RESUMEN
Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19-dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.
Asunto(s)
Ensamble y Desensamble de Cromatina , Sacarosa , Animales , Cromatina/genética , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Homeostasis , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Neoplasias Colorrectales/enzimología , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Complejo Mediador/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sitios de Unión , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Quinasa 8 Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Masculino , Complejo Mediador/antagonistas & inhibidores , Complejo Mediador/genética , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcripción Genética , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Wnt/ß-catenin signaling pathway exerts integral roles in embryogenesis and adult homeostasis. Aberrant activation of the pathway is implicated in growth-associated diseases and cancers, especially as a key driver in the initiation and progression of colorectal cancer (CRC). Loss or inactivation of Adenomatous polyposis coli (APC) results in constitutive activation of Wnt/ß-catenin signaling, which is considered as an initiating event in the development of CRC. Increased Wnt/ß-catenin signaling is observed in virtually all CRC patients, underscoring the importance of this pathway for therapeutic intervention. Prior studies have deciphered the regulatory networks required for the cytoplasmic stabilisation or degradation of the Wnt pathway effector, ß-catenin. However, the mechanism whereby nuclear ß-catenin drives or inhibits expression of Wnt target genes is more diverse and less well characterised. Here, we describe a brief synopsis of the core canonical Wnt pathway components, set the spotlight on nuclear mediators and highlight the emerging role of chromatin regulators as modulators of ß-catenin-dependent transcription activity and oncogenic output.
Asunto(s)
Carcinogénesis/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Transcripción Genética , Proteínas Wnt/genética , beta Catenina/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Carcinogénesis/metabolismo , Carcinogénesis/patología , Cromatina/química , Cromatina/metabolismo , Cromatina/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Epigénesis Genética , Histonas/genética , Histonas/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transducción de Señal , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.
Asunto(s)
Caspasa 8/metabolismo , Inestabilidad Cromosómica , Neoplasias del Colon/enzimología , Fibroblastos/enzimología , Mitosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Aneuploidia , Animales , Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Fibroblastos/patología , Células HT29 , Humanos , Inflamación/enzimología , Inflamación/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal , Quinasa Tipo Polo 1RESUMEN
CDK8 and its paralog, CDK19, collectively termed 'Mediator Kinase,' are cyclin-dependent kinases that have been implicated as key rheostats in cellular homeostasis and developmental programming. CDK8 and CDK19 are incorporated, in a mutually exclusive manner, as part of a 4-protein complex called the Mediator kinase module. This module reversibly associates with the Mediator, a 26 subunit protein complex that regulates RNA Polymerase II mediated gene expression. As part of this complex, the Mediator kinases have been implicated in diverse process such as developmental signaling, metabolic homeostasis and in innate immunity. In recent years, dysregulation of Mediator kinase module proteins, including CDK8/19, has been implicated in the development of different human diseases, and in particular cancer. This has led to intense efforts to understand how CDK8/19 regulate diverse biological outputs and develop Mediator kinase inhibitors that can be exploited therapeutically. Herein, we review both context and function of the Mediator kinases at a molecular, cellular and animal level. In so doing, we illuminate emerging concepts underpinning Mediator kinase biology and highlight certain aspects that remain unsolved.
RESUMEN
Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células de Paneth/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Apoptosis/genética , Células Cultivadas , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIB/genéticaRESUMEN
Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.