RESUMO
Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription-from transcription initiation through to export of mRNA from the nucleus-is influenced by the UPS and that all major arms of the system--from the first step in ubiquitin (Ub) conjugation through to the proteasome-are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitinação , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Fatores de Transcrição/químicaRESUMO
WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias , Repetições WD40 , Animais , Humanos , Camundongos , Cromatina , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Animais , Neoplasias/tratamento farmacológico , Linhagem Celular TumoralRESUMO
BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.
Assuntos
Genes myc , Neuroblastoma , Humanos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Linhagem Celular Tumoral , Segregação de Cromossomos , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neuroblastoma/metabolismoRESUMO
MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at â¼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.
Assuntos
Carcinogênese/metabolismo , Cromatina/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anisotropia , Sítios de Ligação/genética , Carcinogênese/genética , Cromatina/química , Cromatina/genética , Polarização de Fluorescência , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Biossíntese de Proteínas/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Cromatina/metabolismo , Sequência Conservada/genética , Feminino , Humanos , Masculino , Ligação Proteica , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents.
Assuntos
Linfoma de Burkitt/metabolismo , Cromatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linfoma de Burkitt/genética , Carcinogênese , Linhagem Celular Tumoral , Cromatina/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Nus , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
X-linked inhibitor of apoptosis (XIAP) plays an important role in preventing apoptotic cell death. XIAP has been shown to participate in signaling pathways, including Wnt signaling. XIAP regulates Wnt signaling by promoting the monoubiquitylation of the co-repressor Groucho/TLE family proteins, decreasing its affinity for the TCF/Lef family of transcription factors and allowing assembly of transcriptionally active ß-catenin-TCF/Lef complexes. We now demonstrate that XIAP is phosphorylated by GSK3 at threonine 180, and that an alanine mutant (XIAPT180A) exhibits decreased Wnt activity compared to wild-type XIAP in cultured human cells and in Xenopus embryos. Although XIAPT180A ubiquitylates TLE3 at wild-type levels in vitro, it exhibits a reduced capacity to ubiquitylate and bind TLE3 in human cells. XIAPT180A binds Smac (also known as DIABLO) and inhibits Fas-induced apoptosis to a similar degree to wild-type XIAP. Our studies uncover a new mechanism by which XIAP is specifically directed towards a Wnt signaling function versus its anti-apoptotic function. These findings have implications for development of anti-XIAP therapeutics for human cancers.
Assuntos
Treonina/metabolismo , Proteína Wnt3A/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Motivos de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação , Ligação Proteica , Via de Sinalização Wnt , Proteína Wnt3A/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , XenopusRESUMO
Ubiquitin, and components of the ubiquitin-proteasome system, feature extensively in the regulation of gene transcription. Although there are many examples of how ubiquitin controls the activity of transcriptional regulators and coregulators, there are few examples of core components of the transcriptional machinery that are directly controlled by ubiquitin-dependent processes. The budding yeast protein Asr1 is the prototypical member of the RPC (RING, PHD, CBD) family of ubiquitin-ligases, characterized by the presence of amino-terminal RING (really interesting new gene) and PHD (plant homeo domain) fingers and a carboxyl-terminal domain that directly binds the largest subunit of RNA polymerase II (pol II), Rpb1, in response to phosphorylation events tied to the initiation of transcription. Asr1-mediated oligo-ubiquitylation of pol II leads to ejection of two core subunits of the enzyme and is associated with inhibition of polymerase function. Here, we present evidence that Asr1-mediated ubiquitylation of pol II is required for silencing of subtelomeric gene transcription. We show that Asr1 associates with telomere-proximal chromatin and that disruption of the ubiquitin-ligase activity of Asr1--or mutation of ubiquitylation sites within Rpb1--induces transcription of silenced gene sequences. In addition, we report that Asr1 associates with the Ubp3 deubiquitylase and that Asr1 and Ubp3 play antagonistic roles in setting transcription levels from silenced genes. We suggest that control of pol II by nonproteolytic ubiquitylation provides a mechanism to enforce silencing by transient and reversible inhibition of pol II activity at subtelomeric chromatin.
Assuntos
Inativação Gênica , Peptídeo Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero , Ubiquitina-Proteína Ligases/metabolismo , Cromatografia de AfinidadeRESUMO
OBJECTIVE: Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. DESIGN: We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. RESULTS: BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. CONCLUSION: Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.
Assuntos
Carcinogênese/genética , Moléculas de Adesão Celular/genética , Colite Ulcerativa/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Membrana/genética , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Biomarcadores Tumorais/genética , Células CACO-2 , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite Ulcerativa/genética , Colo/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Metilação de DNA , Sulfato de Dextrana , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Via de Sinalização WntRESUMO
Cdc48/p97 is an evolutionary conserved ubiquitin-dependent chaperone involved in a broad array of cellular functions due to its ability to associate with multiple cofactors. Aside from its role in removing RNA polymerase II from chromatin after DNA damage, little is known about how this AAA-ATPase is involved in the transcriptional process. Here, we show that yeast Cdc48 is recruited to chromatin in a transcription-coupled manner and modulates gene expression. Cdc48, together with its cofactor Ubx3 controls monoubiquitylation of histone H2B, a conserved modification regulating nucleosome dynamics and chromatin organization. Mechanistically, Cdc48 facilitates the recruitment of Lge1, a cofactor of the H2B ubiquitin ligase Bre1. The function of Cdc48 in controlling H2B ubiquitylation appears conserved in human cells because disease-related mutations or chemical inhibition of p97 function affected the amount of ubiquitylated H2B in muscle cells. Together, these results suggest a prominent role of Cdc48/p97 in the coordination of chromatin remodeling with gene transcription to define cellular differentiation processes.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ubiquitinação , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Mutação , Mioblastos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Proteína com ValosinaRESUMO
Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et al. challenged this view by reporting that the prototypical yeast activator Gal4 does not dynamically associate with chromatin, but rather 'locks in' to stable promoter complexes that are resistant to competition. Here we present evidence that the assay used to reach this conclusion is unsuitable, and that promoter-bound, active Gal4 is indeed susceptible to competition in vivo. Our data challenge the key evidence that Nalley et al. used to reach their conclusion, and indicate that Gal4 functions in vivo within the context of dynamic promoter complexes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Ligação Competitiva/efeitos dos fármacos , Imunoprecipitação da Cromatina , Estradiol/farmacologia , Galactoquinase/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Transativadores/genéticaRESUMO
It has recently become clear that components of the proteasome are recruited to sites of gene transcription. Prevailing evidence suggests that the transcriptionally relevant form of the proteasome is a subcomplex of 19S base proteins, which functions as an ATP-dependent chaperone that influences transcriptional processes. Despite this notion, compelling evidence for a transcription-dedicated 19S base complex is lacking, and 20S proteasome subunits have been shown to associate with chromatin in some contexts. To gain insight into the form of the proteasome that is recruited to chromatin, we assembled a panel of highly specific antibodies that recognize native yeast proteasome subunits in chromatin immunoprecipitation assays. Using these reagents, we show that components from the three major subassemblies of the proteasome--19S lid, 19S base, and 20S core--associate with the activated GAL10 gene in yeast in a virtually indistinguishable manner. We find that proteasome subunits Rpt1, Rpt4, Rpn8, Rpn12, Pre6, and Pre10 are recruited to GAL10 rapidly upon galactose induction. These subunits associate with the entire transcribed portion of GAL10, display near-identical patterns of distribution, and dissociate from chromatin rapidly once transcription is shut down. We also find that proteasome subunits are enriched at telomeres and at genes transcribed by RNA polymerase III. Our data suggest that the transcriptionally relevant form of the proteasome is the canonical 26S complex.
Assuntos
Cromatina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Western Blotting , Cromatina/genética , Imunoprecipitação da Cromatina , Galactose/farmacologia , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Lipid electrophiles modify cellular targets, altering their function. Here, we describe histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells: four 4-ketoamides to Lys and three Michael adducts to His. A 4-ketoamide adduct residing at H3K27 was identified in stimulated macrophages. Modification of histones H3 and H4 prevented nucleosome assembly.
Assuntos
Aldeídos/química , Adutos de DNA/química , Epigênese Genética/fisiologia , Histonas/química , Estresse Oxidativo/fisiologia , Processamento de Proteína Pós-Traducional , Acilação , Aldeídos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Adutos de DNA/genética , Adutos de DNA/metabolismo , Epigênese Genética/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Estresse Oxidativo/genéticaRESUMO
WDR5 is a conserved nuclear protein that scaffolds the assembly of epigenetic regulatory complexes and moonlights in functions ranging from recruiting MYC oncoproteins to chromatin to facilitating the integrity of mitosis. It is also a high-value target for anti-cancer therapies, with small molecule WDR5 inhibitors and degraders undergoing extensive preclinical assessment. WDR5 inhibitors were originally conceived as epigenetic modulators, proposed to inhibit cancer cells by reversing oncogenic patterns of histone H3 lysine 4 methylation-a notion that persists to this day. This premise, however, does not withstand contemporary inspection and establishes expectations for the mechanisms and utility of WDR5 inhibitors that can likely never be met. Here, we highlight salient misconceptions regarding WDR5 inhibitors as epigenetic modulators and provide a unified model for their action as a ribosome-directed anti-cancer therapy that helps focus understanding of when and how the tumor-inhibiting properties of these agents can best be understood and exploited.
RESUMO
Mutations in the SWI/SNF chromatin remodeling complex occur in ~20% of cancers. In rhabdoid tumors defined by loss of the SWI/SNF subunit SMARCB1, dysregulation of enhancer-mediated gene expression is pivotal in driving oncogenesis. Enhancer dysregulation in this setting is tied to retention of the SWI/SNF ATPase BRG1-which becomes essential in the absence of SMARCB1-but precisely how BRG1 contributes to this process remains unknown. To characterize how BRG1 participates in chromatin remodeling and gene expression in SMARCB1-deficient cells, we performed a genome-wide characterization of the impact of BRG1 depletion in multiple rhabdoid tumor cell lines. We find that although BRG1-regulated open chromatin sites are distinct at the locus level, the biological characteristics of the loci are very similar, converging on a set of thematically related genes and pointing to the involvement of the AP-1 transcription factor. The open chromatin sites regulated by BRG1 colocalize with histone-marked enhancers and intriguingly include almost all super-enhancers, revealing that BRG1 plays a critical role in maintaining super-enhancer function in this setting. These studies can explain the essentiality of BRG1 to rhabdoid tumor cell identity and survival and implicate the involvement of AP-1 as a critical downstream effector of rhabdoid tumor cell transcriptional programs.
RESUMO
The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares , Ribossomos , Proteína Supressora de Tumor p53 , Humanos , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Peptidomiméticos/farmacologiaRESUMO
The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the "WIN" site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small molecule WIN site inhibitors, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anti-cancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anti-cancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.
RESUMO
Degradation of Myc protein is mediated by E3 ubiquitin ligases, including SCF(Fbw7) and SCF(Skp2), but much remains unknown about the mechanism of S-phase kinase-associated protein (Skp2)-mediated Myc degradation. In the present study, we show that upregulated Myc protein, which triggers the G1-S phase progression in response to growth-stimulatory signals, induces reactive oxygen species modulator 1 (Romo1) expression. Romo1 subsequently triggers Skp2-mediated ubiquitylation and degradation of Myc by a mechanism not previously reported in normal lung fibroblasts. We also show that reactive oxygen species (ROS) derived from steady-state Romo1 expression are necessary for cell cycle entry of quiescent cells. From this study, we suggest that the generation of ROS mediated by pre-existing Romo1 protein is required for Myc induction. Meanwhile, Romo1 expression induced by Myc during G1 phase stimulates Skp2-mediated Myc degradation in a negative-feedback mechanism.
Assuntos
Retroalimentação Fisiológica , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular , Meios de Cultura Livres de Soro/metabolismo , Regulação para Baixo , Fase G1 , Humanos , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Transcrição Gênica , UbiquitinaçãoRESUMO
Collectively, the MYC family of oncoprotein transcription factors is overexpressed in more than half of all malignancies. The ability of MYC proteins to access chromatin is fundamental to their role in promoting oncogenic gene expression programs in cancer and this function depends on MYC-cofactor interactions. One such cofactor is the chromatin regulator WDR5, which in models of Burkitt lymphoma facilitates recruitment of the c-MYC protein to chromatin at genes associated with protein synthesis, allowing for tumor progression and maintenance. However, beyond Burkitt lymphoma, it is unknown whether these observations extend to other cancers or MYC family members, and whether WDR5 can be deemed as a "universal" MYC recruiter. Here, we focus on N-MYC amplified neuroblastoma to determine the extent of colocalization between N-MYC and WDR5 on chromatin while also demonstrating that like c-MYC, WDR5 can facilitate the recruitment of N-MYC to conserved WDR5-bound genes. We conclude based on this analysis that N-MYC and WDR5 colocalize invariantly across cell lines at predicted sites of facilitated recruitment associated with protein synthesis genes. Surprisingly, we also identify N-MYC-WDR5 cobound genes that are associated with DNA repair and cell cycle processes. Dissection of chromatin binding characteristics for N-MYC and WDR5 at all cobound genes reveals that sites of facilitated recruitment are inherently different than most N-MYC-WDR5 cobound sites. Our data reveals that WDR5 acts as a universal MYC recruiter at a small cohort of previously identified genes and highlights novel biological functions that may be coregulated by N-MYC and WDR5 to sustain the neuroblastoma state.