Transcriptional Deregulation of Mutated BCL6 Alleles by Loss of Negative Autoregulation in Diffuse Large B Cell Lymphoma

Abstract

The BCL6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B cells and required for GC formation. In ~35% of diffuse large B cell lymphomas (DLBCL) and 5–14% of follicular lymphomas (FL), the BCL6 locus is altered by chromosomal translocations that deregulate its expression by a mechanism known as promoter substitution. In addition, the BCL6 5′ noncoding sequences are targeted by multiple somatic mutations that cluster within ~1.5 kb from the transcription initiation site and are found in 30–40% of normal GC B cells (frequency: 0.14 × 10 –2 /bp) as well as in a fraction of all GC-derived B cell lymphomas. 1,2 The structural features, distribution, and specificity for GC B cells strongly suggest that BCL6 mutations are introduced by the same mechanism that targets immunoglobulin V genes in the GC. Nonetheless, the biological role of these mutations in normal and malignant B cell development remains unclear. The present study was aimed at investigating the effect of BCL6 mutations on gene transcriptional regulation. To this end, the BCL6 5′ noncoding region (~5 kb) from 24 tumor cases [6 DLBCL, 5 Burkitt lymphoma (BL), 4 FL, and 5 chronic lymphocytic leukemia (CLL), corresponding to 35 mutated alleles] or sorted GC B cells (N = 20 mutated alleles) was linked to the luciferase gene, and the activity of the corresponding constructs was evaluated in transient transfection/reporter gene assays in two BCL6 permissive lymphoma lines (Ly1 and MUTUI-BL59). We found that, while mutant alleles derived from normal GC cells, or from BL, FL, and CLL cases, displayed comparable activity to that of a wild-type construct, four (33%) of the 12 DLBCL-associated alleles were significantly overexpressed (4–18-fold) in both transfected lines. Among multiple mutations scattered throughout the BCL6 5′ noncoding sequences of these four alleles, single nucleotide substitutions, all located within the first noncoding exon of the gene, were found to be responsible for the observed overexpression. These mutations affect two adjacent BCL6 binding sites and were found to prevent BCL6 from binding to its own promoter, both in vitro in electrophoretic mobility shift