CAR T-cells targeting FGFR4 and CD276 simultaneously show potent antitumor effect against childhood rhabdomyosarcoma.

Chimeric antigen receptor (CAR) T-cells targeting Fibroblast Growth Factor Receptor 4 (FGFR4), a highly expressed surface tyrosine receptor in rhabdomyosarcoma (RMS), are already in the clinical phase of development, but tumour heterogeneity and suboptimal activation might hamper their potency. Here we report an optimization strategy of the co-stimulatory and targeting properties of a FGFR4 CAR. We replace the CD8 hinge and transmembrane domain and the 4-1BB co-stimulatory domain with those of CD28. The resulting CARs display enhanced anti-tumor activity in several RMS xenograft models except for an aggressive tumour cell line, RMS559. By searching for a direct target of the RMS core-regulatory transcription factor MYOD1, we identify another surface protein, CD276, as a potential target. Bicistronic CARs (BiCisCAR) targeting both FGFR4 and CD276, containing two distinct co-stimulatory domains, have superior prolonged persistent and invigorated anti-tumor activities compared to the optimized FGFR4-specific CAR and the other BiCisCAR with the same 4-1BB co-stimulatory domain. Our study thus lays down the proof-of-principle for a CAR T-cell therapy targeting both FGFR4 and CD276 in RMS.

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.

Genome-wide identification of Fgfr genes and function analysis of Fgfr4 in myoblasts differentiation of Lateolabrax maculatus.

Fibroblast growth factor receptors (Fgfrs) are involved in cell proliferation, differentiation, and migration via complex signaling pathways in different tissues. Our previous studies showed that fibroblast growth factor receptor 4 (fgfr4) was detected in the most significant quantitative trait loci (QTL) for growth traits. However, studies focusing on the function of fgfr4 on the growth of bony fish are still limited. In this study, we identified seven fgfr genes in spotted sea bass (Lateolabrax maculatus) genome, namely fgfr1a, fgfr1b, fgfr2, fgfr3, fgfr4, fgfr5a, and fgfr5b. Phylogenetic analysis, syntenic analysis and gene structure analysis were conducted to further support the accuracy of our annotation and classification results. Additionally, fgfr4 showed the highest expression levels among fgfrs during the proliferation and differentiation stages of spotted sea bass myoblasts. To further study the function of fgfr4 in myogenesis, dual-fluorescence in situ hybridization (ISH) assay was conducted, and the results showed co-localization of fgfr4 with marker gene of skeletal muscle satellite cells. By treating differentiating myoblasts cultured in vitro with BLU-554, the mRNA expressions of myogenin (myog) and the numbers of myotubes formed by myoblasts increased significantly compared to negative control group. These results indicated that Fgfr4 inhibits the differentiation of myoblasts in spotted sea bass. Our findings contributed to filling a research gap on fgfr4 in bony fish myogenesis and the theoretical understanding of growth trait regulation of spotted sea bass.

Discovery of 2,6-Naphthyridine Analogues as Selective FGFR4 Inhibitors for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is responsible for 90% of cases. Approximately 30% of patients diagnosed with HCC are identified as displaying an aberrant expression of fibroblast growth factor 19 (FGF19)-fibroblast growth factor receptor 4 (FGFR4) as an oncogenic-driver pathway. Therefore, the control of the FGF19-FGFR4 signaling pathway with selective FGFR4 inhibitors can be a promising therapy for the treatment of HCC. We herein disclose the design and synthesis of novel FGFR4 inhibitors containing a 2,6-naphthyridine scaffold. Compound 11 displayed a nanomolar potency against Huh7 cell lines and high selectivity over FGFR1-3 that were comparable to that of fisogatinib (8) as a reference standard. Additionally, compound 11 demonstrated remarkable antitumor efficacy in the Huh7 and Hep3B HCC xenograft mouse model. Moreover, bioluminescence imaging experiments with the orthotopic mouse model support that compound 11 can be considered a promising candidate for treating HCC.

Oncofetal SNRPE promotes HCC tumorigenesis by regulating the FGFR4 expression through alternative splicing.

BACKGROUND: Due to insufficient knowledge about key molecular events, Hepatocellular carcinoma (HCC) lacks effective treatment targets. Spliceosome-related genes were significantly altered in HCC. Oncofetal proteins are ideal tumor therapeutic targets. Screening of differentially expressed Spliceosome-related oncofetal protein in embryonic liver development and HCC helps discover effective therapeutic targets for HCC. METHODS: Differentially expressed spliceosome genes were analysis in fetal liver and HCC through bioinformatics analysis. Small nuclear ribonucleoprotein polypeptide E (SNRPE) expression was detected in fetal liver, adult liver and HCC tissues. The role of SNRPE in HCC was performed multiple assays in vitro and in vivo. SNRPE-regulated alternative splicing was recognized by RNA-Seq and confirmed by multiple assays. RESULTS: We herein identified SNRPE as a crucial oncofetal splicing factor, significantly associated with the adverse prognosis of HCC. SOX2 was identified as the activator for SNRPE reactivation. Efficient knockdown of SNRPE resulted in the complete cessation of HCC tumorigenesis and progression. Mechanistically, SNRPE knockdown reduced FGFR4 mRNA expression by triggering nonsense-mediated RNA decay. A partial inhibition of SNRPE-induced malignant progression of HCC cells was observed upon FGFR4 knockdown. CONCLUSIONS: Our findings highlight SNRPE as a novel oncofetal splicing factor and shed light on the intricate relationship between oncofetal splicing factors, splicing events, and carcinogenesis. Consequently, SNRPE emerges as a potential therapeutic target for HCC treatment. Model of oncofetal SNRPE promotes HCC tumorigenesis by regulating the AS of FGFR4 pre-mRNA.

Development of Highly Potent and Selective Covalent FGFR4 Inhibitors Based on SNAr Electrophiles.

Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent "warheads" based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.

Discovery of N-(4-((6-(3,5- Dimethoxyphenyl)-9H-purine derivatives as irreversible covalent FGFR inhibitors.

Fibroblast growth factor receptor (FGFR) is an attractive target for cancer therapy, but existing FGFR inhibitors appear to hardly meet the demand for clinical application. Herein, a number of irreversible covalent FGFR inhibitors were designed and synthesized by selecting several five- and six-membered azaheterocycles as parent scaffold with different substituents to take over the hydrophobic region in the active pocket of FGFR proteins. Among the resulting target compounds, III-30 showed the most potent effect on enzyme activity inhibition and anti-proliferative activity against the tested cancer cell lines. Significantly, III-30 could inhibit the enzyme activity by achieving irreversible covalent binding with FGFR1 and FGFR4 proteins. It could also regulate FGFR-mediated signaling pathway and mitochondrial apoptotic pathway to promote cancer cell apoptosis and inhibit cancer cell invasion and metastasis. Moreover, III-30 had a good metabolic stability and showed relatively potent anti-tumor activity in the MDA-MB-231 xenograft tumor mice model.

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.

Erythropoietin alleviates lung ischemia-reperfusion injury by activating the FGF23/FGFR4/ERK signaling pathway.

Background: The purpose of the present study was to investigate the effect of erythropoietin (EPO) on lung ischemia-reperfusion injury (LIRI). Methods: Sprague Dawley rats and BEAS-2B cells were employed to construct an ischemia-reperfusion (I/R)-induced model in vivo and in vitro, respectively. Afterward, I/R rats and tert-butyl hydroperoxide (TBHP)-induced cells were treated with different concentrations of EPO. Furthermore, 40 patients with LIRI and healthy controls were enrolled in the study. Results: It was observed that lung tissue damage, cell apoptosis and the expression of BAX and caspase-3 were higher in the LIRI model in vivo and in vitro than in the control group, nevertheless, the Bcl-2, FGF23 and FGFR4 expression level was lower than in the control group. EPO administration significantly reduced lung tissue damage and cell apoptosis while also up-regulating the expression of FGF23 and FGFR4. Rescue experiments indicated that EPO exerted a protective role associated with the FGF23/FGFR4/p-ERK1/2 signal pathway. Notably, the expression of serum EPO, FGF23, FGFR4 and Bcl-2 was decreased in patients with LIRI, while the expression of caspase-3 and BAX was higher. Conclusion: EPO could effectively improve LIRI, which might be related to the activation of the FGF23/FGFR4/p-ERK1/2 signaling pathway.

FGFR4-driven plasticity in breast cancer progression and resistance to therapy.

Breast cancer (BCa) is a complex and heterogeneous disease, with different intrinsic molecular subtypes that have distinct clinical outcomes and responses to therapy. Although intrinsic subtyping provides guidance for treatment decisions, it is now widely recognised that, in some cases, the switch of the BCa intrinsic subtype (which embodies cellular plasticity), may be responsible for therapy failure and disease progression. Aberrant FGFR4 signalling has been implicated in various cancers, including BCa, where it had been shown to be associated with aggressive subtypes, such as HER2-enriched BCa, and poor prognosis. More importantly, FGFR4 is also emerging as a potential driver of BCa intrinsic subtype switching, and an essential promoter of brain metastases, particularly in the HER2-positive BCa. Although the available data are still limited, the findings may have far-reaching clinical implications. Here, we provide an updated summary of the existing both pre- and clinical studies of the role of FGFR4 in BCa, with a special focus on its contribution to subtype switching during metastatic spread and/or induced by therapy. We also discuss a potential clinical benefit of targeting FGFR4 in the development of new treatment strategies.

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.

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.

Hypericin Alleviates Chronic Kidney Disease-induced Left Ventricular Hypertrophy by Regulation of FGF23-FGFR4 Signaling Pathway.

ABSTRACT: Chronic kidney disease (CKD) is a significant global health threat that imposes a substantial burden on both individuals and societies. CKD frequently correlates with cardiovascular events, particularly left ventricular hypertrophy (LVH), which contributes to the high mortality rate associated with CKD. Fibroblast growth factor 23 (FGF23), a hormone primarily involved in regulating calcium and phosphorus metabolism, has been identified as a major risk factor for LVH in CKD patients. Elevated serum FGF23 levels are known to induce LVH and myocardial fibrosis by activating the fibroblast growth factor receptor 4 (FGFR4) signal pathway. Therefore, targeting FGFR4 and its downstream signaling pathways holds potential as a treatment strategy for cardiac dysfunction in CKD. In our current study, we have discovered that Hypericin, a key component derived from Hypericum perforatum , has the ability to alleviate CKD-related LVH by targeting the FGFR4/phospholipase C gamma 1 (PLCγ1) signaling pathway. Through in vitro experiments using rat cardiac myocyte H9c2 cells, we observed that Hypericin effectively inhibits FGF23-induced hypertrophy and fibrosis by suppressing the FGFR4/PLCγ1/calcineurin/nuclear factor of activated T-cell (NFAT3) signaling pathway. In addition, our in vivo studies using mice on a high-phosphate diet and rat models of 5/6 nephrectomy demonstrated that Hypericin has therapeutic effects against CKD-induced LVH by modulating the FGFR4/PLCγ1/calcineurin/NFAT3 signaling pathway. In conclusion, our research highlights the potential of Hypericin as a candidate for the treatment of CKD-induced cardiomyopathy. By suppressing the FGFR4/PLCγ1 signaling pathway, Hypericin shows promise in attenuating LVH and myocardial fibrosis associated with CKD.

FGF19-Based Mini Probe Targeting FGFR4 for Diagnosis and Surgical Navigation of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a frequent malignancy that has a high death rate and a high rate of recurrence following surgery, owing to insufficient surgical resection. Furthermore, HCC is prone to peritoneal metastasis (HCC-PM), resulting in a significant number of tiny cancer lesions, making surgical removal more challenging. As a potential imaging target, FGFR4 is highly expressed in tumors, especially in HCC, but is less expressed in the normal liver. In this study, we used computational simulation approaches to develop peptide I0 derived from FGF19, a particular ligand of FGFR4, and labeled it with the NIRF dye, MPA, for HCC detection. In surgical navigation, the TBR was 9.31 ± 1.36 and 8.57 ± 1.15 in HepG2 in situ tumor and HCC-PM models, respectively, indicating considerable tumor uptake. As a result, peptide I0 is an excellent clinical diagnostic reagent for HCC, as well as a tool for surgically resecting HCC peritoneal metastases.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity.

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.

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.

Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

Helicobacter pylori (H. pylori) is the leading risk factor for gastric carcinogenesis. Fibroblast growth factor receptor 4 (FGFR4) is a member of transmembrane tyrosine kinase receptors that are activated in cancer. We investigated the role of FGFR4 in regulating the cellular response to H. pylori infection in gastric cancer. High levels of oxidative stress signature and FGFR4 expression were detected in gastric cancer samples. Gene set enrichment analysis (GSEA) demonstrated enrichment of NRF2 signature in samples with high FGFR4 levels. H. pylori infection induced reactive oxygen species (ROS) with a cellular response manifested by an increase in FGFR4 with accumulation and nuclear localization NRF2. Knocking down FGFR4 significantly reduced NRF2 protein and transcription activity levels, leading to higher levels of ROS and DNA damage following H. pylori infection. We confirmed the induction of FGFR4 and NRF2 levels using mouse models following infection with a mouse-adapted H. pyloristrain. Pharmacologic inhibition of FGFR4 using H3B-6527, or its knockdown, remarkably reduced the level of NRF2 with a reduction in the size and number of gastric cancer spheroids. Mechanistically, we detected binding between FGFR4 and P62 proteins, competing with NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-dependent degradation with subsequent accumulation and translocation to the nucleus. These findings demonstrate a novel functional role of FGFR4 in cellular homeostasis via regulating the NRF2 levels in response to H. pylori infection in gastric carcinogenesis, calling for testing the therapeutic efficacy of FGFR4 inhibitors in gastric cancer models.

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.