RESUMEN
Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
Asunto(s)
Cromosomas Humanos/genética , Elementos de Facilitación Genéticos , Amplificación de Genes , Oncogenes , Acetilación , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cromatina/metabolismo , ADN de Neoplasias/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes Relacionados con las Neoplasias , Sitios Genéticos , Glioblastoma/genética , Glioblastoma/patología , Histonas/metabolismo , Humanos , Neuroglía/metabolismoRESUMEN
Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Terapia Molecular Dirigida/tendencias , Factores de Elongación Transcripcional/antagonistas & inhibidores , Factores de Elongación Transcripcional/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , Ratones , Interferencia de ARN , Transcripción Genética , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma is the most malignant primary brain tumor, the prognosis of which remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared with NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor ß (PDGFRß) proliferative and stem cell signaling pathways through posttranscriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.
Asunto(s)
Glioblastoma , Humanos , Animales , Ratones , Glioblastoma/tratamiento farmacológico , Nexinas de Clasificación/genética , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Proteínas Tirosina Quinasas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Mammalian cells respond to insufficient oxygen through transcriptional regulators called hypoxia-inducible factors (HIFs). Although transiently protective, prolonged HIF activity drives distinct pathological responses in different tissues. Using a model of chronic HIF1a accumulation in pluripotent-stem-cell-derived oligodendrocyte progenitors (OPCs), we demonstrate that HIF1a activates non-canonical targets to impair generation of oligodendrocytes from OPCs. HIF1a activated a unique set of genes in OPCs through interaction with the OPC-specific transcription factor OLIG2. Non-canonical targets, including Ascl2 and Dlx3, were sufficient to block differentiation through suppression of the oligodendrocyte regulator Sox10. Chemical screening revealed that inhibition of MEK/ERK signaling overcame the HIF1a-mediated block in oligodendrocyte generation by restoring Sox10 expression without affecting canonical HIF1a activity. MEK/ERK inhibition also drove oligodendrocyte formation in hypoxic regions of human oligocortical spheroids. This work defines mechanisms by which HIF1a impairs oligodendrocyte formation and establishes that cell-type-specific HIF1a targets perturb cell function in response to low oxygen.
Asunto(s)
Células Precursoras de Oligodendrocitos , Células Madre Pluripotentes , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , OligodendroglíaRESUMEN
Meningiomas are the most common primary intracranial tumor with current classification offering limited therapeutic guidance. Here, we interrogated meningioma enhancer landscapes from 33 tumors to stratify patients based upon prognosis and identify novel meningioma-specific dependencies. Enhancers robustly stratified meningiomas into three biologically distinct groups (adipogenesis/cholesterol, mesodermal, and neural crest) distinguished by distinct hormonal lineage transcriptional regulators. Meningioma landscapes clustered with intrinsic brain tumors and hormonally responsive systemic cancers with meningioma subgroups, reflecting progesterone or androgen hormonal signaling. Enhancer classification identified a subset of tumors with poor prognosis, irrespective of histologic grading. Superenhancer signatures predicted drug dependencies with superior in vitro efficacy to treatment based upon the NF2 genomic profile. Inhibition of DUSP1, a novel and druggable meningioma target, impaired tumor growth in vivo. Collectively, epigenetic landscapes empower meningioma classification and identification of novel therapies. SIGNIFICANCE: Enhancer landscapes inform prognostic classification of aggressive meningiomas, identifying tumors at high risk of recurrence, and reveal previously unknown therapeutic targets. Druggable dependencies discovered through epigenetic profiling potentially guide treatment of intractable meningiomas.This article is highlighted in the In This Issue feature, p. 1611.
Asunto(s)
Epigenómica/métodos , Meningioma/genética , Humanos , Meningioma/patología , PronósticoRESUMEN
Commonly-mutated genes have been found for many cancers, but less is known about mutations in cis-regulatory elements. We leverage gains in tumor-specific enhancer activity, coupled with allele-biased mutation detection from H3K27ac ChIP-seq data, to pinpoint potential enhancer-activating mutations in colorectal cancer (CRC). Analysis of a genetically-diverse cohort of CRC specimens revealed that microsatellite instable (MSI) samples have a high indel rate within active enhancers. Enhancers with indels show evidence of positive selection, increased target gene expression, and a subset is highly recurrent. The indels affect short homopolymer tracts of A/T and increase affinity for FOX transcription factors. We further demonstrate that signature mismatch-repair (MMR) mutations activate enhancers using a xenograft tumor metastasis model, where mutations are induced naturally via CRISPR/Cas9 inactivation of MLH1 prior to tumor cell injection. Our results suggest that MMR signature mutations activate enhancers in CRC tumor epigenomes to provide a selective advantage.
Asunto(s)
Neoplasias Colorrectales/genética , Reparación de la Incompatibilidad de ADN/genética , Elementos de Facilitación Genéticos/genética , Epigenoma , Mutación/genética , Acetilación , Animales , Secuencia de Bases , Línea Celular Tumoral , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Mutación INDEL/genética , Lisina/metabolismo , Ratones , Inestabilidad de Microsatélites , Motivos de Nucleótidos/genética , Fenotipo , Selección Genética , Factores de Transcripción/metabolismoRESUMEN
Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.
Asunto(s)
Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Terapia Molecular Dirigida , Células Madre Neoplásicas/patología , Pirimidinas/biosíntesis , Animales , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Crotonatos/farmacología , Dihidroorotato Deshidrogenasa , Receptores ErbB/metabolismo , Eliminación de Gen , Humanos , Hidroxibutiratos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Nitrilos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Toluidinas/farmacología , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1-CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms. SIGNIFICANCE: Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.This article is highlighted in the In This Issue feature, p. 1469.
Asunto(s)
Factores de Transcripción ARNTL/genética , Neoplasias Encefálicas/tratamiento farmacológico , Proteínas CLOCK/genética , Glioblastoma/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Relojes Circadianos/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Humanos , Ratones , Células Madre Neoplásicas/química , Células Madre Neoplásicas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma is the most lethal primary brain tumor; however, the crosstalk between glioblastoma stem cells (GSCs) and their supportive niche is not well understood. Here, we interrogated reciprocal signaling between GSCs and their differentiated glioblastoma cell (DGC) progeny. We found that DGCs accelerated GSC tumor growth. DGCs preferentially expressed brain-derived neurotrophic factor (BDNF), whereas GSCs expressed the BDNF receptor NTRK2. Forced BDNF expression in DGCs augmented GSC tumor growth. To determine molecular mediators of BDNF-NTRK2 paracrine signaling, we leveraged transcriptional and epigenetic profiles of matched GSCs and DGCs, revealing preferential VGF expression by GSCs, which patient-derived tumor models confirmed. VGF serves a dual role in the glioblastoma hierarchy by promoting GSC survival and stemness in vitro and in vivo while also supporting DGC survival and inducing DGC secretion of BDNF. Collectively, these data demonstrate that differentiated glioblastoma cells cooperate with stem-like tumor cells through BDNF-NTRK2-VGF paracrine signaling to promote tumor growth.