A20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-κB signaling in activated T cells.

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.

Assembly mechanism of the CARMA1-BCL10-MALT1-TRAF6 signalosome.

The CARMA1-BCL10-MALT1 (CBM) signalosome is a central mediator of T cell receptor and B cell receptor-induced NF-κB signaling that regulates multiple lymphocyte functions. While caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1) nucleates B cell lymphoma 10 (BCL10) filament formation through interactions between CARDs, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a paracaspase with structural similarity to caspases, which recruits TNF receptor-associated factor 6 (TRAF6) for K63-linked polyubiquitination. Here we present cryo-electron microscopy (cryo-EM) structure of the BCL10 CARD filament at 4.0-Å resolution. The structure redefines CARD-CARD interactions compared with the previous EM structure determined from a negatively stained sample. Surprisingly, time-lapse confocal imaging shows that BCL10 polymerizes in a unidirectional manner. CARMA1, the BCL10 nucleator, serves as a hub for formation of star-shaped filamentous networks of BCL10 and significantly decreases the lag period of BCL10 polymerization. Cooperative MALT1 interaction with BCL10 filaments observed under EM suggests immediate dimerization of MALT1 in the BCL10 filamentous scaffold. In addition, TRAF6 cooperatively decorates CBM filaments to form higher-order assemblies, likely resulting in all-or-none activation of the downstream pathway. Collectively, these data reveal biophysical mechanisms in the assembly of the CARMA1-BCL10-MALT1-TRAF6 complex for signal transduction.

Genetic errors of the human caspase recruitment domain-B-cell lymphoma 10-mucosa-associated lymphoid tissue lymphoma-translocation gene 1 (CBM) complex: Molecular, immunologic, and clinical heterogeneity.

Three members of the caspase recruitment domain (CARD) family of adaptors (CARD9, CARD10, and CARD11) are known to form heterotrimers with B-cell lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma-translocation gene 1 (MALT1). These 3 CARD-BCL10-MALT1 (CBM) complexes activate nuclear factor κB in both the innate and adaptive arms of immunity. Human inherited defects of the 3 components of the CBM complex, including the 2 adaptors CARD9 and CARD11 and the 2 core components BCL10 and MALT1, have recently been reported. Biallelic loss-of-function mutant alleles underlie several different immunologic and clinical phenotypes, which can be assigned to 2 distinct categories. Isolated invasive fungal infections of unclear cellular basis are associated with CARD9 deficiency, whereas a broad range of clinical manifestations, including those characteristic of T- and B-lymphocyte defects, are associated with CARD11, MALT1, and BCL10 deficiencies. Interestingly, human subjects with these mutations have some features in common with the corresponding knockout mice, but other features are different between human subjects and mice. Moreover, germline and somatic gain-of-function mutations of MALT1, BCL10, and CARD11 have also been found in patients with other lymphoproliferative disorders. This broad range of germline and somatic CBM lesions, including loss-of-function and gain-of-function mutations, highlights the contribution of each of the components of the CBM complex to human immunity.

MiR-539 inhibits thyroid cancer cell migration and invasion by directly targeting CARMA1.

MicroRNAs (miRNAs) are important regulators of multiple cellular processes, and aberrant miRNA expression has been observed in thyroid cancer. However, the role of miRNAs in thyroid cancer metastasis remains largely unknown. In the current study, we found that miR-539 plays a suppressor role in thyroid cancer cell migration and invasion. Luciferase reporter assays confirmed that miR-539 binding to the 3'-UTR region of CARMA1 inhibited the expression of CARMA1 in thyroid cancer cells. Further studies demonstrated that CARMA1 can significantly promote the migration and invasion of thyroid cancer cells. Interestingly, overexpression or knockdown of CARMA1 effectively blocked the effect of miR-539 on the migration and invasion of thyroid cancer cells. Furthermore, we showed that miR-539 expression was frequently downregulated and CARMA1 expression was significantly upregulated in thyroid cancer cell lines and thyroid cancer tissues compared with controls. Taken together, our data indicate that miR-539 is a novel regulator of migration and invasion in human thyroid cancer cells by targeting CARMA1.

The CARD11-BCL10-MALT1 (CBM) signalosome complex: Stepping into the limelight of human primary immunodeficiency.

Next-generation DNA sequencing has accelerated the genetic characterization of many human primary immunodeficiency diseases (PIDs). These discoveries can be lifesaving for the affected patients and also provide a unique opportunity to study the effect of specific genes on human immune function. In the past 18 months, a number of independent groups have begun to define novel PIDs caused by defects in the caspase recruitment domain family, member 11 (CARD11)-B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1 [CBM]) signalosome complex. The CBM complex forms an essential molecular link between the triggering of cell-surface antigen receptors and nuclear factor κB activation. Germline mutations affecting the CBM complex are now recognized as the cause of novel combined immunodeficiency phenotypes, which all share abnormal nuclear factor κB activation and dysregulated B-cell development as defining features. For this "Current perspectives" article, we have engaged experts in both basic biology and clinical immunology to capture the worldwide experience in recognizing and managing patients with PIDs caused by CBM complex mutations.

Crystallization and preliminary X-ray crystallographic studies of the CARD domain of human CARMA1.

The CARMA1 signalosome, which is composed of CARMA1 [caspase recruitment domain (CARD) containing MAGUK protein 1], BCL10 (B-cell lymphoma 10) and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), is a molecular-signalling complex that performs pivotal functions in T-cell receptor (TCR) and B-cell receptor (BCR) mediated NF-κB activation. In this study, the CARD domain of human CARMA1 (CARMA1 CARD), corresponding to amino acids 14-109, was overexpressed in Escherichia coli using an engineered C-terminal His tag. CARMA1 CARD was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 3.2 Å from a crystal belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 45.73, b = 53.37, c = 91.89 Å.

Crosstalk between the CBM complex/NF-κB and MAPK/P27 signaling pathways of regulatory T cells contributes to the tumor microenvironment.

Regulatory T cells (Tregs), which execute their immunosuppressive functions by multiple mechanisms, have been verified to contribute to the tumor microenvironment (TME). Numerous studies have shown that the activation of the CBM complex/NF-κB signaling pathway results in the expression of hypoxia-inducible factor-1 (HIF-1α) and interleukin-6 (IL-6), which initiate the TME formation. HIF-1α and IL-6 promote regulatory T cells (Tregs) proliferation and migration through the MAPK/CDK4/6/Rb and STAT3/SIAH2/P27 signaling pathways, respectively. IL-6 also promotes the production of HIF-1α and enhances the self-regulation of Tregs in the process of tumor microenvironment (TME) formation. In this review, we discuss how the crosstalk between the CARMA1-BCL10-MALT1 signalosome complex (CBM complex)/NF-κB and MAPK/P27 signaling pathways contributes to the formation of the TME, which may provide evidence for potential therapeutic targets in the treatment of solid tumors.

CARMA1 is required for Notch1-induced NF-κB activation in SIL-TAL1-negative T cell acute lymphoblastic leukemia.

The NF-κB signaling pathway is an important downstream pathway of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL) cells. However, the molecular mechanisms underlying the cascade activation of Notch1 in T-ALL cells are poorly understood. Here, we evaluated the role of CARMA1 in Notch1-induced NF-κB activation in T-ALL cells. CARMA1 was highly and specifically expressed in T-ALL cells and correlated with the prognosis of T-ALL patients. Interestingly, CARMA1 knockdown only inhibited the growth and proliferation of SIL-TAL1 fusion gene-negative T-ALL cells. In addition, CARMA1 knockdown arrested T-ALL cells at the G1 phase. Furthermore, CARMA1 knockdown significantly inhibited the proliferation of T-ALL cells in vivo and prolonged the survival of mice. Mechanistically, CARMA1 deficiency abolished Notch1-induced NF-κB transcriptional activation and significantly reduced expression levels of the NF-κB target genes c-Myc, Bcl-2, and CCR7. Taken together, these results of our study identify CARMA1 as one of the crucial mediators of Notch1-induced transformation of T-All cells, suggesting that CARMA1 is a promising therapeutic target for T-ALL due to its specific expression in lymphocytes. KEY MESSAGES: CARMA1 contributes to cell survival only in SIL-TAL1 negative T-ALL cells. CARMA1 is a crucial mediator of Notch1-induced activation of NF-κB pathway. CARMA1 is a promising therapeutic target for T-ALL.

Methods to Study CARD11-BCL10-MALT1 Dependent Canonical NF-κB Activation in Jurkat T Cells.

Jurkat T cells have been of central importance for the discovery of signalling mediators driving NF-κB activation in response to T cell antigen receptor (TCR)/CD28 co-stimulation. The critical function of the key regulators identified in Jurkat T cells has subsequently been verified in primary murine and human T cells. CRISPR/Cas9-mediated genomic editing techniques in combination with viral reconstitution are powerful tools that now enable the investigation of the exact molecular mechanisms that govern T cell signalling, especially the impact of protein-protein interactions, protein modifications, or cancer-associated gain- or loss-of-function mutations. As exemplified by the CARD11 gene encoding a key regulator of NF-κB signalling in T cells, we describe here the detailed workflow for the generation of CRISPR/Cas9 knockout (KO) Jurkat T cells and the subsequent reconstitution using a lentiviral transduction protocol. In addition, we explain the use of a stable NF-κB-dependent EGFP reporter system that enables a reliable quantification of NF-κB transcriptional activation in the reconstituted KO Jurkat T cells.

Targeting BCL10 by small peptides for the treatment of B cell lymphoma.

Rationale: Constitutive activation of the NF-κB signalling pathway plays a pivotal role in the pathogenesis of activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCLs), the most aggressive and chemoresistant form of DLBCL. In ABC-DLBCLs, the CARMA1-BCL10 (CB) complex forms a filamentous structure and functions as a supramolecular organizing centre (CB-SMOC) that is required for constitutive NF-κB activation, making it an attractive drug target for ABC-DLBCL treatment. However, a pharmaceutical approach targeting CB-SMOC has been lacking. Here, we developed Bcl10 peptide inhibitors (BPIs) that specifically target the BCL10 filamentation process. Methods: Electron microscopy and immunofluorescence imaging were used to visualize the effect of the BPIs on the BCL10 filamentation process. The cytotoxicity of the tested BPIs was evaluated in DLBCL cell lines according to cell proliferation assays. Different in vitro experiments (pharmacokinetics, immunoprecipitation, western blotting, annexin V and PI staining) were conducted to determine the functional mechanisms of the BPIs. The in vivo therapeutic effect of the BPIs was examined in different xenograft DLBCL mouse models. Finally, Ki67 and TUNEL staining and histopathology analysis were used to evaluate the antineoplastic mechanisms and systemic toxicity of the BPIs. Results: We showed that these BPIs can effectively disrupt the BCL10 filamentation process, destabilize BCL10 and suppress NF-κB signalling in ABC-DLBCL cells. By examining a panel of DLBCL cell lines, we found that these BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL cells by inducing apoptosis and cell cycle arrest. Moreover, by converting the BPIs to acquire a D-retro inverso (DRI) configuration, we developed DRI-BPIs with significantly improved intracellular stability and unimpaired BPI activity. These DRI-BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL tumors in mouse xenograft models without eliciting discernible adverse effects. Conclusion: We developed novel BPIs to target the BCL10 filamentation process and demonstrated that targeting BCL10 by BPIs is a potentially safe and effective pharmaceutical approach for the treatment of ABC-DLBCL and other CB-SMOC-dependent malignancies.

NF-κB Activation in Lymphoid Malignancies: Genetics, Signaling, and Targeted Therapy.

The NF-κB transcription factor family plays a crucial role in lymphocyte proliferation and survival. Consequently, aberrant NF-κB activation has been described in a variety of lymphoid malignancies, including diffuse large B-cell lymphoma, Hodgkin lymphoma, and adult T-cell leukemia. Several factors, such as persistent infections (e.g., with Helicobacter pylori), the pro-inflammatory microenvironment of the cancer, self-reactive immune receptors as well as genetic lesions altering the function of key signaling effectors, contribute to constitutive NF-κB activity in these malignancies. In this review, we will discuss the molecular consequences of recurrent genetic lesions affecting key regulators of NF-κB signaling. We will particularly focus on the oncogenic mechanisms by which these alterations drive deregulated NF-κB activity and thus promote the growth and survival of the malignant cells. As the concept of a targeted therapy based on the mutational status of the malignancy has been supported by several recent preclinical and clinical studies, further insight in the function of NF-κB modulators and in the molecular mechanisms governing aberrant NF-κB activation observed in lymphoid malignancies might lead to the development of additional treatment strategies and thus improve lymphoma therapy.

Post-translational Modifications of the CARMA1-BCL10-MALT1 Complex in Lymphocytes and Activated B-Cell Like Subtype of Diffuse Large B-Cell Lymphoma.

Piracy of the NF-κB transcription factors signaling pathway, to sustain its activity, is a mechanism often deployed in B-cell lymphoma to promote unlimited growth and survival. The aggressive activated B-cell like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) exploits a multi-protein complex of CARMA1, BCL10, and MALT1 (CBM complex), which normally conveys NF-κB signaling upon antigen receptors engagement. Once assembled, the CBM also unleashes MALT1 protease activity to finely tune the immune response. As a result, ABC DLBCL tumors develop a profound addiction to NF-κB and to MALT1 enzyme, leaving open a breach for therapeutics. However, the pleiotropic nature of NF-κB jeopardizes the success of its targeting and urges us to develop new strategies. In this review, we discuss how post-translational modifications, such as phosphorylation and ubiquitination of the CBM components, as well as, MALT1 proteolytic activity, shape the CBM activity in lymphocytes and ABC DLBCL, and may provide new avenues to restore vulnerability in lymphoma.

BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation.

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.

TCR signaling to NF-κB and mTORC1: Expanding roles of the CARMA1 complex.

Naïve T-cell activation requires signals from both the T-cell receptor (TCR) and the costimulatory molecule CD28. A central mediator of the TCR and CD28 signals is the scaffold protein CARMA1, which functions by forming a complex with partner proteins, Bcl10 and MALT1. A well-known function of the CARMA1 signaling complex is to mediate activation of IκB kinase (IKK) and its target transcription factor NF-κB, thereby promoting T-cell activation and survival. Recent evidence suggests that CARMA1 also mediates TCR/CD28-stimulated activation of the IKK-related kinase TBK1, which plays a role in regulating the homeostasis and migration of T cells. Moreover, the CARMA1 complex connects the TCR/CD28 signals to the activation of mTORC1, a metabolic kinase regulating various aspects of T-cell functions. This review will discuss the mechanism underlying the activation of the CARMA1-dependent signaling pathways and their roles in regulating T-cell functions.

mTOR and its tight regulation for iNKT cell development and effector function.

Invariant NKT (iNKT) cells, which express the invariant Vα14Jα18 TCR that recognizes lipid antigens, have the ability to rapidly respond to agonist stimulation, producing a variety of cytokines that can shape both innate and adaptive immunity. iNKT cells have been implicated in host defense against microbial infection, in anti-tumor immunity, and a multitude of diseases such as allergies, asthma, graft versus host disease, and obesity. Emerging evidence has demonstrated crucial role for mammalian target of rapamycin (mTOR) in immune cells, including iNKT. In this review we will discuss current understanding of how mTOR and its tight regulation control iNKT cell development, effector lineage differentiation, and function.

Oligomerized CARD16 promotes caspase-1 assembly and IL-1ß processing.

Increasing evidence indicates that caspase recruitment domain (CARD)-mediated caspase-1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)-1ß release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo-oligomer. Oligomerized CARD16 formed a filament-like structure with CASP1CARD and a speck with apoptosis-associated speck-like protein containing a CARD. A filament-like structure formed by CARD16 promoted CASP1 filament assembly and IL-1ß release. In contrast, CARD17 did not form a homo-oligomer or filaments and inhibited CASP1-dependent IL-1ß release. Mutated CARD16D27G, mimicking the CARD17 amino acid sequence, formed a homo-oligomer but failed to form a filament-like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL-1ß release. These results suggest that oligomerized CARD16 promotes CARD-mediated molecular assembly and CASP1 activation.

NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.