RESUMEN
The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody-drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Dipéptidos/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Inmunoconjugados/farmacología , Proteolisis/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Dipéptidos/síntesis química , Dipéptidos/farmacocinética , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/farmacocinética , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Oxidorreductasas/inmunología , Células PC-3 , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFß pseudo-receptor BAMBI.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Oncogenes/genética , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Femenino , Edición Génica , Regulación Neoplásica de la Expresión Génica , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their inâ vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low inâ vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained inâ vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor inâ vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.
Asunto(s)
Anticuerpos Monoclonales/química , Proteínas de Ciclo Celular/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Inmunoconjugados/química , Factores de Transcripción/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Femenino , Semivida , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Ratones SCID , Unión Proteica , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Mitogénicos/inmunología , Resonancia por Plasmón de Superficie , Factores de Transcripción/antagonistas & inhibidores , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.
Asunto(s)
Carcinogénesis/metabolismo , Histonas/metabolismo , Células Mieloides/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Arginasa/genética , Arginasa/metabolismo , Línea Celular Tumoral , Células Cultivadas , Elementos de Facilitación Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Regiones Promotoras Genéticas , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Factores de Transcripción p300-CBP/químicaRESUMEN
BACKGROUND: Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed. RESULTS: In this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We find that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks. Lastly, we also find that single nucleotide polymorphisms located in protospacer regions can impair on-target activity as a result of mismatch tolerance. CONCLUSION: We show the impact of multi-target effects on estimating cancer cell dependencies and the impact of off-target effects caused by mismatch tolerance in sgRNA-DNA binding.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Marcación de Gen , Genómica/métodos , ARN Guía de Kinetoplastida , Línea Celular Tumoral , Humanos , Cadenas Ligeras de Miosina/genética , Neoplasias/genética , Factor de Transcripción SOX9/genética , Factores de Transcripción SOXE/genéticaRESUMEN
Acetylation of histone H3 at lysine 27 is a well-defined marker of enhancer activity. However, the functional impact of this modification at enhancers is poorly understood. Here, we use a chemical genetics approach to acutely block the function of the cAMP response element binding protein (CREB) binding protein (CBP)/P300 bromodomain in models of hematological malignancies and describe a consequent loss of H3K27Ac specifically from enhancers, despite the continued presence of CBP/P300 at chromatin. Using this approach to dissect the role of H3K27Ac at enhancers, we identify a critical role for this modification in the production of enhancer RNAs and transcription of enhancer-regulated gene networks.
Asunto(s)
Elementos de Facilitación Genéticos , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Neoplásico/genética , Factores de Transcripción p300-CBP/genética , Acetilación , Sitios de Unión , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Histonas/genética , Humanos , Lisina/metabolismo , Unión Proteica , Dominios Proteicos , ARN Neoplásico/metabolismo , Transcripción Genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.
Asunto(s)
Antineoplásicos/farmacología , Proteína de Unión a CREB/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Proteína de Unión a CREB/química , Perros , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacocinética , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacocinética , ARN/genética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Resistance invariably develops to antiandrogen therapies used to treat newly diagnosed prostate cancers, but effective treatments for castration-resistant disease remain elusive. Here, we report that the transcriptional coactivator CBP/p300 is required to maintain the growth of castration-resistant prostate cancer. To exploit this vulnerability, we developed a novel small-molecule inhibitor of the CBP/p300 bromodomain that blocks prostate cancer growth in vitro and in vivo Molecular dissection of the consequences of drug treatment revealed a critical role for CBP/p300 in histone acetylation required for the transcriptional activity of the androgen receptor and its target gene expression. Our findings offer a preclinical proof of concept for small-molecule therapies to target the CBP/p300 bromodomain as a strategy to treat castration-resistant prostate cancer. Cancer Res; 77(20); 5564-75. ©2017 AACR.
Asunto(s)
Proteína p300 Asociada a E1A/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Proteína p300 Asociada a E1A/deficiencia , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones SCID , Terapia Molecular Dirigida , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Dominios Proteicos , Distribución Aleatoria , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Pirazoles/farmacología , Piridonas/farmacología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Células de Riñón Canino Madin Darby , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Piridonas/síntesis química , Piridonas/química , Relación Estructura-ActividadRESUMEN
Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network.
Asunto(s)
Antineoplásicos/farmacología , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Factores Reguladores del Interferón/metabolismo , Mieloma Múltiple/fisiopatología , Fragmentos de Péptidos/antagonistas & inhibidores , Sialoglicoproteínas/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , HumanosRESUMEN
The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Cinetocoros/fisiología , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Complejos Multiproteicos/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente , Componentes del Gen , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , FosforilaciónRESUMEN
Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.
Asunto(s)
Carcinogénesis/genética , Aberraciones Cromosómicas , Reordenamiento Génico , Genómica/métodos , Animales , Línea Celular Tumoral , Centrómero/genética , Técnica del Anticuerpo Fluorescente , Inestabilidad Genómica , Hibridación Fluorescente in Situ , Ratones , Mitosis/genética , Translocación GenéticaRESUMEN
Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex. Prior work has identified more than 100 different kinetochore components in human cells. However, little is known about the regulatory processes that specify their assembly upon mitotic entry and disassembly at mitotic exit. In this paper, we used a live-cell imaging-based assay to quantify kinetochore disassembly kinetics and systematically analyze the role of potential regulatory mechanisms in controlling kinetochore assembly state. We find that kinetochore assembly and disassembly was driven primarily by mitotic phosphorylation downstream of cyclin-dependent kinase (CDK). In addition, we demonstrate that nuclear exclusion of the Ndc80 complex helped restrict kinetochore formation to mitosis. Combining constitutive CDK-dependent phosphorylation of CENP-T and forced nuclear localization of the Ndc80 complex partially prevented kinetochore disassembly at mitotic exit and led to chromosome segregation defects in subsequent divisions. In total, we find that the coordinated temporal regulation of outer kinetochore assembly is essential for accurate cell division.
Asunto(s)
Núcleo Celular/metabolismo , Segregación Cromosómica/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Cinetocoros/metabolismo , Mitosis/fisiología , Transporte Activo de Núcleo Celular/fisiología , Línea Celular , Núcleo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Quinasas Ciclina-Dependientes/genética , Proteínas del Citoesqueleto , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/fisiologíaRESUMEN
The diverse nature of eukaryotic centromere structure has led to a prevailing view that the kinetochore-chromatin interface is fundamentally different in distinct species. Two studies now challenge this dogma with the identification of budding yeast homologues of the vertebrate centromere DNA-binding proteins CENP-T and CENP-W.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Proteínas del Citoesqueleto , HumanosRESUMEN
The multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the "histone code" beyond canonical nucleosome proteins.
Asunto(s)
Centrómero/química , Centrómero/metabolismo , Pollos/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Animales , Cromatina/química , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Histonas/metabolismo , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Difracción de Rayos XRESUMEN
Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , Nucleosomas/metabolismo , Vertebrados/metabolismo , Secuencia de Aminoácidos , Animales , Proteína A Centromérica , Pollos , Células HeLa , Humanos , Mitosis , Datos de Secuencia Molecular , FosforilaciónRESUMEN
Accurate chromosome segregation requires the interaction of chromosomes with the microtubules from the mitotic spindle. This interaction is mediated by the macro-molecular kinetochore complex, which assembles only at the centromeric region of each chromosome. However, how this site is specified and how assembly of the kinetochore structure is regulated in coordination with cell cycle progression remains unclear. Recent studies have begun to shed light on the mechanisms underlying assembly of this complex structure.
Asunto(s)
Cinetocoros/metabolismo , Animales , Humanos , Proteínas de Microfilamentos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
In 2007, over 12-million people were diagnosed with cancer. According to the American Cancer Society, at least one third of these individuals are not expected to survive the disease, making cancer the second most prevalent cause of death worldwide. Systemic chemotherapy forms the mainstay of cancer treatment, and agents that disrupt mitotic spindle assembly - so called ;anti-mitotics' - are commonly used to treat a wide variety of cancers. Traditional anti-mitotic agents include the microtubule toxins such as taxol, other taxanes and the vinca alkaloids, all of which have proven successful in the clinic. However, patient response remains highly unpredictable, and drug resistance is common. In addition, toxicity is a problem. To address these limitations, a new generation of anti-mitotic drugs is being developed. As the first wave of these new agents enters clinical trails, much hope rests on their outcome. Meanwhile, significant attention is being focused on trying to predict which tumour types are likely to respond. In this Commentary, we outline recent advances in our understanding of how cancer cells respond to anti-mitotic drugs, and discuss the relevance of these studies to their use in the clinic.
Asunto(s)
Antimitóticos/farmacología , Mitosis/efectos de los fármacos , Neoplasias/patología , Muerte Celular , HumanosRESUMEN
Drugs targeting the mitotic spindle are used extensively during chemotherapy, but surprisingly, little is known about how they kill tumor cells. This is largely because many of the population-based approaches are indirect and lead to vague and confusing interpretations. Here, we use a high-throughput automated time-lapse light microscopy approach to systematically analyze over 10,000 single cells from 15 cell lines in response to three different classes of antimitotic drug. We show that the variation in cell behavior is far greater than previously recognized, with cells within any given line exhibiting multiple fates. We present data supporting a model wherein cell fate is dictated by two competing networks, one involving caspase activation, the other protecting cyclin B1 from degradation.