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Key Points Fibroblast growth factor (FGF) signalling controls a myriad of processes in embryonic development and in tissue homeostasis and metabolism in the adult. Recent structural studies have provided a glimpse of the complexity of molecular control that is in place to fine-tune this signalling system to enable it to produce specific signalling outputs in diverse biological contexts. The interaction of FGFs with heparan sulphate glycosaminoglycan chains of heparan sulphate proteoglycans in the pericellular and extracellular matrix defines their mode of action, that is, whether an FGF acts in a paracrine or endocrine fashion. It also determines the shape of gradient formed by a paracrine FGF ligand in the extracellular matrix, which in turn is a determinant of the biological response to that ligand. In addition to mechanisms common to all FGFs, such as the interaction with heparan sulphate, the biological activity of individual ligands or ligand subfamilies is regulated by mechanisms unique to these ligands: amino-terminal alternative splicing controls the activity of FGF8 subfamily ligands; homodimerization autoinhibits the activity of FGF9 subfamily ligands; and site-specific proteolytic cleavage inactivates the phosphaturic hormone FGF23. Alternative splicing in the extracellular immunoglobulin-like domain 3 (D3) of FGF receptor 1 (FGFR1), FGFR2 and FGFR3 primarily determines the ligand-binding specificity of these receptors. This splicing event is fundamental to the establishment of directional paracrine FGF signalling between the epithelium and the mesenchyme, which underlies the coordinated cellular processes that govern organ development. Klotho co-receptors convert FGFRs into specific receptors for endocrine FGFs by a dual mechanism; these co-receptors not only enhance the binding affinity of FGFRs for endocrine FGFs but concomitantly suppress the binding of paracrine FGFs to FGFRs. The finding that heparan sulphate is dispensable for signalling by endocrine FGFs implies that Klotho co-receptors also promote FGFR dimerization upon endocrine FGF binding, which is required for FGFR activation. The structural findings suggest that there may be no functional redundancy among FGF ligands, and genetic data support this conclusion. Hence, future studies should concentrate on identifying novel ligand-specific functions of FGF signalling. Structural data has provided insight into the molecular mechanisms that modulate fibroblast growth factor (FGF) signalling to generate distinct biological outputs in development, tissue homeostasis and metabolism. Mechanisms include alternative splicing of ligand and receptor, homodimerization and site-specific proteolytic cleavage of ligand, and interaction of ligand and receptor with heparan sulphate and Klotho co-receptors. Fibroblast growth factors (FGFs) mediate a broad range of functions in both the developing and adult organism. The accumulated wealth of structural information on the FGF signalling pathway has begun to unveil the underlying molecular mechanisms that modulate this system to generate a myriad of distinct biological outputs in development, tissue homeostasis and metabolism. At the ligand and receptor level, these mechanisms include alternative splicing of the ligand (FGF8 subfamily) and the receptor (FGFR1–FGFR3), ligand homodimerization (FGF9 subfamily), site-specific proteolytic cleavage of the ligand (FGF23), and interaction of the ligand and the receptor with heparan sulphate cofactor and Klotho co-receptor.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK’s kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

Pemigatinib (PEMAZYRE™), a small molecule inhibitor of fibroblast growth factor receptor (FGFR) 1, FGFR2 and FGFR3, received accelerated approval in April 2020 in the USA for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test. Developed by Incyte Corporation, it is the first targeted treatment for cholangiocarcinoma in the USA. The recommended dosage of pemigatinib is 13.5 mg once daily, administered orally with or without food, on days 1–14 of a 21-day cycle until disease progression or unacceptable toxicity. Pemigatinib received orphan designation for the treatment of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2 in August 2019 in the USA. A regulatory assessment for pemigatinib as a treatment for adults with locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or rearrangement that is relapsed or refractory after ≥ 1 line of systemic therapy is underway in the EU. Pemigatinib is also undergoing clinical development in various countries worldwide for use in several other FGFR-driven malignancies (e.g. solid tumour, urothelial carcinoma). This article summarizes the milestones in the development of pemigatinib leading to this first approval for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test.

In the phase 3 CLEAR study, lenvatinib plus pembrolizumab showed improved efficacy versus sunitinib for patients with clear cell renal cell carcinoma (ccRCC). Previous preclinical studies demonstrated that lenvatinib attenuated tumor-associated macrophage (TAM) infiltration into tumor tissues by inhibiting fibroblast growth factor receptor (FGFR). However, the role of the FGFR pathway in ccRCC remains underexplored. This study aims to evaluate FGFR1-4 expression in ccRCC and investigate its relationship with the tumor microenvironment, particularly TAM. We primarily analyzed FGFR1-4 expression and CD163 positive cell count as estimation of TAM infiltration in 57 ccRCC specimens from patients undergoing nephrectomy using immunohistochemistry. Transcriptomic analysis was performed to assess immune-related gene signature and gene expressions. FGFR1 expression was elevated in over 80% of ccRCC samples and was significantly associated with increased CD163-positive TAM infiltration. FGFR1 expression was also negatively correlated with the IMmotion150 Teff gene signature and the expression of interferon-γ signaling targeted genes such as IFNG, GZMB, and CD274, suggesting an immunosuppressive phenotype. In contrast, FGFR2 and FGFR4 expression were less prevalent, and FGFR3 expression was not detected. This study provides the first comprehensive evaluation of FGFR1-4 expression in ccRCC and suggests that FGFR1 expression may contribute to the immunosuppressive tumor microenvironment by recruiting TAM. These findings indicate that FGFR1 could serve as a potential biomarker for therapeutic strategies and highlight the need for further research to explore FGFR-targeted therapies in ccRCC.

Ependymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a “DFG-out” covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. Significance Inhibitors of the FGF receptors (FGFRs) are currently under clinical investigation for the treatment of various cancers. All currently approved kinase inhibitors eventually are rendered useless by the emergence of drug-resistant tumors. We used structure-based drug design to develop the first, to our knowledge, selective, next-generation covalent FGFR inhibitors that can overcome the most common form of kinase inhibitor resistance, the mutation of the so-called “gatekeeper” residue located in the ATP-binding pocket. We also describe a novel kinase inhibitor design strategy that uses a single electrophile to target covalently cysteines that are located in different positions within the ATP-binding pocket. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance.

Fibroblast growth factor receptors (FGFRs) can act as driving oncoproteins in certain cancers, making them attractive drug targets. Here we have characterized tumour cell responses to two new inhibitors of FGFR1–3, AZ12908010 and the clinical candidate AZD4547, making comparisons with the well-characterized FGFR inhibitor PD173074. In a panel of 16 human tumour cell lines, the anti-proliferative activity of AZ12908010 or AZD4547 was strongly linked to the presence of deregulated FGFR signalling, indicating that addiction to deregulated FGFRs provides a therapeutic opportunity for selective intervention. Acquired resistance to targeted tyrosine kinase inhibitors is a growing problem in the clinic but has not yet been explored for FGFR inhibitors. To assess how FGFR-dependent tumour cells adapt to long-term FGFR inhibition, we generated a derivative of the KMS-11 myeloma cell line (FGFR Y373C ) with acquired resistance to AZ12908010 (KMS-11R cells). Basal phosphorylated FGFR and FGFR-dependent downstream signalling were constitutively elevated and refractory to drug in KMS-11R cells. Sequencing of FGFR3 in KMS-11R cells revealed the presence of a heterozygous mutation at the gatekeeper residue, encoding FGFR3 V555M ; consistent with this, KMS-11R cells were cross-resistant to AZD4547 and PD173074. These results define the selectivity and efficacy of two new FGFR inhibitors and identify a secondary gatekeeper mutation as a mechanism of acquired resistance to FGFR inhibitors that should be anticipated as clinical evaluation proceeds.

FGF19 is a hormone that regulates bile acid and glucose homeostasis. Progress has been made in identifying cofactors for receptor activation. However, several functions of FGF19 have not yet been fully defined, including the actions of FGF19 on target tissues, its FGF receptor specificity, and the contributions of other cofactors, such as heparin. Here, we explore the requirements for FGF19-FGFR/co-receptor interactions and signaling in detail. We show that βKlotho was essential for FGF19 interaction with FGFRs 1c, 2c, and 3c, but FGF19 was able to interact directly with FGFR4 in the absence of βKlotho in a heparin-dependent manner. Further, FGF19 activated FGFR4 signaling in the presence or absence of βKlotho, but activation of FGFRs 1c, 2c, or 3c was completely βKlotho dependent. We then generated an FGF19 molecule, FGF19dCTD, which has a deletion of the C-terminal region responsible for βKlotho interaction. We determined that βKlotho-dependent FGFR1c, 2c, and 3c interactions and activation were abolished, and βKlotho-independent FGFR4 activation was preserved; therefore, FGF19dCTD is an FGFR4-specific activator. This unique FGF19 molecule specifically activated FGFR4-dependent signaling in liver and suppressed CYP7A1 expression in vivo, but was unable to activate signaling in adipose where FGFR4 expression is very low. Interestingly, unlike FGF19, treatment of ob/ob mice with FGF19dCTD failed to improve glucose levels and insulin sensitivity. These results suggest that FGF19-regulated liver bile acid metabolism could be independent of its glucose-lowering effect, and direct FGFR activation in adipose tissue may play an important role in the regulation of glucose homeostasis.

Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an Escherichia coli expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in E. coli BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.

Fibroblast growth factor 21 (FGF21) is a cytokine synthesized and released by the liver, muscle and fat and acts both locally and systemically to regulate whole-body metabolism. David Mangelsdorf and his colleagues now show in two separate studies that FGF21 also acts on the region of the brain that regulates circadian rhythm, the suprachiasmatic nucleus, to further regulate whole-body metabolism as well as reproductive function. Fibroblast growth factor 21 (FGF21) is a hepatokine that acts as a global starvation signal to modulate fuel partitioning and metabolism and repress growth 1 ; however, the site of action of these diverse effects remains unclear. FGF21 signals through a heteromeric cell-surface receptor composed of one of three FGF receptors (FGFR1c, FGFR2c or FGFR3c) in complex with β-Klotho 2 , 3 , 4 , a single-pass transmembrane protein that is enriched in metabolic tissues 5 . Here we show that in addition to its known effects on peripheral metabolism, FGF21 increases systemic glucocorticoid levels, suppresses physical activity and alters circadian behavior, which are all features of the adaptive starvation response. These effects are mediated through β-Klotho expression in the suprachiasmatic nucleus of the hypothalamus and the dorsal vagal complex of the hindbrain. Mice lacking the gene encoding β-Klotho ( Klb ) in these regions are refractory to these effects, as well as those on metabolism, insulin and growth. These findings demonstrate a crucial role for the nervous system in mediating the diverse physiologic and pharmacologic actions of FGF21.