its downregulation is required for post-germinal center differentiation or apoptosis. BCL6 is also one of the most commonly involved oncogenes in B-cell lymphomas, in many of which activating mutations of the BCL6 promoter region drive its expression. The reason why BCL6 is required to establish germinal centers is poorly understood. Our previous data indicate that this activity of BCL6 is tightly linked with its effects in lymphomagenesis. Accordingly, a cell permeable peptide that we designed (BCL6 Peptide Inhibitor or BPI) to specifically block BCL6-mediated transcriptional repression, abrogates germinal center formation in vivo and induces apoptosis or G1-cell cycle arrest in Bcl-6 positive Burkitt (BL) and Diffuse Large B-Cell Lymphoma (DLBCL) cell lines. Extensive microarray analysis allowed as to identify genes directly regulated by BCL6, many of which play key roles in DNA repair checkpoints. Based on these and other results, we hypothesize that the specific mechanism of action of BCL6 in both licensing germinal center formation and survival of lymphomas, is to mediate a state of ?tolerance? to DNA damage. This allows affinity maturation to occur in normal B-cells, but also results in lymphomagenesis by mediating genomic instability and evasion of apoptosis. To address this we performed DNA damage response and genomic integrity studies in the context of BCL6 gain of function using WI-38 as a model (human diploid fibroblasts, overexpressing Bcl-6 through lentivirus infection) and loss of BCL6 function in DLBCL and BL cells (with Bcl-6 expression knocked down through a sh-strategy delivered by a lentivirus system). BCL6 impaires cells? ability to sense DNA damage as shown by the analysis of P-Ser139-H2A.X expression by FACS in cells exposed to 5 Gys g-radiation. One hour after DNA damage only 40% of WI-38 Bcl-6 infected were positive for P-H2A.X meanwhile more than 90% of the control cells were. In Bcl-6+ DLBCL cell lines, less than 10% of cells phosphorylated H2A.X as compare with 70% when Bcl-6 expression was knocked down by shRNA. Expression of BCL6 in WI-38 cells delayed DNA repair after radiation as shown by COMET assay. BCL6 is mediating these effects by direct repression of ATR. ATR level is decreased both at mRNA and protein levels in WI-38 cells when BCL6 is overexpressed and the expression of ATR and its downstream susbtrate ChK1 is rescued in DLBCL cell lines when BCL6 is knockn down. We predict that overcoming BCL6 mediated transcriptional repression by either shRNA or BPI, will sensitize lymphoma cells to cytotoxic chemotherapy drugs and is thus likely to be an effective strategy to treat patients with B-cell lymphomas.