RESUMO
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.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular , Quinase 9 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Leucêmica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Complexos Multiproteicos , Proteínas Nucleares/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Estabilidade Proteica , Proteólise , RNA Polimerase II/metabolismo , Fatores de Tempo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Transcrição/genética , Transfecção , Ubiquitina-Proteína Ligases , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Leucemia/metabolismo , Domínios Proteicos , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Edição de Genes , Genoma/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteólise , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Ligação a RNA/química , Sulfonamidas/farmacologia , Motivos de Aminoácidos , Calorimetria , Clonagem Molecular , Fluorometria , Células HCT116 , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Proteínas Nucleares/metabolismo , Peptídeos/química , Mutação Puntual , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteoma , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína 1A de Ligação a Tacrolimo/química , Fatores de Transcrição/genética , Alelos , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Citoplasma/metabolismo , Dimerização , Técnicas de Introdução de Genes , Células HEK293 , Homeostase , Humanos , Ligantes , Camundongos , Mutação , Células NIH 3T3 , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Proteólise , Proteômica , Transdução de Sinais , TransgenesRESUMO
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.
Assuntos
Proteínas de Transporte/química , Células 3T3 , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Ligantes , Células MCF-7 , Camundongos , Mutagênese , Proteínas Nucleares/química , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Domínios Proteicos , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/químicaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Desenho de Fármacos , Leucemia/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Azepinas/administração & dosagem , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Leucemia/patologia , Ligantes , Camundongos , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Triazóis/administração & dosagem , Triazóis/químicaRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Sistemas de Liberação de Medicamentos , Humanos , Ligantes , Estrutura Molecular , Pirróis/químicaRESUMO
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.
Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/imunologia , Janus Quinase 2/imunologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Fator de Transcrição STAT1/imunologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase/imunologia , Ubiquitinação/efeitos dos fármacosRESUMO
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.
Assuntos
Anemia Falciforme , Antidrepanocíticos , Hemoglobina Fetal , Fatores de Transcrição Kruppel-Like , Proteínas do Tecido Nervoso , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Antidrepanocíticos/química , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Cristalografia por Raios X , Descoberta de Drogas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Macaca fascicularis , Proteínas do Tecido Nervoso/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Benzilaminas/farmacologia , Naftiridinas/farmacologia , Proteômica/métodos , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Reparo do DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , TranscriptomaRESUMO
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.
Assuntos
Proteínas Oncogênicas , Proteólise , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzilaminas , Sistemas CRISPR-Cas , Humanos , Fator de Transcrição Ikaros/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Proteólise/efeitos dos fármacos , Quinazolinas , Talidomida/análise , Talidomida/farmacologia , Fatores de TranscriçãoRESUMO
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.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética , Humanos , Luminescência , Ligação Proteica/genética , Transporte Proteico/genética , Ubiquitinação/genéticaRESUMO
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.
Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos , Proteólise , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Ftalimidas/química , Inibidores de Proteínas Quinases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Humanos , Inibidores de Proteínas Quinases/química , Proteólise , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
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.