Ponatinib also Ligustilide supplier inhibited growth of tumors expressing mutated FGFR4 in vivo. Therefore, our results indicate that ponatinib is an effective FDA-approved drug which has the potential to treat RMS with overexpressed or mutated FGFR4. Consistent with these findings, ponatinib inhibited the growth of multiple fusion-positive and fusion-negative RMS cell lines, all with IC50 values in the nanomolar range. Our data confirmed that FGFR4 mRNA expression was significantly higher in fusionpositive cell lines than fusion-negative cell lines. In addition, the 935693-62-2 sensitivity to ponatinib correlated with FGFR4 mRNA expression levels and that fusion-positive cell lines with the higher FGFR4-expressing levels were consistently sensitive to ponatinib. However, cell lines that were fusion-negative or expressed FGFR4 at low levels had a wider variation of ponatinib sensitivity. Therefore, it is possible that a certain threshold of FGFR4 expression is needed for consistent nanomolar sensitivity to ponatinib. In addition, no mutations were found in any of the cell lines by Sanger sequencing of exon 12 and 13 of FGFR4, eliminating the possibility of a mutation conferring sensitivity in any of the studied cell lines. However, since ponatinib is a multikinase inhibitor, which includes inhibition of RET, LYN, LCK, FYN, and ABL at subnanomolar concentrations, it is possible that the activity is related to the inhibition of other kinases. Indeed, even some cell lines with lower levels of FGFR4 expression continue to demonstrate sensitivity to ponatinib and it is possible that this effect may be the result of inhibition of targets other than FGFR4. This is demonstrated in normal skin fibroblast, osteosarcoma, and Ewings sarcoma cell lines. Similar to the RMS cell lines with high expression of wild-type FGFR4, we have shown ponatinib to be effective against a RMS model system with constitutively activating FGFR4 mutations N535K and V550E. After treatment of cells expressing the mutated FGFR4 with ponatinib, IC50 values were achieved in the nanomolar range within 24 hours. We found there was G1/S arrest of cell cycling with an increase in the sub G1 phase fraction indicating cell death, which was confirmed by caspase 3/7 induction. It is interesting to note that the in vitro data shows ponatinib to be effective against wild-type and mutant FGFR4, whereas our in vivo results show that ponatinib only inhibits tumor growth of cells harboring the FGFR4 mutations but not the wild-type FGFR4. One possible reason for this may come from our observation that the murine RMS cells expressing wild-type FGFR4 have a higher IC50 than the cells expressing the two mutant FGFR4s.