RESUMEN
The interdependence of p53 and MDM2 is critical for proper cell survival and cell death and, when altered, can lead to tumorigenesis. Mitogen-activated protein kinase (MAPK) signaling pathways function in a wide variety of cellular processes, including cell growth, migration, differentiation, and death. Here we discovered that transforming growth factor ß-activated kinase 1 (TAK1)-binding protein 1 (TAB1), an activator of TAK1 and of p38α, associates with and inhibits the E3 ligase activity of MDM2 toward p53 and its homolog, MDMX. Depletion of TAB1 inhibits MDM2 siRNA-mediated p53 accumulation and p21 induction, partially rescuing cell cycle arrest induced by MDM2 ablation. Interestingly, of several agents commonly used as DNA-damaging therapeutics, only cell death caused by cisplatin is mitigated by knockdown of TAB1. Two mechanisms are required for TAB1 to regulate apoptosis in cisplatin-treated cells. First, p38α is activated by TAB1 to phosphorylate p53 N-terminal sites, leading to selective induction of p53 targets such as NOXA. Second, MDMX is stabilized in a TAB1-dependent manner and is required for cell death after cisplatin treatment. Interestingly TAB1 levels are relatively low in cisplatin-resistant clones of ovarian cells and in ovarian patient's tumors compared with normal ovarian tissue. Together, our results indicate that TAB1 is a potential tumor suppressor that serves as a functional link between p53-MDM2 circuitry and a key MAPK signaling pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Oncogénicas/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Fosforilación , Proteolisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
Germline mutation of the BRCA1 associated protein-1 (BAP1) gene has been linked to uveal melanoma, mesothelioma, meningioma, renal cell carcinoma and basal cell carcinoma. Germline variants have also been found in familial cutaneous melanoma pedigrees, but their contribution to sporadic melanoma has not been fully assessed. We sequenced BAP1 in 1,977 melanoma cases and 754 controls and used deubiquitinase assays, a pedigree analysis, and a histopathological review to assess the consequences of the mutations found. Sequencing revealed 30 BAP1 variants in total, of which 27 were rare (ExAc allele frequency <0.002). Of the 27 rare variants, 22 were present in cases (18 missense, one splice acceptor, one frameshift and two near splice regions) and five in controls (all missense). A missense change (S98R) in a case that completely abolished BAP1 deubiquitinase activity was identified. Analysis of cancers in the pedigree of the proband carrying the S98R variant and in two other pedigrees carrying clear loss-of-function alleles showed the presence of BAP1-associated cancers such as renal cell carcinoma, mesothelioma and meningioma, but not uveal melanoma. Two of these three probands carrying BAP1 loss-of-function variants also had melanomas with histopathological features suggestive of a germline BAP1 mutation. The remaining cases with germline mutations, which were predominantly missense mutations, were associated with less typical pedigrees and tumours lacking a characteristic BAP1-associated histopathological appearances, but may still represent less penetrant variants. Germline BAP1 alleles defined as loss-of-function or predicted to be deleterious/damaging are rare in cutaneous melanoma.
Asunto(s)
Melanoma/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Adulto , Proteína BRCA1/genética , Femenino , Mutación del Sistema de Lectura , Predisposición Genética a la Enfermedad , Variación Genética , Genética de Población , Mutación de Línea Germinal/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Melanoma/metabolismo , Mutación Missense , Linaje , Neoplasias Cutáneas/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Neoplasias de la Úvea/genética , Melanoma Cutáneo MalignoRESUMEN
MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Ribosómicas/fisiología , Animales , Sitios de Unión , Línea Celular , Regulación hacia Abajo , Humanos , Ratones , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Transducción de Señal , Estrés Fisiológico , Proteína p53 Supresora de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).
Asunto(s)
Neoplasias , Peptidasa Específica de Ubiquitina 7 , Factor A de Crecimiento Endotelial Vascular , Humanos , Proteínas Potenciadoras de Unión a CCAAT/farmacología , Fibroblastos/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Microambiente Tumoral , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidoresRESUMEN
More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin-proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials. However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging. In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration.
Asunto(s)
Enzimas Desubicuitinizantes/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Ubiquitina/metabolismo , Descubrimiento de Drogas/tendencias , Industria Farmacéutica , Drogas en Investigación/uso terapéutico , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Deubiquitylating enzymes or DUBs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. Approximately 100 DUBs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. Several aspects of DUB biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. However, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. A comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. Activity probes have been used to profile classes of enzymes, including DUBs, and allow functional and dynamic properties to be assigned to individual proteins. The ability to directly monitor DUB activity within a native biological system is essential for understanding the physiological and pathological role of individual DUBs. We will discuss the evolution of DUB activity probes, from in vitro assay development to their use in monitoring DUB activity in cells and in animal tissues, as well as recent progress and prospects for assessing DUB inhibition in vivo.
Asunto(s)
Proteómica/métodos , Ubiquitina/metabolismo , Animales , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Activación Enzimática/genética , Activación Enzimática/fisiología , Humanos , Procesamiento Proteico-Postraduccional/genética , Ubiquitina/genéticaRESUMEN
The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors.
Asunto(s)
Ubiquitina/metabolismo , Animales , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Humanos , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activity-based profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.
Asunto(s)
Sondas Moleculares/química , Neoplasias/enzimología , Proteómica/métodos , Pirroles/química , Bibliotecas de Moléculas Pequeñas/química , Proteasas Ubiquitina-Específicas/análisis , Alquinos/química , Línea Celular Tumoral , Humanos , Técnicas de Sonda Molecular , Proteasas Ubiquitina-Específicas/antagonistas & inhibidoresRESUMEN
Changes to the nucleolus, the site of ribosome production, have long been linked to cancer, and mutations in several ribosomal proteins (RPs) have been associated with an increased risk for cancer in human diseases. Relevantly, a number of RPs have been shown to bind to MDM2 and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and cell cycle arrest, thus revealing a RP-Mdm2-p53 signaling pathway that is critical for ribosome biogenesis surveillance. Here, we have identified RPL37, RPS15, and RPS20 as RPs that can also bind Mdm2 and activate p53. We found that each of the aforementioned RPs, when ectopically expressed, can stabilize both co-expressed Flag-tagged Mdm2 and HA-tagged p53 in p53-null cells as well as endogenous p53 in a p53-containing cell line. For each RP, the mechanism of Mdm2 and p53 stabilization appears to be through inhibiting the E3 ubiquitin ligase activity of Mdm2. Interestingly, although they are each capable of inducing cell death and cell cycle arrest, these RPs differ in the p53 target genes that are regulated upon their respective introduction into cells. Furthermore, each RP can downregulate MdmX levels but in distinct ways. Thus, RPL37, RPS15 and RPS20 regulate the Mdm2-p53-MdmX network but employ different mechanisms to do so.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ciclo Celular , Línea Celular Tumoral , Humanos , Inmunoprecipitación , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Proteína p53 Supresora de Tumor/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to regulate myriad intra-cellular processes. The addition and removal of ubiquitin or ubiquitin-like proteins from factors has recently been demonstrated as a key mechanism to modulate DNA damage response (DDR) pathways. It is thus, timely to evaluate the potential for ubiquitin pathway enzymes as DDR drug targets for therapeutic intervention. The synthetic lethal approach provides exciting opportunities for the development of targeted therapies to treat cancer: most tumours have lost critical DDR pathways, and thus rely more heavily on the remaining pathways, while normal tissues are still equipped with all DDR pathways. Here, we review key deubiquitylating enzymes (DUBs) involved in DDR pathways, and describe how targeting DUBs may lead to selective therapies to treat cancer patients.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteasas Ubiquitina-Específicas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Estrés Oxidativo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Morfolinas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Animales , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada , Cristalografía por Rayos X , Descubrimiento de Drogas , Femenino , Células HeLa , Humanos , Ratones , Modelos Moleculares , Morfolinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
High-throughput screening highlighted 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile (1) as an active inhibitor of ubiquitin-specific proteases (USPs), a family of hydrolytic enzymes involved in the removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1, led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Indenos/química , Pirazinas/química , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Indenos/farmacología , Conformación Molecular , Pirazinas/síntesis química , Pirazinas/farmacología , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.
Asunto(s)
Indenos/farmacología , Inhibidores de Proteasas/farmacología , Pirazinas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos/fisiología , Animales , Sitios de Unión/fisiología , Caenorhabditis elegans , Compartimento Celular/fisiología , Línea Celular Tumoral , Evolución Molecular , Humanos , Conformación Molecular , Mutación/genética , Fosforilación , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.