Inhibiting BCL-6


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Toni Hoffmann, Rob Ford, Paul Ko Ferrigno

Leeds Institute of Molecular Medicine, Leeds, West Yorkshire, UK

Abstract

BCL-6 is a transcriptional repressor that plays an essential role in the development of germinal centers of B-cells. The C-terminal domain contains six C-terminal zinc-finger motifs that enables binding to specific DNA sequences. The N-terminal POZ domain mediates protein-protein interactions with other corepressors, e.g. SMRT, NCoR, and BCoR, that subsequently result in the constrained expression of target genes, e.g. Blimp-1. BCL-6 expression is often misregulated in diffuse large B-cell lymphoma, and is abnormally high in other types of non-Hodgkin’s lymphoma thus, preventing normal B-cell differentiation. Inhibiting the function of BCL-6 is therefore an interesting therapeutic target.

Here we present data on the development and application of a high throughput cell-based luciferase reporter assay to screen for small drug-like compounds that inhibit the function of BCL-6. The assay utilizes the observation that the BCL-6 binds to the same DNA sequence motif as STAT transactivators. By using a reporter construct that expresses firefly luciferase under the control of a STAT-activated promoter that is repressed by endogenously highly expressed BCL-6 in a Burkitt’s lymphoma cell line (DG75), we are able to screen for compounds that derepress the luciferase expression and thus may inhibit the function of BCL-6. The assay is being used to screen a library of 480 natural products. The compounds are robotically applied in 96-well-plates in a randomized pattern to ensure screen neutrality.

BCL-6 is a transcriptional repressor that plays an essential role in the development of germinal centers of B-cells. The C-terminal domain contains six C-terminal zinc-finger motifs that enables binding to specific DNA sequences. The N-terminal POZ domain mediates protein-protein interactions with other corepressors, e.g. SMRT, NCoR, and BCoR, that subsequently result in the constrained expression of target genes, e.g. Blimp-1. BCL-6 expression is often misregulated in diffuse large B-cell lymphoma, and is abnormally high in other types of non-Hodgkin’s lymphoma thus, preventing normal B-cell differentiation. Inhibiting the function of BCL-6 is therefore an interesting therapeutic target.

Here we present data on the development and application of a high throughput cell-based luciferase reporter assay to screen for small drug-like compounds that inhibit the function of BCL-6. The assay utilizes the observation that the BCL-6 binds to the same DNA sequence motif as STAT transactivators. By using a reporter construct that expresses firefly luciferase under the control of a STAT-activated promoter that is repressed by endogenously highly expressed BCL-6 in a Burkitt’s lymphoma cell line (DG75), we are able to screen for compounds that derepress the luciferase expression and thus may inhibit the function of BCL-6. The assay is being used to screen a library of 480 natural products. The compounds are robotically applied in 96-well-plates in a randomized pattern to ensure screen neutrality.

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