Target Sequence Accessibility Limits Activation-Induced Cytidine Deaminase Activity in Primary Mediastinal B-Cell Lymphoma Sergey W. Popov, 1 Gerhard Moldenhauer, 2 Beate Wotschke, 1 Silke Bru ¨derlein, 1 Thomas F. Barth, 1 Karola Dorsch, 1 Olga Ritz, 1 Peter Mo ¨ller, 1 and Frank Leitha ¨user 1 1 Department of Pathology, University of Ulm, Ulm, Germany and 2 Department of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Abstract Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)–negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcrip- tion, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID- dependent somatic gene diversification in PMBL. [Cancer Res 2007;67(14):6555–64] Introduction Primary mediastinal B-cell lymphoma (PMBL) is categorized as a subentity of diffuse large B-cell lymphoma (DLBCL) in the current WHO classification. The concept of PMBL as a distinct lymphoma type relies on its characteristic clinical, histomorphologic, immu- nologic, and molecular properties (1). The immunoglobulin (Ig) genes of PMBL are clonally rearranged and typically carry a high load of somatic hypermutation (SHM) with a distribution pattern that indicates selection of a functional B-cell receptor (2, 3). In combination with the finding of switched isotypes (3), this strongly suggests that PMBL originate from B cells that have been exposed to the germinal center reaction. The maturation of a B-cell reaction is achieved by two distinct genetic alterations of the Ig gene. As a hallmark of the germinal center reaction, SHM hones the B-cell receptor for maximum binding affinity by introducing point mutations to the variable region of the Ig heavy and light chain gene. Class switch recombination (CSR) exchanges the Ig isotype via nonhomolo- gous DNA recombination between the targeted switch regions, thereby determining the effector function of the B-cell response (4). For long, the molecular basis of SHM and CSR has remained elusive. Recently, however, it was shown that activation-induced cytidine deaminase (AID) plays a central role in SHM and CSR (5, 6). This finding has triggered intense research activity shedding some light on the initial molecular mechanisms of these two processes, although there is an ongoing debate on whether AID targets RNA or DNA. The DNA hypothesis, which has gained recently some acceptance, holds that AID directly deaminates cytosine residues in DNA (7). Because single- stranded, but not duplex, DNA is the substrate of AID, target sequences require transient denaturation by transcription (8). For CSR, this is achieved by germ-line transcription through the acceptor and donor switch region, flanking the sequence to be looped out (4). To facilitate SHM, AID must be phosphorylated and interact with replication protein A (9), whereas additional, as yet unidentified, cofactors are likely to complex with AID to initiate CSR (10). For this reason, it is obvious that the detection of AID gene products alone does not automatically proof AID to be active. Indeed, a dissociation of AID expression and activity is a well-recognized finding in B-cell neoplasms (11–13). This observation has been substantiated recently by an experimental approach showing that the induction of a mutator phenotype by AID overexpression in vitro critically depends on the targeted B-cell line (14). Thus far, there has been only one report investigating AID expression in PMBL (15). This study, which included six PMBL cases, revealed AID mRNA levels in PMBL that were comparable with follicular lymphoma (FL) and to non-PMBL types of DLBCL. The presence of hypermutated and class switched Ig genes proves that AID has been active in PMBL (3). However, it is presently unclear whether AID continues to be active or shut down due to alterations of the molecule itself or a nonsupportive state of the lymphoma cells. To address this question, we measured AID gene products in 16 primary PMBL cases and 2 PMBL-derived cell lines, confirming AID expression by mRNA quantification and on the protein level. We then searched for evidence of ongoing class switching as an indicator of AID activity. To elucidate the reason for the low rate of CSR despite the prevalent expression of a putatively functional AID molecule in PMBL, we determined the accessibility of the AID target sequences in the Ig switch regions and carried out in vitro experiments to assess whether physiologic stimuli could revert PMBL cells into a class switching mode. Finally, we examined ongoing SHM in PMBL cell lines and determined whether it correlated with target gene transcription. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Peter Mo ¨ller, Department of Pathology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. Phone: 731-50056320; Fax: 731- 50056384; E-mail: peter.moeller@uniklinik-ulm.de. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-2166 www.aacrjournals.org 6555 Cancer Res 2007; 67: (14). 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