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

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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.


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EWS-FLI1 suppression blocks 18F-FLT activity in xenograft models.(A) Treatment of mice bearing a Ewing sarcoma xenograft with EC-8042 suppressed PET avidity of the xenograft at 24 h of treatment. The bladder in cross section (lower panel) shows activity consistent with similar amounts of tracer administered. (B) Suppression of activity correlates with TK1 staining (top) in EC-8042-treated tissue but not control or 5-FU treated tissues (10× view). H&E staining confirms tumor tissue (bottom). Size bar indicates 200 micrometer.
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f5: EWS-FLI1 suppression blocks 18F-FLT activity in xenograft models.(A) Treatment of mice bearing a Ewing sarcoma xenograft with EC-8042 suppressed PET avidity of the xenograft at 24 h of treatment. The bladder in cross section (lower panel) shows activity consistent with similar amounts of tracer administered. (B) Suppression of activity correlates with TK1 staining (top) in EC-8042-treated tissue but not control or 5-FU treated tissues (10× view). H&E staining confirms tumor tissue (bottom). Size bar indicates 200 micrometer.

Mentions: To demonstrate the clinical utility of this assay, we showed that these effects translated to the in vivo setting in our xenograft model of Ewing sarcoma. Mice were implanted with TC71 xenografts in the left gastrocnemius muscle. Once the tumors were established, they were treated with 1 mg/kg mithramycin, 24 mg/kg EC-8042, or 1.5 mg/kg of EC-8105, all by the intraperitoneal route. Again, marked suppression of 18F-FLT activity was observed in vivo (particularly at 24 h) and was most prominent with EC-8042, consistent with an EWS-FLI1-specific effect (Fig. 5A; Supplementary Figs 1 and 2). Cross-section imaging indicated that similar amounts of tracer were administered to the animals (Fig. 5A). Suppression was not observed with 50 mg/kg of intraperitoneal 5-FU, which markedly induced 18F-FLT activity in these Ewing sarcoma xenografts, particularly at 6 h of treatment (Supplementary Fig. 3). Recovery to normal FLT activity occurred at 48 h following treatment (data not shown). Staining of tissue for TK1 showed its suppressed expression upon drug treatment, consistent with in vitro data (Fig. 5B). Interestingly, EC-8042 showed the greatest degree of suppression of 18F-FLT activity and the most marked changes in staining, which correlates with the most profound response to treatment that we recently reported26. In contrast, the staining was more variable with EC-8105 and mithramycin, consistent with the relative activity of these drugs by the intraperitoneal route (Supplementary Fig. 4). Although this correlation with response would need to be tested in a prospective fashion preferably in patients.


18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma
EWS-FLI1 suppression blocks 18F-FLT activity in xenograft models.(A) Treatment of mice bearing a Ewing sarcoma xenograft with EC-8042 suppressed PET avidity of the xenograft at 24 h of treatment. The bladder in cross section (lower panel) shows activity consistent with similar amounts of tracer administered. (B) Suppression of activity correlates with TK1 staining (top) in EC-8042-treated tissue but not control or 5-FU treated tissues (10× view). H&E staining confirms tumor tissue (bottom). Size bar indicates 200 micrometer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: EWS-FLI1 suppression blocks 18F-FLT activity in xenograft models.(A) Treatment of mice bearing a Ewing sarcoma xenograft with EC-8042 suppressed PET avidity of the xenograft at 24 h of treatment. The bladder in cross section (lower panel) shows activity consistent with similar amounts of tracer administered. (B) Suppression of activity correlates with TK1 staining (top) in EC-8042-treated tissue but not control or 5-FU treated tissues (10× view). H&E staining confirms tumor tissue (bottom). Size bar indicates 200 micrometer.
Mentions: To demonstrate the clinical utility of this assay, we showed that these effects translated to the in vivo setting in our xenograft model of Ewing sarcoma. Mice were implanted with TC71 xenografts in the left gastrocnemius muscle. Once the tumors were established, they were treated with 1 mg/kg mithramycin, 24 mg/kg EC-8042, or 1.5 mg/kg of EC-8105, all by the intraperitoneal route. Again, marked suppression of 18F-FLT activity was observed in vivo (particularly at 24 h) and was most prominent with EC-8042, consistent with an EWS-FLI1-specific effect (Fig. 5A; Supplementary Figs 1 and 2). Cross-section imaging indicated that similar amounts of tracer were administered to the animals (Fig. 5A). Suppression was not observed with 50 mg/kg of intraperitoneal 5-FU, which markedly induced 18F-FLT activity in these Ewing sarcoma xenografts, particularly at 6 h of treatment (Supplementary Fig. 3). Recovery to normal FLT activity occurred at 48 h following treatment (data not shown). Staining of tissue for TK1 showed its suppressed expression upon drug treatment, consistent with in vitro data (Fig. 5B). Interestingly, EC-8042 showed the greatest degree of suppression of 18F-FLT activity and the most marked changes in staining, which correlates with the most profound response to treatment that we recently reported26. In contrast, the staining was more variable with EC-8105 and mithramycin, consistent with the relative activity of these drugs by the intraperitoneal route (Supplementary Fig. 4). Although this correlation with response would need to be tested in a prospective fashion preferably in patients.

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