BCL6 Overexpression – Regulated By AhR/ARNT and Wild-Type MEF2B – Drives Expression of Germinal Center Markers MYBL1 and LMO2

The evolution of tumor clones and the clonal architecture of tumors can be followed by the analysis of clone-specific mutations. The diffuse large B-cell lymphoma (DLBCL)-derived cell line U-2932 comprises two subclones (R1 and R2). Immunoglobulin gene hypermutation analysis showed that R1 and R2 represent subclones of the original tumor. Thus, the two U-2932 subclones seemed to be ideal to study the cellular consequences of clonal evolution. Both clones were derived from a presumptive mother clone with genomic BCL2 amplification, which acquired distinct sets of secondary rearrangements leading alternatively to the overexpression of BCL6 (R1) and MYC (R2) in the respective daughter clones. R2 carries t(8;14) a classical activating rearrangement of MYC in B-cells. R1 did not show any of the typical BCL6 translocations responsible for aberrant BCL6 expression. We applied a whole genome array to find out whether numerical aberrations might explain BCL6 expression in R1 and to investigate if subclone-specific gene expression might be attributable to numerical aberrations in general. More than 200 genes showed >10 fold expression differences between R1 and R2. Statistical analysis of results from copy number aberration and expression data analysis revealed that for 58/221 of the differentially expressed genes, numerical differences between the two subclones effectively predict differences in gene expression (sensitivity 0.64; specificity 0.94; accuracy 0.78). Thus, for a sizeable minority of genes numerical aberrations provided an explanation for the differences in gene expression between the U-2932 subclones. However, BCL6 was none of these genes. Thus, we searched for an alternative explanation for the R1-restricted overexpression of this germinal center oncogene. MEF2B point mutations occur in 11% of DLBCL contributing to the genesis of BCL6 positive lymphomas. The U-2932 subclones did not carry MEF2B mutations. Interestingly however, expression levels of MEF2B paralleled those of BCL6 in the U-2932 subclones. Knockdown experiments showed that wild-type MEF2B controlled BCL6 transcription. To test whether MEF2B and BCL6 showed coordinated expression in general, we analyzed the expression and the mutational status of these genes in 23 DLBCL cell lines. Confirming a positive correlation, independence of MEF2B and BCL6 expression levels could be rejected with a p-value according to Fisher´s exact test of 0.0001 against a level of significance of 0.05 (sensitivity 0.92, specificity 0.9, accuracy 0.91).

The MEF2B promoter carries binding sites of the AhR/ARNT transcriptional complex. AhR inhibition and ARNT knockdown experiments with the U-2932 subclones revealed that MEF2B is a downstream target of AhR/ARNT signalling. A positive correlation between AhR and MEF2B expression levels could be shown for the majority of 23 DLBCL cell lines tested (sensitivity 0.61, specificity 1.0, accuracy 0.78). These results indicate that the AhR/ARNT-induced expression of wild-type MEF2B might be an independent regulator for BCL6 expression in DLBCL, besides canonical BCL6 translocations, BCL6 promoter hypermutation and MEF2B mutations. To find out the extent to which BCL6 contributed to the subclone-specific gene expression in the U-2932 subclones, we ectopically expressed BCL6 in subclone R2: 48/221 differentially expressed genes were affected. Interestingly, 28/48 genes were upregulated by BCL6 inducing the germinal center markers MYBL1 and LMO2, although BCL6 is believed to act as a transcriptional repressor.

In summary, the two subclones of the DLBCL cell line U-2932 faithfully model tumor heterogeneity. Significant expression differences were shown for 221 genes, more than half of which were attributable to genomic copy number differences or to clone-specific expression of the signal transducer AhR and the oncogene BCL6. Moreover, we could show that BCL6 overexpression – regulated by AhR/ARNT and wild-type MEF2B – drives expression of subclone-specific germinal center markers MYBL1 and LMO2 in the DLBCL cell line U-2932.