Studies of gene fusions in rhabdomyosarcoma

Division Of Basic Sciences - Nci

Investigators

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Abstract

In our recently published work, we demonstrated that FGF8 influences proliferation and transformation of fusion-positive (FP) rhabdomyosarcoma (RMS) cells as well as tumorigenicity of primary and recurrent tumors in our myoblast model system. My laboratory and other laboratories previously determined that PAX3-FOXO1 (P3F) induces upregulation of both FGF8 and FGFR4, and thus P3F is able to activate expression of both a growth factor and its receptor in FP RMS. To further understand fibroblast growth factor (FGF) signaling in RMS tumors, we analyzed expression of the FGF's and FGF receptors (FGFR1, FGFR2, FGFR3, FGFR4) in human primary RMS tumors and cell lines. Of the four receptors, FGFR1 and FGFR4 are highly expressed in both FP and fusion-negative (FN) RMS tumors, and of the FGFs, FGF8 is most highly expressed in FP tumors. Whereas FGFR1 and FGFR4 expression levels are relatively consistent among FP RMS tumors, there is substantial variability in FGF8 expression in FP RMS tumors. In the human Dbt myoblast model system, FGF8 is induced by P3F in parental and primary tumor-derived (TD) cells and is expressed at very high levels in a P3F-independent manner in several recurrent TD clones. For the FGF receptors, FGFR1 was uniformly present in primary and recurrent TD cells whereas FGFR4 was higher in P3F-induced parental and primary TD cells, but very low to absent in the recurrent TD cells. To study the functional significance of these growth factors and receptors, we selected FGF receptor inhibitors that are specific for FGFR1 (PD173074) and FGFR4 (H3B6527) in addition to a potent pan inhibitor (Erdafitinib). In real-time population growth and colony assays, FP RMS cells with high FGF8 expression are more sensitive to the FGFR4 and pan-FGFR inhibitors than FP RMS cells with low FGF8 whereas there was no consistent difference between the two groups in the effect of the FGFR1 inhibitor. To investigate the relationship of FGF8 to these inhibitors, we knocked out FGF8 in the high FGF8-expressing FP RMS cell line RH30. Comparison of control to knockout subclones revealed a lower sensitivity to the FGFR4 and pan-FGFR inhibitors in the knockout subclones but no difference in FGFR1 inhibitor sensitivity. These findings suggest that FGF8 expression in FP RMS cells is necessary for the sensitivity to FGFR inhibitors. We then assessed the effect of P3F on these effects in three different systems. First, comparison of drug sensitivity between high FGF8-expressing RH30 cells and a spontaneous non-transformed variant expressing very low levels of P3F (as well as FGF8 and FGFR4) revealed a substantial loss in sensitivity to the FGFR4 and pan-FGFR inhibitors in the low P3F variant and only a small change in FGFR1 inhibitor sensitivity. Second, after P3F knockout in two FP RMS lines, these lines demonstrated FGF8 and FGFR4 downregulation and a large loss in sensitivity to the FGFR4 and pan-FGFR inhibitors but only a small change in FGFR1 inhibitor sensitivity. Third, after inducing P3F expression in our Dbt myoblast system, we found the expected increase in FGF8 and FGFR4 expression along with augmented sensitivity to the FGFR4 and pan-FGFR inhibitors but not to the FGFR1 inhibitor. To determine the effect of FGF8 on inhibitor sensitivity in the absence of P3F, we introduced FGF8 into the MYCN-expressing Dbt cells and found increased sensitivity to the FGFR1 and pan-FGFR inhibitors but not the FGFR4 inhibitor. These results suggest that FGF8 is sufficient to activate the FGF pathway and thus sensitize the cells to the FGF receptor inhibitors. In addition, these results suggest that in the presence of P3F, FGF8 signals through FGFR4 but in the absence of P3F, such as in the P3F independent recurrences, FGF8 signals through FGFR1.