FGFR redundancy limits the efficacy of FGFR4-selective inhibitors in hepatocellular carcinoma.

Aberrant fibroblast growth factor 19 (FGF19) signaling mediated by its receptor, FGF receptor 4 (FGFR4), and coreceptor, klotho ß (KLB), is a driver of hepatocellular carcinoma (HCC). Several potent FGFR4-selective inhibitors have been developed but have exhibited limited efficacy in HCC clinical trials. Here, by using HCC cell line models from the Cancer Cell Line Encyclopedia (CCLE) and the Liver Cancer Model Repository (LIMORE), we show that selective FGFR4 inactivation was not sufficient to inhibit cancer cell proliferation and tumor growth in FGF19-positive HCC. Moreover, genetic inactivation of KLB in these HCC cells resulted in a fitness defect more severe than that resulting from inactivation of FGFR4. By a combination of biochemical and genetic approaches, we found that KLB associated with FGFR3 and FGFR4 to mediate the prosurvival functions of FGF19. KLB mutants defective in interacting with FGFR3 or FGFR4 could not support the growth or survival of HCC cells. Genome-wide CRISPR loss-of-function screening revealed that FGFR3 restricted the activity of FGFR4-selective inhibitors in inducing cell death; the pan-FGFR inhibitor erdafitinib displayed superior potency than FGFR4-selective inhibitors in suppressing the growth and survival of FGF19-positive HCC cells. Among FGF19-positive HCC cases from The Cancer Genome Atlas (TCGA), FGFR3 is prevalently coexpressed with FGFR4 and KLB, suggesting that FGFR redundancy may be a common mechanism underlying the de novo resistance to FGFR4 inhibitors. Our study provides a rationale for clinical testing of pan-FGFR inhibitors as a treatment strategy for FGF19-positive HCC.

Targeting the FGF19-FGFR4 pathway for cholestatic, metabolic, and cancerous diseases.

Human fibroblast growth factor 19 (FGF19, or FGF15 in rodents) plays a central role in controlling bile acid (BA) synthesis through a negative feedback mechanism. This process involves a postprandial crosstalk between the BA-activated ileal farnesoid X receptor and the hepatic Klotho beta (KLB) coreceptor complexed with fibrobalst growth factor receptor 4 (FGFR4) kinase. Additionally, FGF19 regulates glucose, lipid, and energy metabolism by coordinating responses from functional KLB and FGFR1-3 receptor complexes on the periphery. Pharmacologically, native FGF19 or its analogs decrease elevated BA levels, fat content, and collateral tissue damage. This makes them effective in treating both cholestatic diseases such as primary biliary or sclerosing cholangitis (PBC or PSC) and metabolic abnormalities such as nonalcoholic steatohepatitis (NASH). However, chronic administration of FGF19 drives oncogenesis in mice by activating the FGFR4-dependent mitogenic or hepatic regenerative pathway, which could be a concern in humans. Agents that block FGF19 or FGFR4 signaling have shown great potency in preventing FGF19-responsive hepatocellular carcinoma (HCC) development in animal models. Recent phase 1/2 clinical trials have demonstrated promising results for several FGF19-based agents in selectively treating patients with PBC, PSC, NASH, or HCC. This review aims to provide an update on the clinical development of both analogs and antagonists targeting the FGF19-FGFR4 signaling pathway for patients with cholestatic, metabolic, and cancer diseases. We will also analyze potential safety and mechanistic concerns that should guide future research and advanced trials.

Predictive significance of FGFR4 p.G388R polymorphism in metastatic colorectal cancer patients receiving trifluridine/tipiracil (TAS-102) treatment.

BACKGROUND: TAS-102 (Lonsurf®) is an oral fluoropyrimidine consisting of a combination of trifluridine (a thymidine analog) and tipiracil (a thymidine phosphorylation inhibitor). The drug is effective in metastatic colorectal cancer (mCRC) patients refractory to fluorouracil, irinotecan and oxaliplatin. This study is a real-world analysis, investigating the interplay of genotype/phenotype in relation to TAS-102 sensitivity. METHODS: Forty-seven consecutive mCRC patients were treated with TAS-102 at the National Cancer Institute of Naples from March 2019 to March 2021, at a dosage of 35 mg/m2, twice a day, in cycles of 28 days (from day 1 to 5 and from day 8 to 12). Clinical-pathological parameters were described. Activity was evaluated with RECIST criteria (v1.1) and toxicity with NCI-CTC (v5.0). Survival was depicted through the Kaplan-Meyer curves. Genetic features of patients were evaluated with Next Generation Sequencing (NGS) through the Illumina NovaSeq 6000 platform and TruSigt™Oncology 500 kit. RESULTS: Median age of patients was 65 years (range: 46-77). Forty-one patients had 2 or more metastatic sites and 38 patients underwent to more than 2 previous lines of therapies. ECOG (Eastern Cooperative Oncology Group) Performance Status (PS) was 2 in 19 patients. The median number of TAS-102 cycles was 4 (range: 2-12). The most frequent toxic event was neutropenia (G3/G4 in 16 patients). There were no severe (> 3) non-haematological toxicities or treatment-related deaths. Twenty-six patients experienced progressive disease (PD), 21 stable disease (SD). Three patients with long-lasting disease control (DC: complete, partial responses or stable disease) shared an FGFR4 (p.Gly388Arg) mutation. Patients experiencing DC had more frequently a low tumour growth rate (P = 0.0306) and an FGFR4 p.G388R variant (P < 0.0001). The FGFR4 Arg388 genotype was associated with better survival (median: 6.4 months) compared to the Gly388 genotype (median: 4 months); the HR was 0.25 (95% CI 0.12- 0.51; P = 0.0001 at Log-Rank test). CONCLUSIONS: This phenotype/genotype investigation suggests that the FGFR4 p.G388R variant may serve as a new marker for identifying patients who are responsive to TAS-102. A mechanistic hypothesis is proposed to interpret these findings.

The anti-tumor effects of AZD4547 on ovarian cancer cells: differential responses based on c-Met and FGF19/FGFR4 expression.

BACKGROUND: The FGF/FGFR signaling pathway plays a critical role in human cancers. We analyzed the anti-tumor effect of AZD4547, an inhibitor targeting the FGF/FGFR pathway, in epithelial ovarian cancer (EOC) and strategies on overcoming AZD4547 resistance. METHODS: The effect of AZD4547 on cell viability/migration was evaluated and in vivo experiments in intraperitoneal xenografts using EOC cells and a patient-derived xenograft (PDX) model were performed. The effect of the combination of AZD4547 with SU11274, a c-Met-specific inhibitor, FGF19-specific siRNA, or an FGFR4 inhibitor was evaluated by MTT assay. RESULTS: AZD4547 significantly decreased cell survival and migration in drug-sensitive EOC cells but not drug-resistant cells. AZD4547 significantly decreased tumor weight in xenograft models of drug-sensitive A2780 and SKOV3ip1 cells and in a PDX with drug sensitivity but not in models with drug-resistant A2780-CP20 and SKOV3-TR cells. Furthermore, c-Met expression was high in SKOV3-TR and HeyA8-MDR cells, and co-administration of SU11274 and AZD4547 synergistically induced cell death. In addition, expressions of FGF19 and FGFR4 were high in A2780-CP20 cells. Combining AZD4547 with FGF19 siRNA or with a selective FGFR4 inhibitor led to significantly reduced cell proliferation in A2780-CP20 cells. CONCLUSIONS: This study showed that AZD4547 has significant anti-cancer effects in drug-sensitive cells and PDX models but not in drug-resistant EOC cells. In drug-resistant cells, the expression level of c-Met or FGF19/FGFR4 may be a predictive biomarker for AZD4547 treatment response, and a combination strategy of drugs targeting c-Met or FGF19/FGFR4 together with AZD4547 may be an effective therapeutic strategy for EOC.

Whole-exome sequencing reveals candidate high-risk susceptibility genes for endometriosis.

BACKGROUND: Endometriosis is a common, chronic disease among fertile-aged women. Disease course may be highly invasive, requiring extensive surgery. The etiology of endometriosis remains elusive, though a high level of heritability is well established. Several low-penetrance predisposing loci have been identified, but high-risk susceptibility remains undetermined. Endometriosis is known to increase the risk of epithelial ovarian cancers, especially of endometrioid and clear cell types. Here, we have analyzed a Finnish family where four women have been diagnosed with surgically verified, severely symptomatic endometriosis and two of the patients also with high-grade serous carcinoma. RESULTS: Whole-exome sequencing revealed three rare candidate predisposing variants segregating with endometriosis. The variants were c.1238C>T, p.(Pro413Leu) in FGFR4, c.5065C>T, p.(Arg1689Trp) in NALCN, and c.2086G>A, p.(Val696Met) in NAV2. The only variant predicted deleterious by in silico tools was the one in FGFR4. Further screening of the variants in 92 Finnish endometriosis and in 19 endometriosis-ovarian cancer patients did not reveal additional carriers. Histopathology, positive p53 immunostaining, and genetic analysis supported the high-grade serous subtype of the two tumors in the family. CONCLUSIONS: Here, we provide FGFR4, NALCN, and NAV2 as novel high-risk candidate genes for familial endometriosis. Our results also support the association of endometriosis with high-grade serous carcinoma. Further studies are required to validate the findings and to reveal the exact pathogenesis mechanisms of endometriosis. Elucidating the genetic background of endometriosis defines the etiology of the disease and provides opportunities for expedited diagnostics and personalized treatments.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

Fibroblast growth factor receptor 4 deficiency in macrophages aggravates experimental colitis by promoting M1-polarization.

OBJECTIVE AND DESIGN: Compelling evidence indicates that dysregulated macrophages may play a key role in driving inflammation in inflammatory bowel disease (IBD). Fibroblast growth factor (FGF)-19, which is secreted by ileal enterocytes in response to bile acids, has been found to be significantly lower in IBD patients compared to healthy individuals, and is negatively correlated with the severity of diarrhea. This study aims to explore the potential impact of FGF19 signaling on macrophage polarization and its involvement in the pathogenesis of IBD. METHODS: The dextran sulfate sodium (DSS)-induced mouse colitis model was utilized to replicate the pathology of human IBD. Mice were created with a conditional knockout of FGFR4 (a specific receptor of FGF19) in myeloid cells, as well as mice that overexpressing FGF19 specifically in the liver. The severity of colitis was measured using the disease activity index (DAI) and histopathological staining. Various techniques such as Western Blotting, quantitative PCR, flow cytometry, and ELISA were employed to assess polarization and the expression of inflammatory genes. RESULTS: Myeloid-specific FGFR4 deficiency exacerbated colitis in the DSS mouse model. Deletion or inhibition of FGFR4 in bone marrow-derived macrophages (BMDMs) skewed macrophages towards M1 polarization. Analysis of transcriptome sequencing data revealed that FGFR4 deletion in macrophages significantly increased the activity of the complement pathway, leading to an enhanced inflammatory response triggered by LPS. Mechanistically, FGFR4-knockout in macrophages promoted complement activation and inflammatory response by upregulating the nuclear factor-κB (NF-κB)-pentraxin3 (PTX3) pathway. Additionally, FGF19 suppressed these pathways and reduced inflammatory response by activating FGFR4 in inflammatory macrophages. Liver-specific overexpression of FGF19 also mitigated inflammatory responses induced by DSS in vivo. CONCLUSION: Our study highlights the significance of FGF19-FGFR4 signaling in macrophage polarization and the pathogenesis of IBD, offering a potential new therapeutic target for IBD.

Bromocriptine sensitivity in bromocriptine-induced drug-resistant prolactinomas is restored by inhibiting FGF19/FGFR4/PRL.

PURPOSE: At present, various treatment strategies are available for pituitary adenomas, including medications, surgery and radiation. The guidelines indicate that pharmacological treatments, such as bromocriptine (BRC) and cabergoline (CAB), are important treatments for prolactinomas, but drug resistance is an urgent problem that needs to be addressed. Therefore, exploring the mechanism of drug resistance in prolactinomas is beneficial for clinical treatment. METHODS: In our research, BRC-induced drug-resistant cells were established. Previous RNA sequencing data and an online database were used for preliminary screening of resistance-related genes. Cell survival was determined by Cell Counting Kit-8 (CCK-8) assay, colony formation assays and flow cytometry. Quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting, immunohistochemistry, immunofluorescence and Co-immunoprecipitation (Co-IP) were used to assess the molecular changes and regulation. The therapeutic efficacy of BRC and FGFR4 inhibitor fisogatinib (FISO) combination was evaluated in drug-resistant cells and xenograft tumors in nude mice. RESULTS: Consistent with the preliminary results of RNA sequencing and database screening, fibroblast growth factor 19 (FGF19) expression was elevated in drug-resistant cells and tumor samples. With FGF19 silencing, drug-resistant cells exhibited increased sensitivity to BRC and decreased intracellular phosphorylated fibroblast growth factor receptor 4 (FGFR4) levels. After confirming that FGF19 binds to FGFR4 in prolactinoma cells, we found that FGF19/FGFR4 regulated prolactin (PRL) synthesis through the ERK1/2 and JNK signaling pathways. Regarding the effect of targeting FGF19/FGFR4 on BRC efficacy, FISO and BRC synergistically inhibited the growth of tumor cells, promoted apoptosis and reduced PRL levels. CONCLUSION: Overall, our study revealed FGF19/FGFR4 as a new mechanism involved in the drug resistance of prolactinomas, and combination therapy targeting the pathway could be helpful for the treatment of BRC-induced drug-resistant prolactinomas.

Discovery of novel FGFR4 inhibitors through a build-up fragment strategy.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. FGFR4 has been implicated in HCC progression, making it a promising therapeutic target. We introduce an approach for identifying novel FGFR4 inhibitors by sequentially adding fragments to a common warhead unit. This strategy resulted in the discovery of a potent inhibitor, 4c, with an IC50 of 33 nM and high selectivity among members of the FGFR family. Although further optimisation is required, our approach demonstrated the potential for discovering potent FGFR4 inhibitors for HCC treatment, and provides a useful method for obtaining hit compounds from small fragments.

Special issue "The advance of solid tumor research in China": FGFR4 alterations predict efficacy of immune checkpoint inhibitors in nonsmall cell lung cancer.

Immune checkpoint inhibitors (ICIs), which represent the new standard of care for advanced nonsmall cell lung cancer (NCSLC), are not effective in many patients. Biomarkers are needed to guide treatment. Sequencing data of an ICI-treated cohort were analyzed to identify genomic signatures predicting ICI efficacy, followed by validation using multiple independent cohorts. Their predictive mechanism was explored by evaluating the tumor immune microenvironment and tumor mutational burden (TMB). In the discovery cohort, patients carrying FGFR4 alterations (FGFR4altered ) had a better objective response rate (ORR) (50.0% vs 19.4%; P = .057) and improved median progression-free survival (mPFS) (13.17 vs 3.17 months; HR 0.37; 95% CI 0.14-1; P = .04) than wild-type patients (FGFR4wt ). In the publicly available validation cohorts, FGFR4 alterations correlated with higher ORR (100% vs 31%; P = .028), longer median overall survival (mOS) (not reached [NR] vs 11 months; HR 0.28, 95% CI 0.09-0.89, P = .02), and mPFS (NR vs 6.07 months; HR 0.05, 95% CI 0-3.94, P = .039). FGFR4 alterations were confirmed as an independent predictor of superior PFS (P = .014) and OS (P = .005). FGFR4altered patients also exhibited a significantly improved disease control rate (100% vs 60%, P = .045) and prolonged mPFS (9.70 vs 3.16 months; P = .095) compared to FGFR4wt patients in our Shanghai Pulmonary Hospital cohort. FGFR4 alterations associated with a higher TMB levels, more CD8+ T cells in the tumor stroma, and a higher M1/M2 ratio for tumor-associated macrophages in the tumor center and stroma. Thus, FGFR4 alterations may serve as a potential independent predictor of ICI efficacy in NSCLC.

Induction of Fibroblast Growth Factor Receptor 4 by Helicobacter pylori via Signal Transducer and Activator of Transcription 3 With a Feedforward Activation Loop Involving SRC Signaling in Gastric Cancer

BACKGROUND & AIMS: Helicobacter pylori (H pylori) infection is the main risk factor for gastric cancer. The role of fibroblast growth factor receptors (FGRFs) in H pylori-mediated gastric tumorigenesis remains largely unknown. This study investigated the molecular and mechanistic links between H pylori, inflammation, and FGFR4 in gastric cancer. METHODS: Cell lines, human and mouse gastric tissue samples, and gastric organoids models were implemented. Infection with H pylori was performed using in vitro and in vivo models. Western blot, real-time quantitative reverse-transcription polymerase chain reaction, flow cytometry, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and luciferase reporter assays were used for molecular, mechanistic, and functional studies. RESULTS: Analysis of FGFR family members using The Cancer Genome Atlas data, followed by validation, indicated that FGFR4 messenger (m)RNA was the most significantly overexpressed member in human gastric cancer tissue samples (P < .001). We also detected high levels of Fgfr4 mRNA and protein in gastric dysplasia and adenocarcinoma lesions in mouse models. Infection with J166, 7.13, and PMSS1 cytotoxin-associated gene A (CagA)+ H pylori strains induced FGFR4 mRNA and protein expression in in vitro and in vivo models. This was associated with a concordant activation of signal transducer and activator of transcription 3 (STAT3). Analysis of the FGFR4 promoter suggested several putative binding sites for STAT3. Using chromatin immunoprecipitation assay and an FGFR-promoter luciferase reporter containing putative STAT3 binding sites and their mutants, we confirmed a direct functional binding of STAT3 on the FGFR4 promoter. Mechanistically, we also discovered a feedforward activation loop between FGFR4 and STAT3 where the fibroblast growth factor 19­FGFR4 axis played an essential role in activating STAT3 in a SRC proto-oncogene non-receptor tyrosine kinase dependent manner. Functionally, we found that FGFR4 protected against H pylori-induced DNA damage and cell death. CONCLUSIONS: Our findings demonstrated a link between infection, inflammation, and FGFR4 activation, where a feedforward activation loop between FGFR4 and STAT3 is established via SRC proto-oncogene non-receptor tyrosine kinase in response to H pylori infection. Given the relevance of FGFR4 to the etiology and biology of gastric cancer, we propose FGFR4 as a druggable molecular vulnerability that can be tested in patients with gastric cancer.

Identification of Novel Genetic Risk Factors for Focal Segmental Glomerulosclerosis in Children: Results From the Chronic Kidney Disease in Children (CKiD) Cohort.

RATIONALE & OBJECTIVE: Focal segmental glomerulosclerosis (FSGS) is a major cause of pediatric nephrotic syndrome, and African Americans exhibit an increased risk for developing FSGS compared with other populations. Predisposing genetic factors have previously been described in adults. Here we performed genomic screening of primary FSGS in a pediatric African American population. STUDY DESIGN: Prospective cohort with case-control genetic association study design. SETTING & PARTICIPANTS: 140 African American children with chronic kidney disease from the Chronic Kidney Disease in Children (CKiD) cohort, including 32 cases with FSGS. PREDICTORS: Over 680,000 common single-nucleotide polymorphisms (SNPs) were tested for association. We also ran a pathway enrichment analysis and a human leucocyte antigen (HLA)-focused association study. OUTCOME: Primary biopsy-proven pediatric FSGS. ANALYTICAL APPROACH: Multivariate logistic regression models. RESULTS: The genome-wide association study revealed 169 SNPs from 14 independent loci significantly associated with FSGS (false discovery rate [FDR]<5%). We observed notable signals for genetic variants within the APOL1 (P=8.6×10-7; OR, 25.8 [95% CI, 7.1-94.0]), ALMS1 (P=1.3×10-7; 13.0% in FSGS cases vs 0% in controls), and FGFR4 (P=4.3×10-6; OR, 24.8 [95% CI, 6.3-97.7]) genes, all of which had previously been associated with adult FSGS, kidney function, or chronic kidney disease. We also highlighted novel, functionally relevant genes, including GRB2 (which encodes a slit diaphragm protein promoting podocyte structure through actin polymerization) and ITGB1 (which is linked to renal injuries). Our results suggest a major role for immune responses and antigen presentation in pediatric FSGS through (1) associations with SNPs in PTPRJ (or CD148, P=3.5×10-7), which plays a role in T-cell receptor signaling, (2) HLA-DRB1∗11:01 association (P=6.1×10-3; OR, 4.5 [95% CI, 1.5-13.0]), and (3) signaling pathway enrichment (P=1.3×10-6). LIMITATIONS: Sample size and no independent replication cohort with genomic data readily available. CONCLUSIONS: Our genetic study has identified functionally relevant risk factors and the importance of immune regulation for pediatric primary FSGS, which contributes to a better description of its molecular pathophysiological mechanisms. PLAIN-LANGUAGE SUMMARY: We assessed the genetic risk factors for primary focal segmental glomerulosclerosis (FSGS) by simultaneously testing over 680,000 genetic markers spread across the genome in 140 children, including 32 with FSGS lesions. Fourteen independent genetic regions were significantly associated with pediatric FSGS, including APOL1 and ALMS1-NAT8, which were previously found to be associated with FSGS and chronic kidney diseases in adults. Novel genes with relevant biological functions were also highlighted, such as GRB2 and FGFR4, which play a role in the kidney filtration barrier and in kidney cell differentiation, respectively. Finally, we revealed the importance of immune regulation in pediatric FSGS through associations involving cell surface proteins presenting antigens to the immune system and interacting with T-cell receptors.

A phase Ib/II study of BLU-554, a fibroblast growth factor receptor 4 inhibitor in combination with CS1001, an anti-PD-L1, in patients with locally advanced or metastatic hepatocellular carcinoma.

OBJECTIVE: Targeted therapy combined with immunotherapy has become the main treatment option for hepatocellular carcinoma (HCC). This trial assessed the safety and efficacy of fibroblast growth factor receptor 4 inhibitor (BLU-554) in combination with the anti-PD-L1 monoclonal antibody (CS1001) in patients with locally advanced or metastatic HCC. PATIENTS AND METHODS: This Phase Ib/II trial enrolled patients with locally advanced or metastatic HCC who were FGF19-positive. The patients were intravenously administered with CS1001 (1200 mg) every three weeks and orally administered with BLU-554 (600 mg) daily. The primary endpoint was objective response rate (ORR), as assessed according to RECISTv1.1. RESULTS: Four patients were treated with BLU-554 combined with CS1001. The trial revealed a 50% ORR and 100% DCR. The main adverse reactions that were attributed to BLU-554 in combination with CS1001 were diarrhoea, liver function impairments and skin rashes. Only one patient had immune-related adverse reactions. CONCLUSION: Preliminary data showed that BLU-554 in combination with CS1001 is safe and effective for treatment of patients with locally advanced or metastatic HCC.

Fibroblast growth factor receptor type 4 as a potential therapeutic target in clear cell renal cell carcinoma.

BACKGROUND: Several clear cell renal cell carcinoma (ccRCC) cases harbour fibroblast growth factor receptor 4 (FGFR4) gene copy number (CN) gains. In this study, we investigated the functional contribution of FGFR4 CN amplification in ccRCC. METHODS: The correlation between FGFR4 CN determined via real-time PCR and protein expression evaluated using western blotting and immunohistochemistry was assessed in ccRCC cell lines (A498, A704, and 769-P), a papillary RCC cell line (ACHN), and clinical ccRCC specimens. The effect of FGFR4 inhibition on ccRCC cell proliferation and survival was assessed via either RNA interference or using the selective FGFR4 inhibitor BLU9931, followed by MTS assays, western blotting, and flow cytometry. To investigate whether FGFR4 is a potential therapeutic target, a xenograft mouse model was administered BLU9931. RESULTS: 60% of ccRCC surgical specimens harboured an FGFR4 CN amplification. FGFR4 CN was positively correlated with its protein expression. All ccRCC cell lines harboured FGFR4 CN amplifications, whereas ACHN did not. FGFR4 silencing or inhibition attenuated intracellular signal transduction pathways, resulting in apoptosis and suppressed proliferation in ccRCC cell lines. BLU9931 suppressed tumours at a tolerable dose in the mouse model. CONCLUSION: FGFR4 contributes to ccRCC cell proliferation and survival following FGFR4 amplification, making it a potential therapeutic target for ccRCC.

Investigation of the Proliferative Potential of FGF21 or FGF19 in Liver-Specific FGFR4-Deficient Mice.

Fibroblast growth factor 21 (FGF21) and FGF15/FGF19 belong to the same subgroup of FGFs and are believed to have therapeutic potential in the treatment of type 2 diabetes and associated metabolic dysfunctionalities and pathological conditions. FGF19 has been proposed to induce hyperplasia and liver tumors in FVB mice (named after its susceptibility to Friend leukemia virus B), mediated by the FGF receptor 4 (FGFR4). The goal of this work was to investigate whether FGF21 might also have a potential proliferative effect mediated via FGFR4 using liver-specific Fgfr4 knockout (KO) mice. We conducted a mechanistic 7-day study involving female Fgfr4 fl/fl and Fgfr4 KO mice with a treatment regimen of twice daily or daily subcutaneous injections of FGF21 or FGF19 (positive control), respectively. The Ki-67 liver labeling index (LI) was evaluated by a semi-automated bioimaging analysis. The results showed a statistically significant increase in FGF21- and FGF19-treated Fgfr4 fl/fl mice. Interestingly, in Fgfr4 KO mice, this effect was absent following both treatments of FGF19 and FGF21, indicating that not only the FGFR4 receptor is pivotal for the mediation of hepatocellular proliferation by FGF19 leading finally to liver tumors but it seems also that FGFR4/FGF21 signaling has an impact on the hepatocellular proliferative activity, which does not promote the formation of hepatocellular liver tumors based on the current knowledge.

Characterization of fibroblast growth factor receptor 4 (FGFR4) from the red swamp crayfish Procambarus clarkii and its role in antiviral and antimicrobial immune responses.

FGFRs involved multiple physiological processes, such as endocrine homeostasis, wound repair, and cellular behaviors including proliferation, differentiation and survival. In the present study, the homologs of fibroblast growth factor receptor 4 (FGFR4) were identified and characterized from the red swamp crayfish Procambarus clarkii for the first time. The full-length cDNAs of pcFGFR4 were 2878 bp with 2451 bp open reading frame (ORF), respectively. The deduced pcFGFR4 protein contained an immunoglobulin, two immunoglobulin C-2 Type, a transmembrane region and a catalytic domain. Real-time PCR analysis showed that pcFGFR4 were highly expressed in muscle and hemocyte. Moreover, the expression levels of pcFGFR4 in the hepatopancreas and hemocyte were positively stimulated after challenge with Aeromonas hydrophila and WSSV, implying the involvement of pcFGFR4 against bacterial and viral infections in innate immune responses. While pcFGFR4 were silenced in vivo, the expression levels of antimicrobial peptide (AMP) genes (pcALF1-5,8 and pcCrustin1-2) and NF-κB signaling components (pcDrosal and pcRelish) were significantly reduced. Additionally, NF-κB signaling could be markedly activated by overexpression of pcFGFR4 in HEK293T cells. Finally, our results indicated that pcFGFR4 regulated crayfish's innate immunity by modulating NF-κB signaling. These findings may provide new insights into pcFGFR4-mediated signaling cascades in crustaceans and provide a better understanding of crustacean innate immune system.

Curtailing FGF19's mitogenicity by suppressing its receptor dimerization ability.

As a physiological regulator of bile acid homeostasis, FGF19 is also a potent insulin sensitizer capable of normalizing plasma glucose concentration, improving lipid profile, ameliorating fatty liver disease, and causing weight loss in both diabetic and diet-induced obesity mice. There is therefore a major interest in developing FGF19 as a therapeutic agent for treating type 2 diabetes and cholestatic liver disease. However, the known tumorigenic risk associated with prolonged FGF19 administration is a major hurdle in realizing its clinical potential. Here, we show that nonmitogenic FGF19 variants that retain the full beneficial glucose-lowering and bile acid regulatory activities of WT FGF19 (FGF19WT) can be engineered by diminishing FGF19's ability to induce dimerization of its cognate FGF receptors (FGFR). As proof of principle, we generated three such variants, each with a partial defect in binding affinity to FGFR (FGF19ΔFGFR) and its coreceptors, i.e., ßklotho (FGF19ΔKLB) or heparan sulfate (FGF19ΔHBS). Pharmacological assays in WT and db/db mice confirmed that these variants incur a dramatic loss in mitogenic activity, yet are indistinguishable from FGF19WT in eliciting glycemic control and regulating bile acid synthesis. This approach provides a robust framework for the development of safer and more efficacious FGF19 analogs.

BLU-554, A selective inhibitor of FGFR4, exhibits anti-tumour activity against gastric cancer in vitro.

BACKGROUND: Fibroblast growth factor receptor 4 (FGFR4) plays a key role in cancer progression, including tumour proliferation, invasion, and metastasis. Recent studies have shown that the FGFR4 selective inhibitor BLU-554 has clinical benefits on tumour regression in hepatocellular carcinoma patients. However, the effect of BLU-554 on gastric cancer remains unknown. METHODS: Changes in cell proliferation, apoptosis and cell cycle, migration, and invasion capabilities of MKN-45 cells treated with FGFR4 selective inhibitors were detected by CCK-8 assay, flow cytometry, transwell assay, and wound healing assay, respectively. Western blotting was used to detect the effect of BLU-554 on the expression of FGFR4, FRS2α, and p-ERK1/2. RESULTS: As the concentration of the inhibitor increased, the survival rate of gastric cancer cells decreased, and the trend of BLU-554 was more obvious; a high dose of BLU-554 caused significant cell apoptosis and cell cycle arrest as well as reduced cell invasion ability. The expression levels of FGFR4, FRS2α, and p-ERK1/2 were also significantly reduced when cells were treated with medium and high doses of BLU-554. CONCLUSION: BLU-554 inhibited the mitogen-activated protein kinase (RAS-RAF-MEK-ERK) pathway by inhibiting FGFR4, ultimately impeding the proliferation and invasion of gastric cancer cells and promoting cell apoptosis and cell cycle arrest.

Monomeric/dimeric forms of Fgf15/FGF19 show differential activity in hepatocyte proliferation and metabolic function.

Human Fibroblast Growth Factor 19 (FGF19) and mouse ortholog Fgf15 play similar roles in liver regeneration and metabolism via the activation of Fgfr4/b-klotho (Klb). Monomeric FGF19 and dimeric Fgf15 are both necessary for liver regeneration and proper bile acid (BA) metabolism. FGF19 elicits stronger effects than Fgf15 on glucose and fatty acid metabolism and only FGF19 induces hepatocellular carcinoma (HCC). However, inhibiting FGF19/FGFR4 signaling in HCC patients is associated with toxicity due to elevated BA levels. Here, we examine the structure/function relationship in Fgf15/FGF19 to better understand the molecular basis for their distinct functions. We demonstrate that FGF19 is a more effective activator of Fgfr4 and of downstream signaling (Erk, Plcg1) than Fgf15. Furthermore, we use site-directed mutagenesis to show that the presence or absence of an unpaired cysteine in Fgf15/19 modulates ligand structure and determines the ability of these molecules to induce hepatocyte proliferation, with monomers being more potent activators. Consistent with these findings, an engineered dimeric variant of FGF19 is less effective than wild-type FGF19 at inducing liver growth in cooperation with the Wnt-enhancer RSPO3. In contrast to effects on proliferation, monomeric and dimeric ligands equally inhibited the expression of Cyp7a1, the enzyme catalyzing the rate limiting step in BA production. Thus, structure and function of Fgf15/FGF19 are intricately linked, explaining why FGF19, but not Fgf15, induces liver tumorigenesis. Our data provide insight into FGF19/FGFR4 signaling and may inform strategies to target this pathway while limiting on-target toxicity due to dysregulation of BA production or induction of hepatocyte proliferation.

Design, synthesis, and biological evaluation of quinazoline derivatives with covalent reversible warheads as potential FGFR4 inhibitors.

Fibroblast growth factor receptor 4 (FGFR4) together with co-receptors modulate the activation of downstream proteins that regulate fundamental processes, and elevated FGFR4 activity is associated with Hepatocellular Carcinoma (HCC). Hence, FGFR4 is a promising therapeutic target for HCC. Based on BLU9931, we designed and synthesized a series of phenylquinazoline derivatives as novel inhibitors of FGFR4 through the covalent reversible strategy. Among them, a novel compound (C3) showed FGFR4 and cell proliferation inhibitory activity. Cellular mechanism studies demonstrated that compound C3 induced apoptosis via the FGFR4 signaling pathway blockage. Further mechanism study showed that C3 has the reversible covalent binding capacity, could be used as a reference for the development of novel FGFR4 covalent reversible inhibitors.