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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.

No MeSH data available.


Related in: MedlinePlus

Identification of 18F-FLT as a pharmacodynamic marker of EWS-FLI1 activity.(A) Schema showing theory of study. Suppression of an EWS-FLI1 target expressed on the cell surface will allow quantitation of EWS-FLI1 activity based on loss of transport of a labeled substrate or loss of binding of a labeled antibody or ligand. (B) Heat map summarizing screen results. EWS-FLI1 was silenced in three different cell lines (TC32, TC71 and EW8) and the induction (red) or suppression (blue) of expression was measured by qPCR. ENT1, ENT2, and TK1 (arrows) were all suppressed with silencing of EWS-FLI1. (C) Confirmatory PCR in 4 cell lines showing suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1 as measured by qPCR. (P for all treatments vs. no change) (D) Western blots confirm suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1.
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f1: Identification of 18F-FLT as a pharmacodynamic marker of EWS-FLI1 activity.(A) Schema showing theory of study. Suppression of an EWS-FLI1 target expressed on the cell surface will allow quantitation of EWS-FLI1 activity based on loss of transport of a labeled substrate or loss of binding of a labeled antibody or ligand. (B) Heat map summarizing screen results. EWS-FLI1 was silenced in three different cell lines (TC32, TC71 and EW8) and the induction (red) or suppression (blue) of expression was measured by qPCR. ENT1, ENT2, and TK1 (arrows) were all suppressed with silencing of EWS-FLI1. (C) Confirmatory PCR in 4 cell lines showing suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1 as measured by qPCR. (P for all treatments vs. no change) (D) Western blots confirm suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1.

Mentions: We reasoned that because EWS-FLI1 alters the expression of a percentage of the transcriptome, a commercially available PET tracer could likely be used to monitor EWS-FLI1 activity14. We hypothesized that we could identify a surface protein driven by EWS-FLI1 in Ewing sarcoma cells that could be labeled with a radioactively tagged transport substrate, ligand, or antibody (Fig. 1A). This would allow the measurement of expression before and after drug treatment to quantify EWS-FLI1 suppression. We linked available tracers to the proteins responsible for their PET activity and used qPCR to see if any were suppressed upon siRNA silencing of EWS-FLI1 in three Ewing sarcoma cell lines. We found limited suppression of the majority of these targets, except for three proteins responsible for 18F-FLT activity, all of which were significantly suppressed with silencing of EWS-FLI1 (see arrows, Fig. 1B; statistics in Supplementary Table 1).


18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma
Identification of 18F-FLT as a pharmacodynamic marker of EWS-FLI1 activity.(A) Schema showing theory of study. Suppression of an EWS-FLI1 target expressed on the cell surface will allow quantitation of EWS-FLI1 activity based on loss of transport of a labeled substrate or loss of binding of a labeled antibody or ligand. (B) Heat map summarizing screen results. EWS-FLI1 was silenced in three different cell lines (TC32, TC71 and EW8) and the induction (red) or suppression (blue) of expression was measured by qPCR. ENT1, ENT2, and TK1 (arrows) were all suppressed with silencing of EWS-FLI1. (C) Confirmatory PCR in 4 cell lines showing suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1 as measured by qPCR. (P for all treatments vs. no change) (D) Western blots confirm suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Identification of 18F-FLT as a pharmacodynamic marker of EWS-FLI1 activity.(A) Schema showing theory of study. Suppression of an EWS-FLI1 target expressed on the cell surface will allow quantitation of EWS-FLI1 activity based on loss of transport of a labeled substrate or loss of binding of a labeled antibody or ligand. (B) Heat map summarizing screen results. EWS-FLI1 was silenced in three different cell lines (TC32, TC71 and EW8) and the induction (red) or suppression (blue) of expression was measured by qPCR. ENT1, ENT2, and TK1 (arrows) were all suppressed with silencing of EWS-FLI1. (C) Confirmatory PCR in 4 cell lines showing suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1 as measured by qPCR. (P for all treatments vs. no change) (D) Western blots confirm suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1.
Mentions: We reasoned that because EWS-FLI1 alters the expression of a percentage of the transcriptome, a commercially available PET tracer could likely be used to monitor EWS-FLI1 activity14. We hypothesized that we could identify a surface protein driven by EWS-FLI1 in Ewing sarcoma cells that could be labeled with a radioactively tagged transport substrate, ligand, or antibody (Fig. 1A). This would allow the measurement of expression before and after drug treatment to quantify EWS-FLI1 suppression. We linked available tracers to the proteins responsible for their PET activity and used qPCR to see if any were suppressed upon siRNA silencing of EWS-FLI1 in three Ewing sarcoma cell lines. We found limited suppression of the majority of these targets, except for three proteins responsible for 18F-FLT activity, all of which were significantly suppressed with silencing of EWS-FLI1 (see arrows, Fig. 1B; statistics in Supplementary Table 1).

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