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. 2013 Oct 4;8(10):e76551.
doi: 10.1371/journal.pone.0076551. eCollection 2013.

Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma With the Inhibitor Ponatinib (AP24534)

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Free PMC article

Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma With the Inhibitor Ponatinib (AP24534)

Samuel Q Li et al. PLoS One. .
Free PMC article

Abstract

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RMS cell lines with overexpressed FGFR4 are more sensitive to ponatinib.
(A) The sensitivity of a panel of six fusion-positive RMS cell lines (RH4, RH28, JR, RH41, RH5, and RH30) and eight fusion-negative RMS cell lines (BIRCH, RH18, TTC-442, CT-10, CTR, TTC-516, RD, and RH36) to ponatinib is correlated to FGFR4 mRNA expression levels by Spearman ranking (p = 0.0261). (B) Comparing the variation in IC50 values of fusion-positive (FP) and fusion-negative (FN) RMS cell lines shows a significant difference by F test (p = 0.0125). (C) A difference in IC50 values can be seen between RMS cell lines expressing low (below a relative level of 6) and high (above a relative level of 6) levels of FGFR4 (p = 0.0344).
Figure 2
Figure 2. RMS772 cell harboring activating FGFR4 mutations V550E or N535K are more sensitive to ponatinib (AP24534) after 24 hour treatment than RMS772 cells expressing wild-type (WT) FGFR4 or the empty vector (VCtrl) (*p = <0.0001, **p = <0.0001).
Figure 3
Figure 3. Ponatinib (AP24534) holds cell cycling at sub G1 phase and induces cell death via apoptosis.
(A) Cell cycle analysis of the two most sensitive cell lines to ponatinib, RH4 and RH5, and the two RMS772 cell lines expressing FGFR4 mutations (N535K and V550E) showed increased time in sub G1 phase and decreased time in S phase across all four cell lines after 24 hours of treatment with 2.5 µM ponatinib. (B) Cell death induced by 2.5 µM ponatinib treatment is mediated via the caspase 3/7 pathway (*p = 0.0029, **p = 0.0027, ***p = 0.0017, ****p = 0.0001).
Figure 4
Figure 4. Western blot analysis of expression and phosphorylation of wild-type and mutated FGFR4 and its downstream target, STAT3, after treatment with 0, 200, and 800 nM concentrations of ponatinib (AP24534) for 8 hours.
(A) A dose-dependent decrease in wild-type FGFR4 phosphorylation as shown by immunoprecipitation of FGFR4 and immunoblotting for phosphotyrosine. (B) A similar dose-dependent inhibition is seen for FGFR4 with the V550E and N535K mutation. (C-D) Western blot shows a dose-dependent decrease in STAT3 phosphorylation after treatment with ponatinib for three fusion-positive (RH4, RH5, and RH41) and one fusion-negative (CTR) RMS cell lines as well as the two RMS772 cell lines expressing the FGFR4 mutations N535K and V550E.
Figure 5
Figure 5. In vivo tumor growth assay with daily treatment of 30 mg/kg of ponatinib after tumor volumes reach 100 mm3.
Arrow indicates the start of treatment. (A) Treatment of tumors harboring the FGFR4 N535K mutation with ponatinib significantly inhibits tumor growth after 10 days of treatment (*p = 0.0165, **p = 0.0048). (B) Treatment of tumors containing the FGFR4 V550E mutation with ponatinib significantly inhibits tumor growth after 6 days of treatment (*p = 0.0185, **p = 0.0087, ***p = 0.0005). (C) Treatment of tumors expressing the wild-type FGFR4 with ponatinib does not affect tumor growth. (D) Treatment of tumors expressing the empty vector with ponatinib does not affect tumor growth.

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