RESUMEN
In this Letter, the sentence beginning "This work was funded ." in the Acknowledgements should have read "CPRIT (RP140105) to J.C.R." rather than "CPRIT (RP150445) to J.C.R." This error has been corrected online.
RESUMEN
Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.
Asunto(s)
Proteína BRCA1/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Conformación de Ácido Nucleico , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Reparación del ADN por Recombinación , Sarcoma de Ewing/genética , Transcripción Genética , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Daño del ADN , Humanos , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/metabolismoRESUMEN
Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.
Asunto(s)
Anilidas/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Anilidas/síntesis química , Anilidas/química , Animales , Proteína Quinasa CDC2/metabolismo , Células Cultivadas , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of â¼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Amplificación de Genes , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Edición Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Proteína Proto-Oncogénica N-Myc/antagonistas & inhibidores , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Proteínas de Ciclo Celular/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , CohesinasRESUMEN
Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Proliferación Celular , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Fosfoproteínas Fosfatasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Ewing sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type TP53, and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for TP53 wild-type Ewing sarcoma, we used a genome-scale CRISPR-Cas9 screening approach and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies. The stapled peptide inhibitor of MDM2 and MDM4, ATSP-7041, showed anti-tumor efficacy in vitro and in multiple mouse models. The USP7 inhibitor, P5091, and the Wip1/PPM1D inhibitor, GSK2830371, decreased the viability of Ewing sarcoma cells. The combination of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic action on the p53 pathway. The effects of the inhibitors, including the specific USP7 inhibitor XL-188, were rescued by concurrent TP53 knockout, highlighting the essentiality of intact p53 for the observed cytotoxic activities.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Genoma Humano , Sarcoma de Ewing/genética , Proteína p53 Supresora de Tumor/genética , Aminopiridinas/farmacología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Dipéptidos/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Ratones Desnudos , Mutación/genética , Proteínas de Neoplasias/metabolismo , Péptidos Cíclicos/farmacología , Reproducibilidad de los Resultados , Sarcoma de Ewing/patología , Tiofenos/farmacología , Transcripción Genética/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively1. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival2-6, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential transcription factors-MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2-are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Transcripción Genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones Desnudos , Factores de Transcripción/genéticaRESUMEN
Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.
Asunto(s)
Quinasas Ciclina-Dependientes/efectos de los fármacos , Fenilendiaminas/farmacología , Proteína Proto-Oncogénica c-fli-1/genética , Pirimidinas/farmacología , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Fusión Oncogénica/efectos de los fármacos , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/efectos de los fármacos , Proteína EWS de Unión a ARN/efectos de los fármacos , Mutaciones Letales Sintéticas/efectos de los fármacos , Mutaciones Letales Sintéticas/genéticaRESUMEN
Drug resistance represents a major challenge to achieving durable responses to cancer therapeutics. Resistance mechanisms to epigenetically targeted drugs remain largely unexplored. We used bromodomain and extra-terminal domain (BET) inhibition in neuroblastoma as a prototype to model resistance to chromatin modulatory therapeutics. Genome-scale, pooled lentiviral open reading frame (ORF) and CRISPR knockout rescue screens nominated the phosphatidylinositol 3-kinase (PI3K) pathway as promoting resistance to BET inhibition. Transcriptomic and chromatin profiling of resistant cells revealed that global enhancer remodeling is associated with upregulation of receptor tyrosine kinases (RTKs), activation of PI3K signaling, and vulnerability to RTK/PI3K inhibition. Large-scale combinatorial screening with BET inhibitors identified PI3K inhibitors among the most synergistic upfront combinations. These studies provide a roadmap to elucidate resistance to epigenetic-targeted therapeutics and inform efficacious combination therapies.
Asunto(s)
Azepinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Indazoles/farmacología , Terapia Molecular Dirigida/métodos , Neuroblastoma/tratamiento farmacológico , Sulfonamidas/farmacología , Triazoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Supervivencia sin Enfermedad , Epigénesis Genética/efectos de los fármacos , Femenino , Humanos , Ratones Desnudos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.