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18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

View Article: PubMed Central - PubMed

ABSTRACT

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.

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Specificity of effect for Ewing sarcoma cells and EWS-FLI1.(A) Treatment of TC32 Ewing sarcoma cells with 50 nM mithramycin (MMA) phenocopied the suppression of ENT1, ENT2, and TK1 expression that occurs with siRNA silencing of EWS-FLI1. (B) Treatment of TC32 Ewing sarcoma cells with the mithramycin analogs EC-8042 and EC-8105 suppressed ENT1, ENT2, and TK1, but 5-FU induced their expression (P for all treatments vs. no change) (C) Expression of ENT1, ENT2, and TK1 was unchanged or induced in 4 control cell lines that do not express EWS-FLI1 with treatment with mithramycin or 5-FU.
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f2: Specificity of effect for Ewing sarcoma cells and EWS-FLI1.(A) Treatment of TC32 Ewing sarcoma cells with 50 nM mithramycin (MMA) phenocopied the suppression of ENT1, ENT2, and TK1 expression that occurs with siRNA silencing of EWS-FLI1. (B) Treatment of TC32 Ewing sarcoma cells with the mithramycin analogs EC-8042 and EC-8105 suppressed ENT1, ENT2, and TK1, but 5-FU induced their expression (P for all treatments vs. no change) (C) Expression of ENT1, ENT2, and TK1 was unchanged or induced in 4 control cell lines that do not express EWS-FLI1 with treatment with mithramycin or 5-FU.

Mentions: We confirmed that silencing of EWS-FLI1 repressed the mRNA expression of ENT1, ENT2, and TK1 in four Ewing sarcoma cell lines (Fig. 1C; statistics in Supplementary Table 2). TK1, the best indicator of 18F-FLT PET activity, was the most suppressed in all lines tested. Furthermore, TK1 has been independently linked to EWS-FLI1 in a previous study25. We confirmed suppression of ENT1, ENT2, and TK1 at the protein level in four Ewing sarcoma cell lines (Figs 1D and 2A). Again, TK1 was the most suppressed; ENT2 and ENT1 showed only modest suppression.


18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma
Specificity of effect for Ewing sarcoma cells and EWS-FLI1.(A) Treatment of TC32 Ewing sarcoma cells with 50 nM mithramycin (MMA) phenocopied the suppression of ENT1, ENT2, and TK1 expression that occurs with siRNA silencing of EWS-FLI1. (B) Treatment of TC32 Ewing sarcoma cells with the mithramycin analogs EC-8042 and EC-8105 suppressed ENT1, ENT2, and TK1, but 5-FU induced their expression (P for all treatments vs. no change) (C) Expression of ENT1, ENT2, and TK1 was unchanged or induced in 4 control cell lines that do not express EWS-FLI1 with treatment with mithramycin or 5-FU.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037393&req=5

f2: Specificity of effect for Ewing sarcoma cells and EWS-FLI1.(A) Treatment of TC32 Ewing sarcoma cells with 50 nM mithramycin (MMA) phenocopied the suppression of ENT1, ENT2, and TK1 expression that occurs with siRNA silencing of EWS-FLI1. (B) Treatment of TC32 Ewing sarcoma cells with the mithramycin analogs EC-8042 and EC-8105 suppressed ENT1, ENT2, and TK1, but 5-FU induced their expression (P for all treatments vs. no change) (C) Expression of ENT1, ENT2, and TK1 was unchanged or induced in 4 control cell lines that do not express EWS-FLI1 with treatment with mithramycin or 5-FU.
Mentions: We confirmed that silencing of EWS-FLI1 repressed the mRNA expression of ENT1, ENT2, and TK1 in four Ewing sarcoma cell lines (Fig. 1C; statistics in Supplementary Table 2). TK1, the best indicator of 18F-FLT PET activity, was the most suppressed in all lines tested. Furthermore, TK1 has been independently linked to EWS-FLI1 in a previous study25. We confirmed suppression of ENT1, ENT2, and TK1 at the protein level in four Ewing sarcoma cell lines (Figs 1D and 2A). Again, TK1 was the most suppressed; ENT2 and ENT1 showed only modest suppression.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus