• BCL6--regulated by AhR/ARNT and wild-type MEF2B--drives expression of germinal center markers MYBL1 and LMO2.

      Ding, Jie; Dirks, Wilhelm G; Ehrentraut, Stefan; Geffers, Robert; MacLeod, Roderick A F; Nagel, Stefan; Pommerenke, Claudia; Romani, Julia; Scherr, Michaela; Vaas, Lea A I; et al. (2015-06)
      Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.
    • Peripheral T-cell lymphoma cell line T8ML-1 highlights conspicuous targeting of PVRL2 by t(14;19)(q11.2;q13.3).

      Ehrentraut, Stefan; Nagel, Stefan; Pommerenke, Claudia; Dirks, Wilhelm G; Quentmeier, Hilmar; Kaufmann, Maren; Meyer, Corinna; Zaborski, Margarete; Geffers, Robert; Fujiwara, Hiroshi; et al. (2017-01-01)
      Focal amplifications and chromosome translocations involving the long arm of chromosome 19 (19q13.3) are recurrent in T-cell lymphoma, where neighboring BCL3 and PVRL2 are competing target genes. Here we present the oncogenomic characterization of a peripheral T-cell lymphoma (PTCL) cell line T8ML-1 to reveal t(14;19)(q11.2;q13.3) juxtaposing TRA@ and PVRL2. Parallel mRNA and protein expression data for the 19q13.3 region of interest pinpointed PVRL2 as the sole conspicuous target therein. Collectively, our findings endorse T8ML-1 as the first proven cell line model for t(14;19)/PTCL.
    • Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation.

      Quentmeier, Hilmar; Pommerenke, Claudia; Ammerpohl, Ole; Geffers, Robert; Hauer, Vivien; MacLeod, Roderick Af; Nagel, Stefan; Romani, Julia; Rosati, Emanuela; Rosén, Anders; et al. (2016-08-23)
      Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies.