FGFR1 mRNA Research Articles

Hypoxia induced cellular and exosomal RPPH1 promotes breast cancer angiogenesis and metastasis through stabilizing the IGF2BP2/FGFR2 axis.

Metastasis is the major cause of breast cancer mortality, with angiogenesis and tumor-released exosomes playing key roles. However, the communication between breast cancer cells and endothelial cells and its role in tumor metastasis remains unclear. Here, we characterize a long noncoding RNA, RPPH1, which is upregulated in breast cancer tissues and positively associated with poor prognosis. Hypoxia microenvironment upregulates the expression of RPPH1 in breast cancer cells, and promotes its packaging into exosomes through hnRNPA1, leading to the maintenance of stemness and aggressive traits in cancer cells and angiogenesis in endothelial cells. The function of cellular and exosomal RPPH1 was confirmed in the MMTV-PyMT mouse model, in which ASO-RPPH1 therapy effectively inhibited tumor progression and metastasis. Mechanistically, RPPH1 protects IGF2BP2 from ubiquitination-induced degradation, stabilizes N6-methyladenosine (m6A)-modified FGFR2 mRNA, and activates the PI3K/AKT pathway. Our research unveils the role of RPPH1 in breast cancer metastasis and highlights its potential as a therapeutic target.

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

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. 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. 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. 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.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [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 6957.

Comparison of tissue based FGFR mutation detection by Therascreen FGFR with UroTyper FGFR and ADC test and relevance for potential co-targeting with TROP2 and NECTIN4: Preview of Bladder BRIDGister.

679 Background: In view of the efficacy of FGFR targeting in early and advanced bladder cancer, as has been shown for erdafitinib in the THOR and NORSE trial, molecular testing of FGFR mutations and fusions will soon become clinical routine worldwide. Therascreen FGFR testing has been approved as companion diagnostic to select patients for erdafitinib. However, technical problems and shortages as well as a limited number of detectable mutations indicate the need of FGFR test systems that can be performed easily & fast worldwide. The objective was to evaluate concordance of UroTyper FGFR with Therascreen FGFR mutation testing and unravel molecular characteristics of FGFR mutated tumors to assess potential co-targeting options for erdafitinib to justify subsequent adopted clinical trials with the Bladder BRIDGister framework. Methods: FFPE tumor tissues from 100 TURB samples were prospectively collected as part of the Bladder BRIDGister. RNA from FFPE tissues were extracted by commercial kits to assess Therascreen FGFR & UroTyper FGFR MODAPLEX (BIOTYPE GmbH, Dresden) in conjunction with relative gene expression of subtyping markers (i.a.. PPARG), ADC targets (TROP2, NECTIN4) and CPI targets by RT-qPCR systems. Hierarchical clustering, Spearman correlation tests were done by JMP 9.0.0 (SAS software). Results: After exclusion of FGFR fusion samples, a total of 94 samples was available for comparative mutation analysis. Both PCR based FGFR tests concordantly identified S249C (n=15), R248C (n=2) and G370C (n=1) mutations, while the results for Y373C were in part discrepant with Therascreen FGFR detecting 9 mutations, while UroTyper FGFR did detect 6 mutations. Further examination of the 3 discrepant Y373C cases by SNaPshot and Uromonitor FGFR test revealed that the Therascreen FGFR test resulted in 3 false positive results. Moreover, one sample was invalid from Therascreen FGFR test. Overall, the concordance was very good and reached a kappa value of 0.92. Interestingly, FGFR3 mutations by UroTyper FGFR test were positively associated not only with FGFR3 mRNA overexpression (r=0.6699, p<0.0001), but also with TROP2 and NECTIN4 (r=0.5026 and r=0.4406, p<0.0001) indicating, that combinations of FGFR targeting with ADC targeting of TROP2 or NECTIN4 could have strong synergistic effects. Hierarchical clustering revealed a homogenous FGFR3 mutated group of tumors simultaneously co-expressing the target genes TROP2, NECTIN4 and PPARG. Conclusions: PCR-based FGFR assessment by Therascreen and UroTyper is highly concordant and enables fast, local FGFR assessment within few hours. FGFR3 mutations are associated with increased TROP2 & NECTIN4 expression indicating potential synergistic options which warrants further exploration as part of molecularly stratified clinical trials.

FUS-Mediated CircFGFR1 Accelerates the Development of Papillary Thyroid Carcinoma by Stabilizing FGFR1 Protein.

Papillary thyroid carcinoma (PTC) is the most prevalent type of thyroid cancer and its incidence is rising globally. The molecular mechanisms of PTC progression remain unclear, hindering the development of effective treatments. This study focuses on hsa_circ_0008016 (circFGFR1), a circular RNA significantly up-regulated in PTC cells. Silencing circFGFR1 inhibited PTC cell proliferation and increased cell apoptosis, suggesting its role in PTC progression. The RNA-binding protein FUS was identified as a promoter of circFGFR1 formation. While circFGFR1 does not influence FGFR1 mRNA translation, it inhibits ubiquitination and degradation of FGFR1 protein, prolonging its half-life. CircFGFR1 also interacts with protein CBL, inhibiting CBL-mediated ubiquitination of FGFR1 proteins. Rescue assays confirmed circFGFR1 promotes PTC cell growth through mediating FGFR1. This study highlights the potential of circFGFR1 as a therapeutic target, offering insights into PTC's molecular mechanisms, and paving the way for novel treatment strategies.

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B092.

The potential pharmacological mechanism of prunetin against osteoporosis: transcriptome analysis, molecular docking, and experimental approaches

Background Prunetin is an O-methylated isoflavone, known for its beneficial properties. However, its specific pharmacological effects in the treatment of osteoporosis (OP) remain poorly understood. This study aims to elucidate the mechanisms underlying the antiosteoporotic effects of prunetin through a combination of bioinformatics analysis and cell experiments. Methods We gathered predicted targets of prunetin from various online platforms. Differential expression analysis of mRNAs in patients with OP was conducted using the Limma package, based on the GSE35959 dataset. A PPI network diagram was visualized and analyzed using Cytoscape 3.7.2 software. Molecular docking was employed to assess the binding affinity between ligands and receptors, and selected key genes were further validated through cell experiments. Results A total of 4062 differentially expressed genes (DEGs) were identified from the GSE35959 dataset. Among these, 58 genes were found to overlap with the targets of prunetin, indicating their potential as therapeutic targets. The enrichment analysis indicated these targets were mainly enriched in MAPK, FoxO, and mTOR signaling pathways. The molecular docking analysis demonstrated that prunetin exhibited strong binding activity with the core targets. Furthermore, cell experiments revealed that prunetin effectively reversed the expression levels of ALB, ESR1, PTGS2, and FGFR1 mRNA in MC3T3-E1 cells treated with dexamethasone (DEX). Conclusion Our research revealed the multi-pathway and multi-target features of prunetin in treating OP, shedding light on the potential mechanisms underlying the effectiveness of prunetin against OP. These findings serve as a theoretical foundation for future drug development in this field.

MiRNA-470-5p suppresses epithelial-mesenchymal transition of embryonic palatal shelf epithelial cells by targeting Fgfr1 during palatogenesis.

MicroRNAs (miRNAs) have been identified as crucial modulators of gene expression and to play a role in palatogenesis. The aim of this study was to explore the potential role and regulatory mechanisms of miRNAs during palatogenesis. RNA-sequencing was performed to compare the RNA expression profiles of mouse embryonic palatal shelf (MEPS) tissue between an all-trans retinoic acid (ATRA)-induced group and control group, followed by reverse transcription-quantitative polymerase chain reaction for validation, demonstrating upregulated expression of miRNA-470-5p and downregulated expression of Fgfr1 in the ATRA-induced group. The specific binding sites of miRNA-470-5p that potentially govern Fgfr1 expression were predicted by miRanda and TargetScan. The relationship between miRNA-470-5p and Fgfr1 was validated in HEK293T cells by luciferase reporter assays, confirming that miR-470-5p acts directly on the Fgfr1 3'-untranslated region. Fgfr1 mRNA and FGFR1 protein levels were markedly downregulated in MEPS epithelial cells over-expressing miRNA-470-5p. Functional experiments in vitro with CCK-8, cell colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) staining assays revealed that upregulated miRNA-470-5p expression could inhibit the epithelial-mesenchymal transition (EMT) of MEPS epithelial cells by targeting Fgfr1. These findings provide a new molecular mechanism of cleft palate formation, which can inform the development of new treatment and/or prevention targets.

Maternal exercise improves markers of metabolic signaling and reduces inflammation in brown adipose tissue of offspring

Maternal obesity impacts approximately one-third of pregnant people and impacts offspring through the transmission of obesogenic and diabetic traits. Pregnant patients often spend considerable time sedentary, which has been associated with harmful cardiometabolic changes. Our recent data show that compared with sedentary conditions, voluntary exercise during pregnancy in mice shifts adipose distribution of offspring away from energy storing white adipose tissue towards energy burning brown adipose tissue (BAT), which could improve energy metabolism over the lifespan. The objective of this study was to test the hypothesis that voluntary exercise in pregnant mice improves metabolic signaling pathways and reduces markers of inflammation in BAT from offspring. To test this, female C57BL/6J mice were placed on control diet and allocated to sedentary versus voluntary running wheel exercise cages. Voluntary wheel running was allowed for two weeks prior to breeding and throughout weaning. When offspring were 16 weeks of age, they were euthanized for collection of BAT. The study groups were male sedentary (n=9), female sedentary (n=11), male exercise (n=12), female exercise (n=8). Quantitative real-time polymerase chain reaction was used to measure mRNA of fibroblast-growth factor receptors (β-klotho, Fgfr1, Fgfr2) and inflammation markers (IL10, IL1β, TNFα) in BAT. Outcomes were analyzed by two-way ANOVA to assess effects of exercise, sex, and their interaction. Offspring from exercised mothers had a significant decrease in mRNA of IL1β (1.4±0.4 male sedentary vs. 0.4±0.1 male exercise vs. 1.1±0.2 female sedentary vs. 0.8 ±0.2 female exercise; PSex=0.668, PEX=0.006, PInt=0.098) and TNFα (0.9±0.1 male sedentary vs. 0.5±0.1 male exercise vs. 1.3±0.3 female sedentary vs. 0.6 ±0.1 female exercise; PSex=0.126, PEX=0.007, PInt=0.562) and an increase in Fgfr2 (1.1±0.2 male sedentary vs. 1.7±0.3 male exercise vs. 1.3±0.3 female sedentary vs. 3.8 ±1.2 female exercise; PSex=0.063, PEX=0.011, PInt=0.096) in BAT, which was largely independent of sex. There was no significant main effect of either sex or exercise on IL10, Fgfr1, or β-klotho mRNA in BAT. These data suggest that maternal exercise is associated with decreased inflammation and increased protective metabolic signaling in BAT. This could impact energy metabolism given increasing evidence for advantageous metabolic adaptations with increased BAT health and signaling. Additional studies are needed to further understand the impact of maternal exercise on adipose health, energy metabolism, and cardiovascular outcomes in offspring over the lifespan. American Heart Association Undergraduate Summer Fellowship, Department of Obstetrics and Gynecology at Penn State Milton S Hershey Medical Center, and Dani Peress MD Memorial Fund from the Society of Maternal Fetal Medicine This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Liver-derived extracellular vesicles from patients with hepatitis B virus-related acute-on-chronic liver failure impair hepatic regeneration by inhibiting on FGFR2 signaling via miR-218-5p.

Impaired liver regeneration in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients is closely related to prognosis; however, the mechanisms are not yet defined. Liver-derived extracellular vesicles (EVs) may be involved in the dysregulation of liver regeneration. Clarifying the underlying mechanisms will improve the treatments for HBV-ACLF. EVs were isolated by ultracentrifugation from liver tissues of HBV-ACLF patients (ACLF_EVs) after liver transplantation, and their function was investigated in acute liver injury (ALI) mice and AML12 cells. Differentially expressed miRNAs (DE-miRNAs) were screened by deep miRNA sequencing. The lipid nanoparticle (LNP) system was applied as a carrier for the targeted delivery of miRNA inhibitors to improve its effect on liver regeneration. ACLF_EVs inhibited hepatocyte proliferation and liver regeneration, with a critical role of miR-218-5p. Mechanistically, ACLF_EVs fused directly with target hepatocytes and transferred miR-218-5p into hepatocytes, acting by suppressing FGFR2 mRNA and inhibiting the activation of ERK1/2 signaling pathway. Reducing the level of miR-218-5p expression in the liver of ACLF mice partially restored liver regeneration ability. The current data reveal the mechanism underlying impaired liver regeneration in HBV-ACLF that promotes the discovery of new therapeutic approaches.

PSUN311 JTV-519 Alleviates the Impairment of FGF21 Axis Signaling after Spinal Cord Contusion in Mice

Abstract Spinal cord injury (SCI) causes adverse changes in body composition and metabolic profile due to dramatic loss of skeletal muscle mass and an extremely sedentary lifestyle. These abrupt and permanent changes increase the risk for cardiovascular disease, liver disorders, and type 2 diabetes compared to the general population. We have reported that metabolic dysregulation following SCI due to transection is associated with impaired fibroblast growth factor 21 (FGF21) and adiponectin (ADPN) signaling [1], two important regulators of metabolism [2, 3]. JTV519, which stabilizes ryanodine receptors and blocks calcium uptake via SERCA [4], has been reported to have a strong cardioprotective effect via stabilization of ryanodine receptor 2. We examined the effects of SCI caused by contusion (a more common cause of SCI), with or without JTV519 administration, on FGF21 and ADPN signaling in tissues collected from mice at 56 days after SCI. SCI reduced serum and hepatic mRNA levels of FGF21 by 75% and 45%, respectively, compared to sham mice. SCI also significantly diminished FGF21 receptor (FGF-R1) and coreceptor (beta-klotho, KLB) mRNA expression in adipose tissue. In addition, SCI decreased serum ADPN levels, particularly its high molecular weight (HMW) isoform which is the most potent form of the protein. These inhibitory effects were associated with decreased hepatic mRNA expression for PPARalpha (55%), type-2 adiponectin receptor (AdipoR2; 70%), and Glut4 (75%). In addition, SCI reduced mRNA expression of leptin (by 85%) and UCP1 (by 95%) in adipose tissue. In skeletal muscle (gastrocnemius), SCI lowered mRNA expression of PGC1alpha (by 38%), ADPN (by 80%), FGF-R1 (by 35%) and AdipoR1 (by 20%) compared to sham mice. Treating sham-mice with JTV519 upregulated FGF21 and ADPN serum and mRNA levels as well as the expression of the associated metabolic genes of interest (mentioned above). Likewise, SCI-mice treated with JTV519 also demonstrated upregulation of serum levels of FGF21, the HMW isoform of ADPN, and hepatic FGF21 mRNA expression compared to vehicle-treated SCI-mice. Decreased hepatic mRNA expression of PPARalpha, AdipoR2, Glut4 and FGF-R1 and KLB mRNA in adipose tissue were also reversed after JTV519 administration, with levels approximately equivalent to those observed in sham-vehicle treated mice but lower than sham-JTV519 treated mice. Moreover, JTV519 treatment protected against SCI-induced decreases of PGC1alpha, ADPN and FGF-R1 mRNA expression in the gastrocnemius. These data imply that JTV-519 is able to alleviate SCI-induced impairments of FGF21/ADPN signaling. Therefore, JTV519 could be a potential pharmacological agent to treat metabolic dysfunction after SCI.

FGFR mRNA Expression in Cholangiocarcinoma and Its Correlation with FGFR2 Fusion Status and Immune Signatures.

Selective FGFR inhibitors are effective against cholangiocarcinomas that harbor gene alterations in FGFR2. Clinical trials suggest that expression of wild-type FGFR mRNA can predict sensitivity to FGFR inhibitors, but this biomarker has not been well characterized in cholangiocarcinoma. This study explores the prevalence of FGFR mRNA overexpression in cholangiocarcinoma, its role in predicting sensitivity to FGFR inhibitors, and its association with immune markers. Tissue microarrays of intrahepatic (ICC) and extrahepatic cholangiocarcinomas (ECC) resected between 2004 and 2015 were used to evaluate FGFR1-4 mRNA expression levels by RNA in situ hybridization (ISH). Expression levels of FGFR2 mRNA were correlated with FGFR2 fusion status and with patient outcomes. Immune markers expression was assessed by IHC and CSF1 and CSF1 receptor expression were examined by RNA ISH. Among 94 patients with resected cholangiocarcinoma, the majority had ICC (77%). FGFR2 fusions were identified in 23% of ICCs and 5% of ECCs. High levels of FGFR mRNA in FGFR2 fusion-negative ICC/ECC were seen for: FGFR1 (ICC/ECC: 15%/0%), FGFR2 (ICC/ECC: 57%/0%), FGFR3 (ICC/ECC: 53%/18%), and FGFR4 (ICC/ECC: 32%/0%). Overall, 62% of fusion-negative cholangiocarcinomas showed high levels of FGFR mRNA. In patients with advanced FGFR2 fusion-positive ICC, high levels of FGFR2 mRNA did not correlate with clinical benefit. FGFR2 fusion-positive tumors showed a paucity of PD-L1 on tumor cells. FGFR mRNA overexpression occurs frequently in cholangiocarcinoma in the absence of genetic alterations in FGFR. This study identifies a molecular subpopulation in cholangiocarcinoma for which further investigation of FGFR inhibitors is merited outside currently approved indications.

1241P Phase Ib study of futibatinib plus pembrolizumab in patients with advanced or metastatic solid tumors: Tolerability results and antitumor activity in esophageal carcinoma

Combination therapy using FGFR inhibitors and Immune checkpoint inhibitors (ICIs) are being explored as a novel strategy for cancer patients (pts). Here, we report the safety and tolerability of FGFR inhibitor futibatinib plus pembrolizumab in pts with advanced (adv) or metastatic solid tumors and first results for preliminary antitumor activity in pts with esophageal carcinoma (EC). This study consists of Feasibility phase (FP) and Expansion phase (EP). In FP, pts with adv or metastatic solid tumors harboring FGF/FGFR abnormalities received futibatinib 20 mg once daily (QD) plus pembrolizumab 200 mg every 3weeks. In EP, pts with ICIs naive (Cohort A) or ICIs refractory (Cohort B) for adv or metastatic EC harboring FGFR mRNA overexpression who have received at least one prior therapy with a fluorouracil and platinum-based drug received futibatinib plus pembrolizumab at the recommended dose (RD) determined in FP. Primary endpoint was dose-limiting toxicity (DLT) in FP and overall response rate (ORR) in EP; Secondary endpoints included treatment-related adverse events (TRAEs) and disease control rate (DCR). As of November 29, 2021, 11 pts in FP, 9 pts (Cohort A) and 10 pts (Cohort B) in EP were enrolled. No DLTs were observed in FP and the RD of futibatinib in combination with pembrolizumab was determined 20 mg QD. Most common TRAEs in FP (N=11; all grade, grade ≥3) were hyperphosphatemia (91%, 27%), nausea (36%, 0%) and diarrhea (27%, 0%). As results of the interim analysis in EP, confirmed partial responses (PRs) were observed in 4 pts with squamous cell carcinoma (sq) in Cohort A; ORR was 44% with DCR of 78%. In Cohort B, confirmed PRs were observed in 2 pts [1 sq pt; 1 adenocarcinoma (adeno) pt]; ORR was 20% with DCR of 60%. Futibatinib plus pembrolizumab showed well-tolerability and manageable safety profile in pts with adv or metastatic solid tumors. Preliminary antitumor activity was observed not only in ICIs naïve but also in ICIs refractory adv or metastatic EC pts regardless of sq and adeno. Enrollment in EP is ongoing including an additional cohort for ICIs naïve 1st line adv EC and ICIs refractory adv NSCLC pts.

Fibroblast growth factor receptor (FGFR) inhibitor rogaratinib in patients with advanced pretreated squamous-cell non-small cell lung cancer over-expressing FGFR mRNA: The SAKK 19/18 phase II study

BackgroundPatients with advanced squamous-cell lung cancer (SQCLC) frequently (46%) exhibit tumor overexpression of fibroblast growth factor receptor (FGFR) messenger ribonucleic acid (mRNA). Rogaratinib is a novel oral pan-FGFR inhibitor with a good safety profile and anti-tumor activity in early clinical trials as a single agent in FGFR pathway-addicted tumors. SAKK 19/18 determined clinical activity of rogaratinib in patients with advanced SQCLC overexpressing FGFR1-3 mRNA. MethodsPatients with advanced SQCLC failing standard systemic treatment and with FGFR1-3 mRNA tumor overexpression as defined in the protocol received rogaratinib 600 mg BID until disease progression or intolerable toxicity. A 6-months progression-free survival rate (6mPFS) ≤15 % was considered uninteresting (H0), whereas a 6mPFS ≥38 % was considered promising (H1). According to a Simon 2-stage design, 2 out of 10 patients of the first stage were required to be progression-free at 6 months. Comprehensive Genomic Profiling was performedusing the Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific). ResultsBetween July 2019 and November 2020, 49 patients were screened and 20 were classified FGFR-positive. Among a total of 15 patients, 6mPFS was reached in 1 patient (6.7 %), resulting in trial closure for futility after the first stage. There were 7 (46.7 %) patients with stable disease and 5 (33.3 %) patients with progressive disease. Median PFS was 1.6 (95 % CI 0.9–3.5) months and median overall survival (OS) 3.5 (95 % CI 1.0–5.9) months. Most frequent treatment-related adverse events (TRAEs) included hyperphosphatemia in 8 (53 %), diarrhea in 5 (33 %), stomatitis in 3 (20 %) and nail changes in 3 (20 %) patients. Grade ≥3 TRAEs occurred in 6 (40 %) patients. No associations between mutational profile and treatment outcome were observed. ConclusionDespite preliminary signals of activity, rogaratinib failed to improve PFS in patients with advanced SQCLC overexpressing FGFR mRNA. FGFR inhibitors in SQCLC remain a challenging field, and more in-depth understanding of pathway crosstalks may lead to the development of drug combinations with FGFR inhibitors resulting in improved outcomes.

EFFECT OF THYMOQUINONE IN COMBINATION WITH NIVOLUMAB ON EXPERIMENTAL RENAL CELL CANCER MODELS

Objectives: Thymoquinone a bioactive component extracted from the seeds of Nigella sativa, has been proved to have antiinflammatory, antioxidant properties anti antitumor efficiency against breast, prostate, bladder, and renal cell cancer. We aimed to investigate the synergistic cytotoxic and apoptotic effects of thymoquinone in combination with nivolumab, an immuno-control inhibitor, and its mechanism in renal cell cancer models. Materials and Methods: In vitro studies: Caki-1 and Renca renal cancer cell lines with mononuclear cells were cocultured and thymoquinone and nivolumab effect were analyzed by MTT and Annexin V + PI. In vivo studies: Renal cell cancer model was provided by subcutaneous injection of RENCA cells to C57BL/6, male, 8 weeks old mice in four groups. Control group, thymoquinone (1,4,7,14days,20mg/kgIP), nivolumab administered (1,6,13days10mg/ kgIP), thymoquinone and nivolumab. Seven animals was randomized in each experimental group. Apoptosis, necrosis, CD4,CD8,C20,CD34,CD31,VEGF-A by immunohistochemistry, FGFR-1,PDGFRbeta,VEGFR-2,c-kit,RET,c-Met,AXL,Flt-3,TIE2, ICAM-1,VCAM-1 and E-selectin mRNA levels by quantitative real-timePCR were evaluated on tumor tissue. Liver, kidney, brain, lung and heart tissues was evaluated for side effects. Plasma biochemistry tests were performed. Statistical analysis was be performed with Mann-Whitney U test with p <0.05 value. Results: Thymoquinone showed antitumor effect in vitro and in vivo. It also showed increase in immune cell death of nivolumab. Tumor tissue showed prominent necrosis or complete response to therapy in combination group. Conclusions: We conclude that thymoquinone has antitumor effect on renal cell carcinoma and increases the effect of nivolumab immune, cell death effects. Besides, it does not have side effect on other organs whether alone or in combination. 

Prediction of Response to Cisplatin-Based Neoadjuvant Chemotherapy of Muscle-Invasive Bladder Cancer Patients by Molecular Subtyping including KRT and FGFR Target Gene Assessment.

Patients with muscle-invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NAC) have improved prognosis. Molecular subtypes of bladder cancer differ markedly regarding sensitivity to cisplatin-based chemotherapy and harbor FGFR treatment targets to various content. The objective of the present study was to evaluate whether preoperative assessment of molecular subtype as well as FGFR target gene expression is predictive for therapeutic outcome—rate of ypT0 status—to justify subsequent prospective validation within the “BladderBRIDGister”. Formalin-fixed paraffin-embedded (FFPE) tissue specimens from transurethral bladder tumor resections (TUR) prior to neoadjuvant chemotherapy and corresponding radical cystectomy samples after chemotherapy of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, Relative gene expression of subtyping markers (e.g., KRT5, KRT20) and target genes (FGFR1, FGFR3) was analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, Kruskal–Wallis, Mann–Whitney and sensitivity/specificity tests were performed by JMP 9.0.0 (SAS software). The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being node-negative during radical cystectomy after 1 to 4 cycles of NAC. When comparing pretreatment with post-treatment samples, the median expression of KRT20 dropped most significantly from DCT 37.38 to 30.65, which compares with a 128-fold decrease. The reduction in gene expression was modest for other luminal marker genes (GATA3 6.8-fold, ERBB2 6.3-fold). In contrast, FGFR1 mRNA expression increased from 33.28 to 35.88 (~6.8-fold increase). Spearman correlation revealed positive association of pretreatment KRT20 mRNA levels with achieving pCR (r = 0.3072: p = 0.0684), whereas pretreatment FGFR1 mRNA was associated with resistance to chemotherapy (r = −0.6418: p < 0.0001). Hierarchical clustering identified luminal tumors of high KRT20 mRNA expression being associated with high pCR rate (10/16; 63%), while the double-negative subgroup with high FGFR1 expression did not respond with pCR (0/9; 0%). Molecular subtyping distinguishes patients with high probability of response from tumors as resistant to neoadjuvant chemotherapy. Targeting FGFR1 in less-differentiated bladder cancer subgroups may sensitize tumors for adopted treatments or subsequent chemotherapy.

Abstract 6199: Fusion genes: Novel therapeutic targets for gastric signet ring cell carcinoma

Abstract Background: Gastric cancer (GC) is one of the most common lethal malignancies in the world. In recent years, clinicopathological characteristics of GC are changing, marked by increasing incidence of gastric signet ring cell carcinoma (GSRCC) histology. However, at present, no effective therapeutic targets for GSRCC. Fusion genes are considered as ideal therapeutic targets for various cancers with the development of sequencing technique. We attempted to target fusion genes to treat GSRCC. Methods: We used STAR-Fusion, STAR-Seqr, Arriba to get fusion genes in 417 GC samples in The Cancer Genome Atlas (TCGA). Fusion genes identified simultaneously by at least two software are considered “real” fusion genes. Next, we included 556 GC samples of our specimen bank, including 64 GSRCC in this study. All histopathologic diagnoses were reviewed by at least two senior pathologists independently. Clinical information was retrospectively collected and the overall survival (OS) was measured from the date of surgery to the date of death or the last follow-up visit. Formalin fixed paraffin-embedded (FFPE) tissue were collected for next-generation sequencing (NGS) in a 450-gene panel assay. Results: Among 417 GC samples in TCGA, we detected fusion genes in 283 (68%) samples. On average, each GC samples contained 7 fusion genes. And, a single sample contained a maximum of 85 fusion genes. We noticed that one GC sample had FGFR2-TACC2 fusion, which is recurrent and meaningful in intrahepatic cholangiocarcinoma and urothelium carcinoma. On the other hand, among 556 GC samples from our specimen bank, GSRCC patients had lower tumor mutation burden (TMB-L) (P=0.001) than non-SRCC patients. However, we detected gene fusions in 20.3% (13/64) GSRCC patients. GSRCC patients with gene fusions had a higher N stage (P=0.001) and a higher tumor stage (P=0.010) defined by 8th American Joint Committee on Cancer (AICC) criterion. Moreover, GSRCC patients harboring gene fusions (n=9) had poorer overall survival (OS) in comparison with patients without any gene fusion (n=19) (16.0 months vs 21.0 months, P=0.043). In addition, FGFR2 gene fusions (FGFR2-VTI1A and TACC2-FGFR2) were recurrently detected in 3.1% GSRCC tumor samples. We stably expressed TACC2-FGFR2 in gastric cancer cell lines. Using qRT-PCR, we found FGFR2 mRNA levels were increased in TACC2-FGFR2-expressing GC cells and IHC showed TACC2-FGFR2 upregulated FGFR2 protein expression in TACC2-FGFR2-expressing GC cells. Also, TACC2-FGFR2-expressing GC cells were more sensitive to FGFR2 inhibitors. Conclusions: Our observation revealed that fusion genes were recurrent in GSRCC and might associate to shorter OS. FGFR2 fusion genes were recurrent in GSRCC and FGFR2 inhibitors might be a new therapeutic target for GSRCC. Citation Format: Yue Wang, Tao Shi, Xuan Wang, Jinwei Hu, Lixia Yu, Qin Liu, Nandie Wu, Baorui Liu, Jia Wei. Fusion genes: Novel therapeutic targets for gastric signet ring cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6199.

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

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

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

FGFR testing and urine-based risk straticfication from matched tissue and urine samples within the prospective real-world clinicopathological register trial: BRIDGister.

469 Background: The objective of the present study was to assess FGFR mutattions and fusions from matched urine and tissue samples from patients suspicious of bladder cancer and undergoing first TURB within BRIDGister RealWorld Experience trial. Methods: FFPE samples from the first TURB of 39 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by Therascreen FGFR IVD kit (Qiagen GmbH, Hilden). In addition extracellular vesicles were centrally isolated for subsequent RNA extraction (exoRNA.Exosome Diagnostics GmbH, Martinsried) and centrall analysis by QIAcuity digital PCR (Qiagen, Hilden). In addition mRNA based profiling of urine was done by dPCR. Concordance, Spearman, Kruskal-Wallis and MannWhitney were analyzed by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 39 patients (median age: 75, male 69% vs female 31%) of diverse clinical stages (Benign lesions/no tumor 23%, pTa 31%, pT1 26%, pT2 21%) and WHO 1973 grade (G1 8%, G2 39%, G3 34%). Based on FFPE tissue testing using Therascreen FGFR IVD kit and exosomal RNA extraction follwoed by dPCR 12 out of 39 patients exhibited FGFR alterations (31%). Comparison with tissue testing as probable gold standard revealed 67% sensitivity, 85% specificity, 67% PPV and 85%NPV. There were 4 patients being FGFR positive for exoRNA from urine with no mutation found in the corresponding TUR biopsy. Determining ERBB2 mRNA by dPCR from urine revealed that high ERBB2 mRNA correlated with higher WHO1973grade, while high FGFR3 mRNA correlated with lower grade tumors (Spearman r=0.4386 p=0.0075 and r=-0.4663 p=0.0042). Similiarly, trends were seen for association of ERBB2 and FGFR3 mRNA with clinical stage tumors in this pilote cohort (Spearman r=0.2359 p=0.0923 and r=-0.2249 p=0.1089). Conclusions: Extraction of exosomal RNA from urine followed by highly sensitive dPCR mutation testing is feasible with good concordance to matched tissue testing. Urine testing might evolve as alternative approach for FGFR3 screening in a non invasive fashion without the need of transurethral biopsy. Interestingly, mRNA assessment of exosomal RNA from urine before TURB correlated with clinical parameters such as WHO Grade 1973 with ERBB2 mRNA being associated with high grade tumors and FGFR3 mRNA being associated with low grade tumors, which is in line with previous tissue tsting results. This indicates the potential to clinically characterize tumor grade and stage before TUR biopsy or surgery which might be helpful for future risk stratification and planning of surgical intervention. Further exploration is warranted and includes the potential of monitoring patients with regard to urine based mutation detection and risk stratification.