Micafungin protects mouse heart against doxorubicin-induced oxidative injury via suppressing MALT1-dependent k48-linked ubiquitination of Nrf2.

Oxidative stress contributes greatly to doxorubicin (DOX)-induced cardiotoxicity. Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor in DOX-induced myocardial oxidative injury. Recently, we found that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1)-dependent k48-linked ubiquitination was responsible for down-regulation of myocardial Nrf2 in DOX-treated mice. Micafungin, an antifungal drug, was identified as a potential MALT1 inhibitor. This study aims to explore whether micafungin can reduce DOX-induced myocardial oxidative injury and if its anti-oxidative effect involves a suppression of MALT1-dependent k48-linked ubiquitination of Nrf2. To establish the cardiotoxicity models in vivo and in vitro, mice were treated with a single dose of DOX (15 mg/kg, i.p.) and cardiomyocytes were incubated with DOX (1 µM) for 24 h, respectively. Using mouse model of DOX-induced cardiotoxicity, micafungin (10 or 20 mg/kg) was shown to improve cardiac function, concomitant with suppression of oxidative stress, mitochondrial dysfunction, and cell death in a dose-dependent manner. Similar protective roles of micafungin (1 or 5 µM) were observed in DOX-treated cardiomyocytes. Mechanistically, micafungin weakened the interaction between MALT1 and Nrf2, decreased the k48-linked ubiquitination of Nrf2 while elevated the protein levels of Nrf2 in both DOX-treated mice and cardiomyocytes. Furthermore, MALT1 overexpression counteracted the cardioprotective effects of micafungin. In conclusion, micafungin reduces DOX-induced myocardial oxidative injury via suppression of MALT1, which decreases the k48-linked ubiquitination of Nrf2 and elevates Nrf2 protein levels. Thus, micafungin may be repurposed for treating DOX-induced cardiotoxicity.

Insights into mechanisms of MALT1 allostery from NMR and AlphaFold dynamic analyses.

Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) is an attractive target for the development of modulatory compounds in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained unexplored. We present here dynamic analyses of the apo MALT1 form along with the E549A mutation. This investigation used NMR 15N relaxation and NOE measurements between side-chain methyl groups. Our findings confirm that MALT1 exists as a monomer in solution, and demonstrate that the domains display semi-independent movements in relation to each other. Our dynamic study, covering multiple time scales, along with the assessment of conformational populations by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, put the side chain of residue W580 in an inward position, shedding light at potential mechanisms underlying the allosteric regulation of this enzyme.

The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway.

MALT1 has been implicated as an upstream regulator of NF-κB signaling in immune cells and tumors. This study determined the regulatory mechanisms and biological functions of MALT1 in non-small cell lung cancer (NSCLC). In cell culture and orthotopic xenograft models, MALT1 suppression via gene expression interference or protein activity inhibition significantly impaired malignant phenotypes and enhanced radiation sensitivity of NSCLC cells. CSN5, the core subunit of COP9 signalosome, was firstly verified to stabilize MALT1 via disturbing the interaction with E3 ligase FBXO3. Loss of FBXO3 in NSCLC cells reduced MALT1 ubiquitination and promoted its accumulation, which was reversed by CSN5 interference. An association between CSN5/FBXO3/MALT1 regulatory axis and poor prognosis in NSCLC patients was identified. Our findings revealed the detail mechanism of continuous MALT1 activation in NF-κB signaling, highlighting its significance as predictor and potential therapeutic target in NSCLC.

MALT1 substrate cleavage: what is it good for?

CARD-BCL10-MALT1 (CBM) signalosomes connect distal signaling of innate and adaptive immune receptors to proximal signaling pathways and immune activation. Four CARD scaffold proteins (CARD9, 10, 11, 14) can form seeds that nucleate the assembly of BCL10-MALT1 filaments in a cell- and stimulus-specific manner. MALT1 (also known as PCASP1) serves a dual function within the assembled CBM complexes. By recruiting TRAF6, MALT1 acts as a molecular scaffold that initiates IκB kinase (IKK)/NF-κB and c-Jun N-terminal kinase (JNK)/AP-1 signaling. In parallel, proximity-induced dimerization of the paracaspase domain activates the MALT1 protease which exerts its function by cleaving a set of specific substrates. While complete MALT1 ablation leads to immune deficiency, selective destruction of either scaffolding or protease function provokes autoimmune inflammation. Thus, balanced MALT1-TRAF6 recruitment and MALT1 substrate cleavage are critical to maintain immune homeostasis and to promote optimal immune activation. Further, MALT1 protease activity drives the survival of aggressive lymphomas and other non-hematologic solid cancers. However, little is known about the relevance of the cleavage of individual substrates for the pathophysiological functions of MALT1. Unbiased serendipity, screening and computational predictions have identified and validated ~20 substrates, indicating that MALT1 targets a quite distinct set of proteins. Known substrates are involved in CBM auto-regulation (MALT1, BCL10 and CARD10), regulation of signaling and adhesion (A20, CYLD, HOIL-1 and Tensin-3), or transcription (RelB) and mRNA stability/translation (Regnase-1, Roquin-1/2 and N4BP1), indicating that MALT1 often targets multiple proteins involved in similar cellular processes. Here, we will summarize what is known about the fate and functions of individual MALT1 substrates and how their cleavage contributes to the biological functions of the MALT1 protease. We will outline what is needed to better connect critical pathophysiological roles of the MALT1 protease with the cleavage of distinct substrates.

Halting multiple myeloma with MALT1 inhibition: suppressing BCMA-induced NF-κB and inducing immunogenic cell death.

ABSTRACT: Because multiple myeloma (MM) poses a formidable therapeutic challenge despite recent progress, exploring novel targets is crucial. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) emerges as a promising paracaspase with druggable potential, especially unexplored in MM. Our study provided compelling evidence demonstrating a statistically significant elevation of MALT1 expression in human primary MM cells. Moreover, elevated MALT1 expression was associated with a poorer prognosis in MM. Genetic deletion of MALT1 reduced cell growth, colony formation, and tumor growth in vivo. Pharmacological inhibition with 1 µM of a small-molecular MALT1 inhibitor, Mi-2, effectively inhibited cell growth, inducing mitochondria-dependent apoptotic cell death. Mechanistically, MALT1 inhibition disrupted diverse signal transduction pathways, notably impeding nuclear factor κB (NF-κB). Significantly, the inhibition of MALT1 demonstrated a substantial suppression of NF-κB activation by elevating inhibitor of NF-κB, disrupting the nuclear localization of p65 and c-REL. This effect was observed in both the basal state and when stimulated by B-cell maturation antigen, highlighting the pivotal role of MALT1 inhibition in influencing MM cell survival. It was noteworthy that Mi-2 induces properties associated with immunogenic cell death (ICD), as evidenced by increased calreticulin, adenosine triphosphate release, and high-mobility group protein B1 upregulation, consequently triggering ICD-associated immune activation and enhancing CD8+ T-cell cytotoxicity in vitro. In conclusion, our research highlights MALT1 as a promising druggable target for therapeutic interventions in MM, providing insights into its molecular mechanisms in MM progression.

Study of individual domains contributing to MALT1 dimerization in BCL10-independent and dependent assembly.

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.

Inhibition of MALT1 and BCL2 Induces Synergistic Antitumor Activity in Models of B-Cell Lymphoma.

The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

Inhibition of the MALT1-LPCAT3 axis protects cartilage degeneration and osteoarthritis.

The proinflammatory cytokines and arachidonic acid (AA)-derived eicosanoids play a key role in cartilage degeneration in osteoarthritis (OA). The lysophosphatidylcholine acyltransferase 3 (LPCAT3) preferentially incorporates AA into the membranes. Our recent studies showed that MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that LPCAT3 expression was up-regulated in both human and mice articular cartilage of OA, and correlated with severity of OA. The IL-1ß-induces cell death via upregulation of LPCAT3, MMP3, ADAMTS5, and eicosanoids via MALT1. Gene silencing or pharmacological inhibition of LPCAT3 or MALT1 in chondrocytes and human cartilage explants notably suppressed the IL-1ß-induced cartilage catabolism through inhibition of expression of MMP3, ADAMTS5, and also secretion of cytokines and eicosanoids. Mechanistically, overexpression of MALT1 in chondrocytes significantly upregulated the expression of LPCAT3 along with MMP3 and ADAMTS5 via c-Myc. Inhibition of c-Myc suppressed the IL-1ß-MALT1-dependent upregulation of LPCAT3, MMP3 and ADAMTS5. Consistent with the in vitro data, pharmacological inhibition of MALT1 or gene silencing of LPCAT3 using siRNA-lipid nanoparticles suppressed the synovial articular cartilage erosion, pro-inflammatory cytokines, and eicosanoids such as PGE2, LTB4, and attenuated osteoarthritis induced by the destabilization of the medial meniscus in mice. Overall, our data reveal a previously unrecognized role of the MALT1-LPCAT3 axis in osteoarthritis. Targeting the MALT1-LPCAT3 pathway with MALT1 inhibitors or siRNA-liposomes of LPCAT3 may become an effective strategy to treat OA by suppressing eicosanoids, matrix-degrading enzymes, and proinflammatory cytokines.

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

MALT1 Protease Regulates T-Cell Immunity via the mTOR Pathway in Oral Lichen Planus.

Oral lichen planus (OLP) is a T cell-mediated immune mucosal disease of unknown pathogenesis. Whether mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), an intracellular signaling protein, is involved in the T-cell immune dysfunction of OLP remains elusive. MALT1 expression in local and peripheral T cells of OLP and controls was analyzed using immunohistochemistry, multiplex immunohistochemistry, and flow cytometry. The expression of MALT1 in activated Jurkat T cells incubated with either OLP plasma or interleukin (IL)-7/IL-15 was determined by flow cytometry. The effects of MALT1 and mechanistic target of rapamycin (mTOR) on T-cell immunity were investigated through western blot, CCK8 assay, and flow cytometry. The expression of MALT1 protein was elevated in local OLP T cells and mucosal-associated invariant T (MAIT) cells, while reduced in peripheral OLP T cells, MAIT cells, and follicular helper-like MAIT (MAITfh) cells. Stimulation with OLP plasma and IL-7/ IL-15 had no effect on MALT1 expression in activated Jurkat T cells. MALT1 protease-specific inhibitor (MI-2) induced mTOR phosphorylation, increased B-cell lymphoma 10 (BCL10) expression, inhibited T-cell proliferation, and promoted T-cell apoptosis. The combination of MI-2 and rapamycin increased MALT1 expression, further suppressed T-cell proliferation, and facilitated T-cell apoptosis. MALT1 expression is aberrant in both local lesions and peripheral blood of OLP. Inhibition of the mTOR pathway further enhances the suppression of T-cell proliferation and the promotion of apoptosis induced by the MALT1 inhibitor MI-2.

Chimeric and mutant CARD9 constructs enable analyses of conserved and diverged autoinhibition mechanisms in the CARD-CC protein family.

Caspase recruitment domain-containing protein (CARD)9, CARD10, CARD11, and CARD14 all belong to the CARD-coiled coil (CC) protein family and originated from a single common ancestral protein early in vertebrate evolution. All four proteins form CARD-CC/BCL10/MALT1 (CBM) complexes leading to nuclear factor-kappa-B (NF-κB) activation after upstream phosphorylation by various protein kinase C (PKC) isoforms. CBM complex signaling is critical for innate and adaptive immunity, but aberrant activation can cause autoimmune or autoinflammatory diseases, or be oncogenic. CARD9 shows a superior auto-inhibition compared with other CARD-CC family proteins, with very low spontaneous activity when overexpressed in HEK293T cells. In contrast, the poor auto-inhibition of other CARD-CC family proteins, especially CARD10 (CARMA3) and CARD14 (CARMA2), is hampering characterization of upstream activators or activating mutations in overexpression studies. We grafted different domains from CARD10, 11, and 14 on CARD9 to generate chimeric CARD9 backbones for functional characterization of activating mutants using NF-κB reporter gene activation in HEK293T cells as readout. CARD11 (CARMA1) activity was not further reduced by grafting on CARD9 backbones. The chimeric CARD9 approach was subsequently validated by using several known disease-associated mutations in CARD10 and CARD14, and additional screening allowed us to identify several previously unknown activating natural variants in human CARD9 and CARD10. Using Genebass as a resource of exome-based disease association statistics, we found that activated alleles of CARD9 correlate with irritable bowel syndrome (IBS), constipation, osteoarthritis, fibromyalgia, insomnia, anxiety, and depression, which can occur as comorbidities.

Identification of an Ortholog of MALT1 from Shrimp That Induces NF-κB-Mediated Antiviral Immunity.

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) serves as a pivotal mediator for NF-κB activation in response to a wide spectrum of transmembrane receptor stimuli. In the present study, a homolog of MALT1, named LvMALT1, is cloned from the Pacific white shrimp (Litopenaeus vannamei) and its potential function in shrimp innate immunity is explored. The open reading frame of LvMALT1 is 2364 bp that encodes 787 amino acids. The predicted LvMALT1 protein structure comprises a death domain, three immunoglobulin domains, and a caspase-like domain, exhibiting remarkable similarity to other homologs. LvMALT1 is a cytoplasmic-localized protein and could interact with LvTRAF6. Overexpression of LvMALT1 induces the activation of promoter elements governing the expression of several key antimicrobial peptides (AMPs), including penaeidins (PENs) and crustins (CRUs). Conversely, silencing of LvMALT1 leads to a reduction in the phosphorylation levels of Dorsal and Relish, along with a concomitant decline in the in vivo expression levels of multiple AMPs. Furthermore, LvMALT1 is prominently upregulated in response to a challenge by the white spot syndrome virus (WSSV), facilitating the NF-κB-mediated expression of AMPs as a defense against viral infection. Taken together, we identified a MALT1 homolog from the shrimp L. vannamei, which plays a positive role in the TRAF6/NF-κB/AMPs axis-mediated innate immunity.

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.

In silico study of polyphenols as potential inhibitors of MALT1 protein in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common cancer types. Deregulated signaling pathways can trigger certain NHL subtypes, including Diffuse Large B-cell lymphoma. NF-ĸB signaling pathway, which is responsible for the proliferation, growth, and survival of cells, has an essential role in lymphoma development. Although different signals control NF-ĸB activation in various lymphoid malignancies, the characteristic one is the CARD11-BCL10-MALT1 (CBM) complex. The CBM complex is responsible for the initiation of adaptive immune response. Our study is focused on the molecular docking of ten polyphenols as potential CARD11-BCL10-MALT1 complex inhibitors, essentially through MALT1 inhibition. Molecular docking was performed by Auto Dock Tools and AutoDock Vina tool, while SwissADME was used for drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of the ligands. Out of 66 ligands that were used in this study, we selected and visualized five. Selection criteria were based on the binding energy score and position of the ligands on the used protein. 2D and 3D visualizations showed interactions of ligands with the protein. Five ligands are considered potential inhibitors of MALT1, thus affecting NF-ĸB signaling pathway. However, additional in vivo and in vitro studies are required to confirm their mechanism of inhibition.

MALT1 Positively Relates to T Helper 1 and T Helper 17 cells, and Serves as a Potential Biomarker for Predicting 30-Day Mortality in Stanford Type A Aortic Dissection Patients.

Mucosa-associated lymphoid tissue 1 (MALT1) regulates inflammation and T helper (Th) cell differentiation, which may participate in the progression of Stanford type A aortic dissection (TAAD). This study intended to assess the association of MALT1 expression with prognosis in TAAD patients. In this prospective study, MALT1 expression was measured by reverse transcription-quantitative polymerase chain reaction assay from peripheral blood samples in 100 TAAD patients and 100 non-AD controls (non-AD patients with chest pain) before treatment. Besides, Th1, Th2, and Th17 cells of TAAD patients before treatment were measured by flow cytometry assay, and their 30-day mortality was recorded. MALT1 expression was ascended in TAAD patients vs. non-AD controls (P < 0.001). In TAAD patients, elevated MALT1 expression was linked with hypertension complication (P = 0.009), increased systolic blood pressure (r = 0.291, P = 0.003), C-reactive protein (CRP) (r = 0.286, P = 0.004), and D-dimer (r = 0.359, P < 0.001). Additionally, MALT1 expression was positively correlated with Th1 cells (r = 0.312, P = 0.002) and Th17 cells (r = 0.397, P < 0.001), but not linked with Th2 cells (r = -0.166, P = 0.098). Notably, the 30-day mortality of TAAD patients was 28.0%. MALT1 expression [odds ratio (OR) = 1.936, P = 0.004], CRP (OR = 1.108, P = 0.002), D-dimer (OR = 1.094, P = 0.003), and surgery timing (emergency vs. selective) (OR = 8.721, P = 0.024) independently predicted increased risk of death within 30 days in TAAD patients. Furthermore, the combination of the above-mentioned independent factors had an excellent ability in predicting 30-day mortality with the area under curve of 0.949 (95% confidence interval: 0.909-0.989). MALT1 expression relates to increased Th1 cells, Th17 cells, and 30-day mortality risk in TAAD patients.

Function and targeting of MALT1 paracaspase in cancer.

The paracaspase MALT1 has emerged as a key regulator of immune signaling, which also promotes tumor development by both cancer cell-intrinsic and -extrinsic mechanisms. As an integral subunit of the CARD11-BCL10-MALT1 (CBM) signaling complex, MALT1 has an intriguing dual function in lymphocytes. MALT1 acts as a scaffolding protein to drive activation of NF-κB transcription factors and as a protease to modulate signaling and immune activation by cleavage of distinct substrates. Aberrant MALT1 activity is critical for NF-κB-dependent survival and proliferation of malignant cancer cells, which is fostered by paracaspase-catalyzed inactivation of negative regulators of the canonical NF-κB pathway like A20, CYLD and RelB. Specifically, B cell receptor-addicted lymphomas rely strongly on this cancer cell-intrinsic MALT1 protease function, but also survival, proliferation and metastasis of certain solid cancers is sensitive to MALT1 inhibition. Beyond this, MALT1 protease exercises a cancer cell-extrinsic role by maintaining the immune-suppressive function of regulatory T (Treg) cells in the tumor microenvironment (TME). MALT1 inhibition is able to convert immune-suppressive to pro-inflammatory Treg cells in the TME of solid cancers, thereby eliciting a robust anti-tumor immunity that can augment the effects of checkpoint inhibitors. Therefore, the cancer cell-intrinsic and -extrinsic tumor promoting MALT1 protease functions offer unique therapeutic opportunities, which has motivated the development of potent and selective MALT1 inhibitors currently under pre-clinical and clinical evaluation.

Proteolytic Activity of the Paracaspase MALT1 Is Involved in Epithelial Restitution and Mucosal Healing.

The paracaspase MALT1 is a crucial regulator of immune responses in various cellular contexts. Recently, there is increasing evidence suggesting that MALT1 might represent a novel key player in mucosal inflammation. However, the molecular mechanisms underlying this process and the targeted cell population remain unclear. In this study, we investigate the role of MALT1 proteolytic activity in the context of mucosal inflammation. We demonstrate a significant enrichment of MALT1 gene and protein expression in colonic epithelial cells of UC patients, as well as in the context of experimental colitis. Mechanistically we demonstrate that MALT1 protease function inhibits ferroptosis, a form of iron-dependent cell death, upstream of NF-κB signaling, which can promote inflammation and tissue damage in IBD. We further show that MALT1 activity contributes to STAT3 signaling, which is essential for the regeneration of the intestinal epithelium after injury. In summary, our data strongly suggests that the protease function of MALT1 plays a critical role in the regulation of immune and inflammatory responses, as well as mucosal healing. Understanding the mechanisms by which MALT1 protease function regulates these processes may offer novel therapeutic targets for the treatment of IBD and other inflammatory diseases.

MALT1 paracaspase is overexpressed in hepatocellular carcinoma and promotes cancer cell survival and growth.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, therapeutic management of HCC remains a challenge, emphasizing the importance of exploring novel targets. MALT1 paracaspase is a druggable signaling molecule whose dysregulation has been linked to hematological and solid tumors. However, the role of MALT1 in HCC remains poorly understood, leaving its molecular functions and oncogenic implications unclear. Here we provide evidence that MALT1 expression is elevated in human HCC tumors and cell lines, and that correlates with tumor grade and differentiation state, respectively. Our results indicate that ectopic expression of MALT1 confers increased cell proliferation, 2D clonogenic growth, and 3D spheroid formation in well differentiated HCC cell lines with relatively low MALT1 levels. In contrast, stable silencing of endogenous MALT1 through RNA interference attenuates these aggressive cancer cell phenotypes, as well as migration, invasion, and tumor-forming ability, in poorly differentiated HCC cell lines with higher paracaspase expression. Consistently, we find that pharmacological inhibition of MALT1 proteolytic activity with MI-2 recapitulates MALT1 depletion phenotypes. Finally, we show that MALT1 expression is positively correlated with NF-kB activation in human HCC tissues and cell lines, suggesting that its tumor promoting functions may involve functional interaction with the NF-kB signaling pathway. This work unveils new insights into the molecular implications of MALT1 in hepatocarcinogenesis and places this paracaspase as a potential marker and druggable liability in HCC.

MALT1-dependent cleavage of CYLD promotes NF-κB signaling and growth of aggressive B-cell receptor-dependent lymphomas.

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.