Dataset Information

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Sachin Singh

LAB HEAD: Jørgen Wesche

PROVIDER: PXD029719 | Pride | 2022-10-19

REPOSITORIES: Pride

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Publications

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma.

Fiorito Elisa E Szybowska Patrycja P Haugsten Ellen M EM Kostas Michal M Øy Geir F GF Wiedlocha Antoni A Singh Sachin S Nakken Sigve S Mælandsmo Gunhild M GM Fletcher Jonathan A JA Meza-Zepeda Leonardo A LA Wesche Jørgen J

British journal of cancer 20220912 11

<h4>Background</h4>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.<h4>Methods</h4>We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction o ...[more]

PMID: 36097178

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Project description:Background. Heat shock protein 90 (Hsp90) is essential for the stability and the function of many client proteins, such as ERB2, C-RAF, CDK4, HIF-1 aplha and AKT. Recent reports demonstrated that inhibition of Hsp90 modulates multiple functions required for survival of human cancer, such as myeloma (Mitsiades et al, Blood:107, 1092, 2006), however, the precise mechanism of anti-cancer effect of Hsp90 inhibition is still uncertain. Aim. The aim of this study is evaluate the effect of Hsp90 inhibition, and to identify molecular pathways responsible for anti-proliferative effect on ATL cells. Method. For Hsp90 inhibition, Geldanamycin derivates, 17AAG (17-allylamino -17-demethoxygeldanamycin) and 17DMAG (17-(dimethylaminoethylamino) 17-demethoxygeldanamycin) were used in this study. Interleukin 2-independent ATL cell lines (MT-2 and MT-4) and an interleukin 2-dependent ATL cell line (TaY) were incubated, with or without Hsp90 inhibitors. Fresh ATL cells obtained from patients were also used after obtaining informed consent. Cell numbers at 48 h after incubation with or without Hsp90 inhibitors were assessed with the Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, MD, USA). For detection of apoptosis, we used Annexin V-biotin apoptosis detection kit (Calbiochem, La Jolla, CA, USA). Gene expression analysis was done using a DNA microarray (NCBI Gene expression omnibus; GPL 2531) and statistical analysis was done by a GeneSifter (VizXlabs, Seattle, WA, USA). Results. We found cell death induced by Hsp90 inhibitors in all the 3 ATL cell lines as well as patient specimens. Inhibitory concentration (IC50) of 17AAG in 3 ATL cell lines was 300 to 700 nM, and that of 17DMAG was 150 to 200 nM. Fresh ATL cells obtained from patients were more sensitive for either 17AAG or 17DMAG. Gene expression analysis of ATL cells revealed that up-regulation of HSPA1A encoding Hsp70, and genes related to cell cycle arrest (i.e. CDKN1A). Genes regulating cell proliferation or anti-apoptosis (i.e. MYC, BCL2 and Cyclin C), genes related to cytokine or chemokine (i.e. IL9, CCL 17, and CCL27), and notably, genes involved in Wnt/-catenin signaling pathway (i.e.TCF7L2 and TCF4), were remarkably repressed. Inhibition of AKT at the protein level was also evident, suggesting the possibility that AKT may down-regulates -catenin/ TCF7L2 pathways in response to Hsp90 inhibitors in ATL cells. Conclusion. Our results have provided new insights into the complex molecular pharmacology of Hsp90 inhibitors, and suggest that Hsp90 inhibitors might be beneficial as anti-proliferative agent in treating ATL patients. Six samples treated with Hsp90 inhibitors (17-AAG or 17-DMAG) were analyzed in biological duplicate.

Dataset Information

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma

Publications

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma.

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