Central neurocytoma exhibits radial glial cell signatures with FGFR3 hypomethylation and overexpression.

We explored the genomic events underlying central neurocytoma (CN), a rare neoplasm of the central nervous system, via multiomics approaches, including whole-exome sequencing, bulk and single-nuclei RNA sequencing, and methylation sequencing. We identified FGFR3 hypomethylation leading to FGFR3 overexpression as a major event in the ontogeny of CN that affects crucial downstream events, such as aberrant PI3K-AKT activity and neuronal development pathways. Furthermore, we found similarities between CN and radial glial cells based on analyses of gene markers and CN tumor cells and postulate that CN tumorigenesis is due to dysregulation of radial glial cell differentiation into neurons. Our data demonstrate the potential role of FGFR3 as one of the leading drivers of tumorigenesis in CN.

Dysregulated FGFR3 signaling alters the immune landscape in bladder cancer and presents therapeutic possibilities in an agent-based model.

Bladder cancer is an increasingly prevalent global disease that continues to cause morbidity and mortality despite recent advances in treatment. Immune checkpoint inhibitors (ICI) and fibroblast growth factor receptor (FGFR)-targeted therapeutics have had modest success in bladder cancer when used as monotherapy. Emerging data suggests that the combination of these two therapies could lead to improved clinical outcomes, but the optimal strategy for combining these agents remains uncertain. Mathematical models, specifically agent-based models (ABMs), have shown recent successes in uncovering the multiscale dynamics that shape the trajectory of cancer. They have enabled the optimization of treatment methods and the identification of novel therapeutic strategies. To assess the combined effects of anti-PD-1 and anti-FGFR3 small molecule inhibitors (SMI) on tumor growth and the immune response, we built an ABM that captures key facets of tumor heterogeneity and CD8+ T cell phenotypes, their spatial interactions, and their response to therapeutic pressures. Our model quantifies how tumor antigenicity and FGFR3 activating mutations impact disease trajectory and response to anti-PD-1 antibodies and anti-FGFR3 SMI. We find that even a small population of weakly antigenic tumor cells bearing an FGFR3 mutation can render the tumor resistant to combination therapy. However, highly antigenic tumors can overcome therapeutic resistance mediated by FGFR3 mutation. The optimal therapy depends on the strength of the FGFR3 signaling pathway. Under certain conditions, ICI alone is optimal; in others, ICI followed by anti-FGFR3 therapy is best. These results indicate the need to quantify FGFR3 signaling and the fitness advantage conferred on bladder cancer cells harboring this mutation. This ABM approach may enable rationally designed treatment plans to improve clinical outcomes.

Monomeric pilose antler peptide improves depression-like behavior in mice by inhibiting FGFR3 protein expression.

ETHNOPHARMACOLOGICAL RELEVANCE: It has been found that pilose antler peptide has an antidepressant effect on depression. However, the exact molecular mechanism of its antidepressant effect is still unclear. AIM OF THE STUDY: The study sought to determine the impact of monomeric pilose antler peptide (PAP; sequence LVLVEAELRE) on depression as well as investigate potential molecular mechanisms. MATERIALS AND METHODS: Chronic unexpected mild stress (CUMS) was used to establish the model, and the effect of PAP on CUMS mice was detected by the behavioral test. The influence of PAP on neuronal cells and dendritic spine density was observed by immunofluorescence and Golgi staining. FGFR3 and the CaMKII-associated pathway were identified using quantitative real-time polymerase chain reaction, and Western blot analysis was utilized to measure their proteins and gene expression levels. Molecular docking and microscale thermophoresis were applied to detect the binding of PAP and FGFR3. Finally, the effect of FGFR3's overexpression on PAP treatment of depression was detected. RESULTS: PAP alleviated the changes in depressive behavior induced by CUMS, promoted the growth of nerve cells, and the density of dendritic spines was increased to its original state. PAP therapy successfully downregulated the expression of FGFR3 and ERK1/2 while upregulating the expression of CREB, BDNF, and CaMKII. CONCLUSION: Based on the current research, PAP has a therapeutic effect on depression brought on by CUMS by inhibiting FGFR3 expression and enhancing synaptic plasticity.

FGFR inhibition augments anti-PD-1 efficacy in murine FGFR3-mutant bladder cancer by abrogating immunosuppression.

The combination of targeted therapy with immune checkpoint inhibition (ICI) is an area of intense interest. We studied the interaction of fibroblast growth factor receptor (FGFR) inhibition with ICI in urothelial carcinoma (UC) of the bladder, in which FGFR3 is altered in 50% of cases. Using an FGFR3-driven, Trp53-mutant genetically engineered murine model (UPFL), we demonstrate that UPFL tumors recapitulate the histology and molecular subtype of their FGFR3-altered human counterparts. Additionally, UPFL1 allografts exhibit hyperprogression to ICI associated with an expansion of T regulatory cells (Tregs). Erdafitinib blocked Treg proliferation in vitro, while in vivo ICI-induced Treg expansion was fully abrogated by FGFR inhibition. Combined erdafitinib and ICI resulted in high therapeutic efficacy. In aggregate, our work establishes that, in mice, co-alteration of FGFR3 and Trp53 results in high-grade, non-muscle-invasive UC and presents a previously underappreciated role for FGFR inhibition in blocking ICI-induced Treg expansion.

N-glycan on N262 of FGFR3 regulates the intracellular localization and phosphorylation of the receptor.

N-glycosylation and proper processing of N-glycans are required for the function of membrane proteins including cell surface receptors. Fibroblast growth factor receptor (FGFR) is involved in a wide variety of biological processes including embryonic development, osteogenesis, angiogenesis, and cell proliferation. Human FGFR3 contains six potential N-glycosylation sites, however, the roles of glycosylation have not been elucidated. The site-specific profiles of N-glycans of the FGFR3 extracellular domain expressed and secreted by CHO-K1 cells were examined, and glycan occupancies and structures of four sites were determined. The results indicated that most sites were fully occupied by glycans, and the dominant populations were the complex type. By examining single N-glycan deletion mutants of FGFR3, it was found that N262Q mutation significantly increased the population with oligomannose-type N-glycans, which was localized in the endoplasmic reticulum. Protein stability assay suggested that fraction with oligomannose-type N-glycans in the N262Q mutant is more stable than those in the wild type and other mutants. Furthermore, it was found that ligand-independent phosphorylation was significantly upregulated in N262Q mutants with complex type N-glycans. The findings suggest that N-glycans on N262 of FGFR3 affect the intracellular localization and phosphorylation status of the receptor.

Non-Small Cell Lung Carcinoma With Clear Cell Features and FGFR3::TACC3 Gene Rearrangement : Clinicopathologic and Next Generation Sequencing Study of 7 Cases.

Seven cases of primary lung tumors characterized histologically by clear cell morphology and a distinctive FGFR3::TACC3 gene rearrangement are described. The tumors arose in 4 women and 3 men, aged 47 to 81 years (mean=68). They occurred in peripheral locations, predominantly subpleural, and ranged in size from 1.4 to 6.5 cm (mean=4.1 cm). All tumors showed a solid growth pattern with abundant central areas of necrosis and marked nuclear pleomorphism. The tumors demonstrated clear cell histology, with large cohesive tumor cells displaying atypical nuclei and abundant clear cytoplasm. Immunohistochemical stains identified a squamous phenotype in 5 cases and an adenocarcinoma phenotype in 2 cases. One case was a squamous cell carcinoma with focal glandular component, and one of the squamous cell carcinomas showed focal sarcomatoid changes. Next generation sequencing identified FGFR3::TACC3 gene rearrangements in all 7 cases. One case demonstrated a concurrent activating FGFR3 mutation and a second case demonstrated concurrent FGFR3 amplification. Two cases harbored a concurrent KRAS G12D mutation. One case harbored both KRAS and EGFR mutations, and 1 case had a concurrent TP53 mutation. Non-small cell lung carcinoma harboring FGFR3::TACC3 gene rearrangements is extremely rare, and this rearrangement may potentially be enriched in tumors that demonstrate clear cell histology. Identification of FGFR3::TACC3 in patients with lung carcinomas with clear cell features may be of importance as they could potentially be candidates for therapy with tyrosine kinase inhibitors.

Targeting Thbs1 reduces bladder remodeling caused by partial bladder outlet obstruction via the FGFR3/p-FGFR3 pathway.

BACKGROUND: Partial bladder outlet obstruction (pBOO) may lead to bladder remodeling, including fibrosis and extracellular matrix (ECM) deposition. Despite the extensive research on the mechanisms underlying pBOO, potential therapeutic targets for the treatment of pBOO require further research. Dysregulated expression of thrombospondin-1 (Thbs1) has been reported in various human fibrotic diseases; however, its relationship with pBOO remains unclear. AIMS: Investigate the effects of Thbs1 on bladder remodeling caused by pBOO. METHODS: We established a pBOO model in Sprague-Dawley rats and performed urodynamic analyses to estimate functional changes in the bladder, validated the histopathological changes in the bladder by using haematoxylin-eosin and Masson's trichrome staining, identified key target genes by integrating RNA sequencing (RNA-seq) and bioinformatics analyses, validated the expression of related factors using Western blot analysis and RT-qPCR, and used immunofluorescence staining to probe the potential interaction factors of Thbs1. RESULTS: Urodynamic results showed that pressure-related parameters were significantly increased in rats with pBOO. Compared with the sham group, the pBOO group demonstrated significant increases in bladder morphology, bladder weight, and collagen deposition. Thbs1 was significantly upregulated in the bladder tissues of rats with pBOO, consistent with the RNA-seq data. Thbs1 upregulation led to increased expression of matrix metalloproteinase (MMP) 2, MMP9, and fibronectin (Fn) in normal human urinary tract epithelial cells (SV-HUC-1), whereas anti-Thbs1 treatment inhibited the production of these cytokines in TGF-ß1-treated SV-HUC-1. Further experiments indicated that Thbs1 affected bladder remodeling in pBOO via the fibroblast growth factor receptor 3 (FGFR3) pathway. CONCLUSIONS: Thbs1 plays a crucial role in bladder remodeling caused by pBOO. Targeting Thbs1 might alleviate ECM damage. Mechanistically, Thbs1 may function via the FGFR signaling pathway by regulating the FGFR3 receptor, identified as the most relevant disease target of pBOO, and FGF2 may be a mediator. These findings suggest that Thbs1 plays a role in BOO development and is a therapeutic target for this condition.

Increased FGFR3 is involved in T-2 toxin-induced lesions of hypertrophic cartilage associated with endemic osteoarthritis.

This study evaluated the effect of fibroblast growth factor receptor 3 (FGFR3) on damaged hypertrophic chondrocytes of Kashin-Beck disease (KBD). Immunohistochemical staining was used to evaluate FGFR3 expression in growth plates from KBD rat models and engineered cartilage. In vitro study, hypertrophic chondrocytes were pretreated by FGFR3 binding inhibitor (BGJ398) for 24 h before incubation at different T-2 toxin concentrations. Differentiation -related genes (Runx2, Sox9, and Col Ⅹ) and ECM degradation -related genes (MMP-13, Col Ⅱ) in the hypertrophic chondrocytes were analyzed using RT-PCR, and the corresponding proteins were analyzed using western blotting. Hypertrophic chondrocytes death was detected by the Annexin V/PI double staining assay. The integrated optical density of FGFR3 staining was increased in knee cartilage of rats and engineered cartilage treated with T-2 toxin. Both protein and mRNA levels of Runx2, Sox9, Col Ⅱ, and Col Ⅹ were decreased in a dose-dependent manner when exposed to the T-2 toxin and significantly upregulated by 1 µM BGJ398. The expression of MMP-1, MMP-9, and MMP-13 increased in a dose-dependent manner when exposed to T-2 toxin and significantly reduced by 1 µM BGJ398. 1 µM BGJ398 could prevent early apoptosis and necrosis induced by the T-2 toxin. Inhibiting the FGFR3 signal could alleviate extracellular matrix degradation, abnormal chondrocytes differentiation, and excessive cell death in T-2 toxin-induced hypertrophic chondrocytes.

P53, CK20, and FGFR3 Overexpression is Associated with the Characteristics of Urothelial Cell Carcinoma of the Bladder

OBJECTIVES: The aim of this study was to investigate the association between the overexpression of tumor protein (P53), cytokeratin 20 (CK20), fibroblast growth factor receptor 3 (FGFR3), biomarkers and the grading, prognosis, heterogeneity, and relapse tendency of urothelial cell carcinomas (UCCs) of the bladder. METHODS: A cross-sectional study was conducted using 413 samples of Iranian patients diagnosed with UCC of the bladder. The tissue microarray technique was used to evaluate the patterns of tumor tissue. Two pathologists scored tissue staining using a semi-quantitative scoring system. RESULTS: The results showed that P53 was a predictor of a high-grade pattern (the area under the curve (AUC)=0.620) with a best cut-off value of 95.0 using the receiver operating characteristic (ROC) curve. CK20 was another predictor of a high-grade pattern (AUC=0.745) with a best cut-off value of 15. However, the overexpression of both biomarkers was not associated with a heterogeneous pattern and could not predict tumor-associated death or relapse. The heterogeneous (odds ratio (OR)=4.535, p-value=0.001) and non-papillary (OR= 6.363, p-value= 0.001) patterns were effective predictors of tumor recurrence among all baseline variables, including patient and tumor characteristics. FGFR3 was positive in all specimens and was not a valuable biomarker for differentiating patterns. None of the variables predicted tumor prognosis. CONCLUSION: The study findings indicate that the intensity and percentage of cell staining for P53 and CK20 in the UCC of the bladder can aid in differentiating the grading patterns. The tendency of tumor relapse can be predicted by demonstrating heterogeneous and non-papillary patterns.

Fibroblast growth factor receptor 3 mutation attenuates response to immune checkpoint blockade in metastatic urothelial carcinoma by driving immunosuppressive microenvironment.

BACKGROUND: Immune checkpoint blockade (ICB) therapy holds promise in metastatic urothelial carcinoma (UC). Fibroblast growth factor receptor 3 (FGFR3) mutation drives T-cell-depleted microenvironment in UC, which led to the hypothesis that FGFR3 mutation might attenuate response to ICB in patients with metastatic UC. The study aims to compare prognosis and response between patients with FGFR3-mutated and FGFR3-wildtype metastatic UC after ICB therapy, and decode the potential molecular mechanisms. METHODS: Based on the single-arm, multicenter, phase 2 trial, IMvigor210, we conducted a propensity score matched (PSM) analysis. After a 1:1 ratio PSM method, 39 patients with FGFR3-mutated and 39 FGFR3-wildtype metastatic UC treated with atezolizumab were enrolled. A meta-analysis through systematical database retrieval was conducted for validation. In addition, we performed single-cell RNA sequencing on three FGFR3-mutated and three FGFR3-wildtype UC tumors and analyzed 58,069 single cells. RESULTS: The PSM analysis indicated FGFR3-mutated patients had worse overall survival (OS) in comparison to FGFR3-wildtype patients (HR=2.11, 95% CI=(1.16 to 3.85), p=0.015) receiving atezolizumab. The median OS was 9.2 months (FGFR3-mutated) versus 21.0 months (FGFR3-wildtype). FGFR3-mutated patients had lower disease control rate than FGFR3-wildtype patients (41.0% vs 66.7%, p=0.023). The meta-analysis involving 938 patients with metastatic UC confirmed FGFR3 mutation was associated with worse OS after ICB (HR=1.28, 95% CI=(1.04 to 1.59), p=0.02). Single-cell RNA transcriptome analysis identified FGFR3-mutated UC carried a stronger immunosuppressive microenvironment compared with FGFR3-wildtype UC. FGFR3-mutated UC exhibited less immune infiltration, and lower T-cell cytotoxicity. Higher TREM2+ macrophage abundance in FGFR3-mutated UC can undermine and suppress the T cells, potentially contributing to the formation of an immunosuppressive microenvironment. Lower inflammatory-cancer-associated fibroblasts in FGFR3-mutated UC recruited less chemokines in antitumor immunity but expressed growth factors to promote FGFR3-mutated malignant cell development. FGFR3-mutated UC carried abundance of malignant cells characterized by high hypoxia/metabolism and low interferon response phenotype. CONCLUSIONS: FGFR3 mutation can attenuate prognosis and response to ICB in patients with metastatic UC. FGFR3-mutated UC carries a stronger immunosuppressive microenvironment in comparison with FGFR3-wildtype UC. Inhibition of FGFR3 might activate the immune microenvironment, and the combination of FGFR inhibitor targeted therapy and ICB might be a promising therapeutic regimen in metastatic UC, providing important implications for UC clinical management.

KLF2 regulates stemness of human mesenchymal stem cells by targeting FGFR3.

Mesenchymal stem cells (MSCs) are an attractive source of pluripotent cells for regenerative therapy; however, maintaining stemness and self-renewal of MSCs during expansion ex vivo is challenging. For future clinical applications, it is essential to define the roles and signaling pathways that regulate the fate of MSCs. Based on our earlier finding that Krüppel-like factor 2 (KLF2) participates in maintaining stemness in MSCs, we examined further the role of this factor in intrinsic signaling pathways. Using a chromatin immunoprecipitation (ChIP)-sequence assay, we found that the FGFR3 gene is a KLF2 binding site. Knockdown of FGFR3 significantly decreased the levels of key pluripotency factors, enhanced the expression of differentiation-related genes and down-regulated colony formation of human bone marrow MSCs (hBMSCs). Using alizarin red S and oil red O staining, we found that knockdown of FGFR3 inhibited the osteogenic and adipogenic ability of MSCs under conditions of differentiation. The ChIP-qPCR assay confirmed that KLF2 interacts with the promoter regions of FGFR3. Our findings suggest that KLF2 promotes hBMSC stemness by direct regulation of FGFR. Our findings may contribute to enhanced MSC stemness by genetic modification of stemness-related genes.

Promotion effect of FGF23 on osteopenia in congenital scoliosis through FGFr3/TNAP/OPN pathway.

BACKGROUND: Congenital scoliosis (CS) is a complex spinal malformation of unknown etiology with abnormal bone metabolism. Fibroblast growth factor 23 (FGF23), secreted by osteoblasts and osteocytes, can inhibit bone formation and mineralization. This research aims to investigate the relationship between CS and FGF23. METHODS: We collected peripheral blood from two pairs of identical twins for methylation sequencing of the target region. FGF23 mRNA levels in the peripheral blood of CS patients and age-matched controls were measured. Receiver operator characteristic (ROC) curve analyses were conducted to evaluate the specificity and sensitivity of FGF23. The expression levels of FGF23 and its downstream factors fibroblast growth factor receptor 3 (FGFr3)/tissue non-specific alkaline phosphatase (TNAP)/osteopontin (OPN) in primary osteoblasts from CS patients (CS-Ob) and controls (CT-Ob) were detected. In addition, the osteogenic abilities of FGF23-knockdown or FGF23-overexpressing Ob were examined. RESULTS: DNA methylation of the FGF23 gene in CS patients was decreased compared to that of their identical twins, accompanied by increased mRNA levels. CS patients had increased peripheral blood FGF23 mRNA levels and decreased computed tomography (CT) values compared with controls. The FGF23 mRNA levels were negatively correlated with the CT value of the spine, and ROCs of FGF23 mRNA levels showed high sensitivity and specificity for CS. Additionally, significantly increased levels of FGF23, FGFr3, OPN, impaired osteogenic mineralization and lower TNAP levels were observed in CS-Ob. Moreover, FGF23 overexpression in CT-Ob increased FGFr3 and OPN levels and decreased TNAP levels, while FGF23 knockdown induced downregulation of FGFr3 and OPN but upregulation of TNAP in CS-Ob. Mineralization of CS-Ob was rescued after FGF23 knockdown. CONCLUSIONS: Our results suggested increased peripheral blood FGF23 levels, decreased bone mineral density in CS patients, and a good predictive ability of CS by peripheral blood FGF23 levels. FGF23 may contribute to osteopenia in CS patients through FGFr3/TNAP / OPN pathway.

[Investigation of the mutational status of the FGFR3 gene in urothelial bladder carcinoma]. / Issledovanie mutatsionnogo statusa gena FGFR3 v urotelial'noi kartsinome mochevogo puzyrya.

OBJECTIVE: To study the somatic mutational status of the FGFR3 gene in urothelial bladder cancer (BC) and evaluate its relationship with the clinical and morphological characteristics of the tumor, deficiency of the DNA mismatch repair (dMMR), PD-L1 tumor status, and immunohistochemical (IHC) expression of the p16 protein. MATERIAL AND METHODS: Surgical material of 40 patients with BC, on which the mutational status of the FGFR3 gene was studied using the molecular genetic method, as well as the MMR status, PD-L1 and p16 expression by the IHC method. RESULTS: FGFR3 mutations, such as G370C, S249C, S371C/Y373C, R248C, were detected in 35.0% of the studied BC samples. FGFR3 status did not depend on the gender and age of patients, as well as on the degree of tumor lymphoid infiltration (TILs). Statistically significant differences were found in the analysis of FGFR3 status depending on the histological structure and degree of tumor differentiation, as well as on the pT stage. The FGFR3 status of BC was not associated with the IHC expression of the studied proteins of the MMR system, as well as with the PD-L1 status. Higher levels of PD-L1 expression were demonstrated by BC tumor cells, in which no aberrations in FGFR3 were detected. There was no significant association between p16 status and the presence of FGFR3 mutations, but for FGFR3-positive carcinomas, the basal pattern of p16 staining by IHC was noted. CONCLUSION: A positive somatic mutational status of the FGFR3 gene was statistically significantly more common in the group of papillary low-grade non-muscle-invasive BC, demonstrating basal p16 IHC staining. In the study sample, there was no statistically significant relationship between the FGFR3 status of BC and gender and age differences, TILs, MMR status, PD-L1 status (SP142 and 22C3), and p16 status. The results of the study indicate the need to determine the FGFR3 status in patients with BC for further prescription of personalized therapy.

Role of FGFR3 in bladder cancer: Treatment landscape and future challenges.

Bladder cancer is a heterogeneous malignancy and is responsible for approximately 3.2% of new diagnoses of cancer per year (Sung et al., 2021). Fibroblast Growth Factor Receptors (FGFRs) have recently emerged as a novel therapeutic target in cancer. In particular, FGFR3 genomic alterations are potent oncogenic drivers in bladder cancer and represent predictive biomarkers of response to FGFR inhibitors. Indeed, overall âˆ¼50% of bladder cancers have somatic mutations in the FGFR3 -coding sequence (Cappellen et al., 1999; Turner and Grose, 2010). FGFR3 gene rearrangements are typical alterations in bladder cancer (Nelson et al., 2016; Parker et al., 2014). In this review, we summarize the most relevant evidence on the role of FGFR3 and the state-of-art of anti-FGFR3 treatment in bladder cancer. Furthermore, we interrogated the AACR Project GENIE to investigate clinical and molecular features of FGFR3-altered bladder cancers. We found that FGFR3 rearrangements and missense mutations were associated with a lower fraction of mutated genome, compared to the FGFR3 wild-type tumors, as also observed in other oncogene-addicted cancers. Moreover, we observed that FGFR3 genomic alterations are mutually exclusive with other genomic aberrations of canonical bladder cancer oncogenes, such as TP53 and RB1. Finally, we provide an overview of the treatment landscape of FGFR3-altered bladder cancer, discussing future perspectives for the management of this disease.

Downregulated FGFR3 Expression Inhibits In Vitro Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells of Mice with TBXT Gene Mutation.

We studied the effect of fibroblast growth factor receptor 3 (FGFR3) inhibitor BGJ-398 on the differentiation of bone marrow mesenchymal stem cells (BM MSC) into osteoblasts in wild type (wt) mice and in animals with mutation in TBXT gene (mt) and possible differences in the pluripotency of these cells. Cytology tests showed that the cultured BM MSC could differentiate into osteoblasts and adipocytes. The effect of different BGJ-398 concentrations on the expression of FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 were studied by quantitative reverse transcription PCR. The expression of RUNX2 protein was evaluated by Western blotting. BM MSC of mt and wt mice did not differ in pluripotency and expressed the same membrane marker antigens. BGJ-398 inhibitor reduced the expression of FGFR3 and RUNX2. In BM MSC from mt and wt mice have similar gene expression (and its changing) in FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 genes. Thus, our experiments confirmed the effect of decreased expression of FGFR3 on osteogenic differentiation of BM MSC from wt and mt mice. However, BM MSC from mt and wt mice did not differ in pluripotency and are an adequate model for laboratory research.

PRMT5/FGFR3/AKT Signaling Axis Facilitates Lung Cancer Cell Metastasis.

Objectives: This study aims to investigate the function of the protein arginine methyltransferase 5 (PRMT5) and fibroblast growth factor receptor 3 (FGFR3)/Akt signaling axis in the epithelial-mesenchymal transition (EMT) of human lung cancer. Methods: The mRNA and protein expression levels of PRMT5, FGFR3, p-Akt, and EMT markers are determined by quantitative real-time PCR and Western blotting, respectively; the expression and localization of PRMT5, p-Akt, and proliferating cell nuclear antigen are detected by immunofluorescence; the human lung cancer cell proliferation is measured by MTS assay. Results: PRMT5 and FGFR3 are highly expressed in human lung cancer tissues and are closely related to lymphatic metastasis. Moreover, down-regulation of PRMT5 by lentivirus-mediated shRNAs or inhibition of PRMT5 by specific inhibitors attenuates FGFR3 expression, Akt phosphorylation, and lung cancer cell proliferation. Further studies show that silencing PRMT5 impairs EMT-related markers, including vimentin, collagen I, and ß-catenin. Conversely, ectopic expression of PRMT5 increases FGFR3 expression, Akt phosphorylation, and EMT-related markers, suggesting that PRMT5 regulates metastasis probably through the FGFR3/Akt signaling axis. Conclusion: PRMT5/FGFR3/Akt signaling axis controls human lung cancer progression and metastasis and also implies that PRMT5 may serve as a prognostic biomarker and therapeutic candidate for treating lung cancer.

Oncogenic driver FGFR3-TACC3 requires five coiled-coil heptads for activation and disulfide bond formation for stability.

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.

Juvenile toxicity study of PF-07256472/recifercept, a recombinant human soluble fibroblast growth factor receptor 3, in 2-3-month-old cynomolgus monkeys.

Achondroplasia is an autosomal disorder caused by point mutation in the gene encoding fibroblast growth factor receptor 3 (FGFR3) and resulting in gain of function. Recifercept is a potential disease modifying treatment for achondroplasia and functions as a decoy protein that competes for ligands of the mutated FGFR3. Recifercept is intended to restore normal bone growth by preventing the mutated FGFR3 from negative inhibitory signaling in pediatric patients with achondroplasia. Here we evaluated the potential effects of twice weekly administration of recifercept to juvenile cynomolgus monkeys (approximately 3-months of age at the initiation of dosing) for 6-months. No adverse effects were noted in this study, identifying the high dose as the no-observed-adverse-effect-level and supporting the use of recifercept in pediatric patients from birth. Considering that juvenile toxicity studies in nonhuman primates are not frequently conducted, and when they are conducted they typically utilize animals ≥9 months of age, this study demonstrates the feasibility of executing a juvenile toxicity study in very young monkeys prior to weaning.

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (monomeric GFP [mGFP]-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer growth factor receptor-bound 2 (GRB2) tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared with previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

Discovery of octahydropyrrolo [3,2-b] pyridin derivative as a highly selective Type I inhibitor of FGFR3 over VEGFR2 by high-throughput virtual screening.

Although the aberrant activity of fibroblast growth factor receptor 3 (FGFR3) is implicated in various cancers, the reported kinase inhibitors of FGFR3 tend to cause side effects resulting from the inhibitory activity on vascular endothelial growth factor receptor 2 (VEGFR2). Therefore, it is necessary to find a novel high-selective inhibitor of FGFR3 over VEGFR2 from the small-molecule compound database. In this study, integrated virtual screening protocols were established to screen for selective inhibitors of FGFR3 over VEGFR2 in Drugbank and Asinex databases by combining three-dimensional pharmacophore model, molecular docking, molecular dynamics (MD) simulation, and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations. Finally, it is found that Asinex-5082, as an octahydropyrrolo[3,2-b] pyridin derivative, has larger binding free energy with FGFR3 (-39.3 kcal/mol) than reference drug Erdafitinib (-29.9 kcal/mol), while cannot bind with VEGFR2, resulting in considerable inhibitory selectivity. This is because Asinex-5082, unlike Erdafitinib, has not m-dimethoxybenzene with large steric hindrance, thus can enter the larger ATP-binding pocket of FGFR3 with DFG-in conformation to form hydrophobic interaction with residues Met529, Ile539, and Tyr557 as well as hydrogen bond with Ala558. On the other hand, due to the fact that the benzodioxane and N-heterocyclic rings are connected by carbonyl (C=O), Asinex-5082 cannot rotate freely so as to enter the smaller ATP binding pocket of VEGFR2 on the DFG-out conformation. The lead molecule Asinex-5082 may facilitate the rational design and development of novel selective inhibitors of FGFR3 over VEGFR2 as anticancer drugs.