Specific Peptide Disruption of the Bcl-6 Repression Complex Reveals Its Transcriptional Mechanism of Action in Normal and Malignant B-Cells and Is a Novel Therapeutic Approach for Diffuse Large B-Cell Lymphoma.

The Bcl-6 transcriptional repressor is required for the establishment of germinal centers during B-cell maturation. Regulatory elements of the BCL6 gene are frequently mutated in diffuse large B-cell lymphomas (DLBCL), leading to inappropriately timed expression of Bcl-6. However, it is unknown whether Bcl-6 is involved in lymphomagenesis. Within Bcl-6, the N-terminal BTB domain is involved in mediating transcriptional repression. We investigated the mechanism of action of the Bcl-6 BTB domain using X-ray crystallography and functional assays. We found that the SMRT, N-CoR and BCoR corepressors bind directly to Bcl-6 through a 16 residue “BBD motif”, which fits into a highly specific “lateral groove” surface feature of the Bcl-6 BTB domain. To determine the contribution of BTB lateral groove recruitment of corepressors to transcriptional and biological functions of Bcl-6, we engineered small Bcl-6 BTB peptide inhibitors (BPI). BPI penetrate cells and localize to the nuclei where they specifically bound to Bcl-6 and blocked its interaction and co-localization with co-repressors. This resulted in dose-dependent blockade of repression by Bcl-6 in reporter assays and reactivation of endogenous Bcl-6 target genes in Bcl-6 expressing B-cells measured by real-time PCR. Loss of repression was caused by disruption of the endogenous Bcl-6 transcriptional repression complex. We found that BPI specifically excluded corepressors from the promoters of endogenous Bcl-6 target genes, resulting in a switch from repressed to activated histone code settings with consequent gene reactivation. From the biological standpoint, BPI injection into mice reproduced the B-cell phenotype of Bcl-6 null animals, as germinal center formation in response to T-cell dependent antigens was abrogated. To address the question of whether Bcl-6 is required to maintain the malignant phenotype of DLBCL, a panel of Bcl-6 positive and negative DLBCL cells were exposed to BPI. BPI induced growth suppression, cell cycle arrest and apoptosis in Bcl-6 positive but not negative DLBCL cells. Expression array analysis showed immediate upregulation of checkpoint genes involved in proliferation and apoptosis, allowing us to identify novel direct Bcl-6 target genes. In contrast, BPI did not upregulate differentiation related genes and did not induce differentiation in lymphoma cells, indicating that the Bcl-6 BTB domain lateral groove controls proliferation and survival pathways but not differentiation. Finally, BPI completely suppressed growth of Bcl-6 positive human DLBCL xenotransplants without any toxicity to other organs. In summary, specific disruption of the Bcl-6 BTB domain repression mechanism allowed us to reveal the contribution of specific corepressor recruitment to the activities of Bcl-6 in B-cells and to demonstrate that Bcl-6 is in fact an oncogene, required to maintain the malignant phenotype of Bcl-6 positive DLBCL. Our pre-clinical studies indicate that Bcl-6 is a bona fide therapeutic target and that BPI is a specific, potent and non-toxic anti-lymphoma therapeutic agent.