Mutations of multiple genes cause deregulation of NF-κB in diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL1. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-κB transcription complex2. However, except for a small fraction of cases3, it remains unclear whether NF-κB activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A (RANK)) regulators of NF-κB. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-κB responses, is most commonly affected, with 30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-κB. Thus, our results demonstrate that NF-κB activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-κB responses.

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Figure 1: The NF-κB pathway is active in ABC-DLBCL and in a smaller fraction of GCB-DLBCL.
Figure 2: Mutations and deletions of the A20 gene in ABC-DLBCL.
Figure 3: Reconstitution of A20 -null cell lines causes apoptosis and cell growth arrest via inhibition of NF-κB.

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Gene Expression Omnibus

Data deposits

The expression data and the 250K SNP data reported here have been deposited in the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) database (series accession number GSE12195 and GSE15127).

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Acknowledgements

We thank P. Smith, P. Chadwick and the Molecular Pathology Facility of the Herbert Irving Comprehensive Cancer Center (HICCC) at Columbia University Medical Center for histology service; V. V. V. Murty and the HICCC Molecular Cytogenetics Service for assistance on the FISH analysis; the HICCC Flow Cytometry Facility for fluorescence-activated cell sorting; V. Miljkovic and J. Pack for help with the Affymetrix gene expression hybridization; U. Klein and D. Dominguez-Sola for suggestions; L. Menard for help with the mutation analysis; and G. Inghirami for the pWPI lentiviral vector. Automated DNA sequencing was performed at Genewiz Inc. L.P. is on leave from the Institute of Hematology, University of Perugia Medical School, Perugia, Italy. This work was supported by NIH grants P01 CA92625-07 (R.D.-F.), NIAID R01AI066116, the National Centers for Biomedical Computing NIH Roadmap initiative U54CA121852 (A.Ca.), and a Leukemia and Lymphoma Society SCOR grant (R.D.-F.). L.P. would like to dedicate this work to the memory of Enrico Pasqualucci.

Author Contributions L.P. and R.D.-F. designed the study. M.C., A.G. and Q.S. performed experiments; L.P. performed the A20 functional assays; W.K.L. and A.Ca. developed tools for genome-wide expression profile analysis; S.V.N. performed the FISH analysis; A.Ch. and G.B. analysed all immunohistochemistry data; A.Ch., G.B., F.B. and M.P. provided DLBCL samples; M.B., F.B. and M.S. performed SNP array analysis; L.P. designed experiments, coordinated the study, analysed data and wrote the manuscript, which was commented on by all authors.

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Correspondence to Laura Pasqualucci.

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This file contains Supplementary Methods, Supplementary Figures S1-S6 with Legends, Supplementary References and Supplementary Tables S1-S9. Supplementary Fig. 4 was amended on 9 July 2009. (PDF 2821 kb)

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Compagno, M., Lim, W., Grunn, A. et al. Mutations of multiple genes cause deregulation of NF-κB in diffuse large B-cell lymphoma. Nature 459, 717–721 (2009). https://doi.org/10.1038/nature07968

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