Identification of Tensin-3 as a MALT1 substrate that controls B cell adhesion and lymphoma dissemination.

The protease MALT1 promotes lymphocyte activation and lymphomagenesis by cleaving a limited set of cellular substrates, most of which control gene expression. Here, we identified the integrin-binding scaffold protein Tensin-3 as a MALT1 substrate in activated human B cells. Activated B cells lacking Tensin-3 showed decreased integrin-dependent adhesion but exhibited comparable NF-κB1 and Jun N-terminal kinase transcriptional responses. Cells expressing a noncleavable form of Tensin-3, on the other hand, showed increased adhesion. To test the role of Tensin-3 cleavage in vivo, mice expressing a noncleavable version of Tensin-3 were generated, which showed a partial reduction in the T cell-dependent B cell response. Interestingly, human diffuse large B cell lymphomas and mantle cell lymphomas with constitutive MALT1 activity showed strong constitutive Tensin-3 cleavage and a decrease in uncleaved Tensin-3 levels. Moreover, silencing of Tensin-3 expression in MALT1-driven lymphoma promoted dissemination of xenografted lymphoma cells to the bone marrow and spleen. Thus, MALT1-dependent Tensin-3 cleavage reveals a unique aspect of the function of MALT1, which negatively regulates integrin-dependent B cell adhesion and facilitates metastatic spread of B cell lymphomas.

The protease activity of the paracaspase MALT1 is controlled by monoubiquitination.

The protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how this activity is controlled remains unknown. Here we identify a monoubiquitination of MALT1 on Lys644 that activated the protease function of MALT1. Monoubiquitinated MALT1 had enhanced protease activity, whereas a ubiquitination-deficient MALT1 mutant with replacement of that lysine with arginine (MALT1(K644R)) had less protease activity, which correlated with impaired induction of interleukin 2 (IL-2) via the T cell antigen receptor in activated T cells. Expression of MALT1(K644R) diminished the survival of cells derived from diffuse large B cell lymphoma of the activated B cell-like subtype (ABC DLBCL), which require constitutive protease activity of MALT1 for survival. Thus, monoubiquitination of MALT1 is essential for its catalytic activation and is therefore a potential target for the treatment of ABC-DLBCL and for immunomodulation.

Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain.

The catalytic activity of the protease MALT1 is required for adaptive immune responses and regulatory T (Treg)-cell development, while dysregulated MALT1 activity can lead to lymphoma. MALT1 activation requires its monoubiquitination on lysine 644 (K644) within the Ig3 domain, localized adjacent to the protease domain. The molecular requirements for MALT1 monoubiquitination and the mechanism by which monoubiquitination activates MALT1 had remained elusive. Here, we show that the Ig3 domain interacts directly with ubiquitin and that an intact Ig3-ubiquitin interaction surface is required for the conjugation of ubiquitin to K644. Moreover, by generating constitutively active MALT1 mutants that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the Ig3-protease interaction surface, and the active site of the protease domain. Finally, we show that MALT1 mutants that alter the Ig3-ubiquitin interface impact the biological response of T cells. Thus, ubiquitin binding by the Ig3 domain promotes MALT1 activation by an allosteric mechanism that is essential for its biological function.

The proteolytic activity of the paracaspase MALT1 is key in T cell activation.

The paracaspase MALT1 is pivotal in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 contains a caspase-like domain, but it is unknown whether this domain is proteolytically active. Here we report that MALT1 had arginine-directed proteolytic activity that was activated after T cell stimulation, and we identify the signaling protein Bcl-10 as a MALT1 substrate. Processing of Bcl-10 after Arg228 was required for T cell receptor-induced cell adhesion to fibronectin. In contrast, MALT1 activity but not Bcl-10 cleavage was essential for optimal activation of transcription factor NF-kappaB and production of interleukin 2. Thus, the proteolytic activity of MALT1 is central to T cell activation, which suggests a possible target for the development of immunomodulatory or anticancer drugs.

Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

B-cell receptor-driven MALT1 activity regulates MYC signaling in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients.

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.

CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas.

A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor-κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL.

Activating mutations in genes related to TCR signaling in angioimmunoblastic and other follicular helper T-cell-derived lymphomas.

Angioimmunoblastic T-cell lymphoma (AITL) and other lymphomas derived from follicular T-helper cells (TFH) represent a large proportion of peripheral T-cell lymphomas (PTCLs) with poorly understood pathogenesis and unfavorable treatment results. We investigated a series of 85 patients with AITL (n = 72) or other TFH-derived PTCL (n = 13) by targeted deep sequencing of a gene panel enriched in T-cell receptor (TCR) signaling elements. RHOA mutations were identified in 51 of 85 cases (60%) consisting of the highly recurrent dominant negative G17V variant in most cases and a novel K18N in 3 cases, the latter showing activating properties in in vitro assays. Moreover, half of the patients carried virtually mutually exclusive mutations in other TCR-related genes, most frequently in PLCG1 (14.1%), CD28 (9.4%, exclusively in AITL), PI3K elements (7%), CTNNB1 (6%), and GTF2I (6%). Using in vitro assays in transfected cells, we demonstrated that 9 of 10 PLCG1 and 3 of 3 CARD11 variants induced MALT1 protease activity and increased transcription from NFAT or NF-κB response element reporters, respectively. Collectively, the vast majority of variants in TCR-related genes could be classified as gain-of-function. Accordingly, the samples with mutations in TCR-related genes other than RHOA had transcriptomic profiles enriched in signatures reflecting higher T-cell activation. Although no correlation with presenting clinical features nor significant impact on survival was observed, the presence of TCR-related mutations correlated with early disease progression. Thus, targeting of TCR-related events may hold promise for the treatment of TFH-derived lymphomas.

The paracaspase MALT1: biological function and potential for therapeutic inhibition.

The paracaspase MALT1 has a central role in the activation of lymphocytes and other immune cells including myeloid cells, mast cells and NK cells. MALT1 activity is required not only for the immune response, but also for the development of natural Treg cells that keep the immune response in check. Exaggerated MALT1 activity has been associated with the development of lymphoid malignancies, and recently developed MALT1 inhibitors show promising anti-tumor effects in xenograft models of diffuse large B cell lymphoma. In this review, we provide an overview of the present understanding of MALT1's function, and discuss possibilities for its therapeutic targeting based on recently developed inhibitors and animal models.

Role of the CARMA1/BCL10/MALT1 complex in lymphoid malignancies.

PURPOSE OF REVIEW: The CARMA1/BCL10/MALT1 (CBM) complex is a multimeric signaling complex controlling several important aspects of lymphocyte activation. Gain-of-function mutations in the genes encoding CBM proteins or their upstream regulators are associated with lymphoid malignancies, whereas loss-of-function mutations lead to immunodeficiency. This review reports on recent findings advancing our understanding of how CBM proteins contribute to malignant and nonmalignant hematological diseases in humans. RECENT FINDINGS: Somatic gain-of-function mutations of CARMA1 (also known as CARD11), originally described for patients with diffuse large B-cell lymphoma, have recently been identified in patients with acute T-cell leukemia/lymphoma or Sézary syndrome, and in patients with a B-cell lymphoproliferative disorder known as BENTA. Loss-of-function mutations of CARMA1 and MALT1, on the other hand, have been reported to underlie human immunodeficiency. Lately, it has become clear that CBM-dependent signaling promotes lymphomagenesis not only via NF-κB activation, but also via the AP-1 family of transcription factors. The identification of new substrates of the protease MALT1 and the characterization of mice expressing catalytically inactive MALT1 have deepened our understanding of how the CBM complex controls lymphocyte proliferation through promoting MALT1's protease activity. SUMMARY: The discovery of CARMA1 gain-of-function mutations in T-cell malignancies and BENTA patients, as well as the association of CARMA1 and MALT1 mutations with human immunodeficiency highlight the importance of CBM proteins in the regulation of lymphocyte functions, and suggest that the protease activity of MALT1 might be targeted to treat specific lymphoid malignancies.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines.

The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

ABSTRACT: The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain-containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

Adapter and enzymatic functions of proteases in T-cell activation.

Proteases control many vital aspects of humoral and cellular immune responses, including the maturation of cytokines and the killing of target cells. Recently, it has become evident that triggering of the T-cell receptor controls T-cell proliferation through proteases such as mucosa-associated lymphoid tissue 1 (MALT1) and Caspase-8 that act both as adapters and enzymes. Here, we discuss the role of these and other proteases that are relevant to the control of the T-cell response and represent interesting targets of therapeutic immunomodulation.

MALT-1 shortens lifespan by inhibiting autophagy in the intestine of C. elegans.

The caspase-like protease MALT1 promotes immune responses and oncogenesis in mammals by activating the transcription factor NF-κB. MALT1 is remarkably conserved from mammals to simple metazoans devoid of NF-κB homologs, like the nematode C. elegans. To discover more ancient, NF-κB -independent MALT1 functions, we analysed the phenotype of C. elegans upon silencing of MALT-1 expression systemically or in a tissue-specific manner. MALT-1 silencing in the intestine caused a significant increase in life span, whereas intestinal overexpression of MALT-1 shortened life expectancy. Interestingly, MALT-1-deficient animals showed higher constitutive levels of autophagy in the intestine, which were particularly evident in aged or starved nematodes. Silencing of the autophagy regulators ATG-13, BEC-1 or LGG-2, but not the TOR homolog LET-363, reversed lifespan extension caused by MALT-1 deficiency. These findings suggest that MALT-1 limits the lifespan of C. elegans by acting as an inhibitor of an early step of autophagy in the intestine.

Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function.

Protective adaptive immune responses rely on TCR-mediated recognition of Ag-derived peptides presented by self-MHC molecules. However, self-Ag (tumor)-specific TCRs are often of too low affinity to achieve best functionality. To precisely assess the relationship between TCR-peptide-MHC binding parameters and T cell function, we tested a panel of sequence-optimized HLA-A(*)0201/NY-ESO-1(157-165)-specific TCR variants with affinities lying within physiological boundaries to preserve antigenic specificity and avoid cross-reactivity, as well as two outliers (i.e., a very high- and a low-affinity TCR). Primary human CD8 T cells transduced with these TCRs demonstrated robust correlations between binding measurements of TCR affinity and avidity and the biological response of the T cells, such as TCR cell-surface clustering, intracellular signaling, proliferation, and target cell lysis. Strikingly, above a defined TCR-peptide-MHC affinity threshold (K(D) < approximately 5 muM), T cell function could not be further enhanced, revealing a plateau of maximal T cell function, compatible with the notion that multiple TCRs with slightly different affinities participate equally (codominantly) in immune responses. We propose that rational design of improved self-specific TCRs may not need to be optimized beyond a given affinity threshold to achieve both optimal T cell function and avoidance of the unpredictable risk of cross-reactivity.