Daniel Nagel

Pharmacologic inhibition of MALT1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive ABC-DLBCL.

Proteolytic activity of the mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) paracaspase is required for survival of the activated B cell subtype of diffuse large B cell lymphoma (ABC-DLBCL). We have identified distinct derivatives of medicinal active phenothiazines, namely mepazine, thioridazine, and promazine, as small molecule inhibitors of the MALT1 protease. These phenothiazines selectively inhibit cleavage activity of recombinant and cellular MALT1 by a noncompetitive mechanism. Consequently, the compounds inhibit anti-apoptotic NF-κB signaling and elicit toxic effects selectively on MALT1-dependent ABC-DLBCL cells in vitro and in vivo. Our data provide a conceptual proof for a clinical application of distinct phenothiazines in the treatment of ABC-DLBCL.

Critical role of PI3K signaling for NF-κB–dependent survival in a subset of activated B-cell–like diffuse large B-cell lymphoma cells

The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.

NF-κB Essential Modulator (NEMO) Interaction with Linear and Lys-63 Ubiquitin Chains Contributes to NF-κB Activation

The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation. We show here that the NEMO C terminus, comprising the ubiquitin binding region and a zinc finger, has a high preference for binding to linear ubiquitin chains. However, immobilization of NEMO, which may be reminiscent of cellular oligomerization, facilitates the interaction with Lys-63 ubiquitin chains. Moreover, selective mutations in NEMO that abolish association with linear ubiquitin but do not affect binding to Lys-63 ubiquitin are only partially compromising NF-κB signaling in response to TNFα stimulation in fibroblasts and T cells. In line with this, TNFα-triggered expression of NF-κB target genes and induction of apoptosis was partially compromised by NEMO mutations that selectively impair the binding to linear ubiquitin chains. Thus, in vivo NEMO interaction with linear and Lys-63 ubiquitin chains is required for optimal IKK activation, suggesting that both type of chains are cooperating in triggering canonical NF-κB signaling.

Flrt Structure: Balancing Repulsion and Cell Adhesion in Cortical and Vascular Development

Summary FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.

Structural Analysis of Phenothiazine Derivatives as Allosteric Inhibitors of the MALT1 Paracaspase

The human mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is responsible for the survival, proliferation, and activation of B and T lymphocytes upon antigen stimulation by means of the canonical NF-kB signaling pathway. MALT1 is constitutively associated with BCL10 and assembles to form the CBM complex with CARMA1, CARMA3, or CARD9 upon activation by their individual receptors (CARMA1: CARD-containing membrane-associated guanylate kinase protein 1; CARD9: caspase-recruitment-domain-containing protein 9). In the activated CBM complex MALT1 acts as a scaffolding platform and promotes the recruitment of signaling factors like tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), transforming growth factor (TGF)-b-activated kinase (TAK1), and the regulatory subunit NEMO (NF-kB essential modulator) of the IKK complex (inhibitor of transcription factor NF-kB (IkB) kinase) which finally leads to IKK activation. Besides its scaffolding function, MALT1 confers proteolytic activity that is required for optimal T-cell activation. Cleavage of the MALT1 substrates BCL10, A20, CYLD, and RelB results in enhanced NF-kB as well as c-Jun N-terminal kinase (JNK) activation and controls T-cell adhesion. 6] Recent studies revealed that MALT1 is activated by ligand binding and dimerization of the paracaspase domain. However, spontaneous dimerization of MALT1 in vitro leads to a catalytically inactive conformation. Hence, an additional structural rearrangement is needed for MALT1 activation that is supported by monoubiquitination in the C-terminal Ig3 domain of MALT1. Modified activity of individual members of the CBM signaling complex is associated with lymphomagenesis. In this context the deregulated expression of CARMA1, BCL10, and MALT1 is critical for the survival of the activated B-cell subtype of diffuse-large B-cell lymphoma (ABC-DLBCL) and constitutive MALT1 paracaspase activity is a common feature of ABC-DLBCL cells. Moreover, two recent studies demonstrated the crucial role of MALT1 in the early phase of experimental autoimmune encephalomyelitis (EAE), the main animal model for multiple sclerosis (MS). These results suggest that MALT1 is an important therapeutic target to treat multiple sclerosis, the most common chronic inflammatory demyelinating disease of the human central nervous system. Thus, there is substantial interest in developing small-molecule compounds that specifically inhibit MALT1, a promising new approach for the treatment of MALT lymphoma, ABC-DLBCL, and multiple sclerosis. Recently, distinct phenothiazines have been identified as potent small-molecule inhibitors of MALT1 that selectively kill ABC-DLBCL in vitro and in vivo. The identified drugs, mepazine, thioridazine, and promazine, have a long clinical history as antipsychotics. Yet, it is still unclear how these substances inhibit MALT1. A detailed understanding of the binding mode is crucial to further optimize the efficacy and selectivity of these compounds for clinical use. Here, we report the crystal structure of ligand-free dimeric human MALT1Casp-Ig3 in complex with the tricyclic phenothiazine derivative thioridazine. Unexpectedly, the structure reveals that the inhibitor binds in a pocket located opposite to the caspase active site, in the interface between

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

BackgroundThe paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-κB. 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.MethodsThe 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.ResultsWe 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.ConclusionsAltogether, 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.

Activity-Based Probes for Detection of Active MALT1 Paracaspase in Immune Cells and Lymphomas

MALT1 paracaspase is activated upon antigen receptor stimulation to promote lymphocyte activation. In addition, deregulated MALT1 protease activity drives survival of distinct lymphomas such as the activated B cell type of diffuse large B cell lymphoma (ABC-DLBCL). Here, we designed fluorophore or biotin-coupled activity based-probes (ABP) that covalently modify the active center of MALT1. MALT1-ABPs are exclusively labeling an active modified full length form of MALT1 upon T cell stimulation. Further, despite the CARMA1 requirement for initial MALT1 activation, the MALT1-ABPs show that protease activity is not confined to the high-molecular CARMA1-BCL10-MALT1 (CBM) complex. Using biotin-coupled ABPs, we developed a robust assay for sensitive and selective detection of active MALT1 in cell lines, primary lymphocytes, and DLBCL tumor biopsies. Taken together, MALT1-ABPs represent powerful chemical tools to measure cellular MALT1 activation, determine efficacy of small molecule inhibitors, and classify lymphomas based on MALT1 activity status.

Inhibition of Canonical NF-κB Signaling by a Small Molecule Targeting NEMO-Ubiquitin Interaction.

The IκB kinase (IKK) complex acts as the gatekeeper of canonical NF-κB signaling, thereby regulating immunity, inflammation and cancer. It consists of the catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO/IKKγ. Here, we show that the ubiquitin binding domain (UBAN) in NEMO is essential for IKK/NF-κB activation in response to TNFα, but not IL-1β stimulation. By screening a natural compound library we identified an anthraquinone derivative that acts as an inhibitor of NEMO-ubiquitin binding (iNUB). Using biochemical and NMR experiments we demonstrate that iNUB binds to NEMOUBAN and competes for interaction with methionine-1-linked linear ubiquitin chains. iNUB inhibited NF-κB activation upon UBAN-dependent TNFα and TCR/CD28, but not UBAN-independent IL-1β stimulation. Moreover, iNUB was selectively killing lymphoma cells that are addicted to chronic B-cell receptor triggered IKK/NF-κB activation. Thus, iNUB disrupts the NEMO-ubiquitin protein-protein interaction interface and thereby inhibits physiological and pathological NF-κB signaling.

Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma

Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances.

Heterogenous nuclear ribonucleoprotein Q increases protein expression from HIV-1 Rev-dependent transcripts.

BackgroundHeterogenous nuclear ribonucleoproteins (hnRNPs) control many processes of the gene expression machinery including mRNA transcription, splicing, export, stability and translation. Recent data show interaction of the HIV-1 Rev regulatory protein with a subset of hnRNP proteins, that includes hnRNP Q, suggesting that hnRNPs can contribute to regulation of HIV-1 gene expression by Rev.FindingsIn this work we address the effect of hnRNP Q on Rev-dependent gene expression. We show that hnRNP Q overexpression increased levels of proteins produced from a Rev-dependent reporter gene in the presence of Rev. Increased protein levels did not correlate with changes in either the levels or the nucleocytoplasmic distribution of Rev-dependent reporter mRNAs. Similar observations were made in persistently HIV-1 infected HeLa cells. In these cells, hnRNP Q overexpression increased levels of the HIV-1 Gag-p24 protein, while levels of viral Rev-dependent mRNAs were not affected.ConclusionOur data indicate that hnRNP Q can stimulate the protein production of Rev-dependent mRNAs without changing mRNA levels and mRNA export, respectively. This suggests that hnRNP Q can boost HIV gene expression at the level of protein production.