RESUMEN
Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.
Asunto(s)
Melanoma , Humanos , Melanoma/tratamiento farmacológico , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/uso terapéutico , Mutación , Transducción de Señal , Inositol Polifosfato 5-Fosfatasas/genéticaRESUMEN
We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Evolución Biológica , Cromosomas/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Eucariontes/genética , Genoma , Complejos Multiproteicos/genética , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/química , Algoritmos , Animales , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Centrómero/ultraestructura , Cromosomas/química , Cromosomas Humanos/química , Cromosomas Humanos/ultraestructura , Proteínas de Unión al ADN/química , Genoma Humano , Genómica , Heterocromatina/ultraestructura , Humanos , Interfase , Mitosis , Modelos Biológicos , Complejos Multiproteicos/química , Telómero/ultraestructuraRESUMEN
Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Adenosina Trifosfato/química , Sitios de Unión/genética , Sitios de Unión/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Cromosomas/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Humanos , Complejos Multiproteicos/fisiología , Unión Proteica/fisiología , Subunidades de Proteína/metabolismo , CohesinasRESUMEN
Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Acetilación , Dominio Catalítico , Ciclo Celular , Cromatina/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
The X shape of chromosomes is one of the iconic images in biology. Cohesin actually connects the sister chromatids along their entire length, from S phase until mitosis. Then, cohesin's antagonist Wapl allows the separation of chromosome arms by opening a DNA exit gate in cohesin rings. Centromeres are protected against this removal activity, resulting in the X shape of mitotic chromosomes. The destruction of the remaining centromeric cohesin by Separase triggers chromosome segregation. We review the two-phase regulation of cohesin removal and discuss how this affects chromosome alignment and decatenation in mitosis and cohesin reloading in the next cell cycle.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Estructuras Cromosómicas/genética , Mitosis , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Estructuras Cromosómicas/metabolismo , Humanos , CohesinasRESUMEN
Chromosomal instability is a driving force for heterogeneity within tumours. A recent study shows that boosting sister chromatid cohesion corrects chromosomal instability in pRB-deficient cancer cells. This key finding provides an important lead to make tumours more susceptible to anti-cancer drugs.
Asunto(s)
Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/genética , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Proteína de Retinoblastoma/genética , HumanosRESUMEN
Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF(-/-), or p53(-/-)), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Bencimidazoles/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales/métodos , Glioblastoma/enzimología , Glioblastoma/genética , Glioblastoma/patología , Humanos , Indanos/farmacología , Ratones , Ratones Noqueados , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Células-Madre Neurales/enzimología , Células-Madre Neurales/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pteridinas/farmacología , Pirazoles/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Tiofenos/farmacología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Quinasa Tipo Polo 1RESUMEN
The classical X shape of mitotic human chromosomes is the consequence of two distinct waves of cohesin removal. First, during prophase and prometaphase, the bulk of cohesin is driven from chromosome arms by the cohesin antagonist WAPL. This arm-specific cohesin removal is referred to as the prophase pathway [1-4]. The subsequent cleavage of the remaining centromeric cohesin by Separase is known to be the trigger for anaphase onset [5-7]. Remarkably the biological purpose of the prophase pathway is unknown. We find that this pathway is essential for two key mitotic processes. First, it is important to focus Aurora B at centromeres to allow efficient correction of erroneous microtubule-kinetochore attachments. In addition, it is required to facilitate the timely decatenation of sister chromatids. As a consequence, WAPL-depleted cells undergo anaphase with segregation errors, including both lagging chromosomes and catenanes, resulting in micronuclei and DNA damage. Stable WAPL depletion arrests cells in a p53-dependent manner but causes p53-deficient cells to become highly aneuploid. Our data show that the WAPL-dependent prophase pathway is essential for proper chromosome segregation and is crucial to maintain genomic integrity.