Fibroblast Growth Factor Receptor Research Articles

Role of liver FGF21-KLB signaling in ketogenic diet-induced amelioration of hepatic steatosis

BackgroundThe effectiveness of ketogenic diet (KD) in ameliorating fatty liver has been established, although its mechanism is under investigation. Fibroblast growth factor 21 (FGF21) positively regulates obesity-associated metabolic disorders and is elevated by KD. FGF21 conventionally initiates its intracellular signaling via receptor β-klotho (KLB). However, the mechanistic role of FGF21-KLB signaling for KD-ameliorated fatty liver remains unknown. This study aimed to delineate the critical role of FGF21 signaling in the ameliorative effects of KD on hepatic steatosis.MethodsEight-week-old C57BL/6 J mice were fed a chow diet (CD), a high-fat diet (HFD), or a KD for 16 weeks. Adeno-associated virus-mediated liver-specific KLB knockdown mice and control mice were fed a KD for 16 weeks. Phenotypic assessments were conducted during and after the intervention. We investigated the mechanism underlying KD-alleviated hepatic steatosis using multi-omics and validated the expression of key genes.ResultsKD improved hepatic steatosis by upregulating fatty acid oxidation and downregulating lipogenesis. Transcriptional analysis revealed that KD dramatically activated FGF21 pathway, including KLB and fibroblast growth factor receptor 1 (FGFR1). Impairing liver FGF21 signaling via KLB knockdown diminished the beneficial effects of KD on ameliorating fatty liver, insulin resistance, and regulating lipid metabolism.ConclusionKD demonstrates beneficial effects on diet-induced metabolic disorders, particularly on hepatic steatosis. Liver FGF21-KLB signaling plays a critical role in the KD-induced amelioration of hepatic steatosis.

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.

Phase 1b Trial Evaluating Tolerability and Activity of Targeted Fibroblast Growth Factor Receptor Inhibition in Localized Upper Tract Urothelial Carcinoma.

We initiated a biomarker-informed preoperative study of infigratinib, a fibroblast growth factor receptor (FGFR) inhibitor, in patients with localized upper tract urothelial carcinoma (UTUC), a population with high unmet needs and tumor with a high frequency of FGFR3 alterations. Patients with localized UTUC undergoing ureteroscopy or nephroureterectomy/ureterectomy were enrolled on a phase 1b trial (NCT04228042). Once-daily infigratinib 125 mg by mouth × 21 days (28-day cycle) was given for 2 cycles. Tolerability was monitored by Bayesian design and predefined stopping boundaries. The primary endpoint was tolerability, and the secondary endpoint was objective response based on tumor mapping, done after endoscopic biopsy and post-trial surgery. Total planned enrollment: 20 patients. Targeted sequencing performed using a NovaSeq 6000 solid tumor panel. From May 2021 to November 2022, 14 patients were enrolled, at which point the trial was closed due to termination of all infigratinib oncology trials. Two patients (14.3%) had treatment-terminating toxicities, well below the stopping threshold. Responses occurred in 6 (66.7%) of 9 patients with FGFR3 alterations. Responders had median tumor size reduction of 67%, with 3 of 5 patients initially planned for nephroureterectomy/ureterectomy converted to ureteroscopy. Median follow-up in responders was 24.7 months (14.9-28.9). In this first trial of targeted therapy for localized UTUC, FGFR inhibition was well tolerated and had significant activity in FGFR3 altered tumors. Renal preservation was enabled in a substantial proportion of participants. These data support the design of a biomarker-driven phase 2 trial of FGFR3 inhibition in this population with significant unmet clinical needs.

Low-Dose Infigratinib Increases Bone Growth And Corrects Growth Plate Abnormalities In An Achondroplasia Mouse Model.

Achondroplasia, the most common form of disproportionate short stature, is caused by gain-of-function point mutations in fibroblast growth factor receptor 3 (FGFR3). Abnormally elevated activation of FGFR3 modulates chondrocyte proliferation and differentiation via multiple signaling pathways, such as the MAPK pathway. Using a mouse model mimicking achondroplasia (Fgfr3Y367C/+), we have previously shown that daily treatment with infigratinib (BGJ398), a selective and orally bioavailable FGFR1-3 inhibitor, at a dose of 2 mg/kg, significantly increased bone growth. In this study, we investigated the activity of infigratinib administered at substantially lower doses (0.2 and 0.5 mg/kg, given once daily) and using an intermittent dosing regimen (1 mg/kg every 3 days). Following a 15-day treatment period, these low dosages were sufficient to observe significant improvement of clinical hallmarks of achondroplasia such as growth of the axial and appendicular skeleton and skull development. Immunohistological labeling demonstrated the positive impact of infigratinib on chondrocyte differentiation in the cartilage growth plate and the cartilage end plate of the vertebrae. Macroscopic and microcomputed analyses showed enlargement of the foramen magnum area at the skull base, thus improving foramen magnum stenosis, a well-recognized complication in achondroplasia. No changes in FGF23 or phosphorus levels were observed, indicating that the treatment did not modify phosphate homeostasis. This proof-of-concept study demonstrates that infigratinib administered at low doses has the potential to be a safe and effective therapeutic option for children with achondroplasia.

Abstract LB162: Tinengotinib in patients with advanced solid tumors harboring actionable FGFR1-3 mutations

Abstract Background: Fibroblast growth factor receptor (FGFR) alterations occur across various malignancies and are potent oncogenic drivers in multiple tumor types. FGFR inhibitors have demonstrated efficacy in several cancers with FGFR alterations. Tinengotinib is a novel kinase inhibitor with high potency to a variety of FGFR 1/2/3 kinase domain mutations and has shown promising efficacy in several ongoing clinical studies. Here, we present pooled data of tinengotinib in previously treated solid tumor patients (pts) with actionable FGFR1-3 mutations from four trials (NCT03654547, NCT04742959, NCT04919642, NCT05253053). Methods: Eligible pts received tinengotinib orally once daily (daily dose ranging from 5-12mg) until disease progression. Analyses of best overall response (BOR) rate, disease control rate (DCR), median progression-free survival (mPFS), median duration of response (mDOR) and treatment-related adverse events (TRAEs) were performed. Results: As of 3-Aug-2023, 53 solid tumor pts with actionable FGFR1-3 mutations were enrolled between Dec-2019 to Aug-2023 in the US and China. The median follow-up time was 14.5 (range, 3.3-43.8) months. Median age was 58 years (range, 29-83). Most pts were female (62%), had ECOG of 1 (68%) and were heavily pretreated with 55% having ≥3 lines of prior antitumor therapy. The most frequent FGFR mutation enrolled was in FGFR2 (77%) followed by FGFR1 (19%) and FGFR3 (11%). 25 pts (47%) had received prior therapy with FGFR inhibitor. A total of 45 pts were efficacy-evaluable, including cholangiocarcinoma (CCA, n=25), colon cancer (n=5), breast cancer (n=3), prostate cancer (n=3), head and neck cancer (n=3), urothelial cancer (n=3), pancreatic cancer (n=1), gastric cancer (n=1) and leiomyosarcoma (n=1). mPFS reached 6.83 (95% CI, 4.90-8.34) months. Partial responses were observed in 16/45 (35.6%) pts across 6 distinct tumor types, including CCA, breast, prostate, urothelium, colon and head and neck cancer. mDOR was 6.74 (95% CI, 3.52-9.46) months. For 44 pts with measurable target lesions per RECISTv1.1, the BOR rate and DCR were 36.4% and 86.4%, respectively. For 22 pts with prior FGFR inhibitor, mPFS was 5.98 (95% CI, 3.75-9.10) months and their BOR rate and DCR were 40.9% and 86.4%. TRAEs were reported in 46 (86.8%) pts, with 13 (24.5%) Grade (G) 1-2, 30 (56.6%) G3, 2 (3.8%) G4, and no G5. The most common TRAEs (≥20%) were hypertension, stomatitis, diarrhea, nausea, blood thyroid stimulating hormone increased, fatigue and anemia. Side effects were manageable and reversible with dose hold and/or dose reduction. Conclusions: We observed promising clinical benefit with tinengotinib in heavily pretreated solid tumor pts with FGFR1-3 mutations. The side effects profile was manageable and no new safety signals have been identified. Further research is planned to evaluate the safety and efficacy of tinengotinib in pts with FGFR1-3 mutations across a wider variety of tumor types. Citation Format: Sarina A. Piha-Paul, Chih-Yi "Andy" Liao, Lionel Fonkoua, Amit MahipaI, Sanjay Goel, Christos Fountzilas, Lin Shen, Daneng Li, Dustin Deming, Farshid Dayyani, Weiqing Han, Zuoxing Niu, Jean Fan, Peng Peng, Hui Wang, Peng Huang, Frank Wu, Milind Javle. Tinengotinib in patients with advanced solid tumors harboring actionable FGFR1-3 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB162.

Ligand bias underlies differential signaling of multiple FGFs via FGFR1.

The differential signaling of multiple FGF ligands through a single fibroblast growth factor (FGF) receptor (FGFR) plays an important role in embryonic development. Here, we use quantitative biophysical tools to uncover the mechanism behind differences in FGFR1c signaling in response to FGF4, FGF8, and FGF9, a process which is relevant for limb bud outgrowth. We find that FGF8 preferentially induces FRS2 phosphorylation and extracellular matrix loss, while FGF4 and FGF9 preferentially induce FGFR1c phosphorylation and cell growth arrest. Thus, we demonstrate that FGF8 is a biased FGFR1c ligand, as compared to FGF4 and FGF9. Förster resonance energy transfer experiments reveal a correlation between biased signaling and the conformation of the FGFR1c transmembrane domain dimer. Our findings expand the mechanistic understanding of FGF signaling during development and bring the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight.

Phase II study of pembrolizumab and lenvatinib in advanced well-differentiated neuroendocrine tumors

Immune checkpoint inhibitors (CPIs) have not been shown to be active in well-differentiated neuroendocrine tumors (NETs), with response rates <5%. Lenvatinib is a multitargeted tyrosine kinase inhibitor which binds to vascular endothelial growth factor and fibroblast growth factor receptors and has demonstrated efficacy in pancreatic and gastrointestinal NETs [44% and 16% objective radiographic response rate (ORR), respectively]. The combination of antiangiogenic and CPI therapies can be synergistic. We therefore evaluated the combination of lenvatinib and pembrolizumab in well-differentiated gastrointestinal (GI) and thoracic NETs. A prospective, phase II trial evaluated patients with advanced GI/thoracic NETs (pancreatic NETs were excluded due to high response rate of lenvatinib monotherapy in this patient population), with evidence of progression within 8 months of study entry and at least two prior lines of systemic therapy. Patients received lenvatinib 20 mg daily and pembrolizumab 200 mg intravenously every 3 weeks until unacceptable toxicity or progression of disease. Primary endpoint was objective response rate, and an interim analysis was planned once 20 patients were enrolled. Four ORRs were required to continue enrollment. Twenty patients were enrolled on protocol from April 2021 to January 2022 (nine small intestine, five lung, two thymic, two unknown primary, one cecal, one presacral primaries). Two patients (10%) achieved a partial response (atypical lung and small intestinal primaries). Median progression-free survival (PFS) was 8 months (95% confidence interval 5.8-10.2 months). Twelve (60%) patients experienced probably or definitely associated grade 3 adverse events (10 hypertension). Fourteen patients (70%) required dose reductions or discontinued one of the medications. Two patients discontinued treatment before radiographic assessment. The combination of pembrolizumab and lenvatinib did not show sufficient response in patients with NETs to warrant continued enrollment on trial.

439 Extracellular-in-frame deletions and kinase domain duplications are novel, gain-of-function mutations in fibroblast growth factor receptor genes in cancer

OBJECTIVES/GOALS: There are gain-of-function genomic alterations in FGFR genes that guide personalized treatment in some patients with cholangiocarcinoma (10%) and bladder cancer (30%) who can benefit from targeted therapies. We sought to evaluate other genomic alterations in cancer involving FGFRs and assess whether they are gain-of-function. METHODS/STUDY POPULATION: We collaborated with Foundation Medicine Inc (FMI), for the assessment of 300,000 sequenced tumors and a retrospective analysis of recent publications, to identify novel candidate FGFR alterations. We propose to transiently transfect HEK293T cells with an empty vector (EV), FGFR1-4 wild-type (WT), and these variants and use a luminescent-proximity based high-throughput assay, AlphaLISA, and Western blot to assess FGFR and phosphorylated downstream signaling proteins, FRS2, AKT and ERK, and their sensitivity to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200. RESULTS/ANTICIPATED RESULTS: Through our collaboration we identified &gt;100 novel candidate FGFR1-4 variants of unknown significance (VUS) including extracellular-in-frame deletions (EIDs), kinase domain duplications (KDDs), insertions/deletions (INDELs), short number variants (SNVs), and truncations. Immunoblot analysis confirmed the presence of desired EV, FGFR WT, and VUS’ in HEK293T cells. We anticipate the FGFR EIDs and KDDs to display an increased presence of in the respective pFGFR, pFRS2, pERK, and pAKT as compared to the EV and FGFR WT by both immunoblot and AlpahLISA analysis. Additionally, we anticipate the VUS’ to be sensitive to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200 using the AlphaLISA assay. DISCUSSION/SIGNIFICANCE: These findings suggest that the novel FGFR VUS’ are capable of constitutive activation of FGFR kinase activity, and they preliminary demonstrate that these newly identified FGFR alterations are therapeutically targetable. Thus, providing rationale for further clinical evaluation to identify new cohorts of FGFR inhibitor responders.

The Role of Fibroblast Growth Factor-Receptor Pathway Aberrations in Pediatric Diseases

The fibroblast growth factor receptors play a crucial role in binding to fibroblast growth factor and are involved in various pathological conditions. These receptors consist of an extracellular ligand domain, a transmembrane helix domain, and an intracellular domain with tyrosine kinase activity. There are over 48 different isoforms of FGFR. Each FGF receptor in ligand-binding properties and kinase domains. The FGF/FGFR signaling pathway is implicated in various pediatric cancers and therefore are both non-selective and selective FGFR inhibitors available. Additionally, there are five distinct membrane FGF receptors dentified in vertebrates. All belonging to the tyrosine kinase superfamily. In this manuscript, the focus is based on an analysis of the role of FGF receptor signaling pathways and aberrations especially in pediatric diseases. Erk 1 and Erk 2 seem to have an important role as a mediator of FGF signaling in different biological and developmental processes. FGF signaling plays an important role in conserved developmental functions in skeletal growth, palate closure, ear development, cranial suture ossification and neuronal development in the child. Aberrant FGF signaling causes different congenital disorders and different forms of cancer in childhood. Modulating FGF signaling is of great importance for the treatment of rare diseases in childhood.

Genomic Profiling and Molecular Characterisation of Metastatic Urothelial Carcinoma

The clinical management of metastatic urothelial carcinoma (mUC) is undergoing a major paradigm shift; the integration of immune checkpoint inhibitors (ICIs) and antibody–drug conjugates (ADCs) into the mUC therapeutic strategy has succeeded in improving platinum-based chemotherapy outcomes. Given the expanding therapeutic armamentarium, it is crucial to identify efficacy-predictive biomarkers that can guide an individual patient’s therapeutic strategy. We reviewed the literature data on mUC genomic alterations of clinical interest, discussing their prognostic and predictive role. In particular, we explored the role of the fibroblast growth factor receptor (FGFR) family, epidermal growth factor receptor 2 (HER2), mechanistic target of rapamycin (mTOR) axis, DNA repair genes, and microsatellite instability. Currently, based on the available clinical data, FGFR inhibitors and HER2-directed ADCs are effective therapeutic options for later lines of biomarker-driven mUC. However, emerging genomic data highlight the opportunity for earlier use and/or combination with other drugs of both FGFR inhibitors and HER2-directed ADCs and also reveal additional potential drug targets that could change mUC management.

Paradoxical cancer cell proliferation after FGFR inhibition through decreased p21 signaling in FGFR1-amplified breast cancer cells

Fibroblast growth factors (FGFs) control various cellular functions through fibroblast growth factor receptor (FGFR) activation, including proliferation, differentiation, migration, and survival. FGFR amplification in ER + breast cancer patients correlate with poor prognosis, and FGFR inhibitors are currently being tested in clinical trials. By comparing three-dimensional spheroid growth of ER + breast cancer cells with and without FGFR1 amplification, our research discovered that FGF2 treatment can paradoxically decrease proliferation in cells with FGFR1 amplification or overexpression. In contrast, FGF2 treatment in cells without FGFR1 amplification promotes classical FGFR proliferative signaling through the MAPK cascade. The growth inhibitory effect of FGF2 in FGFR1 amplified cells aligned with an increase in p21, a cell cycle inhibitor that hinders the G1 to S phase transition in the cell cycle. Additionally, FGF2 addition in FGFR1 amplified cells activated JAK-STAT signaling and promoted a stem cell-like state. FGF2-induced paradoxical effects were reversed by inhibiting p21 or the JAK-STAT pathway and with pan-FGFR inhibitors. Analysis of patient ER + breast tumor transcriptomes from the TCGA and METABRIC datasets demonstrated a strong positive association between expression of FGF2 and stemness signatures, which was further enhanced in tumors with high FGFR1 expression. Overall, our findings reveal a divergence in FGFR signaling, transitioning from a proliferative to stemness state driven by activation of JAK-STAT signaling and modulation of p21 levels. Activation of these divergent signaling pathways in FGFR amplified cancer cells and paradoxical growth effects highlight a challenge in the use of FGFR inhibitors in cancer treatment.

Expression of fibroblast growth factor receptor 2 (FGFR2) in combined hepatocellular-cholangiocarcinoma and intrahepatic cholangiocarcinoma: clinicopathological study.

Genetic alterations including fusions in fibroblast growth factor receptor 2 (FGFR2) are detected in 10-20% of intrahepatic cholangiocarcinoma (iCCA), and FGFR2 inhibitors are effective for the treatment of iCCA. We examined a prevalence of FGFR2 genetic alterations and their clinicopathological significance in combined hepatocellular-cholangiocarcinoma (cHCC-CCA). FGFR2 expression, which is a surrogate marker for FGFR2 genetic alterations, was immunohistochemically assessed in the liver sections from 75 patients with cHCC-CCA, 35 with small duct-type iCCA, 30 with large duct-type iCCA, and 35 with hepatocellular carcinoma (HCC). FGFR2 genetic alterations were detected by reverse transcription-PCR and direct sequence. An association of FGFR2 expression with clinicopathological features was investigated in cHCC-CCAs. FGFR2 expression was detected in significantly more patients with cHCC-CCA (21.3%) and small duct-type iCCA (25.7%), compared to those with large duct-type iCCA (3.3%) and HCC (0%) (p < 0.05). FGFR2-positive cHCC-CCAs were significantly smaller size (p < 0.05), with more predominant cholangiolocarcinoma component (p < 0.01) and less nestin expression (p < 0.05). Genetic alterations of ARID1A and BAP1 and multiple genes were significantly more frequent in FGFR2-positive cHCC-CCAs (p < 0.05). 5'/3' imbalance in FGFR2 genes indicating exon18-truncated FGFR2 was significantly more frequently detected in FGFR2-positive cHCC-CCAs and small duct iCCAs, compared to FGFR2-negative ones (p < 0.05). FGFR2::BICC fusion was detected in a case of cHCC-CCAs. FGFR2 genetic alterations may be prevalent in cHCC-CCAs as well as small duct-type iCCAs, which suggest cHCC-CCAs may also be a possible therapeutic target of FGFR2 inhibitors.

Discovery of a novel homoisoflavonoid derivative 5g for anti-osteoclastic bone loss via targeting FGFR1

Several flavonoids have been shown to exert anti-osteoporosis activity. However, the structure-activity relationship and the mechanism of anti-osteoporosis activity of flavonoids remain unknown. In this study, we prepared a series of novel homoisoflavonoid (HIF) derivatives to evaluate their inhibitory effects on osteoclastogenesis using TRAP-activity in vitro assay. Then, the preliminary structure-activity relationship was studied. Among the evaluated novel flavonoids, derivative 5g exerted the most inhibitory bioactivity on primary osteoclast differentiation without interfering with osteogenesis. It was hence selected for further in vitro, in vivo and mechanism of action investigation. Results show that 5g likely directly binds to the fibroblast growth factor receptor 1 (FGFR1), decreasing the activation of ERK1/2 and IκBα/NF-κB signaling pathways, which in turn blocks osteoclastogenesis in vitro and osteoclastic bone loss in vivo. Our study shows that homoisoflavonoid (HIF) derivatives 5g can serve as a potential novel candidate for treating osteoporosis via inhibition of FGFR1.

Population pharmacokinetic-pharmacodynamic modeling of serum biomarkers as predictors of tumor dynamics following lenvatinib treatment in patients with radioiodine-refractory differentiated thyroid cancer (RR-DTC).

Lenvatinib is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor (VEGF) receptors 1-3, fibroblast growth factor (FGF) receptors 1-4, platelet-derived growth factor receptor-α (PDGFRα), KIT, and RET that have been implicated in pathogenic angiogenesis, tumor growth, and cancer. The primary objective of this work was to evaluate, by establishing quantitative relationships, whether lenvatinib exposure and longitudinal serum biomarker data (VEGF, Ang-2, Tie-2, and FGF-23) are predictors for change in longitudinal tumor size which was assessed based on data from 558 patients with radioiodine-refractory differentiated thyroid cancer (RR-DTC) receiving either lenvatinib or placebo treatment. Lenvatinib PK was best described by a 3-compartment model with simultaneous first- and zero-order absorption and linear elimination from the central compartment with significant covariates (body weight, albumin <30 g/dL, ALP>ULN, RR-DTC, RCC, HCC subjects, and concomitant CYP3A inhibitors). Except for body weight, none of the covariates have any clinically meaningful effect on exposure to lenvatinib. Longitudinal biomarker measurements over time were reasonably well defined by a PK/PD model with common EC50, Emax, and a slope for disease progression for all biomarkers. Longitudinal tumor measurements over time were reasonably well defined by a tumor growth inhibition Emax model, which in addition to lenvatinib exposure, included model-predicted relative changes from baseline over time for Tie-2 and Ang-2 as having significant association with tumor response. The developed PK/PD models pave the way for dose optimization and potential prediction of clinical response.

Hypericin alleviates Chronic Kidney Disease induced Left Ventricular Hypertrophy via regulation of FGF23-FGFR4 signaling pathway.

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 (FGF) 23, 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 FGF 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. Additionally, 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 shoaws promise in attenuating LVH and myocardial fibrosis associated with CKD.

Abstract 5099: Prevalence of fibroblast growth factor receptor (FGFR) alterations (alts) and programmed death-ligand 1 (PD-L1) expression (exp) in Chinese solid tumor patients (pts)

Abstract Background: PD-L1 expression drives anti-PD(L)-1 treatment decisions across many cancer indications. FGFR inhibitors have been approved for the treatment of metastatic urothelial cancer (UC) and cholangiocarcinoma with FGFR alts. BALVERSA (erdafitinib), a selective pan-FGFR kinase inhibitor, has shown clinical activity against FGFR altered solid tumors in pts who exhausted standard treatment options. It is important to understand the prevalence of FGFR alts and relationship between FGFR alts and PD-L1 expression levels in different cancer types. Methods: In this real-world study, 9937 Chinese solid tumor pts with the PD-L1 (22C3) immunohistochemistry and OrigiMed 450-gene next-generation sequencing panel results were selected. Chi-square test and Fisher test were used to explore the association between the FGFR alts and PD-L1 expression. Results: FGFR1-4 somatic alts were found in 9.0% of Chinese cancer pts evaluated. FGFR1 amplification (amp) was the most prevalent alts identified (1.9%). There were no significant differences in frequency of FGFR alts between the Chinese cohort and MSK-IMPACT cohort in each cancer type tested. Up to 40.6% of pts evaluated were PD-L1 positive (CPS ≥1 or TPS ≥1%). The PD-L1 positive rate (TPS ≥1%) in SCLC (12.4%, n=121) and NSCLC adenocarcinoma tumors (LUAD) (26.1%, n=2902) was relatively low. In few tumor types, higher expression of PD-L1 in FGFR altered samples was observed compared with FGFR unaltered samples in pan-tumor (mean TPS 14.8% vs.8.6%, P=6.0*E-9; mean CPS 6.3 vs. 6.1, P=3*E-4). Those includes LUAD (mean TPS 14.2% vs. 7.4%, P = 1.7*E-16), colorectal tumor (mean CPS 4.6 vs. 3.9, P = 4.6*E-8), and uterine tumor (mean CPS 8.1 vs. 6.5, P = 9.6*E-5). While PD-L1 expression is lower in FGFR altered samples than FGFR unaltered samples in UC (n=154; mean CPS 3.9 vs. 6.8, P = 0.004). No relationship was observed between FGFR alts and PD-L1 expression in cholangiocarcinoma (n=610). Specific FGFR alts eligible for erdafitinib UC treatment were further investigated in UC and observed with rate 10.4%, including FGFR3 short variants (9.7%) as main alts. Specific FGFR altered tumors showed similar trend with lower PD-L1 expression than FGFR unaltered tumors in UC. The inverse relationship between FGFR alts and PD-L1 expression in UC compared to other solid tumors may explain variable role of FGFRs since the predominant FGFR alt types vary among different cancer types (FGFR3 short variants enriched in UC vs no such predominant FGFR alt types in other cancer types). Conclusion: The large Chinese solid tumor cohort analysis showed comparable FGFR alts prevalence between Chinese and Western population. Differences in relationship between FGFR alts and PD-L1 expression across tumor types reflect the differential role of predominant FGFR types in each tumor type. Citation Format: Min Qing, Fei Yang, Xiaowei Chen, Xuesong Lyu, Aodi Wang, Yanfei Yu, Shibu Thomas, Longen Zhou. Prevalence of fibroblast growth factor receptor (FGFR) alterations (alts) and programmed death-ligand 1 (PD-L1) expression (exp) in Chinese solid tumor patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5099.

Abstract 1035: Identification of fibroblast growth factor receptors (FGFRs) alterations (alts) at DNA and RNA-level by one-step next-generation sequencing (NGS) panel

Abstract Background: FGFR inhibitors are currently in clinical development or approved for urothelial cancer and cholangiocarcinoma with FGFR fusion (FUS) or/and mutation (MUT). AmoyDx FGFR 1-4 NGS Panel (FGFR-P) was developed for FGFR alts detection based on both DNA and RNA. The goal of this study is to validate and assess the performance of FGFR-P in pan-cancer FGFR alts detection by comparing with an AmoyDx comprehensive genomic profiling test (CGP) and a health authority approved DNA-based NGS panel (DNA-P). Methods: Both FGFR-P and CGP are DNA (MUT) and RNA (FUS) based for detecting FGFR alts with optimized bioinformatics pipeline to eliminate baseline noise caused by deamination events, which is commonly found in aged FFPE samples. Cell lines, cell line/patient derived xenografts (CDXs/PDXs) and 397 samples of 26 cancer types (90 samples &amp;gt;10 years) were used for validation of FGFR-P. Total 382 and 50 pan-cancer samples were respectively tested to compare FGFR-P with CGP and DNA-P. Results: To validate accuracy, 36 FFPE samples from cell lines and CDXs/PDXs with known FGFR status (25 FUSs, 5 MUTs, 6 wild-types) were tested by FGFR-P with concordance rate 100%. In clinical sample testing, FGFR-P also showed high agreement with CGP (99.5%) and DNA-P (98.0%). Conflicting result confirmed by FISH revealed 1 FUS missed by DNA-P but detected by FGFR-P, which demonstrated advantage of FGFR alts detection by DNA+RNA. In addition, an optimized bioinformatics pipeline by mimic samples with deamination events effectively filtered out C:G&amp;gt;T:A false positive signals. FGFR-P achieved high success rate in testing pan-tumor samples (89.9%) including samples &amp;gt;10 years (75.6%). Conclusions: FGFR-P provides a novel opportunity to identify FGFR alt pan-cancer patients using a robust DNA+RNA NGS platform, which shows high success rate even in aged samples and will be a potent tool for sensitive and reliable detection of FGFR alts for clinical diagnostics. CGP FGFR-P MUT Positive Negative Total Positive 29 0 29 Negative 1 194 195 Total 30 194 224 FUS Positive Negative Total Positive 20 1 21 Negative 0 137 137 Total 20 138 158 The overall percent agreement 99.5% Citation Format: Min Qing, Xuejun Chen, Wenqing Su, Xiaofang Zhuo, Ting Shen, Chengjuan Xiong, Xuesong Lyu, Renee Tate, Qibiao Wu, Longen Zhou, Shibu Thomas, Wangwang Ning, Jianqing Wang, Huihui Yan, Zhiqiang Yin, Zhan Huang, Yaxin Xue, Changbin Zhu. Identification of fibroblast growth factor receptors (FGFRs) alterations (alts) at DNA and RNA-level by one-step next-generation sequencing (NGS) panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1035.

Abstract 3755: Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b

Abstract Background: Fibroblast growth factor receptor 2b (FGFR2b) is part of the FGFR transmembrane tyrosine kinase receptor family. This protein has gained more attention in recent years as a tumor progression biomarker in numerous solid tumors. The detection of FGFR2b by immunohistochemistry (IHC) has been used recently in immunotherapy clinical trials with multiple tumor types. During IHC assay development on formalin-fixed paraffin embedded tissue, a thorough study of antigen retrieval through cell conditioning or enzyme incubation, primary antibody incubation, and detection parameters is critical to evaluate staining performance. Design: A study of an investigational anti-FGFR2b (FPR2-D) assay developed on a BenchMark ULTRA automated staining platform explored the effect of antigen retrieval and detection parameters on staining performance. VENTANA System Software was used to modify steps in the IHC staining protocol resulting in 42 permutations. Ten different solid tumors were stained with these modifications to the assay and subsequently evaluated for FGFR2b expression. Result:s The results demonstrate that the staining clarity, intensity, and dynamic range within tissue cases of FGFR2b staining can vary widely between different solid tumor types. While most permutations of the initial assay parameters had minimal impact on the staining performance and readability by a pathologist, the order of the enzyme treatment and the cell conditioning steps (in addition to increased incubation time of primary antibody) had a significant impact when comparing across different solid tumors. Detection of FGFR2b seemed to have a greater susceptibility by tumor type to the antigen retrieval process than has traditionally been observed in other biomarkers. Conclusion: The overall results of this investigation show the importance of an extensive parameter screening approach when evaluating new antibody targets and indication combinations in IHC to ensure optimal staining performance. This is critical to downstream testing and the potential use of new assays in the context of in vitro diagnostics. Citation Format: Autumn Sky Watson, Catherine Le, Anne M. Wertheimer, Spencer Escobedo, Lisa So, Bhavani Bagevalu Siddegowda, John D. Palting, Susan Marion, Steven P. Stratton, Birte Aggeler. Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3755.

Abstract 643: Analysis of the effect of FGFR inhibitor and cannabidiol in colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) remains a serious health problem worldwide. In particular, in the case of stage 4 metastatic cancer, a cure can be expected as multidisciplinary treatment is possible by combining surgery, chemotherapy, and the development of targeted anticancer drugs, but there is still no type of treatment available. It is limited. FGFR (Fibroblast Growth Factor Receptor) has emerged as an important anticancer target in various malignant tumors, and small molecule-based targeted treatments have been developed and clinical trials are underway, and some colon cancer patients are known to have FGFR genetic alterations. Cannabidiol (CBD), a non-psychoactive cannabinoid, has recently been reported to be associated with the ability to induce cell death through various signaling systems in colon cancer, and has anticancer effects and mechanisms related to overcoming anticancer drug resistance. has been identified. Therefore, we sought to analyze the effect of FGFR inhibitors on colon cancer, explore the possibility of developing them as new target substances, and find ways to increase anticancer effects by confirming the effects of combination with cannabidiol (CBD) and related mechanisms. Method: Cell survival was confirmed through the WST-1 assay, an assay for measuring cell viability. The signaling pathway was identified through western blot. Data were compared using two-tailed Student’s t-test or one-way analysis of variance and Tukey’s post hoc test. Result: The FGFR expression pattern was confirmed in various cancer cell lines, and NCI-H716 was confirmed to have high FGFR2 expression in the colorectal cancer cell line. (Figure 1) As a result of treating CBD 4uM and AZD 10nM (Cell proliferation, apoptosis, western blot), cell death occurred more clearly when treated together than when treated alone, confirming the effectiveness of the combination. (Figure 2) An increase in apoptosis of NCI-H716 cells was confirmed when CBD and AZD 4547 were combined. (Figure 3) Through a heatmap of differentially expressed genes in RNA sequencing, it was estimated that ER stress may be related to the combination effect. (Figure 4) The relationship between ER stress and the combination of CBD and AZD 4547 on apoptosis of NCI-H716 cells was confirmed. (Figure 5) Conclusion: It was confirmed that the combined effect of FGFR inhibitor and cannabidiol was effective in colorectal cancer cells, and it was assumed to be related to the ER stress pathway. Citation Format: Yeonuk Ju, Wooyoung Kim, Bugyeom Kim, Jiyoung Kim, Junwoo Bong, Chinock Cheong, Sanghee Kang, Byung Wook Min, Sang Cheul Oh, Sun Il Lee. Analysis of the effect of FGFR inhibitor and cannabidiol in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 643.

Abstract 6497: Molecular characterization and drug sensitivity of a gastric FGFR2-TACC2 and a pancreatic KRAS p.G12C primary culture

Abstract Introduction and Objectives: Gastric and pancreatic cancers lack effective therapies, leading to low survival rates particularly in the metastatic setting. Fibroblast growth factor receptor 2 (FGFR2) amplification and fusions with TACC2 appear in 5.5% and 2.1% of gastric tumors, respectively. In pancreatic cancers, the KRAS p.G12C mutation is limited to 1-2% of cases. FDA has recently approved drugs targeting both alterations, pemigatinib and infigratinib for cholangiocarcinoma with FGFR2 fusions; sotorasib and adagrasib for KRAS p.G12C mutant lung cancers. Here, we report the initiation of primary cultures that can be used as preclinical models for testing antitumor agents in gastric and pancreatic tumor cells. Materials and Methods: Pleural effusions were collected from a gastric and a pancreatic cancer patient, both metastatic. Cells were isolated by centrifugation, cultured for 2-3 months in complete medium and genotyped using next generation sequencing (NGS) and RNA nCounter hybridization. Primary cultures were treated with specific drugs depending on their molecular alterations and clinical guidelines. Cell viability was determined by MTT. Results: Tumor cells from the pleural effusion of the gastric cancer patient were grown in suspension. NGS and nCounter revealed a FGFR2-TACC2 fusion, a FGFR2 amplification (160 copies) and a TP53 p.G245S mutation (100% variant allele fraction, VAF). The fusion and the amplification were confirmed by FISH. Cells were sensitive to erdafitinib, with IC50=40 nM in MTT assays. Irinotecan, 5-fluorouracil and cisplatin were also tested; IC50 were below the described plasma Cmax in the first two cases. The patient was treated with first-line fluoropyrimidine and oxaliplatin with partial response and a 5-month progression-free survival. Due to rapid deterioration, 2nd line treatment was not administered. The tumor cells from the pancreatic cancer patient were adherent, NGS revealed a KRAS amplification (9 copies) accompanied by a KRAS p.G12C mutation (81% VAF) and a TP53 p.R175H mutation (99% VAF). MTT assays were performed to determine the cells’ response to oxaliplatin, 5-fluorouracil, irinotecan, gemcitabine, nab-paclitaxel, adagrasib and sotorasib. IC50s below plasma Cmax were found in the last three cases. Remarkably, the IC50 for sotorasib was 100 nM and for adagrasib was 300 nM. The patient was treated with first-line gemcitabine and nab-paclitaxel with partial response but discontinued after three months due to hematological toxicity. Conclusions: Primary cultures can be initiated from malignant pleural effusions and used for genetic characterization and drug sensitivity profiling. Primaries with specific alterations can be employed as preclinical models to test targeted therapies. Citation Format: Silvia Garcia-Roman, Ekaterina Meshoulam Nikolaeva, Juan José García Mosquera, Mónica Garzón Ibáñez, Ruth Roman, Sonia Rodríguez, Cristina Rodríguez, Clara Mayo de las Casas, Rafael Rosell, Miguel A. Molina-Vila, Cristina Aguado Esteban. Molecular characterization and drug sensitivity of a gastric FGFR2-TACC2 and a pancreatic KRAS p.G12C primary culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6497.