Fibroblast growth factor receptors (FGFRs) are transmembrane tyrosine kinase receptors whose genetic fusions are implicated in a broad range of cancers. FGFR genes encode a family of four homologous tyrosine kinase receptors: FGFR1, FGFR2, FGFR3, and FGFR4. These membrane-bound receptors initiate pathways important for normal organ, skeletal and vascular development by tightly regulating cell proliferation, survival, migration and differentiation(1,2). A consequence of diverse signaling targets, the FGFR genes are some of the most frequently amplified and mutated genes in cancer. Specifically, the constitutive and unregulated activation of FGFRs via genetic translocations, amplifications and point mutations can drive cancerous cell growth through various downstream mechanisms. Cancer-driving genetic mutations and translocations leading to constitutive activation of the FGFR tyrosine kinase domains present particularly promising targets for tyrosine kinase inhibitors (TKIs). The widespread oncogenic implication and potential targeted inhibition of FGFRs are driving further research towards detecting and characterizing these oncogenes.
Fibroblast growth factors (FGFs) and the FGFR signaling pathway play significant roles in cellular processes, including cell survival, adhesion, chemotaxis, apoptosis, migration and proliferation (3). The pathway is initiated by one of the 18 mammalian FGFs binding to one of the 4 homologous tyrosine kinase fibroblast growth receptors (FGFR1, FGFR2, FGFR3 or FGFR4) (4,5). The receptors are composed of an extracellular ligand-binding domain, a single hydrophobic transmembrane domain and a cytoplasmic domain containing the catalytic, split tyrosine kinase core (6,7). A variety of FGFR isoforms can result from alternative splicing of FGFR transcripts. The third immunoglobulin (Ig)-like domain, D3, is particularly sensitive to alternative exon usage which greatly alters ligand-binding between the three isoforms per receptor (8,9). The FGFs bind to FGFRs through simultaneous, low-affinity binding to the surface protein heparin sulphate glycosaminoglycan (HSGAG) (10). The now-functional FGFR-FGF-HSGAG ternary complexes then dimerize, bringing the kinase domains into close proximity to initiate transphosphorylation of tyrosine residues that act as docking sites for downstream effectors.
Figure 1 shows the FGFR-FGF-HSGAG complex. FGF interacts with FGFR through a heparin surface molecule, promoting dimerization of FGFR and transphosphorylation of the kinase domains. Most transcript isoforms of FGFR consist of a 5' untranslated region, three Ig-like domains, the third being especially susceptible to alternative splicing and the peptides of which influence cellular differentiation and FGF specificity. An acid box and heparin-binding motif constitute the region between the first and second Ig domains. A juxtamembrane domain separates the Ig-like domain from the tyrosine kinase domain.
Downstream signaling occurs via two main pathways, beginning with recruitment and phosphorylation of target proteins at the receptor's cytoplasmic tail. Shc, phospholipase-Cγ (PLCγ), Gab1, FRS2, and STAT1 are phosphorylated by FGFR stimulation (5). Activation of these proteins precludes many multifactorial and complex pathways critical in the control of cell migration, survival, shape, differentiation and proliferation. Recognizable pathways include the Ras-independent phosphoinositide 3-kinase (PI3K)-AKT and Ras-dependent mitogen-activated protein kinase (MAPK) signaling pathways, upregulated by the intracellular receptor substrates PLCγ and FRS (21,11,12). FGFR-initiated pathways are tightly regulated by feedback mechanisms and inhibitory proteins like SPRY and SEF13.
Figure 2 shows the major components of the FGFR-mediated signaling pathway. Upon activation of the tyrosine kinase domain activation recruits PLCγ and FRS2, which function as key adaptor proteins for downstream pathways. FRS2 initiates downstream signaling including MAPK and PI3K activation. MAPK3, Sprouty (SPRY) proteins and SEF family members negatively regulate FGFR activity (1,2,12).
Chromosomal translocations that result in subsequent FGFR fusion proteins are lineage-independent and have been directly linked to oncogenesis in a growing number of cancers, including hematological malignancies (14-27), cholangiocarcinomas (28-30), lung cancer (29,31,32), bladder cancer (33), prostate cancer (29), breast cancer (29,34), glioblastoma (35,36), alveolar rhabdosarcomas (37), pleomorphic salivary gland adenomas (37), gastric cancer (38), thyroid cancer (29) and head and neck squamous cell carcinoma (33).
Oncogenic tyrosine kinase translocations can occur at various regions; however, not all gene fusions produce biologically active proteins. Some FGFR translocations result in variants with an N-terminal domain of the fusion partner fused to the C-terminal end of FGFR that includes the kinase domain but not the transmembrane domain. No longer membrane bound or receptor-specific, the fusion proteins reside in the cytosol and are permanently dimerized and continuously signaling. These cytosolic fusion proteins escape normal down-regulation and feedback inhibition routes and are not degraded in lysosomes, resulting in long-term constitutive signaling (39). For example, the FGFR3-TACC3 fusion prevalent in 3% of glioblastoma mulitforme escapes microRNA-mediated regulation because the 3'-untranslated region is lost in the fusion transcript (35,40). The cytosolic tyrosine kinases cause aberrant signaling and unregulated activation of various intracellular pathways, including those discussed previously.
In some cases, chromosomal translocations occur upstream of the transmembrane domain, adopting the partner protein's ligand binding region and changing ligand specificity or ligand binding regulation. This sometimes results in the upregulation of downstream signaling or oligomerization of the tyrosine kinase domain (29). In other cases fusions can occur downstream of the tyrosine kinase domain, leading to aberrant signaling (29). Each unique fusion presents its own challenge for researchers, including detection, understanding the mechanism of action, and the creation of accompanying targeted therapeutics. The fusions associated with FGR1, FGFR2 and FGFR3 are discussed below.
The FGFR1 gene is located on chromosome 8, with 22 exons spanning 57,697 bases from 8p11.23 to p11.22. The canonical isoform of the expressed protein is 822 amino acids long. Translocations involving FGFR1 have been implicated in various types of cancer including hematological malignancies (14-25), glioblastomas (35), breast cancer (29,34), pleomorphic salivary gland adenocarcinomas (37), lung cancer (29), alveolar rhabdomyosarcoma (41) and bladder cancer (42).
A common FGFR1 translocation with an especially poor prognosis is 8p11 myeloproliferative syndrome (EMS), a common precursor of acute myeloid leukemia (AML) and is frequently accompanied by T- and B-cell lymphomas. EMS is characterized by a translocation at the 8p11 locus involving the FGFR1 gene and various fusion partners (43). Typically, the fusion transcripts encode proteins with the N-terminus of the translocation partner and the tyrosine kinase domain of FGFR1 in the C-terminus, resulting in constitutive FGFR1 signaling and promotion of the downstream pathways (34,44).
|BCR-FGFR1||EMS, AML, CML, ALL||14|
|CNTRL-FGFR1||EMS, AML, CML, T-cell lymphoma||15|
|CPSF6-FGFR1||EMS, CMD, MPN, AML||16|
|CUX1-FGFR1||EMS, MPN, ALL, AML||17|
|FGFR1OP2-FGFR1||EMS, AML, MPN||18|
|FGFR1OP-FGFR1||EMS, AML, CMD, ALL, MPD||26|
|HERV-K-FGFR1||EMS, CMD, MPD, AML||20|
|FGFR1-RANBP2||EMS, MPN, AML||21|
|FGFR1-LRRFIP1||ALL, CMD, AML||22|
|MYO18A-FGFR1||EMS, MPN, AML||23|
|TRIM24-FGFR1||EMS, AML, MPN||24|
|TPR-FGFR1||EMS, MPN T-lymphoblastic lymphoma||45|
|ZMYM2-FGFR1||EMS, MPN, ALL, CMD, T-lymphoblastic lymphoma||25|
|FGFR1-PLAG1||Pleomorphic salivary gland adenocarcinomas||37|
The FGFR2 gene is a paralog to FGFR1 and is located on chromosome 10. The gene spans 120,129 bases from 10q25.3 to q26, and the expressed protein comprises 821 amino acids. Translocations involving FGFR2 were only recently identified. However, research within the last few years alone has revealed a large number of FGFR2 translocations, hinting that many more are to follow. So far, fusions have been found in cholangiocarcinoma (29,46), breast cancer (29), prostate cancer (29), lung cancer (29,31), intrahepatic cholangiocarcinoma (28,30), thyroid cancer (29) and gastric cancer (38).
|FGFR2-CCDC6||Cholangiocarcinoma, Breast cancer||29|
|FGFR2-CCAR2||Lung squamous cell carcinoma||29|
The FGFR3 gene is located on chromosome 4p16.3. The gene consists of 15,566 bases and expresses a protein 806 amino acids long. Fusions of the FGFR3 gene have been implicated in peripheral T-cell lymphoma (27), bladder cancer (29,33,42), head and neck squamous cell cancer (29), non-small cell lung cancer (NSCLC) lung cancer (29,32) and glioblastoma (40). FGFR3 is implicated as an oncogene in 80% of low-grade non-invasive bladder tumors and is unregulated in about 40% of invasive bladder tumors (47). The FGFR3-TACC3 fusion is the most prevalent fusion in bladder cancer, but FGFR-BAI1AP2L1 in-frame fusions are also implicated in bladder cancer (33). Transforming acidic coiled-coil-containing protein 3 (TACC3) is also a fusion partner of FGFR3 in glioblastoma multiforme and lung squamous cell carcinomas (29,40). In the latter disease, the rearrangement includes the first 18 exons of FGFR3 upstream of the last 7 exons of TACC3. The resulting fusion protein has the last 226 amino acids of TACC3 directly appended to the FGFR3 protein.
|ETV6-FGFR3||Peripheral T-cell lymphoma||27|
|FGFR3-TACC3||NSCLC, Glioblastoma Multiforme, Oral cancer, HNSCC, SCC||32,33,40|
FGFR genomic abnormalities are common in both hematological malignancies and solid tumors. The widespread amplifications, mutations and translocations of FGFR in various cancers drive research and development of small-molecule tyrosine kinase inhibitors (TKIs), which have the ability to target and inhibit constitutively active FGFR fusions. Most multi-kinase TKIs and selective FGFR inhibitors inhibit the ATP-binding domain, while some compete with FGFs for binding to the extracellular domain.
|AZ12908010||Small molecule FGFR-selective inhibitor targeting kinase insert domain of FGFR 1-3 (48)|
|AZD4547||Small molecule FGFR-selective inhibitor that targets the kinase insert domain receptor of FGFR1-3. Inhibits kinase activity and growth in various cell lines with deregulated FGFR activity. (48)|
|Brivanib||BMS-582664, brivanib alaninate||Small molecule FGFR1 inhibitor that decreases receptor autophosphorylation, inhibits bFGF-induced tyrosine kinase activity and reduces phosphorylation of ERK and AKT in breast cancer with FGFR1 amplified cells (49)|
|Danusertib||PHA-739358||Aurora kinase inhibitor that also inhibits FGFR1 and may exhibit antitumor activity (50)|
|Dasatinib||BMS-354825, Sprycel, BMS 354825||Abl and Src family kinase inhibitor reduced growth in mouse models with pro-B lymphoma cells with FGFR1-ZMT2, FGFR1-BCR, FGFR1-CEP110 fusions (51)|
|Dovitinib||CHIR-258, DOVITINIB LACTATE, TKI258,||Multi-target TKI that inhibits proliferation in FGFR1 and FGFR2 amplified breast cancer cell lines showed inhibited proliferation and survival in EMS-related FGF1OP2-FGFR1, ZNF198-FGFR1 and BCR-FGFR1 positive cell lines (52,53)|
|ENMD-2076||ENMD-981693||Small molecule kinase inhibitor with sustained inhibition of FGFR1 and FGFR2. ENMD-2076 has inhibited the in vitro growth of a range of solid tumor and hematopoietic cancer cell lines (54)|
|Foretinib||GSK1363089||Multi-kinase inhibitor that was found to have inhibitory effects on FGFR2 amplification through the blocking of inter-RTK signaling networks in KATO-III, gastric carcinoma cell lines (55)|
|Lenvatinib||E-7080, LENVATINIB MESYLATE,||Multi-targeted kinase inhibitor that has been shown to inhibit tumor angiogenesis by inhibiting FGFR signaling in endothelial cells (56)|
|Lucitabin||E-3810, AL3810||Small molecule, potent inhibitor of multiple tyrosine kinases involved in angiogenisis, including FGFR1. It has been found that AL3810 inhibited autophosphorylation of FGFR1 in endothelial cells (57)|
|Midostaurin||CGP 41251, N-Benzoyl-Staurosporine, N-Benzoylstaurosporine||Small molecule TKI that has been found to inhibit ZNF198-FGFR1 tyrosine kinase activity (58)|
|Nintedanib||BIBF 1120, Intedanib, Vargatef||Small molecule triple tyrosine kinase inhibitor of FGFR1-3 that has increased overall survival in NSCLC (25119062). Seems to be effective in various other tumor types like prostate, colorectal, hepatocellular carcinoma, and gynecological tumors (59)|
|NVP-BGJ398||BGJ398||Selective FGFR inhibitor that inhibits proliferation in a number of cancer cell lines positive for FGFR genetic alterations, including FGFR1 and FGFR3 translocations (60 61)|
|Orantinib||Orantinibum, SU 006668, Sugen TSU68||Small molecule inhibitor of FGFR1. Has induced inhibition of agiogenic receptor tyrosine kinase activity in vivo, leading to broad antitumor effects (62)|
|Pazopanib||GW786034B, PAZOPANIB HYDROCHLORIDE, Votrient||Potent and selective multi-targeted tyrosine kinase inhibitor FGFR2-TACC3 (28,63)|
|PD173074||ATP-competitive inhibition of FGFR kinase activity in vitro. Inhibits growth and induces apoptosis in ZFN198-FGFR1 expressed cells (64)|
|PKC412||Small molecule FGFR inhibitor found to have inhibitory effects on patient with myeloproliferative disorder positive for ZNF198-FGFR1 fusion (58)|
|Ponatinib||AP24534||Multi-targeted TKI targeting CML patients with BCR-ABL. Found to have anti-tumor effects on 14 FGFR-deregulated cell lines. Reduces FGFR1-mediated signaling and inhibits cell growth65. Accompanied reduction and tumor necrosis in FGFR2-MGEA4 fusion (28)|
|Regorafenib||BAY 73-4506, Stivarga||Small molecule multi-kinase inhibitor that shows survival benefits in metastatic colorectal cancer (66)|
|Sunitinib||SU-11248, Sutent||Multi-targeted TKI that inhibits FGFR1 activity. Sunitinib differentially inhibited growth of cells expressing ZNF198-FGFR1 (64)|
|Vandetanib||AZD-6474, Zactima||Small molecule TKI shown to inhibit growth in cells with ZNF198-FGFR1 fusion (64)|
Although the list of TKIs being studied continues to grow, research is still needed to identify novel fusion partners and understand their mechanisms of action. Fortunately, through the widespread adoption of next-generation sequencing (NGS), research on genetic abnormalities in cancer has significantly accelerated. NGS-based fusion detection has recently benefited from advancements like Anchored Multiplex PCR (AMP™) that allows for discovery of novel fusions in a scalable manner, while bioinformatics tools have helped to convert that information into usable data. Through the clever applications of these types of tools, the cancer research community is poised to make groundbreaking discoveries to further understand the biology and targeting of genes such as the FGFR gene family.
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