Limits...
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

The suppression of 18F-FLT activity only occurs with EWS-FLI1 suppression and can not be explained by non-specific changes in cell cycle progression (A) Cell cycle changes in TC32 Ewing sarcoma cells that occur with siRNA silencing of EWS-FLI1 or treatment with 50 nM mithramycin (MMA) or the mithramycin analogs EC8105 (15 nM) or EC8042 (50 nM). (B) Cell counts as percentage of total (+/− SD). Grey boxes highlight treatments that increase the number of cells in G1 or S. Treatments identical to Fig. 3A. (C) Treatment of TC32 Ewing sarcoma cells with non-specific chemotherapy: 2 nM vincristine (VCR), 80 nM etoposide (etop) or 50 nM doxorubicin (doxo) does not suppress 18F-FLT activity in comparison to mithramycin (MMA). Cells treated equitoxic concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037393&req=5

f4: The suppression of 18F-FLT activity only occurs with EWS-FLI1 suppression and can not be explained by non-specific changes in cell cycle progression (A) Cell cycle changes in TC32 Ewing sarcoma cells that occur with siRNA silencing of EWS-FLI1 or treatment with 50 nM mithramycin (MMA) or the mithramycin analogs EC8105 (15 nM) or EC8042 (50 nM). (B) Cell counts as percentage of total (+/− SD). Grey boxes highlight treatments that increase the number of cells in G1 or S. Treatments identical to Fig. 3A. (C) Treatment of TC32 Ewing sarcoma cells with non-specific chemotherapy: 2 nM vincristine (VCR), 80 nM etoposide (etop) or 50 nM doxorubicin (doxo) does not suppress 18F-FLT activity in comparison to mithramycin (MMA). Cells treated equitoxic concentrations.

Mentions: Therefore, we needed to show that the suppression of FLT activity did not reflect a nonspecific more rapid progression through the cell cycle. In all cases, we found cell cycle arrest. Silencing of EWS-FLI1 with siRNA caused a marked arrest of TC32 cells in G1, from 40.8% (SEM +/− 3.0) to 60.8 (SEM +/− 3.1) (Fig. 4A,B). This G1 arrest was phenocopied with mithramycin and EC-8042 in particular (Fig. 4B). In contrast, EC-8105 did not cause a G1 arrest and instead increased the percentage of cells in S-phase from 17.4 (SEM +/− 1.7) to 25.8 (SEM +/− 3). One would therefore predict an increase in 18F-FLT activity with these treatments based on the accumulation of cells in late G1 or S that would be expected to increase TK1 expression and activity. However, in every instance where EWS-FLI1 was suppressed 18F-FLT activity decreased regardless of which phase in the cell cycle the cells were arrested. These results show that in Ewing sarcoma cells, blockade of EWS-FLI1 leads to an inability of cells to take up and retain 18F-FLT and this effect is the major determinant of activity in this cell type in the setting of EWS-FLI1 directed therapy.


18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma
The suppression of 18F-FLT activity only occurs with EWS-FLI1 suppression and can not be explained by non-specific changes in cell cycle progression (A) Cell cycle changes in TC32 Ewing sarcoma cells that occur with siRNA silencing of EWS-FLI1 or treatment with 50 nM mithramycin (MMA) or the mithramycin analogs EC8105 (15 nM) or EC8042 (50 nM). (B) Cell counts as percentage of total (+/− SD). Grey boxes highlight treatments that increase the number of cells in G1 or S. Treatments identical to Fig. 3A. (C) Treatment of TC32 Ewing sarcoma cells with non-specific chemotherapy: 2 nM vincristine (VCR), 80 nM etoposide (etop) or 50 nM doxorubicin (doxo) does not suppress 18F-FLT activity in comparison to mithramycin (MMA). Cells treated equitoxic concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The suppression of 18F-FLT activity only occurs with EWS-FLI1 suppression and can not be explained by non-specific changes in cell cycle progression (A) Cell cycle changes in TC32 Ewing sarcoma cells that occur with siRNA silencing of EWS-FLI1 or treatment with 50 nM mithramycin (MMA) or the mithramycin analogs EC8105 (15 nM) or EC8042 (50 nM). (B) Cell counts as percentage of total (+/− SD). Grey boxes highlight treatments that increase the number of cells in G1 or S. Treatments identical to Fig. 3A. (C) Treatment of TC32 Ewing sarcoma cells with non-specific chemotherapy: 2 nM vincristine (VCR), 80 nM etoposide (etop) or 50 nM doxorubicin (doxo) does not suppress 18F-FLT activity in comparison to mithramycin (MMA). Cells treated equitoxic concentrations.
Mentions: Therefore, we needed to show that the suppression of FLT activity did not reflect a nonspecific more rapid progression through the cell cycle. In all cases, we found cell cycle arrest. Silencing of EWS-FLI1 with siRNA caused a marked arrest of TC32 cells in G1, from 40.8% (SEM +/− 3.0) to 60.8 (SEM +/− 3.1) (Fig. 4A,B). This G1 arrest was phenocopied with mithramycin and EC-8042 in particular (Fig. 4B). In contrast, EC-8105 did not cause a G1 arrest and instead increased the percentage of cells in S-phase from 17.4 (SEM +/− 1.7) to 25.8 (SEM +/− 3). One would therefore predict an increase in 18F-FLT activity with these treatments based on the accumulation of cells in late G1 or S that would be expected to increase TK1 expression and activity. However, in every instance where EWS-FLI1 was suppressed 18F-FLT activity decreased regardless of which phase in the cell cycle the cells were arrested. These results show that in Ewing sarcoma cells, blockade of EWS-FLI1 leads to an inability of cells to take up and retain 18F-FLT and this effect is the major determinant of activity in this cell type in the setting of EWS-FLI1 directed therapy.

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