Impaired fibroblast growth factor receptor (FGFR) signaling is associated with many human conditions, including growth disorders, degenerative diseases, and cancer. Current FGFR therapeutics are based on chemical inhibitors of FGFR tyrosine kinase activity (TKIs). However, FGFR TKIs are limited in their target specificity as they generally inhibit all FGFRs and other receptor tyrosine kinases. In the search for specific inhibitors of human FGFR1, we identified VZ23, a DNA aptamer that binds to FGFR1b and FGFR1c with a KD of 55 nM and 162 nM, respectively, but not to the other FGFR variants (FGFR2b, FGFR2c, FGFR3b, FGFR3c, FGFR4). In cells, VZ23 inhibited the activation of downstream FGFR1 signaling and FGFR1-mediated regulation of cellular senescence, proliferation, and extracellular matrix homeostasis. Consistent with the specificity toward FGFR1 observed in vitro, VZ23 did not inhibit FGFR2-4 signaling in cells. We show that the VZ23 inhibits FGFR1 signaling in the presence of cognate fibroblast growth factor (FGF) ligands and its inhibitory activity is linked to its capacity to form unusual G-quadruplex structure. Our data suggest that targeting FGFR1 with DNA aptamers could be an effective alternative to TKIs for treating impaired FGFR1 signaling in human craniosynostoses.

Central European Institute of Technology Masaryk University 625 00 Brno Czechia

Department of Biology Faculty of Medicine Masaryk University 62500 Brno Czechia

Department of Materials Science and Engineering Institute for NanoBioTechnology and Program in Molecular Biophysics Johns Hopkins University Baltimore MD 21218 USA

Department of Protein Biotechnology University of Wroclaw 50 383 Wroclaw Poland

Department of Protein Engineering University of Wroclaw 50 383 Wroclaw Poland

Institute of Animal Physiology and Genetics of the CAS 60200 Brno Czechia

International Clinical Research Center St Anne's University Hospital 65691 Brno Czechia

National Centre for Biomolecular Research Masaryk University 625 00 Brno Czechia

Slovenian NMR Centre National Institute of Chemistry 1000 Ljubljana Slovenia

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Itoh N., Ornitz D.M. Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J. Biochem. 2011;149:121–130. doi: 10.1093/jb/mvq121. PubMed DOI PMC

Gong S.G. Isoforms of Receptors of Fibroblast Growth Factors. J. Cell. Physiol. 2014;229:1887–1895. doi: 10.1002/jcp.24649. PubMed DOI

Ornitz D.M., Xu J., Colvin J.S., McEwen D.G., MacArthur C.A., Coulier F., Gao G., Goldfarb M. Receptor Specificity of the Fibroblast Growth Factor Family. J. Biol. Chem. 1996;271:15292–15297. doi: 10.1074/jbc.271.25.15292. PubMed DOI

Zhang X., Ibrahimi O.A., Olsen S.K., Umemori H., Mohammadi M., Ornitz D.M. Receptor Specificity of the Fibroblast Growth Factor Family. J. Biol. Chem. 2006;281:15694–15700. doi: 10.1074/jbc.M601252200. PubMed DOI PMC

Yamaguchi T.P., Rossant J. Fibroblast growth factors in mammalian development. Curr. Opin. Genet. Dev. 1995;5:485–491. doi: 10.1016/0959-437X(95)90053-J. PubMed DOI

Ornitz D.M. FGF signaling in skeletal development. Front. Biosci. 1998;3:d781–d794. doi: 10.2741/A321. PubMed DOI

Gallo L.H., Nelson K.N., Meyer A.N., Donoghue D.J. Functions of Fibroblast Growth Factor Receptors in cancer defined by novel translocations and mutations. Cytokine Growth Factor Rev. 2015;26:425–449. doi: 10.1016/j.cytogfr.2015.03.003. PubMed DOI

Kalyukina M., Yosaatmadja Y., Middleditch M.J., Patterson A.V., Smaill J.B., Squire C.J. TAS-120 Cancer Target Binding: Defining Reactivity and Revealing the First Fibroblast Growth Factor Receptor 1 (FGFR1) Irreversible Structure. ChemMedChem. 2019;14:494–500. doi: 10.1002/cmdc.201800719. PubMed DOI

Subbiah V., Sahai V., Maglic D., Bruderek K., Touré B.B., Zhao S., Valverde R., O’Hearn P.J., Moustakas D.T., Schönherr H., et al. RLY-4008, the First Highly Selective FGFR2 Inhibitor with Activity across FGFR2 Alterations and Resistance Mutations. Cancer Discov. 2023;13:2012–2031. doi: 10.1158/2159-8290.CD-23-0475. PubMed DOI PMC

Kamatkar N., Levy M., Hébert J.M. Development of a Monomeric Inhibitory RNA Aptamer Specific for FGFR3 that Acts as an Activator When Dimerized. Mol. Ther. Nucleic Acids. 2019;17:530–539. doi: 10.1016/j.omtn.2019.06.020. PubMed DOI PMC

Eguchi A., Ueki A., Hoshiyama J., Kuwata K., Chikaoka Y., Kawamura T., Nagatoishi S., Tsumoto K., Ueki R., Sando S. A DNA Aptamer That Inhibits the Aberrant Signaling of Fibroblast Growth Factor Receptor in Cancer Cells. JACS Au. 2021;1:578–585. doi: 10.1021/jacsau.0c00121. PubMed DOI PMC

Kimura T., Bosakova M., Nonaka Y., Hruba E., Yasuda K., Futakawa S., Kubota T., Fafilek B., Gregor T., Abraham S.P., et al. An RNA aptamer restores defective bone growth in FGFR3-related skeletal dysplasia in mice. Sci. Transl. Med. 2021;13 doi: 10.1126/scitranslmed.aba4226. PubMed DOI

Nakamura Y. Multiple Therapeutic Applications of RBM-007, an Anti-FGF2 Aptamer. Cells. 2021;10:1617. doi: 10.3390/cells10071617. PubMed DOI PMC

Aikawa T., Segre G.V., Lee K. Fibroblast Growth Factor Inhibits Chondrocytic Growth through Induction of p21 and Subsequent Inactivation of Cyclin E-Cdk2. J. Biol. Chem. 2001;276:29347–29352. doi: 10.1074/jbc.M101859200. PubMed DOI

Krejci P., Prochazkova J., Smutny J., Chlebova K., Lin P., Aklian A., Bryja V., Kozubik A., Wilcox W.R. FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence. Bone. 2010;47:102–110. doi: 10.1016/j.bone.2010.03.021. PubMed DOI PMC

Dailey L., Laplantine E., Priore R., Basilico C. A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation. J. Cell Biol. 2003;161:1053–1066. doi: 10.1083/jcb.200302075. PubMed DOI PMC

Sokolowska-Wedzina A., Borek A., Chudzian J., Jakimowicz P., Zakrzewska M., Otlewski J. Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells. Protein Expr. Purif. 2014;99:50–57. doi: 10.1016/j.pep.2014.03.012. PubMed DOI

Sokolowska-Wedzina A., Chodaczek G., Chudzian J., Borek A., Zakrzewska M., Otlewski J. High-Affinity Internalizing Human scFv-Fc Antibody for Targeting FGFR1-Overexpressing Lung Cancer. Mol. Cancer Res. 2017;15:1040–1050. doi: 10.1158/1541-7786.MCR-16-0136. PubMed DOI

Sarabipour S., Hristova K. Mechanism of FGF receptor dimerization and activation. Nat. Commun. 2016;7 doi: 10.1038/ncomms10262. PubMed DOI PMC

Fafilek B., Balek L., Bosakova M.K., Varecha M., Nita A., Gregor T., Gudernova I., Krenova J., Ghosh S., Piskacek M., et al. The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases. Sci. Signal. 2018;11 doi: 10.1126/scisignal.aap8608. PubMed DOI

Hadari Y.R., Gotoh N., Kouhara H., Lax I., Schlessinger J. Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways. Proc. Natl. Acad. Sci. USA. 2001;98:8578–8583. doi: 10.1073/pnas.161259898. PubMed DOI PMC

Ornitz D.M., Itoh N. The Fibroblast Growth Factor signaling pathway. WIREs Dev. Biol. 2015;4:215–266. doi: 10.1002/wdev.176. PubMed DOI PMC

Gudernova I., Foldynova-Trantirkova S., Ghannamova B.E., Fafilek B., Varecha M., Balek L., Hruba E., Jonatova L., Jelinkova I., Kunova Bosakova M., et al. One reporter for in-cell activity profiling of majority of protein kinase oncogenes. Elife. 2017;6 doi: 10.7554/eLife.21536. PubMed DOI PMC

Plotnikov A.N., Schlessinger J., Hubbard S.R., Mohammadi M. Structural Basis for FGF Receptor Dimerization and Activation. Cell. 1999;98:641–650. doi: 10.1016/S0092-8674(00)80051-3. PubMed DOI

Kiselyov V.V., Kochoyan A., Poulsen F.M., Bock E., Berezin V. Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1. Protein Sci. 2006;15:2318–2322. doi: 10.1110/ps.062206106. PubMed DOI PMC

Hansson E.M., Teixeira A.I., Gustafsson M.V., Dohda T., Chapman G., Meletis K., Muhr J., Lendahl U. Recording Notch Signaling in Real Time. Dev. Neurosci. 2006;28:118–127. doi: 10.1159/000090758. PubMed DOI

Biadun M., Sochacka M., Kalka M., Chorazewska A., Karelus R., Krowarsch D., Opalinski L., Zakrzewska M. Uncovering key steps in FGF12 cellular release reveals a common mechanism for unconventional FGF protein secretion. Cell. Mol. Life Sci. 2024;81:356. doi: 10.1007/s00018-024-05396-9. PubMed DOI PMC