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KMS-11R cells harbour a Val555Met gatekeeper mutation in FGFR3. (a, b) Genomic DNA from KMS-11 or KMS-11R cells was extracted, and PCR was performed using M13-tagged forward and reverse primers to amplify (a) exon 9 including codon 373 or (b) exon 13 including codon 555 from FGFR3. PCR products were sequenced in both directions by dye-terminator sequencing using the ABI3730 capillary sequencer. Both KMS-11 and KMS-11R cells harbour the driving FGFR3Y373C mutation, whereas only KMS-11R harbour the FGFR3V555M gatekeeper mutation. (c) Cartoon and surface-rendered representation of the ATP-binding pocket of human FGFR1 in complex with PD173074 (sticks) (2fgi.pdb). Gatekeeper residue Val561, equivalent to FGFR3 Val555, is shown in yellow in upper panels. A model of the Val561-to-Met mutation, highlighted in yellow, is shown in the lower panels. Pocket surface contributed by Val561 or Met561 is shown in panels on right.
Source publication
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...
Contexts in source publication
Context 1
... mutations in several tyrosine kinases confer increased activity as well as being drug refractory. 38 Sequencing confirmed that both KMS-11 and KMS-11R contained the FGFR Y373C mutation (Figure 7a), but we also found a heterozygous mutation encoding FGFR V555M in KMS-11R cells that was not detected in parental KMS-11 cells (Figure 7b). Val555 of FGFR3 has the same role as Val561 of FGFR1 and Val564 of FGFR2 as the gatekeeper residue in the ATP-binding site (shown in yellow in Figure 7c, upper panels); mutations at this site frequently impart resistance to TKIs. ...
Context 2
... mutations in several tyrosine kinases confer increased activity as well as being drug refractory. 38 Sequencing confirmed that both KMS-11 and KMS-11R contained the FGFR Y373C mutation (Figure 7a), but we also found a heterozygous mutation encoding FGFR V555M in KMS-11R cells that was not detected in parental KMS-11 cells (Figure 7b). Val555 of FGFR3 has the same role as Val561 of FGFR1 and Val564 of FGFR2 as the gatekeeper residue in the ATP-binding site (shown in yellow in Figure 7c, upper panels); mutations at this site frequently impart resistance to TKIs. ...
Context 3
... Sequencing confirmed that both KMS-11 and KMS-11R contained the FGFR Y373C mutation (Figure 7a), but we also found a heterozygous mutation encoding FGFR V555M in KMS-11R cells that was not detected in parental KMS-11 cells (Figure 7b). Val555 of FGFR3 has the same role as Val561 of FGFR1 and Val564 of FGFR2 as the gatekeeper residue in the ATP-binding site (shown in yellow in Figure 7c, upper panels); mutations at this site frequently impart resistance to TKIs. [38][39][40] As residues comprising the ATP-binding pocket are completely conserved among FGFR1-3, with the exception of Ala559 in FGFR3 (see Methods), we used the structure of PD173074 in complex with FGFR1 to model this mutation. ...
Context 4
... As residues comprising the ATP-binding pocket are completely conserved among FGFR1-3, with the exception of Ala559 in FGFR3 (see Methods), we used the structure of PD173074 in complex with FGFR1 to model this mutation. 41 Our model suggests that Met, with a B25% bulkier side-chain, will restrict access to the cavity adjacent to the adenine ring-binding region (shown in yellow in Figure 7c, lower panel). In particular, the equivalent residue of FGFR1, Val561, makes van der Waals contact with the PD173074, 41 whereas the bulkier side chain of Met561 would disrupt binding of PD173074 (Figure 7c, lower panel). ...
Context 5
... Our model suggests that Met, with a B25% bulkier side-chain, will restrict access to the cavity adjacent to the adenine ring-binding region (shown in yellow in Figure 7c, lower panel). In particular, the equivalent residue of FGFR1, Val561, makes van der Waals contact with the PD173074, 41 whereas the bulkier side chain of Met561 would disrupt binding of PD173074 (Figure 7c, lower panel). Taken together, our results suggest that a secondary gatekeeper mutation accounts for acquired resistance to AZ8010, AZD4547 and PD173074 in KMS-11R cells. ...
Similar publications
Background and aims
About 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutationally inactivated TP53, CDKN2A or BAP1. FFs span residues 1-768 of FGFR2 fused to sequences encoded by any of a long list (>60) of partner genes, a configuration suffici...
Citations
... Due to the fact that patients respond differently to SMIs, such as Erdafitinib, and therapy resistances will inevitably occur in the clinical setting, we focused on this adaptive response. Published reports on how to effectively overcome resistance to FGFR inhibition are scarce [16][17][18][19]. Studies in urothelial, hepatic cholangio and breast carcinomas examined various targeting strategies for mutated FGFR variants and adaptive feedback loops activated after FGFR deactivation. ...
Background
Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC).
Methods
The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts.
Results
Fibroblast growth factor receptor (FGFR 1–4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with β1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions.
Conclusions
This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.
... AZD4547, BGJ398, LY2874455, monoclonal antibodies such as MGFR1877S, and finally FGF traps such as HGS1036/ FP-1039 are selective TKIs. [77][78][79][80][81][82][83][84][85][86][87][88] Selective Anti-FGFR TKIs 1 [85,89] 2. BGJ398 is the next selective inhibitor of FGFR1-3. [86] Successful results of using this type of inhibitor to reduce tumor-induced lesions in amplified lung cancer have been reported in some studies. ...
Artificial intelligence talks about modeling intelligent behavior through a computer with the least human involvement. Drug repositioning techniques based on artificial intelligence accelerate the research process and decrease the cost of experimental studies. Dysregulation of fibroblast growth factor (FGF) receptors as the tyrosine kinase family of receptors plays a vital role in a wide range of malignancies. Because of their functional significance, they were considered promising drug targets for the therapy of various cancers. This review has summarized small molecules capable of inhibiting FGF receptors that progressed using artificial intelligence and repositioning drugs examined in clinical trials associated with cancer therapy. This review is based on a literature search in PubMed, Web of Science, Scopus EMBASE, and Google Scholar databases to gather the necessary information in each chapter by employing keywords like artificial intelligence, computational drug design, drug repositioning, and FGF receptor inhibitors. To achieve this goal, a spacious literature review of human studies in these fields—published over the last 20 decades—was performed. According to published reports, nonselective FGF receptor inhibitors can be used for cancer management, and multitarget kinase inhibitors are the first drug class approved due to more advanced clinical studies. For example, AZD4547 and BGJ398 are gradually entering the consumption cycle and are good options as combined treatments. Artificial intelligence and drug repositioning methods can help preselect suitable drug targets more successfully for future inhibition of carcinogenicity.
... 1,3 Selective small-molecule FGFR inhibitors have shown clinical efficacy in patients with tumors harboring FGFR pathway aberrations; however, most are reversible adenosine triphosphate (ATP)competitive inhibitors, for which the emergence of acquired resistance mutations in the FGFR kinase domain is a major challenge. [4][5][6][7] Futibatinib (TAS-120) is a novel and highly selective inhibitor of all four FGFR subtypes that, via covalent binding, results in the irreversible inhibition of downstream FGF/FGFR signaling. Futibatinib has shown broad antiproliferative activity in FGFR-deregulated cell lines and xenograft models. ...
Futibatinib is a covalently binding FGFR1-4 inhibitor that received US Food and Drug Administration approval for the treatment of patients with previously treated, advanced intrahepatic cholangiocarcinoma harboring FGFR2 gene fusions/rearrangements. This phase I trial evaluated the pharmacokinetics (PKs), safety, and tolerability of futibatinib in subjects with impaired hepatic function and matched healthy volunteers. Twenty-two subjects with hepatic impairment (8 mild [Child-Pugh 5-6], 8 moderate [7-9], and 6 severe [10-15]) and 16 matched healthy control subjects received a single oral dose of futibatinib 20 mg. Futibatinib PKs were compared between subjects with mild/moderate/severe hepatic impairment and each corresponding control cohort and the overall control cohort. Relationships between futibatinib PKs and Child-Pugh scores and liver function tests were examined via scatter/regression plots. Compared with matched controls, the area under the plasma concentration-time curve from time zero to infinity increased by 21%/20%/18% and the maximum plasma concentration (Cmax ) increased by 43%/15%/10% in subjects with mild/moderate/severe hepatic impairment, respectively. Changes were not considered clinically relevant: geometric mean ratios were within 80%-125%, except for Cmax in subjects with mild hepatic impairment (143%). No obvious trends were observed among futibatinib PK parameters versus Child-Pugh scores, bilirubin, albumin, international normalized ratio, and aspartate aminotransferase (all p > 0.05). Futibatinib was well-tolerated, with only four grade 1 treatment-emergent adverse events (mild hepatic impairment = 2 and control = 2). The results demonstrate that futibatinib dose adjustments due to mild/moderate/severe hepatic impairment are not necessary in patients receiving futibatinib 20 mg daily.
... FGFR3 mutations have been reported to implicate in malignant tumors, such as urothelial carcinoma [16,17], lung adenocarcinoma [18] and cervical cancer [19,20]. Just as kinase domain mutations in other tyrosine kinases lead to resistance to targeted inhibitors [21,22], point mutations in the molecular break residue (I538V, N540S/K), gatekeeper residue (V555M) of FGFR3 or TEL-FGFR3 are resistant to FGFR inhibitors, such as AZD4547 and erdafitinib [23][24][25]. However, the underlying mechanism of FGFR3 kinase domain mutations leading to drug resistance remains unclear. ...
FGFR3 kinase mutations are associated with a variety of malignancies, but FGFR3 mutant inhibitors have rarely been studied. Furthermore, the mechanism of pan-FGFR inhibitors resistance caused by kinase domain mutations is still unclear. In this study, we try to explain the mechanism of drug resistance to FGFR3 mutation through global analysis and local analysis based on molecular dynamics simulation, binding free energy analysis, umbrella sampling and community network analysis. The results showed that FGFR3 mutations caused a decrease in the affinity between drugs and FGFR3 kinase, which was consistent with the reported experimental results. Possible mechanisms are that mutations affect drug-protein affinity by altering the environment of residues near the hinge region where the protein binds to the drug, or by affecting the A-loop and interfering with the allosteric communication networks. In conclusion, we systematically elucidated the underlying mechanism of pan-FGFR inhibitor resistance caused by FGFR3 mutation based on molecular dynamics simulation strategy, which provided theoretical guidance for the development of FGFR3 mutant kinase inhibitors.
... In contrast, drug resistance for anti-RTK TKIs occurs largely through secondary mutations in RTK kinase domain as well as moderately through bypass signaling activation of other RTKs or downstream molecules 267 . The secondary mutations consist of a large group of gatekeeper mutations such as T790M in EGFR, T798I in HER2, L1196M in ALK, T670I in c-Kit and those in FGFRs (V561M in FGFR1, V564M in FGFR2, V555M in FGFR3, and V550L/M in FGFR4) as well as some other mutations altering drug-binding pocket, which impair the loading of first-generation TKIs and hence abolish their efficacy 167,168,275,276,169,[268][269][270][271][272][273][274] . This problem has been resolved by the second-or third-generation TKIs that were designed to target the secondary mutations of RTKs. ...
... The KMS-11 myeloma cell line study revealed that the FGFR3 V555M mutant is resistant to AZD4547 and PD173074. Downstream signal transduction and FGFR3 phosphorylation could be correlated (Chell et al., 2013). Looking at the conformational explanation, it could be a steric clash with the PD173074 phenyl ring and structural changes in the P-the loop region (Yoza et al., 2016). ...
ABSTRACT Fibroblast Growth Factor (FGF) ligands and their receptors are crucial factors driving chemoresistance in several malignancies, challenging the efficacy of currently available anti-cancer drugs. The Fibroblast growth factor/receptor (FGF/FGFR) signalling malfunctions in tumor cells, resulting in a range of molecular pathways that may impact its drug effectiveness. Deregulation of cell signalling is critical since it can enhance tumor growth and metastasis. Overexpression and mutation of FGF/FGFR induce regulatory changes in the signalling pathways. Chromosomal translocation facilitating FGFR fusion production aggravates drug resistance. Apoptosis is inhibited by FGFR-activated signalling pathways, reducing multiple anti-cancer medications' destructive impacts. Angiogenesis and epithelial-mesenchymal transition (EMT) are facilitated by FGFRs-dependent signalling, which correlates with drug resistance and enhances metastasis. Further, lysosome-mediated drug sequestration is another prominent method of resistance. Inhibition of FGF/FGFR by following a plethora of therapeutic approaches such as covalent and multitarget inhibitors, ligand traps, monoclonal antibodies, recombinant FGFs, combination therapy, and targeting lysosomes and micro RNAs would be helpful. As a result, FGF/FGFR suppression treatment options are evolving nowadays. To increase positive impacts, the processes underpinning the FGF/FGFR axis' role in developing drug resistance need to be clarified, emphasizing the need for more studies to develop novel therapeutic options to address this significant problem. Communicated by Ramaswamy H. Sarma.
... Although tyrosine kinase inhibitors (TKIs) are often used to treat these dysregulated FGFR driven cancers, this therapeutic method often leads to the emergence of a resistant population of cancer cells exhibiting gatekeeper mutations [9]. Such mutations prevent proper binding of TKIs, giving way to a subset of cancer cells that are resistant to TKI treatment, ultimately leading to relapse in patients despite initial responsiveness to treatment [10]. This developed resistance to TKI therapies highlights the need for new approaches to treating cancers with dysregulated FGFRs. ...
FGFR3-TACC3 represents an oncogenic fusion protein frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Various exon breakpoints of FGFR3-TACC3 have been identified in cancers; these were analyzed to determine the minimum contribution of TACC3 for activation of the FGFR3-TACC3 fusion protein. While TACC3 exons 11 and 12 are dispensable for activity, our results show that FGFR3-TACC3 requires exons 13-16 for biological activity. A detailed analysis of exon 13, which consists of 8 heptads forming a coiled coil, further defined the minimal region for biological activity as consisting of 5 heptads from exon 13, in addition to exons 14-16. These conclusions were supported by transformation assays of biological activity, examination of MAPK pathway activation, analysis of disulfide-bonded FGFR3-TACC3, and by examination of the Endoglycosidase H-resistant portion of FGFR3-TACC3. These results demonstrate that clinically identified FGFR3-TACC3 fusion proteins differ in their biological activity, depending upon the specific breakpoint. This study further suggests the TACC3 dimerization domain of FGFR3-TACC3 as a novel target in treating FGFR translocation driven cancers.
... 2b and 3. PD166866 is considered as an FGFR1 inhibitor, while both BGJ398 and AZD4547 are potent inhibitors of FGFRs 1-3, although they might affect FGFR4 weakly. All the inhibitors affect tyrosine kinase activity, hence autophosphorylation and signaling [26][27][28], Treatment of microglial cells with FGFR inhibitors showed that they had efficacies at different doses. PD166866/FGFR1 inhibitor, in the presence of B. burgdorferi, did not have an appreciable effect at 500 nM concentration while at higher concentrations (≥ 1 µM) it significantly downregulated IL-6, CXCL8 and CCL2 (Fig. 2b). ...
Background
Lyme neuroborreliosis, caused by the bacterium Borrelia burgdorferi affects both the central and peripheral nervous systems (CNS, PNS). The CNS manifestations, especially at later stages, can mimic/cause many other neurological conditions including psychiatric disorders, dementia, and others, with a likely neuroinflammatory basis. The pathogenic mechanisms associated with Lyme neuroborreliosis, however, are not fully understood.
Methods
In this study, using cultures of primary rhesus microglia, we explored the roles of several fibroblast growth factor receptors (FGFRs) and fibroblast growth factors (FGFs) in neuroinflammation associated with live B. burgdorferi exposure. FGFR specific siRNA and inhibitors, custom antibody arrays, ELISAs, immunofluorescence and microscopy were used to comprehensively analyze the roles of these molecules in microglial neuroinflammation due to B. burgdorferi.
Results
FGFR1-3 expressions were upregulated in microglia in response to B. burgdorferi. Inhibition of FGFR 1, 2 and 3 signaling using siRNA and three different inhibitors showed that FGFR signaling is proinflammatory in response to the Lyme disease bacterium. FGFR1 activation also contributed to non-viable B. burgdorferi mediated neuroinflammation. Analysis of the B. burgdorferi conditioned microglial medium by a custom antibody array showed that several FGFs are induced by the live bacterium including FGF6, FGF10 and FGF12, which in turn induce IL-6 and/or CXCL8, indicating a proinflammatory nature. To our knowledge, this is also the first-ever described role for FGF6 and FGF12 in CNS neuroinflammation. FGF23 upregulation, in addition, was observed in response to the Lyme disease bacterium. B. burgdorferi exposure also downregulated many FGFs including FGF 5, 7, 9, 11, 13, 16, 20 and 21. Some of the upregulated FGFs have been implicated in major depressive disorder (MDD) or dementia development, while the downregulated ones have been demonstrated to have protective roles in epilepsy, Parkinson’s disease, Alzheimer’s disease, spinal cord injury, blood–brain barrier stability, and others.
Conclusions
In this study we show that FGFRs and FGFs are novel inducers of inflammatory mediators in Lyme neuroborreliosis. It is likely that an unresolved, long-term (neuro)-Lyme infection can contribute to the development of other neurologic conditions in susceptible individuals either by augmenting pathogenic FGFs or by suppressing ameliorative FGFs or both.
... FRS2 interacts with FGFRs via its phosphotyrosine-binding (PTB) domain [20] and increased expression or activation of FRS2 is involved in tumorigenesis of several tumor entities [21][22][23][24][25]. Selective (AZD4547, NVP-BGJ398 and JNJ-42756493) and non-selective (dovitinib or ponatinib) FGFR inhibitors have been explored for cancer therapy [26,27]. However, resistance to FGFR inhibitors can evolve similarly to other receptor tyrosine kinase (RTK) inhibitors, either by the formation of gatekeeper mutations in the catalytic domain or the activation of bypass mechanisms [28,29]. Targeting the adaptor protein FRS2 instead would likely prevent the evolution of FGFR gatekeeper mutations and could possibly also be effective in gatekeeper mutant FGFR-driven tumors by blocking signaling downstream of the RTK [26]. ...
Purpose:
Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs.
Methods:
We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound.
Results:
We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development.
Conclusions:
Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.
... However, AZD4547 inhibits all members of FGFR and resistance has occurred in some lung cancer. [35][36] We decide to develop a library of FGFR degraders using AZD4547 as the binder and investigate the degradation activity and selectivity against members of FGFRs. The co-crystal structure (Fig. 1b) indicates that the piperazine group is exposed to the solvent and we hypothesize that we can attach a linker to the nitrogen atom of this group for the development of PROTACs. ...
Proteolysis Targeting Chimera (PROTAC) has emerged as a novel therapeutic strategy. The major bottleneck for the development of PROTACs is the need to screen multiple parameters to create an effective degrader. It often involves the synthesis of dozens to hundreds of compounds one by one through a tedious process. We have developed a two-stage approach that allows for the rapid synthesis of PROTACs (Rapid-TAC) using preassembled building blocks to screen multiple parameters simultaneously. We herein report the application of this method to the development of PROTACs for FGFR, a challenging membrane protein target. In the first stage, we prepared 24 potential PROTACs quickly from a hydrazide-containing FGFR inhibitor and our previously reported VHL and CRBN ligand library bearing various linkers and an aldehyde functional group. These 24 PROTACs were then directly used for screening in cellular assay for protein degradation. Multiple hits were identified from the initial screening. We then prepared the corresponding stable analogues by replacing the hydrolytic labile acylhydrazone motif with an amide in the second stage. Among them, PROTAC LG1188 was identified as a potent and selective FGFR1 degrader.
