CARD11 as an Oncogene in Diffuse Large B Cell Lymphoma.

The activated B-cell-like (ABC) molecular subtype of diffuse large B cell lymphoma (DLBCL) is characterized by constitutive activation of the nuclear factor-kB (NF-kB) pathway, which it requires for survival. By contrast, the NF-kB pathway is infrequently activated in the germinal center B cell-like (GCB) subtype of DLBCL. A crucial protein involved in activation of the NF-kB pathway by antigen receptor stimulation in normal B and T lymphocytes is CARD11. A previous loss-of-function genetic screen using RNA interference identified the signaling pathway from CARD11 to IkB kinase b as essential for the constitutive NF-kB activity in ABC DLBCL. However, the oncogenic mechanisms underlying the activity of CARD11 in this lymphoma subtype have yet to be elucidated. To this end, we resequenced some or all of the CARD11 exons in 157 primary DLBCL patient samples and cell lines and, as a control, in 19 MALT lymphoma patient samples. Various missense mutations were discovered in 10.4% of ABC DLBCL samples (8/77), and all mutations were located in exons encoding the coiled-coil domain of CARD11. In contrast, CARD11 coiled-coil domain mutations were detected in only 3.8% of the GCB DLBCL patient samples (3/80) and in no MALT lymphoma samples. ABC and GCB DLBCLs with CARD11 mutations were characterized by high expression of an NF-kB gene expression signature. Experimental introduction of each CARD11 mutant form in lymphoma cell lines altered their NF-kB signaling properties. Six of the CARD11 mutants caused strong constitutive NF-kB activation in the absence of any exogenous stimulus, a phenotype that was not elicited by wild type CARD11. For other CARD11 mutants, the enhancement of NF-kB signaling was most apparent upon antigen receptor stimulation. Fluorescence microscopy of cells bearing GFP-tagged CARD11 mutants revealed one or more prominent cytosolic aggregates in most cells whereas GFP-tagged wild type CARD11 was distributed diffusely in the cytoplasm. Multispectral imaging flow cytometry (Imagestream, Amnis) was used to quantitate the degree of aggregate formation by the CARD11 mutants. This analysis revealed a positive correlation between aggregate formation and NF-kB pathway activation. Immunofluorescent staining revealed that endogenous IkB kinase proteins were co-localized with the CARD11 mutants in the cytosolic aggregates, supporting the hypothesis that aggregate formation plays a central role in the activation of NF-kB by the CARD11 mutants. These results demonstrate that CARD11 is a bone fide oncogene in DLBCL. Mutations in the CARD11 coiled-coil domain caused spontaneous multimerization of the protein leading to IkB kinase recruitment and NF-kB signaling, thereby preventing cell death. These findings provide a strong genetic rationale for the development of CARD11 pathway inhibitors for the therapy of DLBCL.