Inhibition Of MALT1 Protease Activity Research Articles

Abstract C127: Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas

Abstract MALT1 is a key regulator of antigen receptor signaling in B and T cells. MALT1 partners with BCL10 and CARMA1 to form the CBM complex in which protease activity of MALT1 cleaves the negative regulators (A20, CYLD, RelB) leading to activation of canonical NF-kB pathway. Constitutive activation of NF-kB signaling is a key driver in B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). Therapeutic targeting of MALT1 protease activity can therefore be a useful approach for the treatment of B-cell lymphomas with dysregulated NF-kB signaling. We sought to discover and develop MALT1 inhibitors with the “best-in-class” profile with an emphasis on lowering plasma protein binding (PPB) to achieve improved pharmacological outcome. After extensive SAR efforts utilizing various computational methods, AUR-112 has been identified as a potential development candidate. The allosteric inhibitor AUR-112 exhibited potent and selective inhibition of MALT1 protease activity in both biochemical and cellular assays. Inhibition of MALT1 correlated with potent antiproliferative activity in an ABC-DLBCL cell line. The mechanistic activity of AUR-112 is supported by the inhibition of IL-10 in a cell line and IL-2 in human whole blood. Consistent with our objective, AUR112 showed low plasma protein binding across multiple species, which translated to greater potency in whole blood compared to published MALT1 inhibitors, positioning it as a best-in-class contender. AUR-112 has excellent oral bioavailability across species and exhibited desirable selectivity profile in kinase, protease and CEREP safety screen panels. Oral administration of AUR-112 led to dose dependent anti-tumor activity including durable tumor regression in cell line and patient derived xenograft models. Treatment with AUR-112 led to potent pharmacodynamic modulation in xenograft models as measured by significant and sustained reduction in serum IL-10 levels that correlated with the compelling efficacy. AUR-112 also demonstrated dose dependent inhibition of T-reg cells in vitro and in vivo supporting the immune modulatory role of MALT1 inhibition. AUR-112 is identified as a development candidate with potentially best-in-class physico-chemical properties resulting in a high free fraction for treatment of ABC-DLBCL patients. IND enabling studies are currently ongoing to support the clinical development of AUR-112. Citation Format: Wesley Roy Balasubramanian, Sammeta Srinivasa Raju, Vasantha K.Y, Archana Bhumi Reddy, Janith Mary Maben, Sandeep Sadashiv Patil, Raghavendra N.R, Kiran Aithal B, Charamanna K.B, Dinesh Chikkanna, Kavitha Nellore, Samiulla D.S, Girish Daginakatte, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C127.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 inhibition alleviates intestinal impairment induced by chronic heat stress in finisher broilers

Heat stress (HS) in poultry has deleterious effects on intestinal development and barrier function, along with inflammatory outbursts. In the present study, chronic HS reduced body weight of broilers and activated mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) /nuclear factor kappa B (NF-κB) signaling pathways to elicit the inflammatory cytokine response in jejunum. Subsequently, this study investigated the protective effects of the Malt1 inhibitor on the intestine of broilers under HS conditions. The 21-day-old male broilers were allocated to 8 pens housed in HS room (34°C for 7 h/d) until 28 d of age. During this period, 4 birds were selected from each heat-stressed pen and received intraperitoneal injection of 20 mg/kg body weight Mepazine (a Malt1 inhibitor) or the equivalent volume of phosphate buffer saline (PBS) every other day. When compared to PBS broilers, birds received Mepazine injection exhibited increased relative weight and higher villus height in jejunum (both P < 0.05). Mepazine treatment also increased (P < 0.05) the mRNA of zonula occludens-1 (ZO-1), claudin-1, and cadherin 1 of jejunum, which was companied by the reduced caspase-3 transcription under HS condition. Meanwhile, the gene expression levels of toll-like receptor 4 (TLR4), Malt1, NF-κB, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the jejunum were significantly downregulated by Mepazine administration (P < 0.05). Although there were no significant differences in the relative weight of the thymus and bursa, the transcription levels of T helper 1 (Th1)- and Th17-related cytokines were lower in thymus of birds injected with Mepazine. The cytokines of Treg cytokine transforming growth factor beta (TGF-β) and forkhead box protein P3 (Foxp3) in both the thymus and bursa were not influenced. These results suggest that inhibition of Malt1 protease activity can protect intestinal integrity by promoting the production of tight junction proteins and attenuating NF-κB-mediated intestinal inflammation response under HS conditions.

CARD14 Signalling Ensures Cell Survival and Cancer Associated Gene Expression in Prostate Cancer Cells.

Prostate cancer (PCa) is one of the most common cancer types in men and represents an increasing global problem due to the modern Western lifestyle. The signalling adapter protein CARD14 is specifically expressed in epithelial cells, where it has been shown to mediate NF-κB signalling, but a role for CARD14 in carcinoma has not yet been described. By analysing existing cancer databases, we found that CARD14 overexpression strongly correlates with aggressive PCa in human patients. Moreover, we showed that CARD14 is overexpressed in the LNCaP PCa cell line and that knockdown of CARD14 severely reduces LNCaP cell survival. Similarly, knockdown of BCL10 and MALT1, which are known to form a signalling complex with CARD14, also induced LNCaP cell death. MALT1 is a paracaspase that mediates downstream signalling by acting as a scaffold, as well as a protease. Recent studies have already indicated a role for the scaffold function of MALT1 in PCa cell growth. Here, we also demonstrated constitutive MALT1 proteolytic activity in several PCa cell lines, leading to cleavage of A20 and CYLD. Inhibition of MALT1 protease activity did not affect PCa cell survival nor activation of NF-κB and JNK signalling, but reduced expression of cancer-associated genes, including the cytokine IL-6. Taken together, our results revealed a novel role for CARD14-induced signalling in regulating PCa cell survival and gene expression. The epithelial cell type-specific expression of CARD14 may offer novel opportunities for more specific therapeutic targeting approaches in PCa.

MALT1 Degradation with a Proteolysis-Targeting Chimera for the Treatment of Activated B-Cell Type Diffuse Large B-Cell Lymphoma

MALT1 Degradation with a Proteolysis-Targeting Chimera for the Treatment of Activated B-Cell Type Diffuse Large B-Cell Lymphoma

Cyclin D2 overexpression drives B1a-derived MCL-like lymphoma in mice.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Inhibitor as a Novel Therapeutic Tool for Lung Injury.

Pulmonary fibrosis is a progressive disease characterized by lung remodeling due to excessive deposition of extracellular matrix. In this study, the bleomycin experimental model of pulmonary fibrosis was employed to investigate the anti-fibrotic and immunomodulatory activity of the inhibition of MALT1 protease activity. Mice received a single intra-tracheal administration of bleomycin (1 mg/kg) in the presence or absence of MI-2, a selective MALT1 inhibitor, (a dose of 30 mg/kg administered intra-peritoneally 1 h after bleomycin and daily until the end of the experiment). Seven days after bleomycin instillation mice were sacrificed and bronchoalveolar lavage fluid analysis, measurement of collagen content in the lung, histology, molecular analysis and immunohistochemistry were performed. To evaluate mortality and body weight gain a subset of mice was administered daily with MI-2 for 21 days. Mice that received MI-2 showed decreased weight loss and mortality, inflammatory cells infiltration, cytokines overexpression and tissue injury. Moreover, biochemical and immunohistochemical analysis displayed that MI-2 was able to modulate the excessive production of reactive oxygen species and the inflammatory mediator upregulation induced by bleomycin instillation. Additionally, MI-2 demonstrated anti-fibrotic activity by reducing transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and receptor associated factor 6 (TRAF6) expression. The underlying mechanisms for the protective effect of MI-2 bleomycin induced pulmonary fibrosis may be attributed to its inhibition on NF-κB pathway. This is the first report showing the therapeutic role of MALT1 inhibition in a bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

Therapeutic Potential of Targeting Malt1-Dependent TCR Downstream Signaling to Promote the Survival of MHC-Mismatched Allografts.

Strategies targeting T cells are the cornerstone of immunosuppression after solid organ transplantation. The transcription factor NF-κB is a key regulator of downstream T-cell activation and induction of inflammatory mediators; its full activation via antigen receptor engagement requires both the scaffold and the protease activity of the paracaspase Malt1. Experimental studies have highlighted that Malt1-deficient mice were resistant to experimental autoimmune encephalomyelitis, although they lacked peripheral regulatory T cells (Treg). Here, we compared targeting Malt1 versus using calcineurin inhibitors as immunosuppression in a stringent experimental transplantation model. We found that Malt1-deficiency impaired Th1-mediated alloresponses in vitro and in vivo and significantly prolonged MHC-mismatched skin allograft survival, compared to cyclosporine. However, it paradoxically enhanced Th17 differentiation in the transplantation setting. Interestingly, more selective inhibition of Malt1 protease activity in wild-type mouse and human peripheral T cells in vitro led to attenuation of alloreactive Th1 cells, while preserving preexisting Treg in the peripheral T-cell pool, and without promoting Th17 differentiation. Thus, there is a place for further investigation of the role of Malt1 signaling in the setting of transplantation.

Pharmacological inhibition of MALT1 protease activity suppresses endothelial activation via enhancing MCPIP1 expression

Mucosa associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is not only an intracellular signaling scaffold protein but also a paracaspase that plays a key role in the signal transduction and cellular activation of lymphocytes and macrophages. However, its role in endothelial cells remains unknown. Here we report that pharmacological inhibition of MALT1 protease activity strongly suppresses endothelial activation via enhancing MCPIP1 expression. Treatment with MALT1 protease inhibitors selectively inhibited TNFα-induced VCAM-1 expression in HUVECs and LPS-induced VCAM-1 expression in mice. In addition, Inhibition of MALT1 protease activity also significantly inhibited TNFα-induced adhesion of THP-1 monocytic cells to HUVECs. To explore the mechanisms, MALT1 inhibitors does not affect the activation of NF-κB signaling pathway in HUVEC. However, they can stabilize MCPIP1 protein and significantly enhance MCPIP1 protein level in endothelial cells. These results suggest that MALT1 paracaspase also targets MCPIP1 and degrade MCPIP1 protein in endothelial cells similar as it does in immune cells. Taken together, the study suggest inhibition of MALT1 protease activity may represent a new strategy for prevention/therapy of vascular inflammatory diseases such as atherosclerosis.

Central role of myeloid MCPIP1 in protecting against LPS-induced inflammation and lung injury

Although systemic inflammatory responses attributable to infection may lead to significant lung injury, the precise molecular mechanisms leading to lung damage are poorly understood and therapeutic options remain limited. Here, we show that myeloid monocyte chemotactic protein-inducible protein 1 (MCPIP1) plays a central role in protecting against LPS-induced inflammation and lung injury. Myeloid-specific MCPIP1 knockout mice developed spontaneous inflammatory syndromes, but at a late age compared to global MCPIP1 knockout mice. Moreover, mice with a myeloid-specific deletion of MCPIP1 were extremely sensitive to LPS-induced lung injury due to overproduction of proinflammatory cytokines and chemokines. We identified C/EBPβ and C/EBPδ, two critical transcriptional factors that drive cytokine production and lung injury, as targets of MCPIP1 RNase. LPS administration caused MCPIP1 protein degradation in the lungs. Pharmacological inhibition of MALT1, a paracaspase that cleaves MCPIP1, by MI-2 selectively increased the MCPIP1 protein levels in macrophages and in the lungs. Meanwhile, administration of MI-2 protected mice from LPS-induced inflammation, lung injury and death. Collectively, these results indicate that myeloid MCPIP1 is central in controlling LPS-induced inflammation and lung injury. Pharmacological inhibition of MALT1 protease activity may be a good strategy to treat inflammatory diseases by enhancing MCPIP1 expression in myeloid cells.

Abstract 3011: Development of selective small-molecule inhibitors of cellular MALT1 protease activity

Abstract Background: The aim of this study was to develop potent, selective and cell-permeable inhibitors of MALT1 protease activity. MALT1 is an Arg-specific protease that cleaves multiple substrates, acting as a key component in T- and B-cell receptor signaling in lymphocytes. MALT1 protease activity is reported to mediate normal and disease-associated lymphocyte proliferation and survival as part of the CARMA-1/BCL-10/MALT1 (CBM) signaling complex, making MALT1 an attractive therapeutic target. Constitutive MALT1 activity is a hallmark of Diffuse Large B Cell Lymphoma of the activated B cell type (ABC-DLBCL). This aberrant activation is observed as a consequence of mutations in CARMA-1/CARD11, and in multiple regulatory proteins upstream of the CBM complex. Methods: MALT1 enzyme assay with Ac-Leu-Arg-Ser-Arg-AMC as substrate was used to measure Ki. Selectivity towards other Arg-specific proteases was measured by inhibition of Trypsin and Thrombin. For determination of cellular potency the ABC-DLBCL cell line OCI-LY3, carrying the L251P mutation of CARMA-1/CARD11, was used. A MALT1 cell assay to measure A20 substrate cleavage was adapted to capillary electrophoresis and quantification. MALT1 down-stream signaling was measured by inhibition of IL-6 expression. Results: Published inhibitors of MALT1 were profiled for potency, selectivity, DMPK properties and activity in cell-based assays, and showed several drawbacks such as low MALT1 activity, low cell permeability and/or selectivity. We developed several novel reversible small molecule inhibitors of MALT1 with Ki &amp;lt;20 nM and high selectivity against Trypsin and Thrombin. Compounds show inhibition of intracellular MALT1 activity with potencies &amp;lt;300nM in the A20 cleavage assay, as well as inhibition of IL-6 expression. Intracellular inhibition of MALT1 activity requires both potent inhibition of MALT1 and cell permeability (estimated by Caco-2 permeability), e.g. compound A with MALT1 Ki = 16nM, Trypsin Ki&amp;gt;100μM, Thrombin Ki&amp;gt;100μM, and Caco-2 Papp = 1×10−6cm/s shows intracellular A20 cleavage IC50 = 1100nM, whereas compound B with MALT1 Ki = 47nM and Caco-2 Papp = 5.6×10−6cm/s, displays intracellular A20 cleavage IC50 = 600nM. We observe a clear dissociation between intracellular inhibition of MALT1 and inhibition of cell proliferation of DLBCL cell lines of ABC-type with constitutive MALT1 activity, e.g. compound C (MALT1 Ki = 35nM, Trypsin Ki&amp;gt;100μM, Thrombin Ki&amp;gt;100μM and intracellular A20 cleavage IC50 = 260nM) inhibits the proliferation of OCI-LY3 cells and TMD8 cells with CC50 values of 73μM and 96μM respectively, after 5 days exposure. Conclusion: Inhibition of MALT1 protease activity did not translate into significant anti-proliferative effects in DLBCL cell lines. These new MALT1 inhibitors provide the opportunity to test other therapeutic hypotheses involving the targeting of MALT1 protease activity. Citation Format: Fredrik G. Oberg, Sofia Karlstrom, Ian Henderson, Ellen Hewitt, Anders Kallin, Susanne Sedig, Jimmy Lindberg, Anna-Karin Sohlenius-Sternbeck, Richard Bethell, Mark Albertella. Development of selective small-molecule inhibitors of cellular MALT1 protease activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3011.

MALT1 has both protease activity-dependent and -independent roles in innate and adaptive immunity (IRM10P.739)

Abstract Hetero-trimeric CARMA/BCL10/MALT1 CBM complexes play important roles in B and T cell antigen receptor signaling and other pathways. Previous studies have suggested that MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a cysteine protease to cleave and inactivate downstream signaling molecules such as A20 and CYLD. However, little is known about the relative importance of these two distinct activities in orchestrating immune responses. Utilizing mice homozygous for either null or protease-dead C461A mutations in MALT1, we found that some but not all MALT1 functions were dependent upon its protease activity. Although protease-dead mice had normal follicular B cell and T cell populations in the spleen, defects were observed in the generation of splenic marginal zone and peritoneal B1 B cells. Ex vivo, B cell receptor-stimulated proliferation was normal, but CD4+ and CD8+ T cells displayed decreased activation-induced proliferation and IL-2 production. C-type lectin receptor stimulation of cytokine production by dendritic cells was also found to be partially dependent upon protease activity. In vivo, protease-dead mice displayed severely reduced basal immunoglobulin levels, and defective T-dependent and T-independent immune responses. Our findings suggest that pharmacologic inhibition of MALT1 protease activity may be a useful therapeutic approach for diseases linked to uncontrolled immune cell activation.

Inhibition of MALT1 Protease Activity Is Selectively Toxic for Activated B CellÃ,–Like Diffuse Large B Cell Lymphoma Cells.

Abstract 1271Poster Board I-293Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell-like (ABC) subtype of DLBCL is characterized by constitutive NF-κB activity and requires signals from CARD11, BCL10 and the paracaspase MALT1 for survival. CARD11, BCL10 and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11/BCL10/MALT1 (CBM) complexes couple upstream events to IKK/NF-κB activation. MALT1 possesses in addition a recently recognized proteolytic activity that cleaves and inactivates the negative NF-κB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here we demonstrate pre-assembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL but not in germinal center B cell-like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-κB activity and decreases the expression of NF-κB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of MALT1 protease activity is selectively toxic for activated B cell–like diffuse large B cell lymphoma cells

Inhibition of MALT1 protease activity is selectively toxic for activated B cell–like diffuse large B cell lymphoma cells

Inhibition of MALT1 protease activity is selectively toxic for activated B cell–like diffuse large B cell lymphoma cells

Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell–like (ABC) subtype of DLBCL is characterized by constitutive NF-κB activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11–BCL10–MALT1 (CBM) complexes couple upstream events to IκB kinase (IKK)/NF-κB activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-κB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell–like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-κB activity, and decreases the expression of NF-κB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.