Nuclear Factor κB–dependent Gene Expression Profiling of Hodgkin's Disease Tumor Cells, Pathogenetic Significance, and Link to Constitutive Signal Transducer and Activator of Transcription 5a Activity

Constitutive nuclear nuclear factor (NF)-κB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-κB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkin's disease (HD) cell lines to identify NF-κB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-κB. The NF-κB–dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-κB target genes. Using chromatin immunoprecipitation we demonstrate that NF-κB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-κB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-κB in the pathogenesis of HD and potentially of other tumors with constitutive NF-κB activation

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. © 2022, The Author(s)

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

Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation

Translational Studies Using the MALT1 Inhibitor (S)-Mepazine to Induce Treg Fragility and Potentiate Immune Checkpoint Therapy in Cancer

INTRODUCTION: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). METHODS: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). RESULTS: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. CONCLUSIONS: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors

A pervasive role of ubiquitin conjugation in activation and termination of IκB kinase pathways

The nuclear factor (NF)-κB pathway is a paradigm for gene expression control by ubiquitin-mediated protein degradation. In stimulated cells, phosphorylation by the IκB kinase (IKK) complex primes NF-κB-inhibiting IκB molecules for lysine (Lys)-48-linked polyubiquitination and subsequent destruction by the 26S proteasome. However, recent studies indicate that the ubiquitin (Ub) system controls NF-κB pathways at many levels. Ub ligases are activated by different upstream signalling pathways, and they function as central regulators of IKK and c-Jun amino-terminal kinase activation. The assembly of Lys 63 polyUb chains provides docking surfaces for the recruitment of IKK-activating complexes, a reaction that is counteracted by deubiquitinating enzymes. Furthermore, Ub conjugation targets upstream signalling mediators as well as nuclear NF-κB for post-inductive degradation to limit the duration of signalling

Pharmacological inhibition of MALT1 protease activity protects mice in a mouse model of multiple sclerosis

Background: The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-kappa B. Besides functioning as a scaffold signaling protein, MALT1 also acts as a cysteine protease that specifically cleaves a number of substrates and contributes to specific T cell receptor-induced gene expression. Recently, small molecule inhibitors of MALT1 proteolytic activity were identified and shown to have promising anticancer properties in subtypes of B cell lymphoma. However, information on the therapeutic potential of small compound inhibitors that target MALT1 protease activity in autoimmunity is still lacking. Methods: The present study aimed to elucidate whether MALT1 protease inhibitors are also useful in the treatment of lymphocyte-mediated autoimmune pathologies such as multiple sclerosis (MS). For this, we studied the therapeutic potential of a recently identified inhibitor of MALT1 protease activity, the phenothiazine derivative mepazine, in the context of experimental autoimmune encephalomyelitis (EAE), the main animal model for MS. Results: We demonstrate that administration of mepazine prophylactically or after disease onset, can attenuate EAE. Importantly, while complete absence of MALT1 affects the differentiation of regulatory T (Treg) cells in vivo, the MALT1 protease inhibitor mepazine did not affect Treg development. Conclusions: Altogether, these data indicate that small molecule inhibitors of MALT1 not only hold great promise for the treatment of B cell lymphomas but also for autoimmune disorders such as MS