RESUMEN
Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.
Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Proteínas Nucleares/metabolismo , Microscopía por Crioelectrón , Factores de Transcripción/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.
Asunto(s)
Aminoaciltransferasas , Linfocitos T CD8-positivos , Quimiocinas , Neoplasias , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Linfocitos T CD8-positivos/patología , Quimiocinas/metabolismo , Inmunoterapia , Infiltración Leucémica , Ratones , Ratones Noqueados , Monocitos , Neoplasias/inmunologíaRESUMEN
Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we apply multiple proteomic techniques to better understand the role of these histone methyltransferases (HMTs) in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns reveal that DTPs display an increase in methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also find that EZH2, in addition to methylating histone H3K27, also can methylate G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin. These complexes are similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone post-translational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the context of H3 serine 10 phosphorylation, primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance.
Asunto(s)
Neoplasias , Proteómica , Tolerancia a Medicamentos , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histonas/metabolismo , Metilación , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.
Asunto(s)
Carcinogénesis/metabolismo , Histonas/metabolismo , Células Mieloides/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Arginasa/genética , Arginasa/metabolismo , Línea Celular Tumoral , Células Cultivadas , Elementos de Facilitación Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Regiones Promotoras Genéticas , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Factores de Transcripción p300-CBP/químicaRESUMEN
Acetylation of histone H3 at lysine 27 is a well-defined marker of enhancer activity. However, the functional impact of this modification at enhancers is poorly understood. Here, we use a chemical genetics approach to acutely block the function of the cAMP response element binding protein (CREB) binding protein (CBP)/P300 bromodomain in models of hematological malignancies and describe a consequent loss of H3K27Ac specifically from enhancers, despite the continued presence of CBP/P300 at chromatin. Using this approach to dissect the role of H3K27Ac at enhancers, we identify a critical role for this modification in the production of enhancer RNAs and transcription of enhancer-regulated gene networks.
Asunto(s)
Elementos de Facilitación Genéticos , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Neoplásico/genética , Factores de Transcripción p300-CBP/genética , Acetilación , Sitios de Unión , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Histonas/genética , Humanos , Lisina/metabolismo , Unión Proteica , Dominios Proteicos , ARN Neoplásico/metabolismo , Transcripción Genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is associated with a wide spectrum of cancers. We report that tamoxifen-induced BAP1 deletion in adult mice resulted in severe thymic atrophy. BAP1 was critical for T cell development at several stages. In the thymus, BAP1 was required for progression through the pre-T cell receptor checkpoint. Peripheral T cells lacking BAP1 demonstrated a defect in homeostatic and antigen-driven expansion. Deletion of BAP1 resulted in suppression of E2F target genes and defects in cell cycle progression, which was dependent on the catalytic activity of BAP1, but did not require its interaction with host cell factor-1 (HCF-1). Loss of BAP1 led to increased monoubiquitination of histone H2A at Lys119 (H2AK119ub) throughout the T cell lineage, in particular in immature thymocytes, but did not alter trimethylation of histone H3 at Lys27 (H3K27me3). Deletion of BAP1 also abrogated B cell development in the bone marrow. Our findings uncover a nonredundant function for BAP1 in maintaining the lymphoid lineage.
Asunto(s)
Linfocitos T/metabolismo , Timocitos/metabolismo , Timo/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Animales , Atrofia , Ciclo Celular/genética , Perfilación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/patología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , UbiquitinaciónRESUMEN
Epithelial-to-mesenchymal transition is implicated in metastasis, where carcinoma cells lose sessile epithelial traits and acquire mesenchymal migratory potential. The mesenchymal state is also associated with cancer stem cells and resistance to chemotherapy. It might therefore be therapeutically beneficial to promote epithelial identity in cancer. Because large-scale cell identity shifts are often orchestrated on an epigenetic level, we screened for candidate epigenetic factors and identified the histone methyltransferase SUV420H2 (KMT5C) as favoring the mesenchymal identity in pancreatic cancer cell lines. Through its repressive mark H4K20me3, SUV420H2 silences several key drivers of the epithelial state. Its knockdown elicited mesenchymal-to-epithelial transition on a molecular and functional level, and cells displayed decreased stemness and increased drug sensitivity. An analysis of human pancreatic cancer biopsies was concordant with these findings, because high levels of SUV420H2 correlated with a loss of epithelial characteristics in progressively invasive cancer. Together, these data indicate that SUV420H2 is an upstream epigenetic regulator of epithelial/mesenchymal state control.
Asunto(s)
Transición Epitelial-Mesenquimal , N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Pancreáticas/metabolismo , Línea Celular Tumoral , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologíaRESUMEN
The H3K4 demethylase KDM5B is amplified and overexpressed in luminal breast cancer, suggesting it might constitute a potential cancer therapy target. Here, we characterize, in breast cancer cells, the molecular effects of a recently developed small-molecule inhibitor of the KDM5 family of proteins (KDM5i), either alone or in combination with the DNA-demethylating agent 5-aza-2'-deoxycytidine (DAC). KDM5i treatment alone increased expression of a small number of genes, whereas combined treatment with DAC enhanced the effects of the latter for increasing expression of hundreds of DAC-responsive genes. ChIP-seq studies revealed that KDM5i resulted in the broadening of existing H3K4me3 peaks. Furthermore, cells treated with the drug combination exhibited increased promoter and gene body H3K4me3 occupancy at DAC-responsive genes compared with DAC alone. Importantly, treatment with either DAC or DAC+KDM5i induced a dramatic increase in H3K27ac at enhancers with an associated significant increase in target gene expression, suggesting a previously unappreciated effect of DAC on transcriptional regulation. KDM5i synergized with DAC to reduce the viability of luminal breast cancer cells in in vitro assays. Our study provides the first look into the molecular effects of a novel KDM5i compound and suggests that combinatorial inhibition along with DAC represents a new area to explore in translational epigenetics.Significance: This study offers a first look into the molecular effects of a novel KDM5 inhibitory compound, suggesting how its use in combination with DNA methylation inhibitors presents new opportunities to explore in translational cancer epigenetics. Cancer Res; 78(5); 1127-39. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Metilación de ADN , Decitabina/farmacología , Inhibidores Enzimáticos/farmacología , Genoma Humano , Histonas/química , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Apoptosis , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proliferación Celular , Epigénesis Genética , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Células Tumorales CultivadasRESUMEN
A propensity for rewiring genetic and epigenetic regulatory networks, thus enabling sustained cell proliferation, suppression of apoptosis, and the ability to evade the immune system, is vital to cancer cell propagation. An increased understanding of how this is achieved is critical for identifying or improving therapeutic interventions. In this study, using acute myeloid leukemia (AML) human cell lines and a custom CRISPR/Cas9 screening platform, we identify the H3K9 methyltransferase SETDB1 as a novel, negative regulator of innate immunity. SETDB1 is overexpressed in many cancers, and loss of this gene in AML cells triggers desilencing of retrotransposable elements that leads to the production of double-stranded RNAs (dsRNAs). This is coincident with induction of a type I interferon response and apoptosis through the dsRNA-sensing pathway. Collectively, our findings establish a unique gene regulatory axis that cancer cells can exploit to circumvent the immune system.
Asunto(s)
Silenciador del Gen , Interferón Tipo I/metabolismo , Leucemia Mieloide Aguda/enzimología , Proteína Metiltransferasas/metabolismo , Retroelementos , Apoptosis , Sistemas CRISPR-Cas , Línea Celular Tumoral , Supervivencia Celular , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Proteína Metiltransferasas/genética , Interferencia de ARN , ARN Bicatenario/biosíntesis , ARN Bicatenario/genética , Transducción de Señal , Factores de Tiempo , Transfección , Escape del TumorRESUMEN
Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.
Asunto(s)
Cromatina/genética , Resistencia a Antineoplásicos/genética , Represión Epigenética/efectos de los fármacos , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias/patología , Animales , Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Estrés Fisiológico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our proteomic datasets are available for download and provide a comprehensive catalogue of alterations in protein abundance, phosphorylation, and histone modifications in oncogenic HRAS and IDH1 driven astrocytoma cells beyond the transcriptomic level.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Isocitrato Deshidrogenasa/genética , Fosfoproteínas/análisis , Proteómica/métodos , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Código de Histonas , Histonas/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Mapas de Interacción de Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histonas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Inhibidores Enzimáticos/farmacología , Estudio de Asociación del Genoma Completo , Histonas/genética , Humanos , Metilación/efectos de los fármacos , Análisis de Secuencia de ADNRESUMEN
UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Animales , Antibacterianos/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Expresión Génica , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Pruebas de Sensibilidad Microbiana , Mutación , Selección Genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , VirulenciaRESUMEN
The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908).
Asunto(s)
Neoplasias del Colon/genética , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Atlas como Asunto , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Perfilación de la Expresión Génica , Células HCT116 , Humanos , Internet , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Programas InformáticosRESUMEN
The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899).
Asunto(s)
Transformación Celular Neoplásica/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/genética , Procesamiento Proteico-Postraduccional , Antineoplásicos/farmacología , Línea Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Fosfatidilinositol 3-Quinasa Clase I , Colon/citología , Colon/efectos de los fármacos , Colon/metabolismo , Bases de Datos Genéticas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Internet , Mutación , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Transducción de Señal , Programas InformáticosRESUMEN
NLRC4 and NLRP3, of the NOD-like receptor (NLR) family of intracellular proteins, are expressed in innate immune cells and are thought to nucleate distinct inflammasome complexes that promote caspase-1 activation, secretion of the proinflammatory cytokines IL-1ß and IL-18, and a form of cell death termed pyroptosis. We show that NLRP3 associates with NLRC4 in macrophages infected with Salmonella typhimurium or transfected with flagellin. The significance of the interaction between the NLRC4 NACHT domain and NLRP3 was revealed when Nlrc4(S533A/S533A) bone marrow-derived macrophages (BMDMs) expressing phosphorylation site mutant NLRC4 S533A had only a mild defect in caspase-1 activation when compared with NLRC4-deficient BMDMs. NLRC4 S533A activated caspase-1 by recruiting NLRP3 and its adaptor protein ASC. Thus, Nlrc4(S533A/S533A) Nlrp3(-/-) BMDMs more closely resembled Nlrc4(-/-) BMDMs in their response to S. typhimurium or flagellin. The interplay between NLRP3 and NLRC4 reveals an unexpected overlap between what had been considered distinct inflammasome scaffolds.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/inmunología , Células de la Médula Ósea/metabolismo , Proteínas de Unión al Calcio/inmunología , Macrófagos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Sustitución de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células de la Médula Ósea/microbiología , Proteínas de Unión al Calcio/genética , Flagelina/genética , Flagelina/inmunología , Macrófagos/microbiología , Ratones , Ratones Noqueados , Mutación Missense , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Dominios Proteicos , Infecciones por Salmonella/genética , Salmonella typhimurium/genéticaRESUMEN
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteína 2 de Unión a Retinoblastoma/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Many cancer cells show distorted epigenetic landscapes. The Cancer Genome Atlas (TCGA) project profiles thousands of tumors, allowing the discovery of somatic alterations in the epigenetic machinery and the identification of potential cancer drivers among members of epigenetic protein families. METHODS: We integrated mutation, expression, and copy number data from 5943 tumors from 13 cancer types to train a classification model that predicts the likelihood of being an oncogene (OG), tumor suppressor (TSG) or neutral gene (NG). We applied this predictor to epigenetic regulator genes (ERGs), and used differential expression and correlation network analysis to identify dysregulated ERGs along with co-expressed cancer genes. Furthermore, we quantified global proteomic changes by mass spectrometry after EZH2 inhibition. RESULTS: Mutation-based classifiers uncovered the OG-like profile of DNMT3A and TSG-like profiles for several ERGs. Differential gene expression and correlation network analyses revealed that EZH2 is the most significantly over-expressed ERG in cancer and is co-regulated with a cell cycle network. Proteomic analysis showed that EZH2 inhibition induced down-regulation of cell cycle regulators in lymphoma cells. CONCLUSIONS: Using classical driver genes to train an OG/TSG predictor, we determined the most predictive features at the gene level. Our predictor uncovered one OG and several TSGs among ERGs. Expression analyses elucidated multiple dysregulated ERGs including EZH2 as member of a co-expressed cell cycle network.
Asunto(s)
Biología Computacional , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Genes Supresores de Tumor , Humanos , Oncogenes , Complejo Represivo Polycomb 2/genética , Proteoma/genéticaRESUMEN
Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a "one-pot" hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.