RESUMEN
Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Antineoplásicos/farmacología , Proteínas de Ciclo Celular , Quinasa 9 Dependiente de la Ciclina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Regulación Leucémica de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Complejos Multiproteicos , Proteínas Nucleares/genética , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Estabilidad Proteica , Proteolisis , ARN Polimerasa II/metabolismo , Factores de Tiempo , Elongación de la Transcripción Genética/efectos de los fármacos , Factores de Transcripción/genética , Transfección , Ubiquitina-Proteína Ligasas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia/genética , Leucemia/metabolismo , Dominios Proteicos , Transcripción Genética , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Edición Génica , Genoma/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Leucemia/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteolisis , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.
Asunto(s)
Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de Unión al ARN/química , Sulfonamidas/farmacología , Secuencias de Aminoácidos , Calorimetría , Clonación Molecular , Fluorometría , Células HCT116 , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Péptidos y Proteínas de Señalización Intracelular/genética , Cinética , Proteínas Nucleares/metabolismo , Péptidos/química , Mutación Puntual , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteoma , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.
Asunto(s)
Sistemas CRISPR-Cas , Proteínas Nucleares/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína 1A de Unión a Tacrolimus/química , Factores de Transcripción/genética , Alelos , Animales , Proteínas de Ciclo Celular , Proliferación Celular , Citoplasma/metabolismo , Dimerización , Técnicas de Sustitución del Gen , Células HEK293 , Homeostasis , Humanos , Ligandos , Ratones , Mutación , Células 3T3 NIH , Proteínas Nucleares/genética , Unión Proteica , Dominios Proteicos , Proteolisis , Proteómica , Transducción de Señal , TransgenesRESUMEN
The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.
Asunto(s)
Proteínas Portadoras/química , Células 3T3 , Animales , Línea Celular Tumoral , Proliferación Celular , Cristalografía por Rayos X , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Ligandos , Células MCF-7 , Ratones , Mutagénesis , Proteínas Nucleares/química , Complejo de la Endopetidasa Proteasomal/química , Unión Proteica , Dominios Proteicos , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/químicaRESUMEN
Cellular signaling is often propagated by multivalent interactions. Multivalency creates avidity, allowing stable biophysical recognition. Multivalency is an attractive strategy for achieving potent binding to protein targets, as the affinity of bivalent ligands is often greater than the sum of monovalent affinities. The bromodomain and extraterminal domain (BET) family of transcriptional coactivators features tandem bromodomains through which BET proteins bind acetylated histones and transcription factors. All reported antagonists of the BET protein BRD4 bind in a monovalent fashion. Here we describe, to our knowledge for the first time, a bivalent BET bromodomain inhibitor-MT1-which has unprecedented potency. Biophysical and biochemical studies suggest MT1 is an intramolecular bivalent BRD4 binder that is more than 100-fold more potent, in cellular assays, than the corresponding monovalent antagonist, JQ1. MT1 significantly (P < 0.05) delayed leukemia progression in mice, as compared to JQ1. These data qualify a powerful chemical probe for BET bromodomains and a rationale for further development of multidomain inhibitors of epigenetic reader proteins.
Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Diseño de Fármacos , Leucemia/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Azepinas/administración & dosificación , Azepinas/química , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Leucemia/patología , Ligandos , Ratones , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Triazoles/administración & dosificación , Triazoles/químicaRESUMEN
The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound's cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1-9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.
RESUMEN
The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Factores de Transcripción/química , Sistemas de Liberación de Medicamentos , Humanos , Ligandos , Estructura Molecular , Pirroles/químicaRESUMEN
Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat ß cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.
Asunto(s)
Células Secretoras de Insulina/efectos de los fármacos , Interferón gamma/inmunología , Janus Quinasa 2/inmunología , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Factor de Transcripción STAT1/inmunología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/inmunología , Fosforilación/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Ubiquitina Tiolesterasa/inmunología , Ubiquitinación/efectos de los fármacosRESUMEN
Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.
Asunto(s)
Anemia de Células Falciformes , Antidrepanocíticos , Hemoglobina Fetal , Factores de Transcripción de Tipo Kruppel , Proteínas del Tejido Nervioso , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Antidrepanocíticos/química , Antidrepanocíticos/farmacología , Antidrepanocíticos/uso terapéutico , Cristalografía por Rayos X , Descubrimiento de Drogas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Macaca fascicularis , Proteínas del Tejido Nervioso/metabolismo , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside a homology-model-guided design, to develop chemical biology approaches for the study of BRD8 inhibition and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.
Asunto(s)
Bencilaminas/farmacología , Naftiridinas/farmacología , Proteómica/métodos , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Linaje de la Célula , Reparación del ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Subunidades de Proteína , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , TranscriptomaRESUMEN
Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.
Asunto(s)
Proteínas Oncogénicas , Proteolisis , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Bencilaminas , Sistemas CRISPR-Cas , Humanos , Factor de Transcripción Ikaros/metabolismo , Proteínas Oncogénicas/química , Proteínas Oncogénicas/metabolismo , Proteolisis/efectos de los fármacos , Quinazolinas , Talidomida/análisis , Talidomida/farmacología , Factores de TranscripciónRESUMEN
Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.
Asunto(s)
Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Complejo de la Endopetidasa Proteasomal/genética , Ubiquitina/genética , Humanos , Luminiscencia , Unión Proteica/genética , Transporte de Proteínas/genética , Ubiquitinación/genéticaRESUMEN
Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target, resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of the respective targeting ligand as a unique feature of small-molecule degraders.
Asunto(s)
Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos , Proteolisis , Línea Celular Tumoral , Humanos , Simulación del Acoplamiento Molecular , Ftalimidas/química , Inhibidores de Proteínas Quinasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/química , Proteolisis , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL) is the signature lesion in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). pVHL loss causes the transcriptional activation of hypoxia-inducible factor (HIF) target genes, including many genes that encode histone lysine demethylases. Moreover, chromatin regulators are frequently mutated in this disease. We found that ccRCC displays increased H3K27 acetylation and a shift toward mono- or unmethylated H3K27 caused by an HIF-dependent increase in H3K27 demethylase activity. Using a focused short hairpin RNA library, as well as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) and a pharmacological inhibitor, we discovered that pVHL-defective ccRCC cells are hyperdependent on the H3K27 methyltransferase EZH1 for survival. Therefore, targeting EZH1 could be therapeutically useful in ccRCC.