FGFR1 Expression Research Articles

METTL3 suppresses anlotinib sensitivity by regulating m6A modification of FGFR3 in oral squamous cell carcinoma

BackgroundN6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA, but there were few studies on its role in cancer drug sensitivity and resistance. Anlotinib has been proved to have effective antitumor effects in oral squamous cell carcinoma (OSCC) in our previous study. Here, we sought to investigate the treatment target of anlotinib and the function and mechanisms of m6A modification in regulating anlotinib effect in OSCC.MethodsAnlotinib treatment in a dose-dependent manner, western blotting, qRT-PCR and cell lost-of-function assays were used to study the treatment target of anlotinib in OSCC. RNA m6A dot blot assays, the m6A MeRIP-seq and MeRIP-qPCR, RNA and protein stability assays were used to explore the m6A modification of the treatment target of anlotinib. Cell lost-of-function assays after METTL3 depletion were conducted to investigate the effect of m6A modification level on the therapeutic effect of anlotinib in OSCC. Patient-derived tumor xenograft (PDX) models and immunohistochemistry staining were performed to study the relationship of METTL3 and antitumor sensitivity of anlotinib in vivo.ResultsAnlotinib targeted FGFR3 in the treatment of OSCC and inhibited tumor cell proliferation and promoted apoptosis by inactivating the FGFR3/AKT/mTOR signaling pathway. METTL3 was identified to target and modify FGFR3 m6A methylation and then decrease the stability of mRNA. METTL3 expression level was related to the anlotinib sensitivity in OSCC cells in vitro and METTL3 knockdown promoted anlotinib sensitivity of OSCC cells by inhibiting the FGFR3 expression. PDX models samples furthermore showed that METTL3 and FGFR3 levels were tightly correlated with the anlotinib efficacy in OSCC.ConclusionsIn summary, our work revealed that FGFR3 was served as the treatment target of anlotinib and METTL3-mediated FGFR3 m6A modification played a critical function in the anlotinib sensitivity in OSCC.

Abstract B023: Prospective clinical genomic profiling of ewing sarcoma: ERF and FGFR1 mutations as recurrent secondary alterations of potential biological and therapeutic relevance

Abstract Background: Ewing Sarcoma (ES) is a primitive sarcoma defined by EWSR1–ETS fusions as the primary driver alteration. To expand our understanding of the genetic and molecular characterization of ES, we conducted a comprehensive analysis of clinical genomic profiling data on tumors from 113 patients using the MSK-IMPACT platform (Integrated Mutation Profiling of Actionable Cancer Targets). Methods: The dataset consisted of ES patients prospectively tested with the FDA-cleared MSK-IMPACT large panel, hybrid capture-based NGS assay. To assess the functional significance of ERF loss, we generated ES cell lines with increased expression of ERF as well as lines with knockdown of ERF. We assessed cell viability, clonogenic growth, and motility and performed transcriptomic and epigenetic analyses. Finally, we validated our findings in vivo using cell line xenografts. Results: Unlike previous ES genomic cohorts, ours included more adult patients (>18 years of age) and more patients with advanced stage at presentation. TP53, STAG2, and CDKN2A were the most common alterations and were associated with worse overall survival at 5-years. Notably, 3% had activating FGFR1 alterations (1 amplification and 2 hotspot activating kinase domain mutations). Mining data generated using a targeted RNAseq assay that includes FGFR1 based on the Archer Anchored Multiplex PCR technology, FGFR1 was highly expressed in the ES cohort (N=42). The 2 patients with activating FGFR1 mutations had relatively high expression of FGFR1. The second novel subset of patients in our cohort were defined by recurrent secondary alterations in ERF, which encodes an ETS domain transcriptional repressor, in 7% of patients (5 truncating mutations, 1 deep deletion, 2 missense mutations). ERF alterations were non-overlapping with STAG2 alterations, suggesting a potentially important biologic role in ES. As the functional significance of FGFR1 mutation in ES has been previously studied, we focused our functional studies on the role of ERF status in ES. In vitro, increased expression of ERF decreased tumor cell growth, colony formation, and motility in two ES cell lines, while ERF loss induced cellular proliferation and clonogenic growth. Transcriptomic analysis of cell lines with ERF loss revealed increased expression of genes and pathways associated with aggressive tumor biology, and epigenetic, chromatin-based studies revealed that ERF competes with EWSR1-FLI1 at ETS binding sites. Conclusion: Our study reveals a previously unexplored role of ERF loss-of-function in ES. Older age in our cohort, and a higher proportion of patients with advanced disease at presentation, could potentially explain the finding of ERF alterations which were associated with aggressive tumor biology in our preclinical studies. Our functional analyses of how ERF modulates EWSR1-FLI1 oncogenicity may open a new window into the pathobiology of ES. Moreover, our data suggest that 3% of ES patients harbor activating FGFR1 mutations, the first targetable kinase alteration in this sarcoma. Citation Format: Arielle Elkrief, Koichi Ogura, Anita S. Bowman, Richard P. Koche, Ryma Benayed, Audrey Mauguen, Marissa S. Mattar, Inna Khodos, Elisa de Stanchina, Paul A. Meyers, John H. Healey, William D. Tap, Neerav Shukla, Meera Hameed, Ahmet Zehir, Charles Sawyers, Rohit Bose, Emily Slotkin, Marc Ladanyi. Prospective clinical genomic profiling of ewing sarcoma: ERF and FGFR1 mutations as recurrent secondary alterations of potential biological and therapeutic relevance [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B023.

FGFR1 Antibody Validation and Characterization of FGFR1 Protein Expression in ER+ Breast Cancer.

Clinical trials in patients with ER+ breast cancer with or without FGFR pathway somatic alterations have shown limited clinical benefit from treatment with FGFR tyrosine kinase inhibitors alone or in combination with endocrine therapy. This is likely because of an inadequate predictive biomarker to select appropriate patients. In this study, we evaluated 4 anti-FGFR1 antibodies in breast cancer cell lines and patient-derived xenografts with FGFR1 amplification. We correlated D8E4 expression in 209 tumors from postmenopausal patients with stage I-III operable ER+ breast cancer with FGFR1 amplification status as determined by fluorescence in situ hybridization. FGFR1 amplification was identified in 10% of tumors (21/209), 80% of which exhibited membranous FGFR1 expression; however, only 50% of amplified cases showed strong, complete membranous staining (3+) based on established criteria to score HER2 by immunohistochemistry. These findings suggest the combined evaluation of FGFR1 status by immunohistochemistry and fluorescence in situ hybridization may need to be incorporated into the selection of patients for trials with FGFR inhibitors.

FGFR1-4 RNA-Based Gene Alteration and Expression Analysis in Squamous Non-Small Cell Lung Cancer.

While fibroblast growth factor receptors (FGFRs) are involved in several biological pathways and FGFR inhibitors may be useful in the treatment of squamous non-small cell lung cancer (Sq-NSCLC), FGFR aberrations are not well characterized in Sq-NSCLC. We comprehensively evaluated FGFR expression, fusions, and variants in 40 fresh-frozen primary Sq-NSCLC (stage IA3–IV) samples and tumor-adjacent normal tissues using real-time PCR and next-generation sequencing (NGS). Protein expression of FGFR1–3 and amplification of FGFR1 were also analyzed. FGFR1 and FGFR4 median gene expression was significantly (p < 0.001) decreased in tumors compared with normal tissue. Increased FGFR3 expression enhanced the recurrence risk (hazard ratio 4.72, p = 0.029), while high FGFR4 expression was associated with lymph node metastasis (p = 0.036). Enhanced FGFR1 gene expression was correlated with FGFR1 protein overexpression (r = 0.75, p = 0.0003), but not with FGFR1 amplification. NGS revealed known pathogenic FGFR2,3 variants, an FGFR3::TACC3 fusion, and a novel TACC1::FGFR1 fusion together with FGFR1,2 variants of uncertain significance not previously reported in Sq-NSCLC. These findings expand our knowledge of the Sq-NSCLC molecular background and show that combining different methods increases the rate of FGFR aberrations detection, which may improve patient selection for FGFRi treatment.

Comprehensive analysis of the prognostic value and immune infiltration of FGFR family members in gastric cancer

BackgroundFibroblast growth factor receptors (FGFRs) modulate numerous cellular processes in tumor cells and tumor microenvironment. However, the effect of FGFRs on tumor prognosis and tumor-infiltrating lymphocytes in gastric cancer (GC) remains controversial.MethodsThe expression of four different types of FGFRs was analyzed via GEPIA, TCGA-STAD, and GTEX databases and our 27 pairs of GC tumor samples and the adjacent normal tissue. Furthermore, the Kaplan–Meier plot and the TCGA database were utilized to assess the association of FGFRs with clinical prognosis. The R software was used to evaluate FGFRs co-expression genes with GO/KEGG Pathway Enrichment Analysis. In vitro and in vivo functional analyses and immunoblotting were performed to verify FGFR4 overexpression consequence. Moreover, the correlation between FGFRs and cancer immune infiltrates was analyzed by TIMER and TCGA databases. And the efficacy of anti-PD-1 mAb treatment was examined in NOG mouse models with overexpressed FGFR1 or FGFR4.ResultsThe expression of FGFRs was considerably elevated in STAD than in the normal gastric tissues and was significantly correlated with poor OS and PFS. ROC curve showed the accuracy of the FGFRs in tumor diagnosis, among which FGFR4 had the highest ROC value. Besides, univariate and multivariate analysis revealed that FGFR4 was an independent prognostic factor for GC patients. According to a GO/KEGG analysis, the FGFRs were implicated in the ERK/MAPK, PI3K-AKT and extracellular matrix (ECM) receptor signaling pathways. In vivo and in vitro studies revealed that overexpression of FGFR4 stimulated GC cell proliferation, invasion, and migration. In addition, FGFR1 expression was positively correlated with infiltrating levels of CD8+ T-cells, CD4+ T-cells, macrophages, and dendritic cells in STAD. In contrast, FGFR4 expression was negatively correlated with tumor-infiltrating lymphocytes. Interestingly, overexpression of FGFR1 in the NOG mouse model improved the immunotherapeutic impact of GC, while overexpression of FGFR4 impaired the effect. When combined with an FGFR4 inhibitor, the anti-tumor effect of anti-PD-1 treatment increased significantly in a GC xenograft mouse model with overexpressed FGFR4. Conclusions FGFRs has critical function in GC and associated with immune cell infiltration, which might be a potential prognosis biomarker and predictor of response to immunotherapy in GC.

The regulation mechanism of different hair types in inner Mongolia cashmere goat based on PI3K-AKT pathway and FGF21.

There is genetic diversity of hair types in the Inner Mongolia cashmere goat population. Previous studies have found that fibroblast growth factor 21 (FGF21) and PI3K-AKT signal pathways may be related to different hair types in Inner Mongolia cashmere goats. Therefore, the purpose of this study was to explore the effects of the PI3K-AKT signal pathway on different hair types, the expression of mRNA and protein expression sites of FGF21 in the hair follicles of cashmere goats with different hair types, so as to lay a foundation for understanding the molecular mechanism of different hair types and the role of skin hair follicle development. In this experiment, the skin tissues of long hair type (LHG) and short hair type (SHG) of Inner Mongolia cashmere goat were collected in three key periods of secondary hair follicle growth, namely, anagen (September), catagen (December), and telogen (March). The relative expression of FGF21 and PI3K-AKT signal pathway candidate gene mRNA in different periods and different hair types was detected by real-time fluorescence quantitative technique (qRT-PCR), and the expression site of FGF21 protein was located by immunohistochemical technique. Through qRT-PCR, it was found that the relative expression of FGF21, FGFR1, AKT3, BRCA1, PKN3, SPP1, and GNG4 was significantly different between LHG and SHG. The expression of FGF21 in the skin of LHG was significantly higher than that of SHG in the three periods. Through immunohistochemical test, it was found that FGF21 protein was mainly expressed in primary hair follicle connective tissue sheath, primary hair follicle outer root sheath, secondary hair follicle outer root sheath, and sebaceous glands. It was also found that the expression of LHG skin tissue in the outer root sheath of primary hair follicles was higher than that of SHG in three periods. In summary, it is suggested that the PI3K-AKT signal pathway may play an important role in the formation of different hair types in Inner Mongolia cashmere goats.

Hydroxysteroid Dehydrogenase Like 2 Promotes the Invasion and Migration of Osteosarcoma by Regulating Fibroblast Growth Factor Receptor 4

We aimed to explore the expression characteristics of HSDL2 in osteosarcoma (OS) as well as the underlying mechanism. A total of 42 OS patients’ tissue samples were collected. HSDL2 level was markedly higher in OS tissues as well as OS cell lines. Besides, patients with high HSDL2 expression had a higher incidence of distant metastasis and a lower overall survival rate. Furthermore, the ability of OS cells to proliferate, invade and metastasize was markedly reduced after HSDL2 knockdown; however, the overexpression of HSDL2 could markedly increase the proliferative, invasive and metastasis ability of OS cells. In addition, HSDL2 can target FGFR4. Furthermore, FGFR4 expression was markedly decreased after HSDL2 knockdown, besides, HSDL2 and FGFR4 expressions were positively correlated in OS tissues. In addition, the recovery experiments suggested that HSDL2 and FGFR4 had a mutual regulation, thereby jointly promoting the invasive and migration ability of OS. HSDL2 expression was markedly increased in OS tissues as well as OS cell lines, which was markedly related to distant metastasis along with poor prognosis of OS patients. Besides, HSDL2 may promote OS progression by increasing the FGFR4 expression.

Mesenchymal FGFR1 and FGFR2 control patterning of the ureteric mesenchyme by balancing SHH and BMP4 signaling.

The coordinated development of the mesenchymal and epithelial progenitors of the murine ureter depends on a complex interplay of diverse signaling activities. We have recently shown that epithelial FGFR2 signaling regulates stratification and differentiation of the epithelial compartment by enhancing epithelial Shh expression, and mesenchymal SHH and BMP4 activity. Here, we show that FGFR1 and FGFR2 expression in the mesenchymal primordium impinges on the SHH/BMP4 signaling axis to regulate mesenchymal patterning and differentiation. Mouse embryos with conditional loss of Fgfr1 and Fgfr2 in the ureteric mesenchyme exhibited reduced mesenchymal proliferation and prematurely activated lamina propria formation at the expense of the smooth muscle cell program. They also manifested hydroureter at birth. Molecular profiling detected increased SHH, WNT and retinoic acid signaling, whereas BMP4 signaling in the mesenchyme was reduced. Pharmacological activation of SHH signaling in combination with inhibition of BMP4 signaling recapitulated the cellular changes in explant cultures of wild-type ureters. Additional experiments suggest that mesenchymal FGFR1 and FGFR2 act as a sink for FGF ligands to dampen activation of Shh and BMP receptor gene expression by epithelial FGFR2 signaling.

Loss of FGFR4 promotes the malignant phenotype of PDAC

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.

Encapsulation of berberine decorated ZnO nano-colloids into injectable hydrogel using for diabetic wound healing.

Chronic wound healing in diabetic patients had been considered a major clinical challenge, so there was an urgent need to establish more effective treatment methods. In this study, we prepared berberine-modified ZnO nano-colloids hydrogel (ZnO-Ber/H) and evaluated its wound healing performance in a diabetic rat. The prepared ZnO-Ber/H had excellent moisturizing, anti-inflammatory and anti-oxidative stress abilities. In vitro, ZnO-Ber/H could effectively up-regulate antioxidant stress factors (Nrf2, HO-1, NQO1) by 4.65-fold, 2.49-fold, 2.56-fold, respectively. In vivo experiments have shown that ZnO-Ber/H could effectively improve the wound healing rate (92.9%) after 15 days of treatment. Meanwhile, the ability of anti-oxidative stress had also been verified in vivo. ZnO-Ber/H down-regulated inflammatory factor (TNF-α, IL-1β, and IL-6) by 72.8%, 55% and 71% respectively, up-regulated vascular related factors VEGF and CD31 by 3.9-fold and 3.2-fold by Western blot. At the same time, ZnO-Ber/H could promote the expression of EGFR and FGFR, thereby affecting the generation of new epithelial tissue. Based on extensive characterization and biological evaluation, ZnO-Ber/H was expected to be a potential candidate for promoting diabetic wound healing.

Novel protein kinase inhibitor TT-00420 inhibits gallbladder cancer by inhibiting JNK/JUN-mediated signaling pathway.

This study aimed to investigate the efficiency of our chemically synthesized TT-00420, a novel spectrum-selective multiple protein kinase inhibitor, in cultured cells and animal models of gallbladder cancer (GBC) and explore its potential mechanism. Multiple GBC models were established to assess the anti-tumor efficiency, toxicity, and pharmacokinetics of TT-00420. Integrated transcriptomic, proteomic and phosphoproteomic analysis was conducted to identify potential downstream effectors of TT-00420. Western blotting, qRT-PCR, nuclear-cytoplasm separation, and immunofluorescence were performed to confirm the multi-omic results and explore the molecular mechanism of TT-00420. Immunohistochemistry was used to detect FGFR1 and p-FGFR1 expression levels in GBC samples. Autodock software was utilized to investigate the potential binding mode between the TT-00420 and the human FGFR1. We found that TT-00420 exerted potent growth inhibition of GBC cell lines and multiple xenograft models. Treatment of mice with 15mg/kg TT-00420 via gavage displayed a half-life of 1.8h in the blood and rapid distribution to the liver, kidneys, lungs, spleen, and tumors at 0.25h, but no toxicity to these organs over 2weeks. Multi-omic analysis revealed c-Jun as a potential downstream effector after TT-00420 treatment. Mechanistically, TT-00420 showed rigorous ability to block FGFR1 and its downstream JNK-JUN (S63/S73) signaling pathway, and induce c-Jun S243-dependent MEK/ERK reactivation, leading to FASLG-dependent tumor cell death. Finally, we found that FGFR1 and p-FGFR1 expression was elevated in GBC patients and these levels correlated with decreased patient survival. TT-00420 shows potent antitumor efficacy and may serve as a novel agent to improve GBC prognosis.

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.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein–umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

FGFR1 promotes tumor immune evasion via YAP-mediated PD-L1 expression upregulation in lung squamous cell carcinoma

BackgroundVariations in FGFR1 are common driver mutations of LSQCC. And immune checkpoint inhibitors targeting PD-1 and PD-L1 are powerful anticancer weapons. Activation of FGFR1 leads to tumorigenesis through multiple downstream molecules, including YAP, but whether and how FGFR1 regulates tumor immune evasion remain largely unclear. MethodsLSQCC cells were modified to increase or decrease the expression of FGFR1, YAP and PD-L1, as assessed by molecular assays. After FGFR1 knockdown, cancer cells were assessed after cocultured with Jurkat T cells in vitro, and the tumor microenvironment were analyzed in C57BL/6 mice. The effect of the combination of FGFR1 knockdown and PD-1 blockade was also explored. ResultsIn human LSQCC, activation of FGFR1 was positively correlated with transcription of PD-L1. In H520 and HCC95 cells, FGFR1 upregulated PD-L1 expression via YAP, and YAP initiated the transcription of PD-L1 after binding to its promoter region. FGFR1 knockdown decreased tumor growth, reduced immune escape and induced reactivation of CD8+ T cells. The combination of FGFR1 knockdown and PD-1 blockade synergistically exerted antitumor effects. ConclusionsThe FGFR1/YAP/PD-L1 regulatory axis mediates tumor-associated immune suppression in lung squamous cell carcinoma, and FGFR1 knockdown reactivates T cells in the tumor microenvironment. Synergistic inhibition of both FGFR1 and PD-1/PD-L1 pathways may be a possible treatment for lung cancer patients.

Abstract 3481: Pharmacokinetics of alofanib and biomarker analysis in patients with advanced gastric cancer: A phase 1b study

Abstract Alofanib is a potent, small molecule, allosteric inhibitor that binds to the non-active extracellular site of IIIc and IIIb FGFR2 isoforms. Phase 1b clinical study (RPT835GC1B) met its primary endpoints and recommended phase 2 dose was described early. Here, we present pharmacokinetics (PK) and results of biomarker analysis. Alofanib was administered daily intravenously for 5-days followed by a 2-day interval (rest). There were five dose levels using a 3 + 3 design. 21 patients have been enrolled in the study. Patients were Caucasian (100%), predominantly male (71%), 67% had 2 and more metastatic sites, including liver (43%) and bone (14.3%) metastases, 19% had ECOG PS 2, and were heavily pretreated (86% had previous 3 and more lines of therapy). The PK and biomarker analysis set included 18 patients. FGFR2 amplification was accessed by FISH with ZytoLight SPEC FGFR2/CEN 10 Dual Color Probe and FGFR2 expression was accessed by IHC with antibody 1G3 (Abcam (ab 5820). Table summarizes PK data. The geometric mean values of Cmax, AUC0-t, T1/2, Vd increased and CL, Kel decreased approximately dose-proportionally after single dosing, similar to previous preclinical studies. The decrease in the mean value of the Vd for a dose of 350 mg/m2 may be associated with a significant increase in AUC0-t. No correlations between PK values and objective response rate (n=2; 9.5%), progression-free (median 3.63 months (95% CI, 1.58 - 5.68) and overall (median 7.0 months (3.82 - 10.18) survival as well as in patients with liver metastases were found (all P&amp;gt;0.1). A positive FGFR2 IHC expression was observed in all tumor cells and a weak positive reaction in normal epithelium. FGFR2 amplification was confirmed by FISH in 1 (5.6%) patient.Alofanib PK in a gastric patient population is well characterized, supporting the use of a once-daily 350 mg/m2 dose. In further studies, the evaluation of FGFR2 amplification seems to be important. Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 50 mg/m2 100 mg/m2 165 mg/m2 250 mg/m2 350 mg/m2 Cmax, mcg/ml (CV%) 21.4 (32.3) 23.7 (18.4) 44.7 (15.5) 72.8 (64.3) 145.9 (42.7) AUC0-t, mcg*h/ml (CV%) 2.3 (31.9) 6.6 (14.2) 13.3 (49.9) 23.8 (8.7) 74.0 (57.5) Vd, ml/m2 (CV%) 4006.1 (28.3) 4907.5 (14.7) 5676.8 (26.6) 6686.7 (11) 3823.0 (63.8) CL, ml/h/m2 (SD) 19609.5 (7740) 14028.2 (1990) 11910.5 (8740) 10011.0 (827) 4183.0 (2420) Kel, 1/h (CV%) 4.9 (15.3) 2.9 (2.8) 2.1 (41.2) 1.5 (3.8) 1.1 (47.0) T1/2, h (SD) 0.1 (0.024) 0.2 (0.01) 0.3 (0.118) 0.5 (0.0171) 0.6 (0.293) Citation Format: Grigory Raskin, Vasily Kazey, Svetlana Gorbacheva, Aiyyna Nikiforova, Galina Statsenko, Elena Artamonova, Liubov Vladimirova, Natalia Besova, Anastasia Mochalova, Ivan Rykov, Vladimir Moiseyenko, Igor Utyashev, Sergei Iugai, Nadezhda Dragun, Dmitry Reznikov, Evgenia Gavrilova, Sergei Tjulandin, Ilya Tsimafeyeu. Pharmacokinetics of alofanib and biomarker analysis in patients with advanced gastric cancer: A phase 1b study [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 3481.

Abstract 579: Anti-myeloid poly-pharmacy allows FGFR4-targeted chimeric antigen receptors to effectively treat an orthotopic model of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children, yet treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The lack of novel therapies and limited immune checkpoint blockade efficacy suggests that CAR-T therapy would be a promising therapeutic approach for RMS and other sarcomas. The key to CAR specificity is to guide engineered T cells to a molecular target that is tumor specific, expressed on the cell surface, and expressed at high enough levels for CAR-T activation. Previous work identified Fibroblast Growth Factor Receptor 4 (FGFR4, CD334) as being specifically and consistently upregulated in RMS, making it a candidate target for CAR-T cells. We tested the feasibility of an FGFR4 targeted CAR for treating RMS using an NSG mouse with RH30 orthotopic (intramuscular) tumors. A previous CAR designed to target FGFR4 was active in vitro but failed to control orthotopic tumors in the NSG mouse model. A new generation of FGFR4 binders was produced targeting the membrane proximal domain of FGFR4. When engineered as CARs, these binders exceeded the activity of previous generation binders in vitro with regard to cellular cytotoxicity and cytokine production. Nevertheless, new candidate binders failed to control orthotopic tumors in vivo. We then interrogated the specific tumor defenses employed by RMS to evade immune control. First, we optimized CAR signaling domains to target low density antigen. Quantitative flow analysis determined FGFR4 expression to be a very low 700 molecules per cell on in vivo RH30 tumors, as opposed to 2-3,000 in tissue culture. We also found that RMS tumors produced a collagen-rich stroma, replete with immunosuppressive myeloid cells. Stroma was induced by T cell therapy and absent in untreated mice. This stroma sequesters CAR-T cells, and produces an immune-excluded phenotype, as assessed by immunohistochemical analysis. Immunohistochemistry identified that M2 macrophages, and to a lesser degree MDSC, were the major cellular constituents of the therapy-induced stroma. RNA expression panel analysis (Nanostring) identified the induction of tumor defense-associated transcripts, including MIF, IDO1, and TGFβ, upon T cell therapy. Based on these results, we devised a strategy to augment CAR-T activity while removing the immunosuppressive barriers. The exposure of mice to anti-myeloid poly-pharmacy (targeting CSF1R (PLX3397), IDO1 (epacadostat), iNOS (L-NAME), TGFβ (SD208), PDL1 (αPD1 antibody), MIF (gene knockout), and myeloid misdifferentiation (ATRA)) allowed FGFR4 CAR-T to successfully clear orthotopic RMS tumors. Our results demonstrate that RMS tumors, even with low copy number targets, can be targeted by CAR-T upon reversal of an immunosuppressive microenvironment, modeling an approach to treating pediatric sarcomas with CAR-T therapy. Citation Format: Peter M. Sullivan, Rajesh Kumar, Wei Li, Lingyang Wang, Yue Zhang, Sophie Jamet, Adam Cheuk, Javed Khan, Dimiter S. Dimitrov, Rimas J. Orentas. Anti-myeloid poly-pharmacy allows FGFR4-targeted chimeric antigen receptors to effectively treat an orthotopic model of rhabdomyosarcoma [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 579.

Genomic Relevance of FGFR2 on the Prognosis of HCV-Induced Hepatocellular Carcinoma Patients.

The Fibroblast Growth Factor Receptors (FGFRs) are known to regulate cancer metabolism in different tumor types, including hepatocellular carcinoma (HCC). Several risk factors are associated with HCC, of which viral infections (Hepatitis B and C) and cirrhosis are prominent. In Pakistan as well as in highly developed countries like the United States, hepatitis C virus HCV infections are most commonly reported in HCC. Here, we aimed to investigate the clinical relevance of FGFR receptors in HCC and their role in HCV-positive HCC cases. 264 HCC samples along with their clinical information and 96 normal liver samples were collected. qPCR was done to estimate the expression of FGFR1, FGFR2, FGFR3 and FGFR4. Three independent HCV-induced HCC cohorts (containing 293 HCC samples) were used for validation. According to in vitro results, FGFR1 was upregulated in HCV+ HCC patients. However, in all three independent cohorts of HCC, significant a down-regulation of FGFR1 was observed. FGFR2 overexpression was observed in the in vitro cohort as well as in three independent HCC cohorts. Interestingly, a strong correlation of FGFR2 expression was observed between cirrhosis and HCV in all four HCC cohorts. Our study suggested that FGFR2 expression can be used to classify HCC patients based on HCV infection. This FGFR2-based classification may lead to new therapeutic strategies against HCV-positive HCC subtypes.

Prospective analysis of the expression status of FGFR2 and HER2 in colorectal and gastric cancer populations: DS-Screen Study

PurposeFibroblast growth factor receptor 2 (FGFR2) and human epidermal growth factor receptor 2 (HER2) proteins are both molecular targets for cancer therapy. The objective of this study was to evaluate the expression status of FGFR2 and HER2 in patients with gastric cancer (GC) or colorectal cancer (CRC).MethodsArchived tumor tissue samples from patients with histologically-confirmed GC or CRC suitable for chemotherapy were analyzed for FGFR2 and HER2 expression using immunohistochemistry and fluorescence in situ hybridization (HER2 in CRC only).ResultsA total of 176 GC patients and 389 CRC patients were enrolled. Among patients with GC, 25.6% were FGFR2-positive and 26.1% were HER2-positive. Among patients with CRC, 2.9% were FGFR2-positive and 16.2% were HER2-positive. No clear relationship was found between FGFR2 and HER2 status in either GC or CRC. In GC, FGFR2 and HER2 statuses did not differ between different primary cancer locations, whereas there were some differences between histological types. Based on FGFR2- and/or HER2-positive status, 117 patients were identified as potentially suitable for inclusion in clinical trials of therapeutic agents targeting the relevant protein (GC = 45, CRC = 72; FGFR = 56, HER2 = 62), of whom 7 were eventually enrolled into such clinical trials.ConclusionsThis study indicated the prevalence of FGFR2 and HER2 in GC and CRC in the Japanese population. The screening performed in this study could be useful for identifying eligible patients for future clinical trials of agents targeting these proteins.Trial registrationClinical trial registration Japic CTI No.: JapicCTI-163380. https://www.clinicaltrials.jp/cti-user/trial/ShowDirect.jsp?directLink=RNlzx1PPCuT.PrVNPxPRwA.

PD53-01 MOLECULAR SUBTYPING OF N-METHYL-N-NITROSOUREA (MNU) RAT MODEL OF UROTHELIAL CARCINOMA REVEALS BOTH LUMINAL AND BASAL SIGNATURES

You have accessJournal of UrologyCME1 May 2022PD53-01 MOLECULAR SUBTYPING OF N-METHYL-N-NITROSOUREA (MNU) RAT MODEL OF UROTHELIAL CARCINOMA REVEALS BOTH LUMINAL AND BASAL SIGNATURES Kara Lombardo, Takahiro Yoshida, Kate Fitzgerald, Kate Johnson, David McConkey, Alok Singh, Ridwan Alam, Max Kates, Alex Baras, and Trinity Bivalacqua Kara LombardoKara Lombardo More articles by this author , Takahiro YoshidaTakahiro Yoshida More articles by this author , Kate FitzgeraldKate Fitzgerald More articles by this author , Kate JohnsonKate Johnson More articles by this author , David McConkeyDavid McConkey More articles by this author , Alok SinghAlok Singh More articles by this author , Ridwan AlamRidwan Alam More articles by this author , Max KatesMax Kates More articles by this author , Alex BarasAlex Baras More articles by this author , and Trinity BivalacquaTrinity Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002630.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Intrinsic subtypes of urothelial carcinoma have been identified with relevant genetic alterations and distinct clinical behavior. Carcinogen induced rodent models are clinically relevant to study urothelial carcinogenesis. In the present study, we sought to identify molecular profiles of the MNU-tumor induced rat model of urothelial carcinoma and provide additional insight into how experimental pre-clinical models may parallel human non-muscle invasive bladder cancer (NMIBC). METHODS: Intravesical MNU instillations were administered in 16 Fisher female rats every other week for a total of 4 instillations. Rats were sacrificed at either 16 (n=10) or 30 (n=6) weeks after initial instillation and bladders were harvested for analysis. Pathologic staging was assessed on H&E, and IHC was performed for basal (KRT5, KRT14, p63) and luminal (CK20, FOXA1, Pparg, Gata3) markers. Bulk RNA sequencing was performed on fresh whole bladders using an Illumina Sequencing platform and cluster analyses were performed for luminal and basal markers using Rstudio. RESULTS: MNU induced development of non-invasive tumors in 10/10 (100%,3 CIS, 3 Ta, 4 T1) bladders at week 16 and 3/6 (50%,3 T1) at week 30, while 3/6 (50%, 3 T2) progressed to muscle invasive disease at week 30. IHC analysis showed retention of luminal markers CK20, FOXA1, Pparg, and Gata3 in CIS, Ta and T1 tumor cells but loss of CDK20, Pparg and Gata3 expression in T2 disease. Basal IHC markers KRT5, KRT14 and p63 were highly expressed in all stages of MNU tumors. Sequencing data show MNU tumors cluster separately from basal-like BBN murine tumors and are more closely clustered with luminal-like UPPL mice tumors when looking at luminal signature markers KRT8 and KRT18 as well as with BASE47 subtype classifier. Cluster analysis including human tumors show MNU clusters more closely with urobasal A and B subtypes which have features including elevated expression of FGFR3, CCND1, and TP63 as well as KRT5 expression (Figure 1). CONCLUSIONS: This study is the first to characterize molecular subtype of the MNU-carcinogen induced rat model of urothelial cancer revealing a complex signature of both basal and luminal markers and provides insight into guiding clinically relevant investigations aimed at improving therapies for urothelial carcinoma. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e910 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kara Lombardo More articles by this author Takahiro Yoshida More articles by this author Kate Fitzgerald More articles by this author Kate Johnson More articles by this author David McConkey More articles by this author Alok Singh More articles by this author Ridwan Alam More articles by this author Max Kates More articles by this author Alex Baras More articles by this author Trinity Bivalacqua More articles by this author Expand All Advertisement PDF DownloadLoading ...

FGFR1 is a potential therapeutic target in neuroblastoma

BackgroundFGFR1 regulates cell–cell adhesion and extracellular matrix architecture and acts as oncogene in several cancers. Potential cancer driver mutations of FGFR1 occur in neuroblastoma (NB), a neural crest-derived pediatric tumor arising in sympathetic nervous system, but so far they have not been studied experimentally. We investigated the driver-oncogene role of FGFR1 and the implication of N546K mutation in therapy-resistance in NB cells.MethodsPublic datasets were used to predict the correlation of FGFR1 expression with NB clinical outcomes. Whole genome sequencing data of 19 paired diagnostic and relapse NB samples were used to find somatic mutations. In NB cell lines, silencing by short hairpin RNA and transient overexpression of FGFR1 were performed to evaluate the effect of the identified mutation by cell growth, invasion and cologenicity assays. HEK293, SHSY5Y and SKNBE2 were selected to investigate subcellular wild-type and mutated protein localization. FGFR1 inhibitor (AZD4547), alone or in combination with PI3K inhibitor (GDC0941), was used to rescue malignant phenotypes induced by overexpression of FGFR1 wild-type and mutated protein.ResultsHigh FGFR1 expression correlated with low relapse-free survival in two independent NB gene expression datasets. In addition, we found the somatic mutation N546K, the most recurrent point mutation of FGFR1 in all cancers and already reported in NB, in one out of 19 matched primary and recurrent tumors. Loss of FGFR1 function attenuated invasion and cologenicity in NB cells, whereas FGFR1 overexpression enhanced oncogenicity. The overexpression of FGFR1N546K protein showed a higher nuclear localization compared to wild-type protein and increased cellular invasion and cologenicity. Moreover, N546K mutation caused the failure in response to treatment with FGFR1 inhibitor by activation of ERK, STAT3 and AKT pathways. The combination of FGFR1 and PI3K pathway inhibitors was effective in reducing the invasive and colonigenic ability of cells overexpressing FGFR1 mutated protein.ConclusionsFGFR1 is an actionable driver oncogene in NB and a promising therapy may consist in targeting FGFR1 mutations in patients with therapy-resistant NB.