FGFR-TKI resistance in cancer: current status and perspectives

Sohl and colleagues utilized structural and kinetic characteristics of FGFR1 to explain affinity changes for the FGFR inhibitors E3810 (Lucitanib) and AZD4547 due to the FGFR1 gatekeeper mutation V561M [29]. These studies showed that V561M mutation reduces affinity for E3810 (a double FGFR-VEGFR inhibitor), but that V561 mutant maintains nanomolar affinity for AZD4547 (a selective FGFR1-3 inhibitor) [29, 30]. Structurally, E3810 lacks flexibility, whereas the conformational flexibility of AZD4547 allows this TKI to adapt to the mutation [29, 31]. In addition, Sohl and colleagues used in vivo and in vitro binding assays to prove that the V561M mutant strongly activated STAT3 and produced significant resistance to AZD4547, thereby driving cancer progression [30]. By knocking out STAT3, cancer cells expressing FGFR1 V561M display restored sensitivity to AZD4547 [30]. These results suggest that knowledge of kinase-inhibitor complex structures alone is insufficient for a comprehensive understanding of the mechanisms of FGFR-TKI resistance. Downstream signaling pathways must also be considered.

FGFR3 resembles FGFR1 structurally. The V561M gatekeeper mutation of FGFR1 corresponds to the V555M gatekeeper mutation of FGFR3. TKI258 (Dovitinib), a poly-kinase inhibitor can simultaneously retain its inhibitory effect in patients with FGFR1 and FGFR3 gatekeeper mutations, but the FGFR1 V561M mutant is markedly less sensitive to PD173074 and AZD4547 [32, 33]. Initially, patients with the FGFR3-TACC3 fusion are highly sensitive to AZD4547, but this TKI becomes ineffective after the appearance of FGFR3 gatekeeper mutations [32, 34]. Chell and his colleagues sequenced a KMS-11 myeloma cell line resistant to AZ12908010, and found the FGFR3 gatekeeper mutation V555M, which conferred cross-resistance to AZD4547 and PD173074 by an increase in FGFR3 phosphorylation and an improvement of downstream signaling transduction [34]. The molecular structural explanation is that the FGFR3 V555M mutation generates steric clashes with the phenyl ring of PD173074 by structural modeling, resulting in enhanced resistance [32, 34]. An additional factor is steric clashes caused by conformational changes in the P-loop region [22].

In a clinical study of three patients with FGFR2-fusion cholangiocarcinoma receiving BGJ398 therapy, all patients developed acquired resistance with FGFR2 gatekeeper V564F mutation [35]. In the BaF3 cell line, which has high FGFR2 expression, researchers identified several Dovitinib-resistant mutations, including gatekeeper mutation [23]. Ponatinib (AP24534), a third-generation TKI, effectively inhibits all Dovitinib-resistant FGFR2 mutants except gatekeeper mutation [23]. Ponatinib, as a type II inhibitor, targets the inactive and DFG-out conformation, but can also target the active conformation [36].

Goyal et al. reported that the irreversible covalent FGFR inhibitor TAS-120 overcomes drug resistance induced by ATP-competitive FGFR inhibitors (such as BGJ398 and Debio 1347) in patients with FGFR2 fusion-positive intrahepatic cholangiocarcinoma [37]. Structurally, the FGFR2 gatekeeper V564F mutation induces steric clashes with the dichloro dimethoxy phenyl ring of BGJ398 [32, 35]. Debio 1347 generally retains activity against the FGFR2 V564F gatekeeper mutation, due to Debio 1347 partially replacing the large dimethoxyphenyl group with a benzimidazole moiety that makes stable contacts with V564F. Debio 1347 also confers resistance to other FGFR2 mutations [37, 38]. TAS-120 binds its target covalently, but it cannot resist FGFR2 gatekeeper mutations, due to steric clashes [37, 39].

It is worth noting that mutations in FGFR4 are rare, but that Hatlen et al. identified gatekeeper mutations (V550M/L) and Hinge-1 (C552) mutations in the FGFR4 kinase domain from hepatocellular carcinoma patients after treatment with the selective FGFR4 inhibitor Fisogatinib (BLU-554) [40]. These mutants prevented Fisogatinib from covalently binding to FGFR4 [37]. Besides, FGFR4 V550M mutation was found in 13% of neuroendocrine breast cancers, and FGFR4 V550L mutation was found in 9% of embryonal rhabdomyosarcoma tumors [41, 42]. Huang et al. found that FGFR4 V550L mutant forms steric clashes with the imidazo[1,2-b]pyridazine scaffold of Ponatinib. Both preventing covalent binding and conflict formation reduced the inhibitory activity of TKI [43]. Surprisingly, our group found by in vivo and in vitro experiments that the Pan-FGFR inhibitor LY2874455 has a remarkable ability to overcome FGFR4 gatekeeper mutation-induced TKI resistance [44]. Further crystallographic experiment proved that LY2874455 binding site is distant from the gatekeeper residue, which avoids steric clashes with the ATP-binding pocket of FGFR4 [44, 45].

When designing TKIs for FGFR gatekeeper mutations, we should take into account the following considerations: (1) The binding site for inhibitors can be far from the hinge region and, especially, from gatekeeper residues. For example, the pan-FGFR inhibitor LY2874455 is reported to be the most effective compound for all the different resistance mutations (Fig. 1a) [35, 40, 44]. The clinical phase I study showed that LY2874455 had nice tolerability and activity with an effective half-life of 12 h, weak toxicities, and RP2D (recommended phase 2 dosing) of 16 mg BID (bis in die) in patients with advanced solid-organ cancer [46]; (2) The inhibitors can selectively inhibit the active conformation of the kinase. Ponatinib binds to FGFR kinases in an inactive and DFG-out conformation (Fig. 1b) with IC50 of nanomolar in preclinical studies. And it is in phase II (NCT02272998) to treat advanced solid tumor patients with FGFR2-activated mutations [47]. However, Ponatinib is a multi-target kinase inhibitor that may cause strong side effects; (3) Cys-mediated covalent inhibitors can overcome gatekeeper mutations. FIIN-2 and FIIN-3 are the first covalent inhibitors based on the PD173074 scaffold. They can effectively bind covalently to C477 of the P-loop to overcome FGFR TKI resistance caused by FGFR gatekeeper mutations (Fig. 1c) [48]. Another case is FGF401, which covalently binds the C552 of Hinge to specifically overcome FGFR4 gatekeeper mutations (Fig. 1D) [49]. At present, FGF401 is in clinical phase I/II trials for hepatocellular carcinoma patients with FGF19/FGFR4 signal abnormity [50]. These covalent inhibitors with both selectivity and flexibility may be the main focus of targeted drug development in the future [51‐53].

After lipophilic weakly basic TKIs enter the cell, they can diffuse freely into lysosomes, driven by the pH gradient change between lysosomes and cytoplasm. The drug is then protonated in an acidic environment and cannot re-cross the lysosomal membrane. It is sequestrated in the lysosomal vesicle, which prevents the drug from reaching its target, resulting in a decrease in drug concentration and drug resistance [55]. Sunitinib and Gefitinib (a selective EGFR-TKI) were reported to have a significant sequestration effect in lysosomes [55, 57].

Englinger and colleagues have documented lysosome-induced drug sequestration in lung cancer with FGFR changes. They analyzed the cell-free fluorescence, intracellular accumulation, and distribution of the multi-kinase inhibitor Nintedanib and the FGFR kinase inhibitor PD173074 using three-dimensional fluorescence spectroscopy, together with analytical chemistry and molecular biology methods. These studies revealed selective accumulation of drugs in the lysosome, and verified the lipophilic and weakly basic drug characteristics of Nintedanib and PD173074 [58‐60]. However, this mechanism is reversible and has a less stable genetic resistance effect [61]. Targeting lysosomes in tumors therapy can be a promising strategy to overcome drug resistance. Besides, autophagy is closely related to lysosome-mediated TKI resistance [62]. Rui Peng and colleagues found that activation of the AMPK/mTOR signaling pathway induced survival autophagy to resist drug therapy in FGFR-TKI resistant gastric cancer cell lines. And TAK1 (TGF-β-activated kinase 1) over-expression enhanced the activation of AMPK signaling and autophagy. Further in vitro and in vivo studies demonstrated that the TAK1 inhibitor NG25 and FGFR inhibitor AZD4547 synergistically inhibited proliferation and autophagy in AZD4547-resistant cell lines and patient-derived xenograft tumor model [63]. It suggests that TAK1 inhibitors cooperated with FGFR inhibitors may be a new therapeutic strategy to overcome autophagy-mediated FGFR-TKI resistance.

ATP-binging cassette (ABC) superfamily transporters are located on cytoplasmic and lysosomal membranes. Cytoplasmic ABC transporters pump TKIs out of the cytoplasm. Lysosomal ABC transporters pump TKI into lysosomes. This process promotes the sequestration of TKIs in lysosomes [56, 64]. For example, ABCG2 can mediate active transport of Imatinib into lysosomes [65]. Moreover, ABC transporters can cooperate with lysosome sequestration to aggravate drug resistance [66].

TFEB, as a key transcription factor, regulates the biogenesis of lysosomes. Under physiological conditions, Ser142 of TFEB is phosphorylated by lysosomal mammalian target rapamycin complex 1 (mTORC1), and the phosphorylated TFEB is retained in the cytoplasm by forming a complex with YWHA (14-3-3) protein, resulting in inactive transcription. In contrast, lysosome-sequestrated TKI is protonated in an acidic environment, which increases the permeability of lysosomal membrane via fluidization and inhibits the activity of mTORC1 kinase. The high permeability of the lysosomal membrane increases the release of Ca²⁺ into cytoplasm to activate calcineurin, which further dephosphorylates TFEB. Dephosphorylated TFEB then dissociates from the 14-3-3/TFEB complex, and enters into the nucleus to initiate transcription of lysosome-associated proteins. In this way, TFEB promotes lysosome generation and increases TKI sequestration and resistance [67‐69].

The most direct way to prevent lysosome sequestration-induced drug resistance is to change the structure of the TKI [56], such as by assembling the TKI onto nanoparticles for delivery into the cell [70]. Another way is targeting the lysosome to eliminate lysosome-mediated TKI sequestration [56, 71]. This approach can involve: (1) Targeting the H + ATP enzyme (maintaining lysosome acidity) to alkalize lysosomes; (2) Lysosomotropic agents, like chloroquine, disturb lysosomal sequestration of TKIs by inhibiting autophagy or de-acidification [56, 66]; (3) Imidazoacridones (IAS) can cause lysosomal photodestruction, which destroys the internal structure of lysosomes [72]; (4) Acid sphingomyelinase (ASM) inhibitors and lysosomal membrane protein (LMP) inhibitors can destroy the stability of the lysosomal membrane and alter membrane permeability [59], which is essential to maintain the pH gradient between lysosome and cytoplasm. Blocking TFEB-mediated lysosomal generation should also be an effective strategy to reduce FGFR-TKI sequestration. Lastly, we can combine kinase inhibitors with autophagy inhibitors and ABC transporter-related inhibitors to overcome resistance. For example, ABCB1 has been reported to be a key player in resistance to the multiple-target kinase inhibitor Nintedanib [73, 74]. Inhibition of ABCB1 and kinases simultaneously should be a promising strategy for overcoming resistance [75‐77].

Secondary activation of the PI3K-AKT signaling pathway is a classical mechanism for FGFR-TKI resistance [78]. Jharna Datta and colleagues found that the phosphorylation levels of AKT and downstream GSK3 were up-regulated in BGJ398 resistant DMS114 (FGFR1-amplificated SCLC) and RT112 (urothelial carcinoma with FGFR aberrations) cell lines. And the drug-resistant cell lines recovered their sensitivity to BGJ398 after blocking the PI3K-AKT signal by AKT inhibitor GSK2141795 or siRNA intervention [79].

The PI3K-AKT signaling pathway can be directly regulated by Pleckstrin Homology-Like Domain family A member 1 (PHLDA1) and Phosphatase and tensin homolog (PTEN), resulting in TKI-resistance (Fig. 3) [78]. PHLDA1 can competitively bind PIP3 with AKT to inhibit the activity of AKT. Once PHLDA1 is knocked down, the continuous activation of AKT signaling is sufficient to sustain cell proliferation and survival, which can induce new resistance to FGFR inhibitors PD173074 and AZD4547 in endometrial cancer cells [80]. PTEN belongs to the protein tyrosine phosphatases (PTP) gene family. Knockout of PTEN can enhance the phosphorylation of AKT, and up-regulate the expression of PIK. It suggests that PTEN deletion is a potential mechanism to resist FGFR inhibition in endometrial cancer cells [81‐83]. Besides, GSK3, as a downstream molecule of PI3K-AKT signaling, can also be activated by PKC signal in an AKT-independent manner, leading to resistance to FGFR inhibitor AZD4547 in Diffuse-Type Gastric Cancer [84].

Increased activation of the RAS-MAPK pathway plays a critical role in FGFR-TKI resistance [78, 85] Kas and colleagues showed that Ras p21 protein activator 1 (RASA1) is a negative regulator of RAS, whose inactivation directly causes activation of the RAS-MAPK pathway [86]. For instance, knockdown of RASA1 activated downstream signaling of RAS, promoting cell growth and causing resistance to FGFR inhibition. In contrast, the recovery of RASA1 expression in RASA1-mutated cells reduced MAPK and PI3K signaling pathways [86, 87].

In addition, as positive and negative factors in regulating MAPK signaling pathways, abnormalities of NRAS and DUSP6 may affect FGFR-TKI resistance. For example, in drug-resistant lung cancer cells, NRAS amplification [88] and DUSP6 deletion lead to a reactivation of the MAPK pathway, thereby resisting FGFR inhibitors [89]. Co-inhibition of FGFR and MAPK pathway by FGFR inhibitors and MEK inhibitor Trametinib induced tumor degradation in tumor xenografts derived from mesenchymal-like KRAS mutant cancer cell lines as well as patient-derived xenograft model with a typical mesenchymal phenotype [90].

Alternative activation of membrane RTKs, such as ErbB3 [91, 92], MET [93, 94], EGFR [95], EphB3 [96, 97], KIT [98], and the crosstalk between RTK and FGFR, account for the resistance of FGFR targeted therapies [99, 100]. A functional genetic screen has found that feedback activation of ErbB3 (Erb-B2 receptor tyrosine kinase 3) signaling pathway can reduce the sensitivity of AZD4547 through activation of downstream PI3K pathway in urothelial carcinoma [91]. Notably, ligand-mediated activation of ErbB2/3 can directly lead to BGJ398 resistance in FGFR3-dependent cancer cells [92].

Singleton et al. found that the activation of MET plays a crucial role in resistance to the FGFR inhibitor AZ8010 [93]. Smurm Kim et al. proved that the enhanced MET can activate PI3K/AKT signaling pathway in an ErbB3-dependent or independent manner to obtain resistance to FGFR-TKIs [94]. Moreover, MET and FGFR can compensate for each other by regulating the activation of downstream signaling pathways [101].

Using parallel RNA interference genetic screens, Maria and colleagues demonstrated that the activation of EGFR can limit the sensitivity of PD170374 in bladder cancer. Combination of FGFR and EGFR inhibitors by PD173074 and Gefitinib overcome this resistance in vitro and in vivo, but with poor tolerance in mice [95]. On the contrary, activation of the FGFR signaling pathway also associates with resistance to EGFR inhibitors [102].

Lee et al. reported that the EphB3 signaling pathway was alternatively activated in FGFR inhibitor AZD4547 resistant gastric cancer cell line SNU-16R, thereby promoting epithelial-to-mesenchymal transition (EMT) of gastric cancer cell through activation of Ras-ERK1/2-mTOR pathway [96, 97]. Blocking EphB3 by LDN-211904 reduced the phosphorylation of the Ras-ERK1/2-mTOR signal and inhibited EMT in SNU-16R cells [96]. The reactivation of mTOR signaling also reduced the sensitivity of FGFR2-amplified tumors to AZD4547 [96].

Bauer and colleagues found that KIT activation was 3–6 folds higher in GIST430 and GIST48 (Imatinib-resistant gastrointestinal stromal tumor) than in GIST882 (Imatinib-sensitive) [98]. And targeting downstream signaling molecular PI3K resulted in substantial apoptosis in the Imatinib-resistant gastrointestinal stromal tumor [98]. Besides, signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to Imatinib. Co-inhibition of KIT and FGFR3 synergistically blocked the growth of Imatinib-resistant cells [103].

To overcome drug resistance, maintaining a high response rate to kinase inhibitors is essential. One approach employs combination therapy, which can block multiple activation pathways simultaneously [104]. For example, in ovarian cancer xenografted mice, co-inhibition of FGFR and mTOR pathway simultaneously by BGJ398 and rapamycin-induced tumor regression, cell cycle arrest, and apoptosis [105]. Intriguingly, FGFR2-TACC3 fusion protein identified in cholangiocarcinoma appears to be a client of heat shock protein 90 (HSP90). The HSP90 inhibitor Ganetespib combined with BGJ398 can greatly inhibit signaling transduction of FGFR2-TACC3 fusion protein [106]. In addition, combination therapy with immune checkpoint inhibitors and combination endocrine therapy with hormonal changes in cancer patients have been reported [107, 108]. For example, co-inhibition of FGFR and PD-1 by Erdafitinib and Cetrelimab (PD-1 targeted monoclonal antibody) is in phase Ib /phase II (NCT03473743) against previously untreated cisplatin-ineligible patients [109].

Combination therapy has great promise, but it may elicit undesirable drug–drug interactions. Therefore, another approach is rationally designing single compounds with dual targets [110]. As a pan-inhibitor of FGFR1-3, 3D185 not only inhibits FGFR in tumor cells but also target CSF-1R (the main survival factor of macrophages), which is vital to the immunosuppressive microenvironment [111]. Another dual-targeted inhibitor is MPT0L145, which targets PIK3C3 and FGFR simultaneously [112]. These double active inhibitors provide new approaches to drug design.

FGFR-TKI resistance events can be directly triggered by FGFR gene fusion [114]. Kim and colleagues identified a novel FGFR2-ACSL5 fusion from a metastatic gastric cancer patient with FGFR2 amplification through RNA sequencing [115]. Intriguingly, at the beginning of FGFR inhibitor treatment, the patient showed strong sensitivity to LY2874455, and no FGFR2-ACSL5 fusion was found in vivo, but eventually, drug resistance was detected along with the FGFR2-ACSL5 fusion gene [115]. Additionally, PIK3-AKT-mTOR pathways were greatly activated in gene fusion- expressing cell lines [115]. However, this study represents an individual case and is not generalized. The function of FGFR2-ASCL5 fusion proteins is not yet clear and maybe affected by tumor heterogeneity and body environment.

The occurrence of FGFR-TKI resistance events can also be triggered by other related gene fusion. Sase et al. performed a study on the mechanism of resistance to FGFR small molecule inhibitors in FGFR2-amplified gastric cancer. They found that the fusion kinase JHDM1D-BRAF located on chromosome 7 confers resistance to AZD4547 in a monoclonal gastric cancer cell line SUN-16. Jumonji C domain-containing histone demethylase 1 homolog D (JHDM1D) is a histone demethylase that plays a major role in neural differentiation [116]. BRAF, which regulates the MAPK/ERK signaling pathway, encodes the RAF family of serine/threonine protein kinases [117]. After JHDM1D-BRAF fusion, constructive dimerization of the fusion protein was enhanced, accompanied by activation of the downstream MAPK pathway, the disappearance of FGFR2 phosphorylation, and a decrease in FGFR2 expression in SUN-16 cells. These results suggest that co-treatment of RAF dimer inhibitors with downstream signaling molecule MEK inhibitors may be an option for avoiding resistance [118] (Table 3).