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.

Patient tissue-derived FGFR4-variant and wild-type colorectal cancer organoid development and anticancer drug sensitivity testing.

Objectives: FGFR4-variant and wild-type colorectal cancer (CRC) organoids were developed to investigate the effects of FGFR4-targeted drugs, including FGFR4-IN and erdafitinib, on CRC and their possible molecular mechanism. Methods: Clinical CRC tissues were collected, seven CRC organoids were developed, and whole exome sequencing (WES) was performed. CRC organoids were cultured and organoid drug sensitivity studies were conducted. Finally, an FGFR4-variant (no wild-type) CRC patient-derived orthotopic xenograft mouse model was developed. Western blot measured ERK/AKT/STAT3 pathway-related protein levels. Results: WES results revealed the presence of FGFR4-variants in 5 of the 7 CRC organoids. The structural organization and integrity of organoids were significantly altered under the influence of targeted drugs (FGFR4-IN-1 and erdafitinib). The effects of FGFR4 targeted drugs were not selective for FGFR4 genotypes. FGFR4-IN-1 and erdafitinib significantly reduced the growth, diameter, and Adenosine Triphosphate (ATP) activity of organoids. Furthermore, chemotherapeutic drugs, including 5-fluorouracil and cisplatin, inhibited FGFR4-variant and wild-type CRC organoid activity. Moreover, the tumor volume of mice was significantly reduced at week 6, and p-ERK1/2, p-AKT, and p-STAT3 levels were down-regulated following FGFR4-IN-1 and erdafitinib treatment. Conclusions: FGFR4-targeted and chemotherapeutic drugs inhibited the activity of FGFR4-variant and wild-type CRC organoids, and targeted drugs were more effective than chemotherapeutic drugs at the same concentration. Additionally, FGFR4 inhibitors hindered tumorigenesis in FGFR4-variant CRC organoids through ERK1/2, AKT, and STAT3 pathways. However, no wild-type control was tested in this experiment, which need further confirmation in the next study.

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.

Immobilized cells on microcarriers for efficient and biomimetic screening of active compounds acting on FGFR4 from Fructus evodiae.

Cell membrane coating strategies have been increasingly researched in new drug discovery from complex herb extracts. However, these systems failed to maintain the functionality of the coated cells because cell membranes, not whole cells were used. Original source cells can be used as a vector for active compound screening in a manner that mimics in vivo processes. In this study, we established a novel approach to fabricate high-density fibroblast growth factor receptor 4 (FGFR4)-HEK293 cells on microcarriers covered with collagen through cell culture and covalent immobilization between proteins. This method enables the efficient screening of active compounds from herbs. Two compounds, evodiamine and limonin, were obtained from Fructus evodiae, which were proven to inhibit the FGFR4 target. Enhanced immobilization effects and negligible damage to FGFR4-HEK293 cells treated with paraformaldehyde were successfully confirmed by immunofluorescence assays and transmission electron microscopy. A column was prepared and used to analyze different compounds. The results showed that the method was selective, specific, and reproducible. Overall, the high density of cells immobilized on microcarriers achieved through cell culture and covalent immobilization represents a promising strategy for affinity screening. This approach highlights the potential of the affinity screening method to identify active compounds from an herbal matrix against designed targets and its prospects for use in drug discovery from herbs.

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.

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.

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.

Design, synthesis, and biological evaluation of selective covalent inhibitors of FGFR4.

Aberrant signaling via fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR4) has been identified as a driver of tumorigenesis and the development of many solid tumors, making FGFR4 is a promising target for anticancer therapy. Herein, we designed and synthesized a series of bis-acrylamide covalent FGFR4 inhibitors and evaluated their inhibitory activity against FGFRs, FGFR4 mutants, and their antitumor activity. CXF-007, verified by mass spectrometry and crystal structures to form covalent bonds with Cys552 of FGFR4 and Cys488 of FGFR1, exhibited stronger selectivity and potent inhibitory activity for FGFR4 and FGFR4 cysteine mutants. Moreover, CXF-007 exhibited significant antitumor activity in hepatocellular carcinoma cell lines and breast cancer cell lines through sustained inhibition of the FGFR4 signaling pathway. In summary, our study highlights a novel covalent FGFR4 inhibitor, CXF-007, which has the potential to overcome drug-induced FGFR4 mutations and might provide a new strategy for future anticancer drug discovery.

Association of the rs1966265 and rs351855 FGFR4 Variants with Colorectal Cancer in a Mexican Population and Their Analysis In Silico.

The aim of this study was to associate FGFR4 rs1966265 and rs351855 variants with colorectal cancer (CRC) in a Mexican population and to perform in silico analysis. Genomic DNA from 412 healthy individuals and 475 CRC patients was analyzed. In silico analysis was performed using the PolyPhen-V2, GEPIA, GTEx, and Cytoscape platforms. The GA genotype dominant model (GAAA) of rs1966265 and the AA genotype dominant and recessive models of rs351855 were identified as CRC risk factors (p < 0.05). CRC patients aged ≥ 50 years at diagnosis who consumed alcohol had a higher incidence of the rs351855 GA genotype than the control group (p < 0.05). Associations were observed between the rs1966265 GA genotype and patients with rectal cancer and stage III-IV disease. The rs351855 AA genotype was a risk factor for partial chemotherapy response, and the GA + AA genotype for age ≥ 50 years at diagnosis and rectal cancer was associated with a partial response to chemotherapy (p < 0.05). The AA haplotype was associated with increased susceptibility to CRC. In silico analysis indicated that the rs351855 variant is likely pathogenic (score = 0.998). Genotypic expression analysis in blood samples showed statistically significant differences (p < 0.05). EFNA4, SLC3A2, and HNF1A share signaling pathways with FGFR4. Therefore, rs1966265 and rs351855 may be potential CRC risk factors.

Blocking of FGFR4 signaling by F30 inhibits hepatocellular carcinoma cell proliferation through HMOX1-dependent ferroptosis pathway.

Excessive activation of FGF19/fibroblast growth factor receptor 4 (FGFR4) signaling is associated with poor survival of patients with hepatocellular carcinoma (HCC). FGFR4 inhibitors show promise for HCC treatment. F30, an indazole derivative designed through computer-aided drug design targeting FGFR4, demonstrated anti-HCC activity as described in our previous studies. However, the precise molecular mechanisms underlying F30's anticancer effects remain largely unexplored. We report here that F30 could effectively induce ferroptosis in HCC cells. The concentrations of cellular ferrous iron, the peroxidation of cell membranes and the homeostasis of reduced glutathione (GSH)/oxidized glutathione disulfide (GSSG) were dysregulated by F30, thereby affecting cellular redox status. Induction of ferroptosis in HCC by F30 was inhibited by specific ferroptosis inhibitor ferrostatin-1. F30 upregulates various ferroptosis-related genes, including the heme oxygenase enzymes 1 (HMOX1), a key mediator of redox regulation. Surprisingly, F30-induced ferroptosis in HCC is dependent on HMOX1. The dysregulation of cellular ferrous iron concentrations and cell membrane peroxidation was rescued when knocking down HMOX1 with specific small interfering RNA. These findings shed light on the molecular mechanisms underlying FGFR4-targeting F30's anti-HCC effects and suggest that FGFR4 inactivation could be beneficial for HCC treatment involving ferroptosis.

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.

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.

FGF19/FGFR4 signaling contributes to hepatocellular carcinoma survival and immune escape by regulating IGF2BP1-mediated expression of PD-L1.

Immune-checkpoint blockade (ICB) therapies have been widely used in clinical treatment of cancer patients, but only 20-30% of patients benefit from immunotherapy. Therefore, it is important to decipher the molecular mechanism of resistance to ICB and develop new combined treatment strategies. PD-L1 up-regulation in tumor cells contributes to the occurrence of immune escape. Increasing evidence shows that its transcription level is affected by multiple factors, which limits the objective response rate of ICB. Fibroblast growth factor 19 (FGF19), a member of the fibroblast growth factor family, is widely involved in the malignant progression of many tumors by binding to fibroblast growth factor receptor 4 (FGFR4). In this study, we confirmed that FGF19 acts as a driver gene in hepatocellular carcinoma (HCC) progression by binding to FGFR4. The up-regulation of FGF19 and FGFR4 in HCC is associated with poor prognosis. We found that FGF19/FGFR4 promoted the proliferation and invasion of HCC cells by driving IGF2BP1 to promote PD-L1 expression. Knockdown of FGFR4 significantly reduced the expression of IGF2BP1/PD-L1 and inhibited the proliferation and invasion of HCC cells. These biological effects are achieved by inhibiting the PI3K/AKT pathway. The combination of FGFR4 knockdown and anti-PD-1 antibody greatly suppressed tumor growth and enhanced the sensitivity of immunotherapy, highlighting the clinical significance of FGF19/FGFR4 activation in immunotherapy.

CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo. METHODS: Four different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models. RESULTS: CD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice. CONCLUSIONS: CD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.

The leukemia inhibitory factor regulates fibroblast growth factor receptor 4 transcription in gastric cancer.

PURPOSE: The gastric adenocarcinoma (GC) represents the third cause of cancer-related mortality worldwide, and available therapeutic options remain sub-optimal. The Fibroblast growth factor receptors (FGFRs) are oncogenic transmembrane tyrosine kinase receptors. FGFR inhibitors have been approved for the treatment of various cancers and a STAT3-dependent regulation of FGFR4 has been documented in the H.pylori infected intestinal GC. Therefore, the modulation of FGFR4 might be useful for the treatment of GC. METHODS: To investigate wich factors could modulate FGFR4 signalling in GC, we employed RNA-seq analysis on GC patients biopsies, human patients derived organoids (PDOs) and cancer cell lines. RESULTS: We report that FGFR4 expression/function is regulated by the leukemia inhibitory factor (LIF) an IL-6 related oncogenic cytokine, in JAK1/STAT3 dependent manner. The transcriptomic analysis revealed a direct correlation between the expression of LIFR and FGFR4 in the tissue of an exploratory cohort of 31 GC and confirmed these findings by two external validation cohorts of GC. A LIFR inhibitor (LIR-201) abrogates STAT3 phosphorylation induced by LIF as well as recruitment of pSTAT3 to the promoter of FGFR4. Furthermore, inhibition of FGFR4 by roblitinib or siRNA abrogates STAT3 phosphorylation and oncogentic effects of LIF in GC cells, indicating that FGFR4 is a downstream target of LIF/LIFR complex. Treating cells with LIR-201 abrogates oncogenic potential of FGF19, the physiological ligand of FGFR4. CONCLUSIONS: Together these data unreveal a previously unregnized regulatory mechanism of FGFR4 by LIF/LIFR and demonstrate that LIF and FGF19 converge on the regulation of oncogenic STAT3 in GC cells.

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.

Molecular Mechanisms Involved in MAFLD in Cholecystectomized Patients: A Cohort Study.

Gallstone disease and metabolic dysfunction-associated fatty liver disease (MAFLD) share numerous common risk factors and progression determinants in that they both manifest as organ-specific consequences of metabolic dysfunction. Nevertheless, the precise molecular mechanisms underlying fibrosis development in cholecystectomized MAFLD patients remain inadequately defined. This study aimed to investigate the involvement of farnesoid X receptor 1 (FXR1) and fibroblast growth factor receptor 4 (FGFR4) in the progression of fibrosis in cholecystectomized MAFLD patients. A meticulously characterized cohort of 12 patients diagnosed with MAFLD, who had undergone liver biopsies during programmed cholecystectomies, participated in this study. All enrolled patients underwent a follow-up regimen at 1, 3, and 6 months post-cholecystectomy, during which metabolic biochemical markers were assessed, along with elastography, which served as indirect indicators of fibrosis. Additionally, the hepatic expression levels of FGFR4 and FXR1 were quantified using quantitative polymerase chain reaction (qPCR). Our findings revealed a robust correlation between hepatic FGFR4 expression and various histological features, including the steatosis degree (r = 0.779, p = 0.023), ballooning degeneration (r = 0.764, p = 0.027), interphase inflammation (r = 0.756, p = 0.030), and steatosis activity score (SAS) (r = 0.779, p = 0.023). Conversely, hepatic FXR1 expression did not exhibit any significant correlations with these histological features. In conclusion, our study highlights a substantial correlation between FGFR4 expression and histological liver damage, emphasizing its potential role in lipid and glucose metabolism. These findings suggest that FGFR4 may play a crucial role in the progression of fibrosis in cholecystectomized MAFLD patients. Further research is warranted to elucidate the exact mechanisms through which FGFR4 influences metabolic dysfunction and fibrosis in this patient population.

The RNA methyltransferase METTL16 enhances cholangiocarcinoma growth through PRDM15-mediated FGFR4 expression.

BACKGROUND: RNA N6-Methyladenosine (m6A) modification is implicated in the progression of human cancers including cholangiocarcinoma (CCA). METTL16 is recently identified as a new RNA methyltransferase responsible for m6A modification, although the role of METTL16 in CCA has not yet been examined. The current study aims to investigate the effect and mechanism of the RNA methyltransferase METTL16 in CCA. METHODS: The expression of METTL16 in CCA was examined by analyzing publicly available datasets or by IHC staining on tumor samples. siRNA or CRISPR/Cas9-mediated loss of function studies were performed in vitro and in vivo to investigate the oncogenic role of METTL16 in CCA. MeRIP-Seq was carried out to identify the downstream target of METTL16. ChIP-qPCR, immunoprecipitation, and immunoblots were used to explore the regulation mechanisms for METTL16 expression in CCA. RESULTS: We observed that the expression of METTL16 was noticeably increased in human CCA tissues. Depletion of METTL16 significantly inhibited CCA cell proliferation and decreased tumor progression. PRDM15 was identified as a key target of METTL16 in CCA cells. Mechanistically, our data showed that METTL16 regulated PRDM15 protein expression via YTHDF1-dependent translation. Accordingly, we observed that restoration of PRDM15 expression could rescue the deficiency of CCA cell proliferation/colony formation induced by METTL16 depletion. Our subsequent analyses revealed that METTL16-PRDM15 signaling regulated the expression of FGFR4 in CCA cells. Specifically, we observed that PRDM15 protein was associated with the FGFR4 promoter to regulate its expression. Furthermore, we showed that the histone acetyltransferase p300 cooperated with the transcription factor YY1 to regulate METTL16 gene expression via histone H3 lysine 27 (H3K27) acetylation in CCA cells. CONCLUSIONS: This study describes a novel METTL16-PRDM15-FGFR4 signaling axis which is crucial for CCA growth and may have important therapeutic implications. We showed that depletion of METTL16 significantly inhibited CCA cell proliferation and decreased tumor progression.

KDM6A promotes hepatocellular carcinoma progression and dictates lenvatinib efficacy by upregulating FGFR4 expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the major causes of death from cancer and has a very poor prognosis with few effective therapeutic options. Despite the approval of lenvatinib for the treatment of patients suffering from advanced HCC, only a small number of patients can benefit from this targeted therapy. METHODS: Diethylnitrosamine (DEN)-CCL4 mouse liver tumour and the xenograft tumour models were used to evaluate the function of KDM6A in HCC progression. The xenograft tumour model and HCC cell lines were used to evaluate the role of KDM6A in HCC drug sensitivity to lenvatinib. RNA-seq and ChIP assays were conducted for mechanical investigation. RESULTS: We revealed that KDM6A exhibited a significant upregulation in HCC tissues and was associated with an unfavourable prognosis. We further demonstrated that KDM6A knockdown remarkably suppressed HCC cell proliferation and migration in vitro. Moreover, hepatic Kdm6a loss also inhibited liver tumourigenesis in a mouse liver tumour model. Mechanistically, KDM6A loss downregulated the FGFR4 expression to suppress the PI3K-AKT-mTOR signalling pathway, leading to a glucose and lipid metabolism re-programming in HCC. KDM6A and FGFR4 levels were positively correlated in HCC specimens and mouse liver tumour tissues. Notably, KDM6A knockdown significantly inhibited the efficacy of lenvatinib therapy in HCC cells in vitro and in vivo. CONCLUSIONS: Our findings revealed that KDM6A promoted HCC progression by activating FGFR4 expression and may be an essential molecule for influencing the efficacy of lenvatinib in HCC therapy.

Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma.

Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.