Elisa Fiorito1,2, Patrycja Szybowska1,2, Ellen M Haugsten1,2, Michal Kostas1,2, Geir F Øy1, Antoni Wiedlocha2,3, Sachin Singh1,2, Sigve Nakken1,2, Gunhild M Mælandsmo1,4, Jonathan A Fletcher5, Leonardo A Meza-Zepeda1,2,6, Jørgen Wesche7,8,9. 1. Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0379, Oslo, Norway. 2. Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Montebello, 0379, Oslo, Norway. 3. Department of Molecular Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0379, Oslo, Norway. 4. Institute of Medical Biology, Faculty of Health Sciences, The Arctic University of Norway - University of Tromsø, 9037, Tromsø, Norway. 5. Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. 6. Genomics Core Facility, Department of Core Facilities, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0379, Oslo, Norway. 7. Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0379, Oslo, Norway. jorgen.wesche@rr-research.no. 8. Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Montebello, 0379, Oslo, Norway. jorgen.wesche@rr-research.no. 9. Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 0372, Oslo, Norway. jorgen.wesche@rr-research.no.
Abstract
BACKGROUND: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. METHODS: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. RESULTS: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. CONCLUSIONS: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.
BACKGROUND: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. METHODS: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. RESULTS: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. CONCLUSIONS: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.
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