Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by a fusion protein (e.g. PAX3-FOXO1) or by mutations in key signalling molecules (e.g. RAS or FGFR4). Despite the latter giving potential for precision medicine approaches in RMS, there are no such treatments implemented in the clinic yet. In order to identify and test novel precision therapy strategies, appropriate cellular and mouse models are crucial. We have here thoroughly characterized a RMS patient-derived cell line model, RMS559, which harbours a FGFR4 V550L activating mutation with high allelic frequency (0.8). Importantly, we show that RMS559 cells are oncogenically dependent on FGFR4 signalling by treatment with the pan-FGFR inhibitor LY2874455. Phosphoproteomic analysis identified RAS/MAPK and PI3K/AKT as the major druggable signalling pathways downstream of FGFR V550L. Inhibitors against these pathways inhibited cell proliferation. Furthermore, we found that FGFR4 V550L is dependent on HSP90 and inhibitors targeting HSP90 efficiently restrain proliferation. Recently, FGFR4 specific inhibitors have been developed. While two of these, BLU-9931 and H3B-6527, did not efficiently inhibit FGFR4 V550L, probably because of the gatekeeper mutation (V550L), one of them, FGF401 inhibited FGFR4 V550L and cell proliferation at low nanomolar concentrations. Finally, we developed a mouse model using RMS559 cells and tested the in vivo efficacy of LY2874455 and FGF401. While LY2874455 inefficiently inhibited growth, FGF401 completely abrogated tumour growth in vivo.

横纹肌肉瘤（Rhabdomyosarcoma, RMS）是一种儿童恶性肿瘤，其致病驱动机制可分为两类：一类由融合蛋白（如PAX3-FOXO1）介导，另一类则由关键信号分子的突变（如RAS或FGFR4）引发。尽管后者为横纹肌肉瘤的精准医疗提供了潜在可行方向，但目前临床尚未有相关治疗方案获批应用。为识别并验证新型精准治疗策略，合适的细胞与小鼠模型至关重要。本研究已对一株患者来源的横纹肌肉瘤细胞系模型RMS559开展了全面表征，该细胞系携带有等位基因频率高达0.8的FGFR4 V550L激活突变。尤为重要的是，我们通过泛FGFR抑制剂LY2874455处理实验，证实RMS559细胞的致癌增殖依赖于FGFR4信号通路。磷酸化蛋白质组学分析显示，RAS/MAPK与PI3K/AKT是FGFR4 V550L下游的主要可靶向信号通路，针对这两条通路的抑制剂可有效抑制细胞增殖。此外，我们发现FGFR4 V550L的信号活化依赖于热休克蛋白90（HSP90），靶向HSP90的抑制剂可有效抑制细胞增殖。近期已有FGFR4特异性抑制剂问世：其中BLU-9931与H3B-6527因存在守门人突变（gatekeeper mutation，V550L）而未能有效抑制FGFR4 V550L的活性，但FGF401可在低纳摩尔浓度下抑制FGFR4 V550L活性与细胞增殖。最后，我们利用RMS559细胞构建了小鼠异种移植模型，并验证了LY2874455与FGF401的体内抗肿瘤疗效：LY2874455的抑瘤效果不佳，而FGF401可完全阻断体内肿瘤的生长。