RESUMO
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.
Assuntos
Proteína BRCA1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Reparo de DNA por Recombinação , Sarcoma de Ewing/genética , Transcrição Gênica , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Amplificação de Genes , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Edição de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Anilidas/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Anilidas/síntese química , Anilidas/química , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteínas de Ciclo Celular/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/genética , CoesinasRESUMO
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.
Assuntos
Transformação Celular Neoplásica/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fosfoproteínas Fosfatases/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Genoma Humano , Sarcoma de Ewing/genética , Proteína Supressora de Tumor p53/genética , Aminopiridinas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Dipeptídeos/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Camundongos Nus , Mutação/genética , Proteínas de Neoplasias/metabolismo , Peptídeos Cíclicos/farmacologia , Reprodutibilidade dos Testes , Sarcoma de Ewing/patologia , Tiofenos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Quinases Ciclina-Dependentes/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Proteína Proto-Oncogênica c-fli-1/genética , Pirimidinas/farmacologia , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Fusão Oncogênica/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/efeitos dos fármacos , Proteína EWS de Ligação a RNA/efeitos dos fármacos , Mutações Sintéticas Letais/efeitos dos fármacos , Mutações Sintéticas Letais/genéticaRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Transcrição Gênica , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Amplificação de Genes , Humanos , Camundongos , Camundongos Nus , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Azepinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indazóis/farmacologia , Terapia de Alvo Molecular/métodos , Neuroblastoma/tratamento farmacológico , Sulfonamidas/farmacologia , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Epigênese Genética/efeitos dos fármacos , Feminino , Humanos , Camundongos Nus , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.