Inhibition of FGF‐FGFR and VEGF‐VEGFR signalling in cancer treatment

Abstract The sites of targeted therapy are limited and need to be expanded. The FGF‐FGFR signalling plays pivotal roles in the oncogenic process, and FGF/FGFR inhibitors are a promising method to treat FGFR‐altered tumours. The VEGF‐VEGFR signalling is the most crucial pathway to induce angiogenesis, and inhibiting this cascade has already got success in treating tumours. While both their efficacy and antitumour spectrum are limited, combining FGF/FGFR inhibitors with VEGF/VEGFR inhibitors are an excellent way to optimize the curative effect and expand the antitumour range because their combination can target both tumour cells and the tumour microenvironment. In addition, biomarkers need to be developed to predict the efficacy, and combination with immune checkpoint inhibitors is a promising direction in the future. The article will discuss the FGF‐FGFR signalling pathway, the VEGF‐VEGFR signalling pathway, the rationale of combining these two signalling pathways and recent small‐molecule FGFR/VEGFR inhibitors based on clinical trials.


| INTRODUC TI ON
Targeted therapies interfering with oncogenic driver alterations have achieved great success in chronic myeloid leukaemia (CML) with BCR-ABL fusions, 1 melanoma with BRAF V600E mutations, 2 lung cancer with EGFR mutations 3 and breast cancer with HER2 amplification. 4 However, approved targeted agents can only block limited types of cancer with specific driver gene alterations. The development of novel therapeutics targeting other cancer driver alterations is extremely urgent to improve patients' prognosis.
The fibroblast growth factor (FGF)-FGF receptor (FGFR) signalling cascade plays a pivotal role in driving cancer growth. Anti-FGF or FGFR therapy is a promising way to treat tumours with FGF and (or) FGFR alterations. 5 With the accelerated approval of erdafitinib for FGFR-altered urothelial carcinoma in April 2019 and pemigatinib for cholangiocarcinoma with FGFR2 fusion or other rearrangements in April 2020, 6,7 the FGF-FGFR signalling pathway has received more attention. However, patients often received limited clinical benefits in treatment with agents that only block the FGF-FGFR signalling cascade. 5 Combination of the inhibitory of the FGF-FGFR signalling pathway with other mechanisms is a promising way to solve this puzzle.
Tumours growth relies on blood supply, and vascular endothelial growth factors (VEGFs) are essential angiogenesis stimulators. 8 Through inhibiting the VEGF-VEGF receptor (VEGFR) signalling, anti-VEGF or VEGR agents have been approved for use in various solid tumours, but they lead only to mild clinical benefits in most situations. 9 Herein, in this review, we mainly focus on the FGF-FGFR signalling pathway, the VEGF-VEGFR signalling pathway, the rationale of combining these two pathways and recent small-molecule FGFR/ VEGFR inhibitors based on clinical trials.

| FGFs
Fibroblast growth factor was first extracted from bovine pituitary in 1973, partially purified in 1975, and finally purified to homogeneity in 1983. [10][11][12] The mammalian FGF family comprises 22 members, including FGF1-FGF23. Human FGF19 and mouse FGF15 are analogs. Phylogenetic and gene locus analyses divide the FGF family into seven subfamilies. Their action mechanisms classify these subfamilies into three groups, the canonical FGF subfamily including the In contrast to other family members, FGFR4 has only one isoform. 17 The FGF-binding pocket is formed by the II and III subregions. 18 The FGFR TK domains are the heart of the action, responsible for offering ATP-binding area and phosphorylating tyrosine residues to gradually increase catalytic activity tens to thousands of times. Finally, the specific phosphorylation site can bind and phosphorylate substrate proteins to activate multiple signal transduction pathways. 19 Take FGFR1 as an example; seven phosphorylatable tyrosine residues have been identified, that is, Y463, Y583, Y585, Y653, Y654, Y730 and Y766. 20 Among these, Y653 and Y654 are essential for kinase activity, and phospho-Y766 serves as a binding site for downstream protein. 21 There are several critical functional loops in the intracellular domain, one of which is an activation loop (A-loop). The conformation of the highly conserved Asp-Phe-Gly motif (DFG-motif) in the A-loop is an indicator of kinase activity status. The DFG-motif exists in two states: the active DFG-in and inactive DFG-out conformations, relating to the mechanism of FGFR inhibitors, which we will describe more below. 22

| Extracellular FGF associated cofactors
Heparin and heparan sulphate proteoglycans (HSPG) act as essential cofactors for the binding of canonical FGFs. 23 Unlike the canonical FGFs, endocrine FGFs require Klotho co-receptors instead to act as cofactors for FGFR activation. αKlotho is a cofactor for FGF23 and βKlotho for FGF15/19 and FGF21. 24 All cofactors are single-pass TM proteins, binding to extracellular Ig-like domain II of FGFR. This 1:1:1 FGF-HS/Klotho-FGFR ternary complex structure leads to conformational changes that stabilize a symmetric 2:2:2dimer. 25
Subsequently, GRB2 recruits SOS and GAB1 to activate the RAS-MAPK and PI3K-AKT pathways, respectively. 26,27 Phosphorylation of Y766 is linked to the initiation of the phospholipase C (PLCγ) pathway. Activated PLCγ catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 production elevates the level of intracellular calcium ion while DAG stimulates protein kinase C (PKC). 28 The STAT pathway is triggered by Y677 phosphorylation. 29

| Roles of FGF-FGFR signalling in physiology
Through triggering downstream signalling pathways, the FGF-FGFR signalling participates in various vital physiological processes. 15,30 By regulating key cell behaviours, such as proliferation, differentiation and survival, the FGF-FGFR signalling pathway can mediate the development of multicellular organisms to ensure proper morphogenesis in the whole development process and also can regulate angiogenesis and wound repair in adults. 31 Besides, endocrine FGFs can regulate bile acid metabolism in the liver, lipid metabolism in the white adipose tissue, and phosphate and vitamin D levels in serum. 15 In contrast, intracellular FGFs, independent of FGFRs, exert their biological activity in their original cells via interaction with cytoplasmic domains of ion-gated sodium channels and mainly play roles in neuronal functions in the postnatal stages. 32

| FGF-FGFR signalling in cancer
FGFRs are not constitutively active in non-malignant cells. The oncogenic role of FGF-FGFR signalling in driving cancer cell proliferation, survival, migration and invasion is mediated by the upregulation of FGF, FGFR genetic alterations, angiogenesis and immune evasion in the tumour microenvironment. 5 Amplification of the FGFR1 gene is the most common in all types of FGFR gene alterations. It has been described in a plethora of human tumour types with different ratios. 33 Recent studies described that the rate of FGFR1 amplification was significantly higher in squamous cell lung cancer (SqCLC) and Asians, and FGFR1 amplification may be a potential new therapeutic target for individual patients with specific lung cancer subtypes such as EGFR TKI for Asian patients with lung adenocarcinoma. 35 FGFR1-amplified lung cancer models respond to FGFR inhibitors in preclinical studies in both non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), especially in SqCLC, with 9.3% in stage I, 22% in stage II, and 19% in stage IV with brain metastasis. 36 However, several phase II clinical trials found its limited activity in FGFR1amplified lung cancer patients with an acceptable safety profile. 37 The relationship between amplification of FGFR1 and prognosis is still in doubt in NSCLC. Maybe, it is because of the FGFR1 amplicon co-amplified with other genes that could contribute to carcinogenesis. 38 In HR (+)/HER2 (-) breast cancers, increased expression of FGFR1 was found in hormone-resistant breast cancer and in patients who received CDK4/6 inhibitors, and these patients can receive 19% of the objective response rate (ORR) treated by lucitanib. 39,40 Combination of FGFR1 and CDK4/6 inhibitors can effectively suppress FGFR1 and aromatase activities and prolong median progression-free survival (PFS) by 5.4 months in FGFR1 amplified group in a phase II clinical trial. 39 FGFR1 amplification is an independent biomarker of a poor prognosis in patients with ER (+) breast cancer. 41 Moreover, FGFR1 and/or FGF3 gene amplification is associated with resistance to HER2 targeted therapy, a shorter PFS survival and a lower pathological complete response (CR) in HER2 (+) early breast cancer treated with neoadjuvant anti-HER2 therapy. 42 What is more, allelic loss and amplification of FGFR1 can predict chemo-and radiotherapy response in breast cancer. 43 FGFR1 amplification correlating with inadequate response to traditional treatments also happens in osteosarcoma, 44  At the same time, FGFR3 S249C mutation in urinary cell-free DNA could predict early-stage (≤pT1) of upper muscle-invasive urothelial carcinoma with 100% positive predictive value. 66 Besides, FGFR3 mutations also occur in cervical, vulvar squamous cell carcinoma and breast cancer. [67][68][69] The kinase domain mutations of FGFR4 (V550E/L and N535D/K) were described in 7% of rhabdomyosarcoma, leading to tumour growth in vivo and drug resistance to all type I and some type II inhibitors in patients. 70 Besides, variant rs351855-G/A can lead to germline FGFR4 G388R substitution, subsequently expose a membrane-proximal STAT3-binding site and trigger STAT3 signalling cascade, which can accelerate cancer progression and also contribute to tumour-extrinsic immune evasion. 71  rearrangements achieved an objective response. 7 Interestingly, FGFR2 fusions also have been found in breast, prostate and thyroid cancer. 33 In addition to the presence of FGFR3 amplification and mutations in urothelial carcinoma, FGFR2/3 fusions have also been detected.

| Upregulation of FGFs
Genetic alterations mentioned above mainly lead to constitutive receptor activation and ligand-independent signalling. However, the ligand-dependent signalling triggered by FGFs also contributes to the pathogenesis of cancer. The increased amount of FGFs comes from the secretion of cancer cells and (or) the surrounding stromal cells, also referred to as autocrine and paracrine ligand signalling. 37

| Angiogenic effects
Although FGF-FGFR signalling plays a significant role in tumour growth, as discussed above, actually FGFs were firstly found as an- Neutralizing FGF2 and FGFRs inhibit neovascularization and tumour growth in vivo models. 90 Though not required for vascular homeostasis or physiological function, FGF-FGFR signalling plays a pivotal role in tissue repair and neovascularization following injury, which validates endothelial cell FGFRs as a target for diseases associated with aberrant vascular proliferation. 91 Among these methods, VEGF-VEGFR signalling deserves attention.

| VEGFs
One hundred years ago, the growth of tumours had already been thought to rely on blood supply. It was not until 1939 that tumour cells were supposed to release a blood vessel growth factor by themselves. 9 And then, in 1971, Folkman speculated that tumours could be treated through anti-angiogenesis. 95 Inspired by these hypotheses, vascular permeability factor (VPF) was found by Senger, and his colleagues in 1983. 96 Ferrara and co-workers isolated VEGFA in 1989. What is more, cDNA and protein sequence analyses proved that VPF and VEGFA were the same molecules. 9 In mammals, the VEGF family consists of five members, VEGFA, B, C, D and placenta growth factor (PLGF), encoded from the same gene and organized in an anti-parallel fashion to form a dimer. 97 In particular, VEGF, referred to as VEGFA, is a major regulator of normal and abnormal angiogenesis. Because of alternative splicing, several variants of VEGFA have been detected, mainly VEGFA121, VEGFA165, VEGFA189 and VEGFA206. 98 The ability to interact with VEGFR co-receptors and proteolytic processing decide the bioactivities of the VEGFA isoforms. 99 Lacking the HSPG-and neuropilin-binding domains, VEGFA121 is a diffuse molecule and cannot remain on the cell surface and in the extracellular matrix (ECM). VEGFA165 has two properties: it can be secreted or stored in the vicinity of the producer cell.
On the other hand, VEGFA189 and VEGFA206 include HSPG-and neuropilin (NRP)-binding domains and can bind to co-receptors with greater affinity than VEGFA165. In addition, protease cleavage of VEGFA189 allows the release of an active, freely diffusible VEGFA110. In other words, VEGFA165 is the most active of all subtypes. 99,100 Hypoxia is the primary inducer of VEGF gene transcription via hypoxia-inducible factor (HIF). Besides, growth factors, hormones, cytokines and oncogenic mutations can also influence the production of VEGF. 101

| VEGFRs
These ligands bind in an overlapping pattern to VEGFR1-3 and have

| Intracellular signal transduction
Among the downstream pathways of VEGFR1-3, VEGFR2 is the most thoroughly studied ( Figure 2). Y1175, Y951 and Y1214 are the three major VEGFA-dependent phosphorylation sites in VEGFR2. 105

| VEGF-VEGFR signalling in cancer
A tumour needs angiogenesis to ensure oxygen and nutrients for its growth. VEGF secreted by tumour cells and their microenvironment, binding to VEGFR2, plays the most crucial role in vascular permeability and neo-angiogenesis. 95 What is more, the capillary and

| Targeting VEGF-VEGFR signalling in cancer
In 1993, the finding that a monoclonal antibody can target and neutralize VEGFA and inhibit tumour growth in the xenograft model led to the translational possibility for targeting VEGF-VEGFR signalling. 114 These agents can be divided into two broad classes: agents targeting the VEGF ligand and agents designed to target the cell surface receptor. 115 As bevacizumab (Avastin) was demonstrated to improve the response rate and survival of patients with CRC combined with chemotherapy, it became the first approved anti-VEGF monoclonal antibody by the FDA in 2004. 116 Since then, bevacizumab, in combination with standard treatments, has gained more and more indications. 117 The treatment with those anti-angiogenic drugs has shown benefit in some patients with advanced cancers, but more drugs lead only to mild clinical benefits. The primary or acquired resistance mediated by both tumour cells and stromal cells may explain the minimal benefits. 9 The resistant mechanisms derived from anti-angiogenic drugs are different from the inhibitors of well-defined oncogenic pathways. So far, there is no definitive evidence of pre-existing or acquired mutations in VEGFA or its signalling pathway. 125 Upregulation of alternative angiogenic factors, including FGF, plays a vital role in the induction of resistance to VEGF/VEGFR inhibitors. 126

| Combination rationale
The prominent roles of the FGF-FGFR and VEGF-VEGFR signalling in tumour cells and angiogenesis have been described in detail earlier in this article. Except for those, other mechanisms, especially combined or interactive mechanisms, deserve further exploration.
As mentioned above, FGF-FGFR and VEGF-VEGFR signalling pathways can promote angiogenesis. Interestingly, both FGF and VEGF can be stored on the ECM-associated HSPGs, and studies have shown that these two pathways have synergistic effects as inducers of angiogenesis. 127 Researchers have found the combination of FGF-1 and VEGF induced a more significant angiogenic effect than the additive effects of FGF-1 or VEGF alone in vitro quantitative fibrin-based 3-dimensional angiogenesis system. 128 Besides, FGFR regulated the secretion of VEGF in a MAPK-dependent manner, and VEGF, in turn, upregulates the expression of FGF. FGF can also induce the VEGFR2 expression in an ERK1/2-dependent pathway, and the expression of VEGFR2 rapidly declines without this interaction. 129 What' more, neutralizing the VEGF antibody reduced FGF-driven angiogenesis, implying that VEGF is a crucial mediator that existed downstream of FGF. 127 It is not surprising that targeting both VEGFR and FGFR resulted in synergistic anti-angiogenic effects in vivo. A similar synergism is found in lymphangiogenesis, and inhibition of it by dual FGFR/VEGFR inhibitor could prevent metastasis easier. 130 In addition, upregulation of FGF expression, expressed by pericytes, has been described as a significant mechanism in resistance to anti-VEGF/VEGFR therapy. 131 In patients with metastatic RCC who progressed after or were intolerant to sorafenib or sunitinib, dual FGFR and VEGFR inhibitors, including anlotinib, dovitinib and lenvatinib with promising results in phase I or II clinical trials bring them another chance to overcome resistance. [132][133][134] Lenvatinib and nintedanib also offer opportunities for patients with HCC who progressed on sorafenib treatment. 135,136 The roles of VEGF-VEGFR signalling in suppressing tumour im- The FGFR/VEGFR inhibitors are also reported to arrest the cell cycle in the G0/G1 phase and cause tumour cell apoptosis. 141 In general, the dual blockade of FGF-FGFR and VEGF-VEGFR signalling cascade is reasonable due to the mechanisms mentioned above ( Figure 3). Small-molecule FGFR/VEGFR inhibitors are preferable because of convenience and economy and are well studied.

| Small-molecule FGFR/VEGFR inhibitors
The small molecular drugs that inhibit FGFR and VEGFR are divided into selective and non-selective FGFR/VEGFR TKIs according to whether the value of IC50 of inhibitory activity to other kinases is <10 nM. 5

| Non-selective FGFR/VEGFR TKIs
The values of IC50 and critical clinical trials of multi-TKIs are listed in Tables 2 and 3, respectively. The details of these drugs will be discussed below.

| Anlotinib
Anlotinib (AL3818) is a multi-TKI that is designed to inhibit VEGFR1-3, FGFR1-4, PDGFRα/β, c-Kit and Ret and has been approved by the CFDA as a third-line or beyond therapy for stage IV NSCLC in 2018. 142 In phase III ALTER-0303 trial, anlotinib significantly improved median OS from 6.3 months in the placebo group to 9.6 months in the anlotinib group (HR, 0.68; 95%CI, 0.54 to 0.87; P =.002) and median PFS from 1.6 months to 5.4 months (HR,0.25; 95%CI, 0.19 to 0.31; P =.001). 143 Besides, anlotinib also showed promising efficacy in patients with metastatic RCC, advanced or metastatic medullary thyroid carcinoma and refractory metastatic soft-tissue sarcoma (STS) progressed after anthracycline-based chemotherapy, naïve from angiogenesis inhibitor. 132,144,145 Interestingly, the incidence of grade 3 or higher side effects is much lower than that of other TKIs.

| E7090
E7090 is an orally non-selective inhibitor of FGFR1-3 and has a slightly lower inhibitory activity on VEGFR2. 155  in unresectable HCC. 160 What' more, lenvatinib plus everolimus also showed promising results in a phase II trial, leading to the FDA approval of this combination in advanced RCC following one prior anti-angiogenic therapy. 134 Interestingly, many efforts have been made to find the relationship between the outcome and biomarkers based on the REFLECT trial. For example, baseline Ang2, upregulated FGF23 and treatment-emergent hypertension correlated with improved PFS, and diarrhoea were significantly associated with OS in lenvatinib-treated patients. 160 In other words, the factors mentioned above may predict the efficacy of lenvatinib. Nowadays, as lenvatinib was reported to decrease tumour-associated mac-

| Selective FGFR/VEGFR TKIs
Nowadays, dual inhibitors of FGFR and VEGFR have been developed. In addition to the basic information listed in Tables 2 and 3, distinct features of these drugs are discussed as follows.

| AZD4547
AZD4547 is a selective and reversible TKI of FGFR1-3 and also shows activity against VEGFR2 at nM concentration with IC50 equal to 24 nM. 182 Its antitumour effect has been confirmed in some preclini-

| ASP5878
ASP5878 is a selective pan-FGFR inhibitor that exerts its antitumour activity towards tumours with FGFR genetic alterations. 186 Researchers have demonstrated the role of ASP5878 in

| Erdafitinib
Erdafitinib (JNJ-42756493) is a highly selective and reversible inhibitor of FGFR1-4 and can inhibit VEGFR2 with IC50 equal to 37 nM. 196 In phase I clinical trials, it showed clinical benefits in glioblastoma, cholangiocarcinoma, urothelial and endometrial cancer with FGFR mutations or fusions, while ORRs in other tumour types were below 10%. 6,197,198 In April 2019, erdafitinib received accelerated approval by the FDA to treat patients with FGFR3 mutated or FGFR2/3 fusion-positive advanced or metastatic urothelial carcinoma after at least one prior platinum-based regimen. The ORR reached 40%, and a median PFS was 5.5 months. At the same time, treatment-related grade 3 or higher adverse events also happened in nearly half the patients, including hyponatremia, stomatitis and asthenia in phase II BLC2001 clinical trial. 6 Erdafitinib also received three black-box warnings by Janssen pharmaceutical company for the risks of ocular disorders, hyperphosphataemia and embryo-foetal toxicity. 199

| Ly2874455
Ly2874455 is a selective pan-FGFR inhibitor, with similar values of IC50 in inhibiting FGFR1-4, which also has inhibitory activity towards VEGFR2 with IC50 equal to 7 nM. 200 Interestingly, as the inhibition of FGF-induced Erk phosphorylation by Ly2874455 is much easier than that of VEGF-mediated target signalling in vivo, LY2874455 can avoid VEGFR2-mediated hypertension at efficacious doses. 201 Until now, a phase I clinical trial has published its results demonstrating the excellent tolerability and activity in patients with advanced cancer, especially for patients with gastric cancer and NSCLC. 201 In addition, some drugs are in the preclinical development stage.
For example, ODM-203 is a selective and equipotent inhibitor of FGFR and VEGFR, which exhibits its equal inhibitory activity towards FGFR and VEGFR families in biochemical assays, cellular assays and in vivo. 141 203,204 Besides, FGF and VEGF induce immunosuppressive microenvironment by inhibiting immune effector cells and recruiting immunosuppressive cells, and FGFR/VEGFR dual inhibitors can revert the TME from immunologically 'cold' tumours into 'hot' tumours. 205 At the same time, immune checkpoint inhibitors (ICIs) have been approved in many types of tumours, working through restoring antitumour T-cell functions. 206 However, lacking pre-existing immune cells in TME leads to inadequate response to monotherapy with ICIs. The combination of lenvatinib and pembrolizumab has received accelerated approval in patients with advanced endometrial cancer and is undergoing phase III clinical trial in HCC and RCC (NCT03713593, NCT02811861). 161,207,208 Combining FGFR/VEGFR dual inhibitors with ICIs is a promising treatment in the future.

ACK N OWLED G EM ENTS
This work is supported by the National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

AUTH O R S' CO NTR I B UTI O N S
YW and XW offered direction and guidance of the manuscript. GL and TC drafted the initial manuscript. ZD revised the manuscript.
GL and YW illustrated the figures and tables for the manuscript. All authors approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.