In addition no mutations were found in any of the cell lines by Sanger sequencing of exon of FGFR4 eliminating the possibility of a mutation conferring sensitivity in any of the studied cell lines. However, since ponatinib is a Midostaurin 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 Ewing��s 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. Therefore a higher inhibitory dosage than what was used may be necessary for the treatment of wild-type FGFR4 in order to observe an effect on tumor xenograft growth. Another possible reason for this may be due to the model system we use: our murine RMS772 cell line which artificially 1345982-69-5 expresses human wild-type FGFR4. Although this models human embryonal rhabdomyosarcoma most closely, expressing human wild-type FGFR4 in a mouse cell or growing in an environment with murine stromal growth factors may alter its behavior differently. For example, we have previously shown that human wild-type FGFR4 does not increa
