RESUMO
Ewing sarcoma is a bone and soft-tissue tumor that depends on the activity of the EWS-FLI1 transcription factor for cell survival. Although a number of compounds have been shown to inhibit EWS-FLI1 in vitro, a clinical EWS-FLI1-directed therapy has not been achieved. One problem plaguing drug development efforts is the lack of a suitable, non-invasive, pharmacodynamic marker of EWS-FLI1 activity. Here we show that 18F-FLT PET (18F- 3'-deoxy-3'-fluorothymidine positron emission tomography) reflects EWS-FLI1 activity in Ewing sarcoma cells both in vitro and in vivo. 18F-FLT is transported into the cell by ENT1 and ENT2, where it is phosphorylated by TK1 and trapped intracellularly. In this report, we show that silencing of EWS-FLI1 with either siRNA or small-molecule EWS-FLI1 inhibitors suppressed the expression of ENT1, ENT2, and TK1 and thus decreased 18F-FLT PET activity. This effect was not through a generalized loss in viability or metabolic suppression, as there was no suppression of 18F-FDG PET activity and no suppression with chemotherapy. These results provide the basis for the clinical translation of 18F-FLT as a companion biomarker of EWS-FLI1 activity and a novel diagnostic imaging approach for Ewing sarcoma.
RESUMO
There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657-68. ©2016 AACR.
Assuntos
Camptotecina/análogos & derivados , Proteínas de Fusão Oncogênica/genética , Proteínas Oncogênicas/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/tratamento farmacológico , Animais , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Irinotecano , Camundongos , Camundongos Nus , Sarcoma de Ewing/patologiaRESUMO
Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.
Assuntos
Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Éxons , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA , RNA Interferente Pequeno/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Proteína EWS de Ligação a RNA/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Sarcoma de Ewing/patologia , Transativadores , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients. EXPERIMENTAL DESIGN: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. RESULTS: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo CONCLUSIONS: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105-18. ©2016 AACR.