RESUMEN
WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks-if any-that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.
Asunto(s)
Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Biosíntesis de Proteínas/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Cromatina/metabolismo , Secuencia Conservada/genética , Femenino , Humanos , Masculino , Unión Proteica , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Quinolonas/síntesis química , Quinolonas/farmacología , Repeticiones WD40/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Cromatina/efectos de los fármacos , Cromatina/genética , Cristalografía por Rayos X , Diseño de Fármacos , Descubrimiento de Drogas , Represión Epigenética/efectos de los fármacos , Genes myc/efectos de los fármacos , Humanos , Relación Estructura-ActividadRESUMEN
The treatment of tumors driven by overexpression or amplification of MYC oncogenes remains a significant challenge in drug discovery. Here, we present a new strategy toward the inhibition of MYC via the disruption of the protein-protein interaction between MYC and its chromatin cofactor WD Repeat-Containing Protein 5. Blocking the association of these proteins is hypothesized to disrupt the localization of MYC to chromatin, thus disrupting the ability of MYC to sustain tumorigenesis. Utilizing a high-throughput screening campaign and subsequent structure-guided design, we identify small-molecule inhibitors of this interaction with potent in vitro binding affinity and report structurally related negative controls that can be used to study the effect of this disruption. Our work suggests that disruption of this protein-protein interaction may provide a path toward an effective approach for the treatment of multiple tumors and anticipate that the molecules disclosed can be used as starting points for future efforts toward compounds with improved drug-like properties.
Asunto(s)
Descubrimiento de Drogas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Ácido Salicílico/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Repeticiones WD40RESUMEN
WDR5 is a component of multiple epigenetic regulatory complexes, including the mixed lineage leukemia (MLL)/SET complexes that deposit histone H3 lysine 4 methylation. Inhibitors of an arginine-binding cavity in WDR5, known as the WDR5-interaction (WIN) site, have been proposed to selectively kill MLL-rearranged malignancies via an epigenetic mechanism. We discovered potent WIN site inhibitors and found that they kill MLL cancer cells not through changes in histone methylation, but by displacing WDR5 from chromatin at protein synthesis genes, choking the translational capacity of these cells, and inducing death via a nucleolar stress response. The mechanism of action of WIN site inhibitors reveals new aspects of WDR5 function and forecasts broad therapeutic utility as anti-cancer agents.
RESUMEN
The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.
Asunto(s)
Cromatina/metabolismo , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sitios de Unión , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Femenino , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Masculino , Unión Proteica/efectos de los fármacosRESUMEN
Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.
Asunto(s)
Replicación del ADN , Recombinación Homóloga , Poli(ADP-Ribosa) Polimerasas/metabolismo , Línea Celular , Sitios Frágiles del Cromosoma , Roturas del ADN , Daño del ADN , Reparación del ADN , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Interferente Pequeño/genética , Fase SRESUMEN
All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.