FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs.

BACKGROUND: Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis. METHODS: The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value. RESULTS: We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients. CONCLUSIONS: This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.

FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.

Elucidating the Role of Wildtype and Variant FGFR2 Structural Dynamics in (Dys)Function and Disorder.

The fibroblast growth factor receptor 2 (FGFR2) gene is one of the most extensively studied genes with many known mutations implicated in several human disorders, including oncogenic ones. Most FGFR2 disease-associated gene mutations are missense mutations that result in constitutive activation of the FGFR2 protein and downstream molecular pathways. Many tertiary structures of the FGFR2 kinase domain are publicly available in the wildtype and mutated forms and in the inactive and activated state of the receptor. The current literature suggests a molecular brake inhibiting the ATP-binding A loop from adopting the activated state. Mutations relieve this brake, triggering allosteric changes between active and inactive states. However, the existing analysis relies on static structures and fails to account for the intrinsic structural dynamics. In this study, we utilize experimentally resolved structures of the FGFR2 tyrosine kinase domain and machine learning to capture the intrinsic structural dynamics, correlate it with functional regions and disease types, and enrich it with predicted structures of variants with currently no experimentally resolved structures. Our findings demonstrate the value of machine learning-enabled characterizations of structure dynamics in revealing the impact of mutations on (dys)function and disorder in FGFR2.

Cancer-associated fibroblasts secrete FGF5 to inhibit ferroptosis to decrease cisplatin sensitivity in nasopharyngeal carcinoma through binding to FGFR2.

Cisplatin (DDP)-based chemoradiotherapy is one of the standard treatments for nasopharyngeal carcinoma (NPC). However, the sensitivity and side effects of DDP to patients remain major obstacles for NPC treatment. This research aimed to study DDP sensitivity regulated by cancer-associated fibroblasts (CAFs) through modulating ferroptosis. We demonstrated that DDP triggered ferroptosis in NPC cells, and it inhibited tumor growth via inducing ferroptosis in xenograft model. CAFs secreted high level of FGF5, thus inhibiting DDP-induced ferroptosis in NPC cells. Mechanistically, FGF5 secreted by CAFs directly bound to FGFR2 in NPC cells, leading to the activation of Keap1/Nrf2/HO-1 signaling. Rescued experiments indicated that FGFR2 overexpression inhibited DDP-induced ferroptosis, and CAFs protected against DDP-induced ferroptosis via FGF5/FGFR2 axis in NPC cells. In vivo data further showed the protective effects of FGF5 on DDP-triggered ferroptosis in NPC xenograft model. In conclusion, CAFs inhibited ferroptosis to decrease DDP sensitivity in NPC through secreting FGF5 and activating downstream FGFR2/Nrf2 signaling. The therapeutic strategy targeting FGF5/FGFR2 axis from CAFs might augment DDP sensitivity, thus decreasing the side effects of DDP in NPC treatment.

Genetic insights into the 'sandwich fusion' subtype of Klippel-Feil syndrome: novel FGFR2 mutations identified by 21 cases of whole-exome sequencing.

BACKGROUND: Klippel-Feil syndrome (KFS) is a rare congenital disorder characterized by the fusion of two or more cervical vertebrae during early prenatal development. This fusion results from a failure of segmentation during the first trimester. Although six genes have previously been associated with KFS, they account for only a small proportion of cases. Among the distinct subtypes of KFS, "sandwich fusion" involving concurrent fusion of C0-1 and C2-3 vertebrae is particularly noteworthy due to its heightened risk for atlantoaxial dislocation. In this study, we aimed to investigate novel candidate mutations in patients with "sandwich fusion." METHODS: We collected and analyzed clinical data from 21 patients diagnosed with "sandwich fusion." Whole-exome sequencing (WES) was performed, followed by rigorous bioinformatics analyses. Our focus was on the six known KFS-related genes (GDF3, GDF6, MEOX1, PAX1, RIPPLY2, and MYO18). Suspicious mutations were subsequently validated through in vitro experiments. RESULTS: Our investigation revealed two novel exonic mutations in the FGFR2 gene, which had not previously been associated with KFS. Notably, the c.1750A > G variant in Exon 13 of FGFR2 was situated within the tyrosine kinase domain of the protein, in close proximity to several established post-translational modification sites. In vitro experiments demonstrated that this certain mutation significantly impacted the function of FGFR2. Furthermore, we identified four heterozygous candidate variants in two genes (PAX1 and MYO18B) in two patients, with three of these variants predicted to have potential clinical significance directly linked to KFS. CONCLUSIONS: This study encompassed the largest cohort of patients with the unique "sandwich fusion" subtype of KFS and employed WES to explore candidate mutations associated with this condition. Our findings unveiled novel variants in PAX1, MYO18B, and FGFR2 as potential risk mutations specific to this subtype of KFS.

A Phase I Study of KIN-3248, an Irreversible Small-molecule Pan-FGFR Inhibitor, in Patients with Advanced FGFR2/3-driven Solid Tumors.

PURPOSE: Despite efficacy of approved FGFR inhibitors, emergence of polyclonal secondary mutations in the FGFR kinase domain leads to acquired resistance. KIN-3248 is a selective, irreversible, orally bioavailable, small-molecule inhibitor of FGFR1-4 that blocks both primary oncogenic and secondary kinase domain resistance FGFR alterations. EXPERIMENTAL DESIGN: A first-in-human, phase I study of KIN-3248 was conducted in patients with advanced solid tumors harboring FGFR2 and/or FGFR3 gene alterations (NCT05242822). The primary objective was determination of MTD/recommended phase II dose (RP2D). Secondary and exploratory objectives included antitumor activity, pharmacokinetics, pharmacodynamics, and molecular response by circulating tumor DNA (ctDNA) clearance. RESULTS: Fifty-four patients received doses ranging from 5 to 50 mg orally daily across six cohorts. Intrahepatic cholangiocarcinoma (48.1%), gastric (9.3%), and urothelial (7.4%) were the most common tumors. Tumors harbored FGFR2 (68.5%) or FGFR3 (31.5%) alterations-23 (42.6%) received prior FGFR inhibitors. One dose-limiting toxicity (hypersensitivity) occurred in cohort 1 (5 mg). Treatment-related, adverse events included hyperphosphatemia, diarrhea, and stomatitis. The MTD/RP2D was not established. Exposure was dose proportional and concordant with hyperphosphatemia. Five partial responses were observed; 4 in FGFR inhibitor naïve and 1 in FGFR pretreated patients. Pretreatment ctDNA profiling confirmed FGFR2/3 alterations in 63.3% of cases and clearance at cycle 2 associated with radiographic response. CONCLUSION: The trial was terminated early for commercial considerations; therefore, RP2D was not established. Preliminary clinical data suggest that KIN-3248 is a safe, oral FGFR1-4 inhibitor with favorable pharmacokinetic parameters, though further dose escalation was required to nominate the MTD/RP2D. SIGNIFICANCE: KIN-3248 was a rationally designed, next generation selective FGFR inhibitor, that was effective in interfering with both FGFR wild-type and mutant signaling. Clinical data indicate that KIN-3248 is safe with a signal of antitumor activity. Translational science support the mechanism of action in that serum phosphate was proportional with exposure, paired biopsies suggested phospho-ERK inhibition (a downstream target of FGFR2/3), and ctDNA clearance may act as a RECIST response surrogate.

The Irreversible FGFR Inhibitor KIN-3248 Overcomes FGFR2 Kinase Domain Mutations.

PURPOSE: FGFR2 and FGFR3 show oncogenic activation in many cancer types, often through chromosomal fusion or extracellular domain mutation. FGFR2 and FGFR3 alterations are most prevalent in intrahepatic cholangiocarcinoma (ICC) and bladder cancers, respectively, and multiple selective reversible and covalent pan-FGFR tyrosine kinase inhibitors (TKI) have been approved in these contexts. However, resistance, often due to acquired secondary mutations in the FGFR2/3 kinase domain, limits efficacy. Resistance is typically polyclonal, involving a spectrum of different mutations that most frequently affect the molecular brake and gatekeeper residues (N550 and V565 in FGFR2). EXPERIMENTAL DESIGN: Here, we characterize the activity of the next-generation covalent FGFR inhibitor, KIN-3248, in preclinical models of FGFR2 fusion+ ICC harboring a series of secondary kinase domain mutations, in vitro and in vivo. We also test select FGFR3 alleles in bladder cancer models. RESULTS: KIN-3248 exhibits potent selectivity for FGFR1-3 and retains activity against various FGFR2 kinase domain mutations, in addition to being effective against FGFR3 V555M and N540K mutations. Notably, KIN-3248 activity extends to the FGFR2 V565F gatekeeper mutation, which causes profound resistance to currently approved FGFR inhibitors. Combination treatment with EGFR or MEK inhibitors potentiates KIN-3248 efficacy in vivo, including in models harboring FGFR2 kinase domain mutations. CONCLUSIONS: Thus, KIN-3248 is a novel FGFR1-4 inhibitor whose distinct activity profile against FGFR kinase domain mutations highlights its potential for the treatment of ICC and other FGFR-driven cancers.

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway.

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Activated FGFR2 signalling as a biomarker for selection of intrahepatic cholangiocarcinoma patients candidate to FGFR targeted therapies.

FGFR inhibitors have been developed to inhibit FGFR activation and signal transduction; notwithstanding, currently the selection of intrahepatic cholangiocarcinoma (iCCA) patients for these drugs only relies on the detection of FGFR2 genetic alterations (GAs) in tumor tissues or circulating tumor DNAs, without concomitant assessment of FGFR2 signalling status. Accordingly, we performed multi-omic analyses of FGFR2 genes and FGFR2 signalling molecules in the tissue samples from 36 iCCA naïve patients. Gain-of-function FGFR2 GAs were detected in 7 patients, including missense mutations (n = 3; p.F276C, p.C382R and p.Y375C), translocations (n = 1) and copy number gain (n = 4; CNV ≥ 4). In contrast, among 29 patients with wild-type FGFR2, 4 cases showed activation of FGFR2 signalling, as they expressed the FGFR2 ligand FGF10 and phosphorylated FGFR2/FRS2α proteins; the remaining 25 cases resulted negative for activated FGFR2 signalling, as they lacked FGFR2 (n = 8) or phosphorylated FRS2α (n = 17) expression. Overall, we found that activation of FGFR2 signalling occurs not only in iCCA naïve patients with FGFR2 GAs, but also in a subgroup carrying wild-type FGFR2. This last finding entails that also this setting of patients could benefit from FGFR targeted therapies, widening indication of these drugs for iCCA patients beyond current approval. Future clinical studies are therefore encouraged to confirm this hypothesis.

Bemarituzumab as first-line treatment for locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma: final analysis of the randomized phase 2 FIGHT trial.

BACKGROUND: We report the final results of the randomized phase 2 FIGHT trial that evaluated bemarituzumab, a humanized monoclonal antibody selective for fibroblast growth factor receptor 2b (FGFR2b), plus mFOLFOX6 in patients with FGFR2b-positive (2 + /3 + membranous staining by immunohistochemistry), HER-2-negative gastric or gastroesophageal junction cancer (GC). METHODS: Patients received bemarituzumab (15 mg/kg) or placebo once every 2 weeks with an additional bemarituzumab (7.5 mg/kg) or placebo dose on cycle 1 day 8. All patients received mFOLFOX6. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate, and safety. Efficacy was evaluated after a minimum follow-up of 24 months. RESULTS: In the bemarituzumab-mFOLFOX6 (N = 77) and placebo-mFOLFOX6 (N = 78) arms, respectively, 59.7% and 66.7% of patients were FGFR2b-positive in ≥ 10% of tumor cells. The median PFS (95% confidence interval [CI]) was 9.5 months (7.3-13.7) with bemarituzumab-mFOLFOX6 and 7.4 months (5.7-8.4) with placebo-mFOLFOX6 (hazard ratio [HR], 0.72; 95% CI 0.49-1.08); median OS (95% CI) was 19.2 (13.6-24.2) and 13.5 (9.3-15.9) months, respectively (HR 0.77; 95% CI 0.52-1.14). Observed efficacy in FGFR2b-positive GC in ≥ 10% of tumor cells was: PFS: HR 0.43 (95% CI 0.26-0.73); OS: HR 0.52 (95% CI 0.31-0.85). No new safety findings were reported. CONCLUSIONS: In FGFR2b-positive advanced GC, the combination of bemarituzumab-mFOLFOX6 led to numerically longer median PFS and OS compared with mFOLFOX6 alone. Efficacy was more pronounced with FGFR2b overexpression in ≥ 10% of tumor cells. Confirmatory phase 3 trials are ongoing (NCT05052801, NCT05111626). CLINICAL TRIAL REGISTRATION: NCT03694522.

Investigating FGFR2 gene as a blood-based epigenetic biomarker in gastric cancer.

BACKGROUND: Gastric adenocarcinoma is a prevalent form of cancer that often remains undetected in its early stages due to the lack of specific symptoms. This delayed diagnosis leads to poor clinical outcomes, underscoring the need for an effective and non-invasive method for early detection. Recent advances in cancer epigenetics have led to the identification of biomarkers that have the potential to revolutionize the early detection and monitoring of this disease. One such promising biomarker is the methylation of the FGFR2 promoter. This study aims to measure the methylation levels of a specific CpG site in the FGFR2 promoter gene in DNA extracted from blood leukocytes from patients with intestinal metaplasia, gastric cancer, and healthy control. MATERIAL AND METHODS: The CpG site of the FGFR2 gene promoter was identified in its control region. Methylation alteration of the selected FGFR2 CpG site was determined through the (methylation-sensitive restriction enzyme) MSRE-qPCR. Genomic DNA was extracted from one hundred twenty-five participants. RESULTS: The normal group had mean methylation levels of 93.23 ± 4.929%, while the IM group had a level of 69.85 ± 27.15%. In GC patients, the levels varied, with 25.96 ± 18.98% in the intestinal type and 28.30 ± 16.07% in the diffuse type. The methylation levels in the IM and GC patients were significantly lower than those in the normal control group. However, no significant difference was observed between the methylation status of the intestinal type of GC and the diffuse type. The Receiver operating characteristic (ROC) curve analysis showed that FGFR2 CpG methylation levels in GC patients compared to normal controls had a high sensitivity of 100% and specificity of 100%, with a cut-off of < 74.25%; when GC patients were compared to IM patients, the sensitivity was 85%, and the specificity was 80%, with a cut-off < 44.45%. CONCLUSIONS: The potential of the FGFR2 methylation status as a non-invasive biomarker lies in its ability to be detected in blood leukocytes, which makes it a promising tool for the early detection of intestinal metaplasia and gastric cancer. This could significantly improve the detection and management of these gastric conditions.

Discovery of lirafugratinib (RLY-4008), a highly selective irreversible small-molecule inhibitor of FGFR2.

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2-altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea.

Superficial acral calcified chondroid mesenchymal neoplasm harboring an FN1::FGFR2 fusion and review of the literature.

Calcified chondroid mesenchymal neoplasm is a recently recognized bone and soft tissue entity primarily found in the extremities and the temporomandibular joint. This neoplasm is typically driven by the fusion of the FN1 gene with a kinase. In this case report, we provide a detailed account of a rare superficial calcified chondroid mesenchymal neoplasm located on the left big toe, characterized by an FN1::FGFR2 fusion. The tumor exhibited a peripheral collarette and consisted of large intradermal histiocytoid to epithelioid cells with no mitotic activity. These cells displayed fine chromatin and abundant pale eosinophilic cytoplasm, forming a swirling syncytium. They were interspersed with localized areas of glassy chondromyxoid matrix containing randomly mineralized calcific material and isolated osteoclast-like giant cells. RNA sequencing confirmed the presence of an FN1 (exon 29)::FGFR2 (exon 7) gene fusion. Our report emphasizes the importance for dermatopathologists to consider this entity when evaluating superficial lesions displaying mesenchymal, chondroid, and calcified attributes.

Sunitinib in Patients With Breast Cancer With FGFR1 or FGFR2 Amplifications or Mutations: Results From the Targeted Agent and Profiling Utilization Registry Study.

PURPOSE: The Targeted Agent and Profiling Utilization Registry Study is a phase II basket trial evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancer and genomic alterations known to be drug targets. Results from cohorts of patients with metastatic breast cancer (BC) with FGFR1 and FGFR2 alterations treated with sunitinib are reported. METHODS: Eligible patients had measurable disease, Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. Simon's two-stage design was used with a primary end point of disease control (DC), defined as objective response (OR) or stable disease of at least 16 weeks duration (SD16+) according to RECIST v1.1. Secondary end points included OR, progression-free survival, overall survival, duration of response, duration of stable disease, and safety. RESULTS: Forty patients with BC with FGFR1 (N = 30; amplification only n = 26, mutation only n = 1, both n = 3) or FGFR2 (N = 10; amplification only n = 2, mutation only n = 6, both n = 2) alterations were enrolled. Three patients in the FGFR1 cohort were not evaluable for efficacy; all patients in the FGFR2 cohort were evaluable. For the FGFR1 cohort, two patients with partial response and four with SD16+ were observed for DC and OR rates of 27% (90% CI, 13 to 100) and 7% (95% CI, 1 to 24), respectively. The null hypothesis of 15% DC rate was not rejected (P = .169). No patients achieved DC in the FGFR2 cohort (P = 1.00). Thirteen of the 40 total patients across both cohorts had at least one grade 3-4 adverse event or serious adverse event at least possibly related to sunitinib. CONCLUSION: Sunitinib did not meet prespecified criteria to declare a signal of antitumor activity in patients with BC with either FGFR1 or FGFR2 alterations. Other treatments and clinical trials should be considered for these patient populations.

The molecular interaction pattern of lenvatinib enables inhibition of wild-type or kinase-mutated FGFR2-driven cholangiocarcinoma.

Fibroblast growth factor receptor (FGFR)-2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.

Pemigatinib for patients with previously treated, locally advanced or metastatic cholangiocarcinoma harboring FGFR2 fusions or rearrangements: A joint analysis of the French PEMI-BIL and Italian PEMI-REAL cohort studies.

BACKGROUND: Pemigatinib is approved for patients with pretreated, locally advanced or metastatic CCA harboring FGFR2 rearrangements or fusions. We aim to assess the effectiveness and safety of pemigatinib in real-world setting. MATERIAL AND METHODS: A joint analysis of two multicentre observational retrospective cohort studies independently conducted in France and Italy was performed. All consecutive FGFR2-positive patients affected by CCA and treated with pemigatinib as second- or further line of systemic treatment in clinical practice, within or outside the European Expanded Access Program, were included. RESULTS: Between July 2020 and September 2022, 72 patients were treated with pemigatinib in 14 Italian and 25 French Centres. Patients had a median age of 57 years, 76% were female, 81% had ECOG-PS 0-1, 99% had intrahepatic CCA, 74% had ≥ 2 metastatic sites, 67% had metastatic disease at diagnosis, while 38.8% received ≥ 2 previous lines of systemic treatment. At data cut-off analysis (April 2023), ORR and DCR were 45.8% and 84.7%, respectively. Median DoR was 7 months (IQR: 5.8-9.3). Over a median follow-up time of 19.5 months, median PFS and 1-year PFS rate were 8.7 months and 32.8%. Median OS and 1-year OS rate were 17.1 months and 60.6%. Fatigue (69.4%), ocular toxicity (68%), nail toxicities (61.1%), dermatologic toxicity (41.6%) hyperphosphataemia (55.6%), stomatitis (48.6%), and diarrhea (36.1%) were the most frequent, mainly G1-G2 AEs. Overall incidence of G3 AEs was 22.2%, while no patient experienced G4 AE. Dose reduction and temporary discontinuation were needed in 33.3% and 40.3% of cases, with 1 permanent discontinuation due to AEs. CONCLUSIONS: These results confirm the effectiveness and safety of pemigatinib in a real-world setting.

Sp1-activated FGFR2 is involved in early-life exposure to nickel-induced craniosynostosis by regulating the ERK1/2 signaling pathway.

Nickel, a common environmental hazard, is a risk factor for craniosynostosis. However, the underlying biological mechanism remains unclear. Here, we found that early-life nickel exposure induced craniosynostosis in mice. In vitro, nickel promoted the osteogenic differentiation of human mesenchymal stem cells (hMSCs), and its osteogenic ability in vivo was confirmed by an ectopic osteogenesis model. Further mRNA sequencing showed that ERK1/2 signaling and FGFR2 were aberrantly activated. FGFR2 was identified as a key regulator of ERK1/2 signaling. By promoter methylation prediction and methylation-specific PCR (MSP) assays, we found that nickel induced hypomethylation in the promoter of FGFR2, which increased its binding affinity to the transcription factor Sp1. During pregnancy and postnatal stages, AZD4547 rescued nickel-induced craniosynostosis by inhibiting FGFR2 and ERK1/2. Compared with normal individuals, nickel levels were increased in the serum of individuals with craniosynostosis. Further logistic and RCS analyses showed that nickel was an independent risk factor for craniosynostosis with a nonlinear correlation. Mediated analysis showed that FGFR2 mediated 30.13% of the association between nickel and craniosynostosis risk. Collectively, we demonstrate that early-life nickel exposure triggers the hypomethylation of FGFR2 and its binding to Sp1, thereby promoting the osteogenic differentiation of hMSCs by ERK1/2 signaling, leading to craniosynostosis.

Ser252Trp mutation in fibroblast growth factor receptor 2 promotes branching morphogenesis in mouse salivary glands.

OBJECTIVES: The purpose of this study was to perform morphological and immunohistochemical (IHC) analysis of the submandibular glands (SMGs) in early development in Apert syndrome model mice (Ap mice). METHODS: ACTB-Cre homozygous mice were mated with fibroblast growth factor receptor 2 (Fgfr2+/Neo-S252W) mice; ACTB-Cre heterozygous mice (ACTB-Cre mice) at embryonic day (E) 13.5 served as the control group, and Fgfr2+/S252W mice (Ap mice) served as the experimental group. Hematoxylin and eosin (H&E) staining was performed on SMGs; Total SMG area and epithelial area were determined, and the epithelial occupancy ratio was calculated. Immunostaining was performed to assess the localization of FGF signaling-related proteins. Next, bromodeoxyuridine (BrdU)-positive cells were evaluated to assess cell proliferation. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess apoptosis in SMGs. RESULTS: The epithelial occupancy ratio was significantly higher in SMGs of Ap mice compared with that in SMGs of controls. FGF7 and bone morphogenetic protein 4 (BMP4) exhibited different localizations in SMGs of Ap mice compared with SMGs of controls. Cell proliferation was higher in SMGs of Ap mice compared with that of controls; however, apoptosis did not different significantly between the two groups. CONCLUSION: Our results suggest that enhanced FGF signaling conferred by missense mutations in FGFR2 promotes branching morphogenesis in SMGs of Ap mice.

Expansion of the complex genotypic and phenotypic spectrum of FGFR2-associated neurocutaneous syndromes.

The fibroblast growth factor receptors comprise a family of related but individually distinct tyrosine kinase receptors. Within this family, FGFR2 is a key regulator in many biological processes, e.g., cell proliferation, tumorigenesis, metastasis, and angiogenesis. Heterozygous activating non-mosaic germline variants in FGFR2 have been linked to numerous autosomal dominantly inherited disorders including several craniosynostoses and skeletal dysplasia syndromes. We report on a girl with cutaneous nevi, ocular malformations, macrocephaly, mild developmental delay, and the initial clinical diagnosis of Schimmelpenning-Feuerstein-Mims syndrome, a very rare mosaic neurocutaneous disorder caused by postzygotic missense variants in HRAS, KRAS, and NRAS. Exome sequencing of blood and affected skin tissue identified the mosaic variant c.1647=/T > G p.(Asn549=/Lys) in FGFR2, upstream of the RAS signaling pathway. The variant is located in the tyrosine kinase domain of FGFR2 in a region that regulates the activity of the receptor and structural mapping and functional characterization revealed that it results in constitutive receptor activation. Overall, our findings indicate FGFR2-associated neurocutaneous syndrome as the accurate clinical-molecular diagnosis for the reported individual, and thereby expand the complex genotypic and phenotypic spectrum of FGFR-associated disorders. We conclude that molecular analysis of FGFR2 should be considered in the genetic workup of individuals with the clinical suspicion of a mosaic neurocutaneous condition, as the knowledge of the molecular cause might have relevant implications for genetic counseling, prognosis, tumor surveillance and potential treatment options.

Synthesis and Evaluation of Novel 99mTc-Labeled FGFR2-Targeting Peptides.

To develop radiolabeled FGFR2-targeting probes for visualizing fibroblast growth factor receptor (FGFR) expression levels in the tumor microenvironment, four novel 99mTc-labeled FGFR2-targeting peptides ([99mTc]Tc-FGFR2-1, [99mTc]Tc-FGFR2-2, [99mTc]Tc-FGFR2-3, and [99mTc]Tc-FGFR2-4) with different amino acid linkers between the targeted peptide moiety and the 99mTc chelating group were designed and synthesized. The in vitro cellular inhibition, internalization, and efflux results demonstrated that the four 99mTc complexes exhibited FGFR2-specific binding and prolonged cellular retention in DU145 human prostate cancer cells, which indicated that modification from the glycine side (N-terminal) of CH02 was feasible. Among them, [99mTc]Tc-FGFR2-1 exhibited the highest in vitro cellular uptake and in vivo tumor uptake at 30 min postinjection, and tumor uptake could be significantly inhibited by the competitor CH02 (53% inhibited, p < 0.05), suggesting the tumor-specific targeting ability of [99mTc]Tc-FGFR2-1. The DU145-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 30 min postinjection of [99mTc]Tc-FGFR2-1, highlighting its potential as a SPECT imaging probe for tumor FGFR2 detection.