RESUMO
Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Expressão Gênica , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Apoptose/genética , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Epigênese Genética , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/terapia , Fenótipo , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/genéticaRESUMO
The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas Musculares/antagonistas & inibidores , Fatores de Transcrição Box Pareados/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Rabdomiossarcoma Alveolar/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Adolescente , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Criança , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Proteína Forkhead Box O1 , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Homeostase , Humanos , Masculino , Proteínas Musculares/fisiologia , Fator de Transcrição PAX3 , Proteínas Ligases SKP Culina F-Box/fisiologiaRESUMO
Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.
Assuntos
Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Complexo Repressor Polycomb 2/genética , Rabdomiossarcoma/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Neoplásica da Expressão Gênica/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Miogenina/genética , Miogenina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Proteína da Leucemia Promielocítica , Rabdomiossarcoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We perform high harmonic generation spectroscopy of aligned nitrogen molecules to characterize the attosecond dynamics of multielectron rearrangement during strong-field ionization. We use the spectrum and ellipticity of the harmonic light to reconstruct the relative phase between different ionization continua participating in the ionization, and thus determine the shape and location of the hole left in the molecule by strong-field ionization. Our interferometric technique uses transitions between the ionic states, induced by the laser field on the subcycle time scale.
