Studies of gene fusions in rhabdomyosarcoma

Division Of Basic Sciences - Nci

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Abstract

In our previous studies, we elucidated the mechanisms related to fibroblast growth factor (FGF) signaling in RMS tumors. Within this pathway, the genes for both FGF8 (an FGF ligand) and FGFR4 (an FGF receptor) are downstream transcriptional targets of PAX3::FOXO1 (P3F). We previously used a myoblast model of fusion-positive rhabdomyosarcoma (FP-RMS) to show that FGF8 is required for P3F-driven tumorigenicity and, when aberrantly expressed, can maintain tumorigenicity in P3F-independent recurrent tumors. Comparison of FP-RMS lines with high FGFR4 and FGF8 expression to lines with low FGFR4 and FGF8 expression revealed higher FGFR4-phosphotyrosine levels in the former lines. These FGFR4-phosphotyrosine differences could be due to intrinsic differences in FGFR4 and/or FGF8 expression and may predict differential vulnerability to FGFR inhibitors. High FGF8 expression in FP-RMS cells is associated with high sensitivity to an FGFR4 inhibitor and a pan-FGFR inhibitor, and FGF8 downregulation results in loss of sensitivity to these inhibitors. In addition, FGFR4 knockdown also resulted in decreased sensitivity to the FGFR4-specific inhibitor. Based on recent single cell RNA sequencing studies that identified multiple subpopulations within FP-RMS tumors, we found that FGF8 and FGFR4 are highly expressed in the same subpopulations (Proliferative and Ground) whereas FGFR1 is most highly expressed in a different subpopulation (Neuronal) which is also the subpopulation with the highest expression of the P3F target signature. The effects of FGF8 upregulation also involve a reciprocal decrease in FGFR4 expression in a signaling pathway that is reverted by inhibitors of FGFR1, MEK or ERK. To better observe the effect of FGF8 on FGFR4 expression in P3F-expressing cells, we performed an experiment in our human myoblast system in which we monitored FGF8-induced changes in FGFR4 expression over a range of P3F induction doses. The magnitude of FGF8-induced inhibition of FGFR4 is highest at low P3F levels and gradually decreases until there is no noticeable FGFR4 inhibition at the highest P3F levels. These findings indicate that the inhibition of FGFR4 expression by FGF8 is antagonized by the stimulation of FGFR4 expression by P3F, and thus the effect of FGF8 will depend on the P3F level in the tumor cells and will certainly be most prominent in tumors that have lost or have low residual P3F expression. These results are consistent with a scenario in which the induction of FGF8 expression by P3F is involved in a feedback loop to lower and fine-tune the expression level of FGFR4 that is induced by P3F. These collected findings are also consistent with our hypothesis that FGF8 exerts oncogenic effects in FP-RMS via FGFR4 and exerts oncogenic effects in P3F-independent relapses via FGFR1.