Transcriptional signature with differential expression of BCL6 target genes accurately identifies BCL6-dependent diffuse large B cell lymphomas

Abstract

Diffuse large B cell lymphomas (DLBCLs) often express BCL6, a transcriptional repressor required for the formation of normal germinal centers. In a subset of DLBCLs, BCL6 is deregulated by chromosomal translocations or aberrant somatic hypermutation; in other tumors, BCL6 expression may simply reflect germinal center lineage. DLBCLs dependent on BCL6-regulated pathways should exhibit differential regulation of BCL6 target genes. Genomic array ChIP-on-chip was used to identify the cohort of direct BCL6 target genes. This set of genes was enriched in modulators of transcription, chromatin structure, protein ubiquitylation, cell cycle, and DNA damage responses. In primary DLBCLs classified on the basis of gene expression profiles, these BCL6 target genes were clearly differentially regulated in "BCR" tumors, a subset of DLBCLs with increased BCL6 expression and more frequent BCL6 translocations. In a panel of DLBCL cell lines analyzed by expression arrays and classified according to their gene expression profiles, only BCR tumors were highly sensitive to the BCL6 peptide inhibitor, BPI. These studies identify a discrete subset of DLBCLs that are reliant on BCL6 signaling and uniquely sensitive to BCL6 inhibitors. More broadly, these data show how genome-wide identification of direct target genes can identify tumors dependent on oncogenic transcription factors and amenable to targeted therapeutics.

Fig. 1.

Identification of BCL6 target genes. ChIP on chip was performed in triplicate on a 24,000 promoter array. (A) Selection of BCL6 target genes. (Left) Density plot of the normalized log ratio of fluorescence intensity (BCL6 vs. input) of the oligonucleotide probes. The probes showing relative enrichment by BCL6 antibodies are clustered in the right tail of the distribution curve. (Right) Density plot of the maximal enrichment peak of each promoter (black line). The gray line represents a similar plot generated by using a random distribution of probes. The indicated cutoff point for selection of positive hits (shaded) was set at the 95th percentile of the random probe curve (see also SI Fig. 4). The y axis for both panels represents the local relative frequency of events within each level of fluorescence intensity represented on the x axis, corresponding to probe frequency (Right) and peak frequency (Left). (B) Representative BCL6 target genes. Shown are the peak BCL6 vs. input enrichment at negative and positive control promoters (CD20 and FCER2, respectively) and four selected gene promoters (SUB1, CR1, CBX3, and MBD1) that met the following criteria: (i) inclusion in an enriched GO category; (ii) validation by single-locus Q-ChIP; (iii) up-regulation after BPI treatment; and (iv) inclusion in the leading edge of the BCL6 target gene set in GSEA (for details, see Materials and Methods). In each graph, the y axis shows fold enrichment by BCL6 antibodies (gray field) vs. a control IgG (black field). The x axis indicates the relative position of the different oligonucleotides relative to the transcriptional start site as annotated in the National Center for Biotechnology Information (NCBI) human genome assembly version 35 (May 2004).

Fig. 2.

BCL6 target genes in primary BCR and OXP DLBCLs and DLBCL cell lines. The top-scoring BCL6 target genes from the GSEA leading edge were clustered with respect to the DLBCL BCR and OxPhos phenotypes and represented visually. Each individual column represents a tumor, and each individual row corresponds to a gene. For comparison, the relative expression of these BCL6 target genes in normal GC B cells is also shown. The color scale at the bottom indicates relative expression. (A) Primary DLBCLs (11). (B) DLBCL cell lines (BCR lines: Ly1, Ly7, SU-DHL4, SU-DHL6, and Farage; OxPhos lines: Ly4, Toledo, Kaspas 422, and Pfeiffer). (C) BCL6 target gene abundance in BCR and OxPhos cell lines after BPI treatment. BCR (SU-DHL6, SU-DHL4) and OxPhos (Toledo, Ly4) cell lines were treated with 20 μM BPI or control peptide for 8 h, and the transcript abundance of the indicated BCL6 targets was evaluated with real-time PCR thereafter. The y axis indicates fold activation of genes after treatment with BPI vs. control peptide based on the ΔΔ^CT normalized to the expression of hypoxanthine-guanine phosphoribosyl transferse (HPRT). BPI treatment increased the expression of each BCL6 target gene in the BCR cell lines but did not alter the expression of these genes in OxPhos lines. BPI treatment increased the abundance of BCL6 targets that were less abundant in BCR than OxPhos cells at baseline (SUB1, ZNF443, CR1, and CBX3; shaded in blue) and others that were more abundant in BCR tumors at baseline (CD74, CCN1, and MBD1; shaded in red). A known BCL6 target gene FCER2 was used as a positive control (shaded in pink).

Fig. 3.

BCR and OxPhos DLBCL cell lines exhibit differential sensitivity to BPI. (A) BPI IC₅₀ for BCR and OxPhos DLBCL cell lines. BCR and OxPhos cell lines were exposed to increasing doses of BPI, and cellular proliferation was assessed at 48 h. The IC₅₀ ± SEM for triplicate samples of each cell line in a representative experiment are shown. (B) Mean BPI IC₅₀ (±SD) for BCR and OxPhos DLBCL cell lines. (C) Proliferation of BPI-treated BCR and OxPhos DLBCL cell lines after BPI treatment. Cell lines were exposed to 20 μM BPI for 48 h, and cellular proliferation was evaluated thereafter.