MOLECULAR MECHANISMS OF MUTANT CARD11 SIGNALING TO NF-κB IN HUMAN DIFFUSE LARGE B CELL LYMPHOMA
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Upon antigen receptor stimulation in both B and T lymphocytes, the signaling adapter CARD11 transforms from a closed inactive state to an open active scaffold that assembles into a multiprotein complex, leading to the activation of NF-κB, a critical pleiotropic transcription factor of the immune system. This scaffold transition is dysregulated by diffuse large B cell lymphoma (DLBCL)-associated oncogenic CARD11 mutations in humans that induce constitutive NF-κB activation and allow for the uncontrolled proliferation of cancer cells. Since these mutations had only been discovered within the Coiled-coil domain of CARD11, according to the existing signaling model, we speculate that similar oncogenic mutations also exist within the CARD domain. Using PCR-based domain random mutagenesis and a high-throughput quantitative signaling screen, we indeed identify 24 novel gain-of-function CARD11 missense mutations in the CARD domain and define a 19-residue mutational hotspot between the CARD and the Coiled-coil domain as the LATCH domain. Further mechanistic studies reveal that these hyperactive CARD11 mutants disrupt inhibitory domain binding, enhance Bcl10 association and its K63-linked ubiquitination, and promote human lymphoma cell survival. We also identify TRAF2 and TAK1 as required cofactors during the induction of K63-linked ubiquitination of Bcl10. When combining the most active CARD11 missense mutations in both the CARD and the Coiled-coil domain, we identify a super mutant that synergizes the signaling activities of two oncogenic CARD11 missense mutations – C49Y and L251P. Protein complex purification of this super mutant along with mass spectrometry analysis identifies several CARD11 signaling cofactors including Bcl10, Malt1, IKKγ, CK1α and STUB1, in addition to a list of putative cofactors such as USP7, UBE3C, CKIIα, IGBP1, 14-3-3ε and EFHD2. Altogether, our study develops a highly efficient method for the discovery of mutational variants that promote tumorigenesis of NF-κB-dependent lymphomas, establishes a library of hyperactive CARD11 mutations with oncogenic potential, and provides a mechanistic understanding of how these mutants dysregulate the NF-κB signaling pathway. In addition, our data uncover critical components during the assembly of Bcl10 ubiquitination, a novel super-hyperactive CARD11 mutant, and several putative protein regulators of the CARD11 scaffold complex that are potentially dysregulated by oncogenic CARD11 mutations.