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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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18F-FLT (but not 18F-FDG) PET activity is suppressed with EWS-FLI1 silencing.(A) 18F-FLT PET activity decreased in vitro in TC32 Ewing sarcoma cells with siRNA silencing of EWS-FLI1(siEWS) but not with a non-targeting control (siNeg); EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105 decreases 18F-FLT activity (B) 18F-FDG activity does not change with siRNA silencing of EWS-FLI1 and with EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105. 5FU suppresses 18F-FDG activity likely due to a loss of cell viability.
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f3: 18F-FLT (but not 18F-FDG) PET activity is suppressed with EWS-FLI1 silencing.(A) 18F-FLT PET activity decreased in vitro in TC32 Ewing sarcoma cells with siRNA silencing of EWS-FLI1(siEWS) but not with a non-targeting control (siNeg); EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105 decreases 18F-FLT activity (B) 18F-FDG activity does not change with siRNA silencing of EWS-FLI1 and with EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105. 5FU suppresses 18F-FDG activity likely due to a loss of cell viability.

Mentions: Next, we showed that the suppression of EWS-FLI1 and of ENT1, ENT2, and TK1 expression translated into suppression of 18F-FLT activity in Ewing sarcoma cells in vitro. 18F-FLT activity was markedly suppressed with silencing of EWS-FLI1 by siRNA, but not when a non-targeting siRNA (control) was used (Fig. 3A). As additional evidence for the specificity of this effect for EWS-FL1, we showed that the suppression of 18F-FLT was phenocopied with EWS-FLI1-directed small molecules. We showed a highly significant suppression of 18F-FLT activity upon treatment with 50 nM mithramycin, 50 nM EC-8042, and 15 nM EC-8105 (Fig. 3A; statistics in Supplementary Table 4a). As further evidence of specificity, we treated cells with the chemotherapeutic agent 5-FU. Again, no suppression of 18F-FLT activity was found; in fact, its PET activity was induced consistent with known effects of 5-FU28 (Fig. 3A).


18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma
18F-FLT (but not 18F-FDG) PET activity is suppressed with EWS-FLI1 silencing.(A) 18F-FLT PET activity decreased in vitro in TC32 Ewing sarcoma cells with siRNA silencing of EWS-FLI1(siEWS) but not with a non-targeting control (siNeg); EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105 decreases 18F-FLT activity (B) 18F-FDG activity does not change with siRNA silencing of EWS-FLI1 and with EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105. 5FU suppresses 18F-FDG activity likely due to a loss of cell viability.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037393&req=5

f3: 18F-FLT (but not 18F-FDG) PET activity is suppressed with EWS-FLI1 silencing.(A) 18F-FLT PET activity decreased in vitro in TC32 Ewing sarcoma cells with siRNA silencing of EWS-FLI1(siEWS) but not with a non-targeting control (siNeg); EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105 decreases 18F-FLT activity (B) 18F-FDG activity does not change with siRNA silencing of EWS-FLI1 and with EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105. 5FU suppresses 18F-FDG activity likely due to a loss of cell viability.
Mentions: Next, we showed that the suppression of EWS-FLI1 and of ENT1, ENT2, and TK1 expression translated into suppression of 18F-FLT activity in Ewing sarcoma cells in vitro. 18F-FLT activity was markedly suppressed with silencing of EWS-FLI1 by siRNA, but not when a non-targeting siRNA (control) was used (Fig. 3A). As additional evidence for the specificity of this effect for EWS-FL1, we showed that the suppression of 18F-FLT was phenocopied with EWS-FLI1-directed small molecules. We showed a highly significant suppression of 18F-FLT activity upon treatment with 50 nM mithramycin, 50 nM EC-8042, and 15 nM EC-8105 (Fig. 3A; statistics in Supplementary Table 4a). As further evidence of specificity, we treated cells with the chemotherapeutic agent 5-FU. Again, no suppression of 18F-FLT activity was found; in fact, its PET activity was induced consistent with known effects of 5-FU28 (Fig. 3A).

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