RESUMEN
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.
Asunto(s)
Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , FN-kappa B/metabolismo , Linfocitos T/inmunología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Inmunomodulación , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Proteolisis , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.
Asunto(s)
Antineoplásicos/farmacología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Piperidinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Humanos , Células Jurkat , Microsomas/efectos de los fármacos , Estructura Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Piperidinas/síntesis química , Piperidinas/química , Proteolisis/efectos de los fármacos , Ratas , Relación Estructura-ActividadRESUMEN
The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.
Asunto(s)
Linfocitos B Reguladores/inmunología , Caspasas/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos B Reguladores/patología , Caspasas/genética , Diferenciación Celular/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Humanos , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Ratones , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Linfocitos T Reguladores/patología , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/ß sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8 + T cell clonal expansion (CD8 EXP ), independent of HLA mismatch or IS type. Subcloning of TCRα/ß cDNAs from CD8 EXP into Jurkat76 cells (TCR -/- ) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8 EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8 EXP , while CD8 EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples. Overall, our data define the clonal CD8 + T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.
RESUMEN
Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/ß sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/ß cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.
Asunto(s)
Trasplante de Riñón , Transcriptoma , Receptores de Antígenos de Linfocitos T alfa-beta/genética , ARN , Aloinjertos , Rechazo de Injerto/genéticaRESUMEN
We developed a bioinformatics-led substrate discovery workflow to expand the known substrate repertoire of MALT1. Our approach, termed GO-2-Substrates, integrates protein function information, including GO terms from known substrates, with protein sequences to rank substrate candidates by similarity. We applied GO-2-Substrates to MALT1, a paracaspase and master regulator of NF-κB signalling in adaptive immune responses. With only 12 known substrates, the evolutionarily conserved paracaspase functions and phenotypes of Malt1 -/- mice strongly implicate the existence of undiscovered substrates. We tested the ranked predictions from GO-2-Substrates of new MALT1 human substrates by co-expression of candidates transfected with the oncogenic constitutively active cIAP2-MALT1 fusion protein or CARD11/BCL10/MALT1 active signalosome. We identified seven new MALT1 substrates by the co-transfection screen: TANK, TAB3, CASP10, ZC3H12D, ZC3H12B, CILK1 and ILDR2. Using catalytically inactive cIAP2-MALT1 (Cys464Ala), a MALT1 inhibitor, MLT-748, and noncleavable P1-Arg to Ala mutant versions of each substrate in dual transfections, we validated the seven new substrates in vitro. We confirmed the cleavage of endogenous TANK and the RNase ZC3H12D in B cells by Western blotting and mining TAILS N-terminomics datasets, where we also uncovered evidence for these and 12 other candidate substrates by endogenous MALT1. Thus, protein function information improves substrate predictions. The new substrates and other high-ranked MALT1 candidate substrates should open new biological frontiers for further validation and exploration of the function of MALT1 within and beyond NF-κB regulation.
RESUMEN
Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D(254)G and K(325)N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.
Asunto(s)
Convertasas de Complemento C3-C5/fisiología , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Síndrome Hemolítico-Urémico/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Niño , Preescolar , Estudios de Cohortes , Activación de Complemento/genética , Convertasas de Complemento C3-C5/genética , Proteínas del Sistema Complemento/genética , Familia , Femenino , Síndrome Hemolítico-Urémico/inmunología , Síndrome Hemolítico-Urémico/metabolismo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Modelos Moleculares , Proteínas Mutantes/fisiología , Linaje , Adulto JovenRESUMEN
Genetic studies have shown that mutations of complement inhibitors such as membrane cofactor protein, Factors H, I, or B and C3 predispose patients to atypical hemolytic uremic syndrome (aHUS). Factor I is a circulating serine protease that inhibits complement by degrading C3b and up to now only a few mutations in the CFI gene have been characterized. In a large cohort of 202 patients with aHUS, we identified 23 patients carrying exonic mutations in CFI. Their overall clinical outcome was unfavorable, as half died or developed end-stage renal disease after their first syndrome episode. Eight patients with CFI mutations carried at least one additional known genetic risk factor for aHUS, such as a mutation in MCP, CFH, C3 or CFB; a compound heterozygous second mutation in CFI; or mutations in both the MCP and CFH genes. Five patients exhibited homozygous deletion of the Factor H-related protein 1 (CFHR-1) gene. Ten patients with aHUS had one mutation in their CFI gene (Factor I-aHUS), resulting in a quantitative or functional Factor I deficiency. Patients with a complete deletion of the CFHR-1 gene had a significantly higher risk of a bad prognosis compared with those with one Factor I mutation as their unique vulnerability feature. Our results emphasize the necessity of genetic screening for all susceptibility factors in patients with aHUS.
Asunto(s)
Fibrinógeno/genética , Síndrome Hemolítico-Urémico/genética , Mutación , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Adulto JovenRESUMEN
A series of novel benzoxazole derivatives has been designed and shown to exhibit attractive JAK2 inhibitory profiles in biochemical and cellular assays, capable of delivering compounds with favorable PK properties in rats. Synthesis and structure-activity relationship data are also provided.
Asunto(s)
Benzoxazoles/química , Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Administración Oral , Animales , Benzoxazoles/síntesis química , Benzoxazoles/farmacocinética , Janus Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
We have designed and synthesized a novel series of 2,8-diaryl-quinoxalines as Janus kinase 2 inhibitors. Many of the inhibitors show low nanomolar activity against JAK2 and potently suppress proliferation of SET-2 cells in vitro. In addition, compounds from this series have favorable rat pharmacokinetic properties suitable for in vivo efficacy evaluation.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinoxalinas/química , Quinoxalinas/farmacología , Administración Oral , Animales , Línea Celular , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacocinética , Quinoxalinas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.
Asunto(s)
Sangre/metabolismo , Inhibidores de Caspasas/uso terapéutico , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Urea/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/metabolismo , Inhibidores de Caspasas/farmacocinética , Línea Celular Tumoral , Femenino , Semivida , Humanos , Ratones Endogámicos BALB C , Ratones SCID , Microsomas Hepáticos/metabolismo , Neoplasias/tratamiento farmacológico , Pirazoles/síntesis química , Pirazoles/metabolismo , Pirazoles/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratas Sprague-Dawley , Ovinos , Urea/síntesis química , Urea/metabolismo , Urea/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.
Asunto(s)
Inhibidores de Caspasas/uso terapéutico , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Urea/análogos & derivados , Urea/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/farmacología , Descubrimiento de Drogas , Femenino , Humanos , Inmunidad Humoral/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Urea/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Receptores de IgG/inmunología , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Plaquetas/metabolismo , Citocinas/inmunología , Modelos Animales de Enfermedad , Humanos , Ratones , Células Mieloides/metabolismoRESUMEN
Tumour necrosis factor receptor associated factor 4 (TRAF4) is a member of the TRAF family of proteins which are cytoplasmic adaptor molecules strongly implicated in multiple immune functions. A previous investigation of TRAF4 biological functions by gene targeting in mice has shown a role for TRAF4 in embryonic development and neurulation in vivo. However, unlike other TRAF family members, the role of TRAF4 in the immune system is still unknown. To address this question, we performed an extensive characterization of the immune development and immune functions of TRAF4-deficient mice. Our analyses did not reveal any defects in development of T and B lymphocytes, granulocytes, macrophages and dendritic cells, and no defects in reactive oxygen species production and phagocytosis by neutrophils. Cellular and humoral responses against T-cell-dependent antigens were normal, as was dendritic cell maturation in response to microbial components and antigen uptake by dendritic cells. However, we demonstrated that dendritic cells from TRAF4-deficient mice exhibited reduced migration both in transwell experiments and in vivo. These results suggest that TRAF4 is not strictly required for immune development and functions but could participate in immune functions by facilitating immune cell migration.
Asunto(s)
Células Dendríticas/inmunología , Factor 4 Asociado a Receptor de TNF/inmunología , Animales , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/inmunología , Proliferación Celular , Células Cultivadas , Citocinas/biosíntesis , Sistema Inmunológico/crecimiento & desarrollo , Inmunidad Celular , Inmunoglobulina G/biosíntesis , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Ovalbúmina/inmunología , Toxinas Shiga/inmunología , Subgrupos de Linfocitos T/inmunología , Factor 4 Asociado a Receptor de TNF/deficienciaRESUMEN
The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.
Asunto(s)
Caspasas/metabolismo , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/metabolismo , Linfocitos T/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Western Blotting , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasas/genética , Línea Celular , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Células HEK293 , Humanos , Immunoblotting , Células Jurkat , Linfocitos/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Mutagénesis , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/genética , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Interleukin-2 (IL-2) immunotherapy is an attractive approach in treating advanced cancer. However, by binding to its IL-2 receptor α (CD25) subunit, IL-2 exerts unwanted effects, including stimulation of immunosuppressive regulatory T cells (Tregs) and contribution to vascular leak syndrome. We used a rational approach to develop a monoclonal antibody to human IL-2, termed NARA1, which acts as a high-affinity CD25 mimic, thereby minimizing association of IL-2 with CD25. The structure of the IL-2-NARA1 complex revealed that NARA1 occupies the CD25 epitope of IL-2 and precisely overlaps with CD25. Association of NARA1 with IL-2 occurs with 10-fold higher affinity compared to CD25 and forms IL-2/NARA1 complexes, which, in vivo, preferentially stimulate CD8+ T cells while disfavoring CD25+ Tregs and improving the benefit-to-adverse effect ratio of IL-2. In two transplantable and one spontaneous metastatic melanoma model, IL-2/NARA1 complex immunotherapy resulted in efficient expansion of tumor-specific and polyclonal CD8+ T cells. These CD8+ T cells showed robust interferon-γ production and expressed low levels of exhaustion markers programmed cell death protein-1, lymphocyte activation gene-3, and T cell immunoglobulin and mucin domain-3. These effects resulted in potent anticancer immune responses and prolonged survival in the tumor models. Collectively, our data demonstrate that NARA1 acts as a CD25-mimobody that confers selectivity and increased potency to IL-2 and warrant further assessment of NARA1 as a therapeutic.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoterapia/métodos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Interleucina-2/antagonistas & inhibidores , Neoplasias/terapia , Animales , Sitios de Unión , Linfocitos T CD8-positivos/citología , Proliferación Celular , Epítopos/química , Silenciador del Gen , Humanos , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/inmunología , Conformación Proteica , Recombinación Genética , Linfocitos T Citotóxicos/citología , Linfocitos T Reguladores/inmunologíaRESUMEN
Metastatic Lymph Node 51 (MLN51) cDNA was isolated by differential screening of a human breast cancer metastasis cDNA library. MLN51 cDNA encodes a novel human protein of 703 residues that shares no significant homology to any known protein. However MLN51 is well conserved between vertebrate and invertebrate species suggesting an important biological function. The amino terminal half of the protein contains a coiled-coil domain and two potential nuclear localization signals (NLS). The carboxy terminal half contains one SH2 and four SH3 binding motifs. The coiled-coil domain promotes MLN51 oligomerization in transfected cells. When transiently expressed, the MLN51 protein is mainly found in the cytoplasm with a weak nuclear staining. However, deletion of the carboxy terminal half of the protein allows the targeting of the protein to the nucleus, demonstrating that the NLSs are functional. MLN51 is ubiquitously expressed in normal tissues. Human breast carcinomas show MLN51 overexpression in malignant epithelial cells. The uncommon association of protein-protein interaction domains often found either in nuclear or in cytoplasmic signaling proteins raises a possible nucleo-cytoplasmic function for MLN51.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 17/genética , Clonación Molecular , Citoplasma/metabolismo , Cartilla de ADN/química , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ganglios Linfáticos/patología , Metástasis Linfática , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Pruebas de Precipitina , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND: The JAK-STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. Dysregulated JAK-STAT signaling has been implicated in the pathogenesis of multiple human malignancies. OBJECTIVE: Given this pivotal role of JAK-STAT dysregulation, it is important to identify patients with an overactive JAK-STAT pathway for possible treatment with JAK inhibitors. METHODS: We developed a gene signature assay to detect overactive JAK-STAT signaling. The cancer cell line encyclopedia and associated gene-expression data were used to correlate the activation status of STAT5 with the induction of a set of STAT5 target genes. RESULTS: Four target genes were identified (PIM1, CISH, SOCS2, and ID1), the expression of which correlated significantly with pSTAT5 status in 40 hematologic tumor cell lines. In pSTAT5-positive models, the expression of the gene signature genes decreased following ruxolitinib treatment, which corresponded to pSTAT5 downmodulation. In pSTAT5-negative cell lines, neither pSTAT5 modulation nor a change in signature gene expression was observed following ruxolitinib treatment. CONCLUSIONS: The gene signature can potentially be used to stratify or enrich for patient populations with activated JAK-STAT5 signaling that might benefit from treatments targeting JAK-STAT signaling. Furthermore, the 4-gene signature is a predictor of the pharmacodynamic effects of ruxolitinib.
Asunto(s)
Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Animales , Línea Celular Tumoral , Femenino , Neoplasias Hematológicas/tratamiento farmacológico , Xenoinjertos , Humanos , Quinasas Janus/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Nitrilos , Pirazoles/farmacología , Pirimidinas , Transducción de Señal/efectos de los fármacosRESUMEN
Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.
Asunto(s)
Caspasas/genética , Síndromes de Inmunodeficiencia/genética , Linfocitos/inmunología , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Ubiquitina-Proteína Ligasas/metabolismo , Adolescente , Adulto , Animales , Células Presentadoras de Antígenos , Linfocitos B/inmunología , Caspasas/inmunología , Caspasas/metabolismo , Familia , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Activadoras de GTPasa/metabolismo , Técnicas de Sustitución del Gen , Humanos , Quinasa I-kappa B/metabolismo , Immunoblotting , Síndromes de Inmunodeficiencia/inmunología , Inmunoprecipitación , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucocitos Mononucleares , Masculino , Espectrometría de Masas , Ratones , Microscopía Confocal , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Mutación , FN-kappa B/inmunología , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Tonsila Palatina , Proteómica , Proteínas de Unión al ARN/metabolismo , Linfocitos T/inmunología , Espectrometría de Masas en Tándem , Factores de Transcripción , Ubiquitinación/inmunologíaRESUMEN
Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited.