An Activating Mutation (Ser525Pro) within the Transactivation Domain of REL in Two Patients with Human B-Cell Lymphomas Enhances REL’s In Vitro Transforming Activity.

REL/NF-κB transcription factors are implicated in the control of apoptosis and cell growth particular in hematopoetic lineages. The REL locus at chromosomal region 2p13–16 is frequently amplified in B-cell lymphomas including diffuse-large B-cell lymphoma (DLBCL) and may play a role in lymphomagenesis. Overexpression of wild-type REL can transform chicken lymphoid cells in culture, and several experimentally-generated mutations within the REL C-terminal transactivation domain (TAD) have been previously shown to enhance REL’s transforming ability.

We analysed 83 B-cell lymphomas included in the ‘Deutsche Krebshilfe’ funded network „Molecular Mechanisms in Malignant Lymphoma“ for the presence of activating mutations in the coding region of REL. We performed a systematic dHPLC screening for mutation discovery and identified an identical point mutation in two human B-cell lymphomas (a t(14;18)-positive follicular lymphoma and a mediastinal B-cell lymphoma) that changes Ser525 to Pro within the REL TAD. In the mediastinal B-cell lymphoma, the mutation in REL was proven to be of germline origin. FISH showed an amplification of the REL locus in the tumor cells of this case. Quantitative allelic discrimination of S525P indicates that the mutant REL gene was over-represented in both cases. By in vitro experiments we could show that the S525P mutation enhances the in vitro transforming ability of REL in chicken spleen cells. In addition, REL-S525P differs from wild-type REL in its ability to activate certain κB site-containing reporter plasmids in transient transfection assays. In particular, REL-S525P has a reduced ability to activate the human manganese superoxide dismutase (MnSOD) promoter in A293 cells; however, the MnSOD protein is over-expressed in REL-S525P-transformed chicken spleen cells as compared to wild-type REL-transformed cells. Ser525 of REL falls within a sequence that is similar to other known phosphorylation sites of the IκB kinase, and REL-S525P shows a reduced ability to be phosphorylated by IKKα in vitro. The S525P mutation reduces IKKα- and TNFα-stimulated transactivation by REL, as measured in GAL4 reporter assays. Furthermore, REL-S525P-transformed chicken spleen cells are more resistant to TNFα-induced cell death than cells transformed by wild-type REL. These results represent the first identification of a tumor-derived activating mutation in the REL proto-oncogene, and they suggest that the S525P mutation contributes to the development of human B-cell lymphomas by altering REL’s ability to induce target gene expression by affecting an IKKα-regulated transactivation activity.