FGFR1 Overexpression Research Articles

Assessment of prognostic implication of a panel of oncogenes in bladder cancer and identification of a 3-gene signature associated with recurrence and progression risk in non-muscle-invasive bladder cancer

This study evaluated the prognostic value of a panel of 29 oncogenes derived from the analysis of The Cancer Genome Atlas (TCGA data) or from the recent literature on bladder tumors on a well-characterized series of muscle-invasive bladder cancer (MIBC) and non-MIBC (NMIBC) samples and tried to identify molecular prognostic markers. Mutations of HRAS, FGFR3, PIK3CA and TERT were found in 2.9%, 27.2%, 14.9% and 76.7% of tumor samples, respectively. Concerning NMIBC, on multivariate analysis, RXRA and FGFR3 levels were associated with recurrence-free survival (RFS) (p = 0.0022 and p = 0.0069) and RXRA level was associated with progression to muscle-invasive disease (p = 0.0068). We identified a 3-gene molecular signature associated with NMIBC prognosis. FGFR3 overexpression was associated with reduced response to Bacillus Calmette–Guerin treatment (p = 0.037). As regards MIBC, on multivariate analysis, ERCC2 overexpression was associated with RFS (p = 0.0011) and E2F3 and EGFR overexpression were associated with overall survival (p = 0.014 and p = 0.035). RT-PCR findings were confirmed by IHC for FGFR3. Genomic alterations in MIBC revealed in TCGA data also concern NMIBC and seem to be associated with prognosis in terms of recurrence and progression. Correcting these alterations by targeted therapies seems a promising pharmacological approach.

Circulating tumor cells with FGFR2 expression might be useful to identify patients with existing FGFR2-overexpressing tumor.

Fibroblast growth factor receptor (FGFR) is associated with proliferation, migration, and angiogenesis of carcinomas, and FGFR signaling inhibitors are considered a key drug for the treatment of solid tumors with FGFR overexpression. Amplification of FGFR2 is reportedly identified in 3%‐10% of gastric cancers (GCs). The aim of this study is to clarify whether the identification of the circulating tumor cells (CTCs) with FGFR2 overexpression is useful to detect patients with FGFR2‐overexpressing GC. One hundred GC patients who underwent gastrectomy were enrolled. A total volume of 8 mL of peripheral blood was collected from each patient just before gastrectomy, and mononuclear cells were enriched by Ficol density gradient centrifugation. These cells were immunostained with PI/CD45/EpCAM/FGFR2. The number of CTCs with FGFR2 expression in each sample was enumerated by FACScan. The FGFR2 expression level of the resected primary tumor was assessed by immunohistochemistry. The number of FGFR2‐positive CTCs in the GC patients' peripheral blood was significantly correlated with the FGFR2 expression level of the primary GC. The relapse‐free survival of the patients with FGFR2‐positive CTCs (≥5 cells/10 mL blood) was significantly poorer (P = .018, log‐rank) than that of the patients without FGFR2‐positive CTCs (<5 cell/10 mL blood). These findings suggested that the determination of FGFR2‐positive CTCs might help identify an existing tumor with FGFR2 overexpression.

FGFR1 overexpression renders breast cancer cells resistant to metformin through activation of IRS1/ERK signaling

Metformin has been suggested as an anti-cancer agent. However, increasing reports show that some tumors are resistant to metformin. Identification of factors affecting metformin mediated cancer therapy is of great significance. FGFR1 is a receptor-tyrosine-kinase that is frequently overexpressed in breast cancer, which is associated with poor-prognosis. To investigate the effect of FGFR1 overexpression on metformin-induced inhibition of breast cancer cells, we demonstrated that FGFR1 overexpression rendered MCF-7 and T47D cells resistant to metformin. In particular, we found that, in addition to AKT and ERK1/2 activation, FGFR1-induced activation of IRS1 and IGF1R, key regulators connecting metabolism and cancer, was associated with metformin resistance. Targeting IRS with IRS1 KO or IRS inhibitor NT157 significantly sensitized FGFR1 overexpressing cells to metformin. Combination of NT157 with metformin induced enhanced inhibition of p-IGF1R, p-ERK1/2 and p-mTOR. Moreover, we demonstrated that IRS1 functions as a critical mediator of the crosstalk between FGFR1 and IGF1R pathways, which involves a feedback loop between IRS1 and MAPK/ERK. Our study highlights the significance of FGFR1 status and IRS1 activation in metformin-resistance, which will facilitate the development of strategies targeting FGFR overexpression-associated metformin resistance.

961P Genomic and transcriptomic profiling in head and neck tumours to identify treatment options: The WINTHER trial experience

Most personalized medicine studies in head and neck (HN) tumors focus on DNA alterations. There is limited data on the role of transcriptomics to guide treatment selection and its correlation with genomic data. As part of the WINTHER trial, we performed targeted NGS (FoundationOne) on FFPE and gene expression profiling (customized Agilent assay on both tumor and analogous normal tissue) for DNA and RNA-matched drug ranks. Here we perform in-depth analysis of genomic and transcriptomic findings in a cohort of 29 pts with HN cancers. The HN cohort included 13 HNSCC, 9 adenoid cystic carcinoma (ACC) and 7 others (including 3 nasophagyngeal carcinoma (NPC)]. In total, 12 genomic- and 13 transcriptomic-matched therapies were identified. Based on genomic analysis, the proportion of targetable cases ranged from 77% in HNSCC, 55% in ACC and 28% in others, mostly PI3K pathway, receptor tyrosine kinase and DNA damage repair alterations. ACC had lower copy number alterations than others. TMB was low across all histologies, with the exception of one NPC (12 mut/Mb). There were few cases of oncogene amplifications, namely 2 cases with increased PIK3CA copy number in HNSCC (both with coexisting high PIK3CA expression in tumor and tumor/normal) and 1 HNSCC with FGFR2 amplification (without FGFR2 overexpression). On transcriptomic analysis, we found 7 cases with high expression of oncogenes in tumor samples, (>1.5 times the interquartile range above the third quartile). We identified high tumor expression of FGFR1 and/or FGFR2 in ACC (4/7 cases each) and HNSCC (1/11 each), all cases without coexisting genomic alterations in FGFR1 or FGFR2. In ACC, we found two-fold increase in mean number of potentially targetable alterations per sample with genomics + transcriptomics as compared to genomics (1.67 vs. 0.78). In other histologies, no significant changes in targetability rate were seen with transcriptomics added to genomics. The pattern of genomic alteration differs by histology in HN cancer. The addition of transcriptomics to genomic analysis increases the targetability rate, especially in ACC, given high expression of FGFR1 and FGFR2, potential drivers that are often missed with genomic level analysis alone.

Abstract 745: Landscape of FGFRactivating aberrations in Chinese non-small cell lung cancer

Abstract Background Dysfunction in fibroblast growth factor receptor (FGFR) signaling has been reported in non-small cell lung cancer (NSCLC), particularly in lung squamous cell carcinoma (LUSC). We hereby assessed the frequency of FGFR aberrations in a large Chinese NSCLC population. Methods A total of 10,966 NSCLCs whose tumor specimen and/or circulating cell-free DNA (cfDNA) underwent hybridization capture-based next-generation sequencing (NGS) of 400+ cancer-related genes were retrospectively reviewed. Patients' clinical characteristics and treatment histories were further evaluated. Results FGFR aberrations, including mutations, fusions, and gene amplifications, were detected in approximately 1.9% (210/10,966) of the population. FGFR abnormalities were more frequently observed in LUSCs (6.8%, 65/954) than lung adenocarcinomas (1.3%, 128/9596). Previously reported FGFR oncogenic mutations were identified in 19 patients (~0.17%), of which, 68% were male LUSC patients. Eleven out of the 19 patients (58%) had concurrent altered PI3K signaling, thus, highlighting an intriguing molecular characteristic and potential combination therapeutic strategy of dual-targeting FGFR and PI3K signaling in those patients. Furthermore, FGFR fusions retaining the intact kinase domain were identified in 12 patients (0.11%), including nine FGFR3-TACC3 cases, one FGFR2-INA fusion, one novel FGFR4-RAPGEFL1 rearrangement event, and one novel gene fusion between FGFR1 and the SLC20A2 5'-untranslated region, which may result in FGFR1 overexpression. Concomitant EGFR mutations or EGFR amplifications were observed in six patients, four of whom were previously treated with EGFR tyrosine kinase inhibitors, suggesting that FGFR fusions may act as acquired resistance to anti-EGFR therapies. In addition, FGFR amplification was detected in 24 patients, with FGFR1 amplification being the most common event. Notably, FGFR oncogenic mutations, fusions, and gene amplifications were almost always mutually exclusive events. Conclusion We herein report an incidence of FGFR anomalies in a large Chinese NSCLC population, including mutations, gene amplifications, and previously characterized and novel FGFR fusions involving TACC3, INA, RAPGEFL1, and SLC20A2. Keywords: FGFR, activating mutation, fusion, gene amplification, NSCLC, FGFR inhibitors Citation Format: Zhen Zhou, Zichuan Liu, Qiuxiang Ou, Xue Wu, Xiaonan Wang, Yang Shao, Hongyan Liu, Yu Yang. Landscape of FGFRactivating aberrations in Chinese non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 745.

Comprehensive Analysis of Fibroblast Growth Factor Receptor (FGFR) Family Genes in Breast Cancer by Integrating Online Databases and Bioinformatics.

BackgroundFibroblast growth factor receptors (FGFRs) play vital roles in the development and progression of human cancers. This study aimed to comprehensively understand the prognostic performances of FGFR1–4 expression in breast cancer (BC) by mining databases.Material/MethodsThe levels of FGFR1–4 expression in BC were analyzed by online databases, GEPIA (Gene Expression Profiling Interactive Analysis) and UALCAN. Survival analysis of FGFR1–4 was carried out by Kaplan-Meier plotter. GSE74146 was downloaded from Gene Expression Omnibus (GEO) and analyzed by GEO2R to screen the differentially expressed genes (DEGs) between FGFR2-silenced BC cells and control. Over-presentation for DEGs were done by Enrichr tool. Networks of DEGs were obtained by using Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape software. Hub genes were identified by cytoHubba Cytoscape plugin.ResultsThe online databases showed that FGFR1 was significantly downregulated whereas FGFR3 was upregulated in BC. Kaplan-Meier plotter demonstrated the upregulation of both FGFR1 and FGFR3 indicated favorable relapse free survival (RFS) whereas FGFR4 overexpression predicted unfavorable overall survival (OS) in BC patients. Importantly, our results showed FGFR2 overexpression robustly predicted favorable OS and RFS in BC. Further bioinformatics analysis of GSE74146 suggested FGFR2 mainly participated in regulating degradation and organization of the extracellular matrix and signaling of retinoic acid. Moreover, CXCL8, CD44, MMP9, and BMP7 were identified as crucial FGFR2-related hub genes.ConclusionsOur study comprehensively analyzed the prognostic values of FGFR1–4 expression in BC and proposed FGFR2 might serve as a promising biomarker. However, the underlying mechanisms remain to be elucidated.

Molecular mechanism of FGFR activation and resulting differences in the tumor-immune environment of bladder cancer.

432 Background: Activation of FGFR signals in bladder cancer is frequently observed. Previous study reported that infiltration of tumor immune cells is suppressed in FGFR3 mutated bladder cancer. In this study, we elucidate the relationship between molecular mechanism of FGFR activation and tumor immune environment in bladder cancer. Methods: We analyzed 103 tissues of bladder cancer patients. Detection of somatic mutations were performed by target sequencing and gene expressions were analyzed by RNA-seq. Immunohistochemical staining of CD8 was performed for evaluating tumor-infiltrating lymphocytes. We compared the relationship between molecular mechanism of FGFR activation and tumor immune environment. Results: FGFR3 activated mutation or fusion was found in 37 cases (36%). FGFR1 overexpression was found in 10 cases (10%). FGFR3 activated mutation or fusion and FGFR1 overexpression were mutually exclusive. Tumors harboring FGFR3 mutation showed less infiltration of CD8 positive cells ( p = 0.01) and low PD-L1 expression ( p = 0.08) compared to those without. On the other hand, tumors with FGFR1 overexpression showed more infiltration of CD8 positive cells ( p = 0.06) and high PD-L1 expression ( p = 0.02). Conclusions: Clinicopathological features and intratumor immune environment of bladder cancer is different depending on the molecular mechanism of FGFR activation. These results suggest a possible association between molecular mechanism of FGFR activation and sensitivity of immunotherapy.

Resistance to MET/VEGFR2 Inhibition by Cabozantinib Is Mediated by YAP/TBX5-Dependent Induction of FGFR1 in Castration-Resistant Prostate Cancer

The overall goal of this study was to elucidate the role of FGFR1 induction in acquired resistance to MET and VEGFR2 inhibition by cabozantinib in prostate cancer (PCa) and leverage this understanding to improve therapy outcomes. The response to cabozantinib was examined in mice bearing patient-derived xenografts in which FGFR1 was overexpressed. Using a variety of cell models that reflect different PCa disease states, the mechanism underpinning FGFR1 signaling activation by cabozantinib was investigated. We performed parallel investigations in specimens from cabozantinib-treated patients to confirm our in vitro and in vivo data. FGFR1 overexpression was sufficient to confer resistance to cabozantinib. Our results demonstrate transcriptional activation of FGF/FGFR1 expression in cabozantinib-resistant models. Further analysis of molecular pathways identified a YAP/TBX5-driven mechanism of FGFR1 and FGF overexpression induced by MET inhibition. Importantly, knockdown of YAP and TBX5 led to decreased FGFR1 protein expression and decreased mRNA levels of FGFR1, FGF1, and FGF2. This association was confirmed in a cohort of hormone-naïve patients with PCa receiving androgen deprivation therapy and cabozantinib, further validating our findings. These findings reveal that the molecular basis of resistance to MET inhibition in PCa is FGFR1 activation through a YAP/TBX5-dependent mechanism. YAP and its downstream target TBX5 represent a crucial mediator in acquired resistance to MET inhibitors. Thus, our studies provide insight into the mechanism of acquired resistance and will guide future development of clinical trials with MET inhibitors.

Expression of growth factors and their receptors in the primary renal cell carcinoma: new data and review.

IntroductionThe aim of our study was to investigate expression levels and the prognostic value of multiple growth factors and their receptors in the primary tumor cells of renal cell carcinoma (RCC).Material and methodsExpression of vascular endothelial growth factor (VEGF)A, fibroblast growth factor (FGF)2, vascular endothelial growth factor receptor (VEGFR)1, VEGFR2, FGFR1, FGFR2, platelet-derived growth factor receptor (PDGFR)α, and PDGFRβ was investigated in 65 primary RCC specimens by immuhistochemical staining using the appropriate antibodies. Expression levels were evaluated by the semi-quantitative method. A search for correlations of expression levels of investigated growth factors and receptors with RCC features and patients outcomes was performed.ResultsExpression of all growth factors and their receptors was detected both on the surface and in the cytoplasm of the primary tumor cells in RCC patients. The expression of all analyzed factors was interconnected. FGFR2 expression correlated with the largest number of other growth factors and receptors. A strong correlation was revealed between high expression of the studied markers, high Fuhrman grade, and advanced RCC stages. In a univariate analysis overexpression of VEGFR2 (p <0.0001) and FGFR2 (p = 0.014) had negative influence on cancer-specific survival.ConclusionsExpression of growth factors and tyrosine kinase receptors in the primary tumor cells is strongly interconnected and associated with unfavorable features of RCC.

Abstract A033: Evaluation of FGFR2b in context of relevant biomarkers supports potential combination of anti-FGFR2b with anti-PD-(L)1 therapy in gastric cancer

Abstract Background: Gastric cancer is the third leading cause of global cancer-related death, with 782,685 deaths in 2018 (Jemal, A. et al. 2018). Currently available treatments have limited benefits. Hence there’s a pressing need for more effective treatment strategies. FGFR2 gene amplification or protein overexpression in gastric cancer tumors is associated with poor prognosis (Su et al., 2014, Ahn et al., 2016). Five Prime has developed an FGFR2b-specific first-in-class humanized monoclonal IgG1 antibody, bemarituzumab (FPA144, or bema), that selectively blocks FGFR2b and triggers antibody-dependent cell-mediated cytotoxicity that showed monotherapy efficacy in a phase 1 trial (Lee et al. ASCO 2016, Catenacci et al. ASCO 2017). In this study, we evaluated the expression of FGFR2b in the context of relevant biomarkers for other approved and potential drug combinations. Methods: RNA and IHC analysis were performed using public datasets and procured tumor samples, respectively. 97 tissue samples from gastric cancer patients were assessed for expression of FGFR2b, PD-(L)1, and CD8 by IHC. In addition, TCGA-STAD database was evaluated for the status of FGFR2 overexpression and the expression of relevant targeted combination drug partners, e.g., ERBB2, PD-(L)1 and cytolytic score. For PD-(L)1 and CD8 staining, SP263 and SP57 antibodies were used, and results were reported as percent positive cells. For FGFR2b staining, the FPR2-D antibody was used (Kim, KM. et al. 2016). Staining with intensity greater than 2+ was considered FGFR2b-positive and anything less than or equal to 2 was considered negative for FGFR2b overexpression. We also investigated the literature and past and ongoing clinical trials to assess PD-(L)1 prevalence in gastric cancer and its correlation to efficacy. Results: Based on RNA expression and copy number data from samples in the TCGA-STAD dataset, combination of bemarituzumab and HER2 targeting agents would not be expected to have synergistic activity, as both populations appear mutually exclusive. In contrast, FGFR2 positive/overexpressing tumors showed good representation of high RNA expression levels of PD-(L)1, PD-1 and cytolytic score. Clinical studies using anti-PD-(L)1 therapies such as pembrolizumab indicate that most benefit (higher ORR) was observed in patients that express PD-(L)1 above a predefined threshold, measured either as a combined positive score of immune and tumor cell staining, or tumor cell staining alone (&amp;gt;1 as cutoff). In our dataset, we calculated % positive PD-(L)1 for both tumor cells and immune cells. Both FGFR2b+ and FGFR2b- tumors showed similar distribution of CD8 and PD-(L)1 positive cells. 60% of FGFR2b+ tumors were PD-(L)1 positive using a greater than 1% PD-(L)1 (immune cell + tumor cell) cut off. Conclusion: In our dataset, 60% of FGR2b+ population is also PD-(L)1 positive (&amp;gt;1%) suggesting that a combination of bemarituzumab with checkpoint inhibitors such as anti-PD-(L)1 could potentially benefit patients with FGFR2b+ tumors. Further mechanistic insight and support comes from preclinical studies (Pierce et al. AACR 2016), which showed an increase in efficacy with the combination treatment of bemarituzumab and anti-PD1 in an orthotopic mouse model. Future studies will be performed to evaluate and get a better understanding of the underlying mechanism of action of combination activity, and confirm co-expression in a larger, confirmatory dataset. Citation Format: Khushboo Sharma, Maike Schmidt, Kevin Hestir, Nerissa Mendoza, Janine Powers, James Hnatyszyn. Evaluation of FGFR2b in context of relevant biomarkers supports potential combination of anti-FGFR2b with anti-PD-(L)1 therapy in gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A033. doi:10.1158/1535-7163.TARG-19-A033

Abstract 3009: FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling

Abstract Palbociclib is the first CDK4/6 inhibitor approved by the FDA for treating ER+/HER2- breast cancer. Palbociclib resistance is emerging as a clinical challenge. Identification of the factors that determine the sensitivity to Palbociclib is of pivotal significance. Fibroblast Growth Factor Receptor (FGFR) overexpression is frequently detected in breast cancer, which has been associated with poor prognosis. To investigate the impact of FGFR overexpression on Palbociclib resistance, we first established stable sublines of MCF7 and T47D cells that overexpress FGFR1, and examined their responses to Palbociclib. Data from MTT and clonogenic assays demonstrated that overexpression of FGFR1 rendered ER+ MCF7 and T47D cells resistant to Palbociclib significantly. Cell cycle analysis showed that the percentage of cells in S phase in Palbociclib treated MCF7/FGFR1 and T47D/FGFR1 cells was significantly higher than that of the control. FGFR overexpression also significantly attenuated Palbociclib-associated inhibition of cancer cell invasion. We further found that Palbociclib-induced inhibition of cancer cell stemness, as indicated by colony formation in soft agar and mammosphere assays, was significantly reduced in MCF7/FGFR1 and T47D/FGFR1 cells, as compared to control. The data indicate that FGFR1 overexpression in ER+ breast cancer cells are resistant to Palbociclib. We next examined the signaling of the key pathways in the paired cell lines treated with Palbociclib. We found that Palbociclib-induced inhibition of phospho-Rb, E2F, cyclin D3, cyclin B1, cdc-2, c-myc, and cdc-25 was significantly attenuated in MCF-7/FGFR1 and T47D/FGFR1 cells. The protein levels of p-ERK, p-AKT, and p-p38 in MCF-7/FGFR1 and T47D/FGFR1 cells were significantly higher than the control cells with/without Palbociclib treatment. The results also showed that ER alpha phosphorylation, estrogen response element (ERE)-luciferase activity and mRNA levels of ER target genes, including PS2, c-myc, E2F, FGF2 and FGFR2, in MCF-7/FGFR1 and T47D/FGFR1 cells were maintained at higher levels post Palbociclib, as compared to control. We then treated control and FGFR1 overexpressing MCF-7 and T47D cells with Palbociclib in combination with FGFR inhibitor, AZD 4547. The combined drug treatment resulted in remarkably synergistic effect on MCF-7/FGFR1 and T47D/FGFR1 cells, as indicated by MTT and clonogenic assays. Key markers in cell cycle regulation, RTK signaling and ER pathway were significantly inhibited in the cells treated with both Palbociclib and AZD 4547. Taken together, our results demonstrated that FGFR1 overexpression is a critical factor affecting Palbociclib sensitivity. The underlying mechanisms involve FGFR1 overexpression-associated RTK and ER signaling. Combination of Palbociclib with FGFR targeting agents may overcome FGFR-associated Palbociclib resistance. Citation Format: Yujie Shi, Zhikun Ma, Yudan Wu, Amanda B. Parris, Qiong Cheng, Lingfei Kong, Xiaohe Yang. FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3009.

Abstract 3818: BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression

Abstract Introduction: Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC. Materials and Methods: UC cell lines: UMUC-14, RT4, T24, J82, HTB5 and HTB9 cells were used. Cells were treated with various concentration of PTX (0 to 20 nM) and BGJ398 (0 to 6 μM). The half-maximal inhibitory concentrations (IC50) were measured by the CellTiter-Glo assay. Cell cycle was analyzed after propidium iodide (PI) staining via flow cytometry. Changes in cell cycle, apoptosis and EMT-associated protein levels were assessed by Western Blot analysis. The migratory and colony formative property were analyzed by wound healing assay and colony forming assay, respectively. RNA interference was performed to assess the dependence of PTX sensitivity on pro-apoptotic proteins. Results: We showed that mesenchymal-type T24 and J82 cell lines were highly resistant to PTX (0 to 20 nM) or BGJ398 (0 to 6 μM) monotherapy compared to FGFR3-positive epithelial-type (i.e., RT4 and UMUC-14) UC cell lines. We further found that PTX (10 nM) combined with BGJ398 (6 μM) caused synergistic effects on sub G1 accumulation and apoptosis compared to PTX or BGJ398 monotherapy in T24 and J82 cells, which were accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Additionally, 10 nM of PTX combined with 6 μM of BGJ398 synergistically suppressed mesenchymal-type UC cell migration and colony formation via downregulation of EMT-associated factors including snail, slug and ZEB1. FGFR1 knockdown enhanced the antitumor effect of PTX (10 nM) by upregulation of apoptosis-associated markers, cleaved caspase-9 and PARP. Conclusions: We investigated the mechanism of resistance to PTX in UC mediated by FGFR1 and found that combined treatment with BGJ398 can enhance the efficacy of PTX in UC cell lines with mesenchymal features. Our results demonstrate that combination with FGFR1-targeted therapy improves the antitumor efficacy of standard cytotoxic chemotherapy, which is a strategy that warrants further investigation in clinical trials in selected UC patients with FGFR1 overexpression. Citation Format: Haram Ryu, Se Hyun Kim, Chan-Young Ock, Koung Jin Suh, Ji Yun Lee, Ji-Won Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Keun-Wook Lee, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Joong Bae Ahn, Kui-Jin Kim, Sun Young Rha. BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3818.

Abstract 4853: M6123 is an ADCC-enhanced and selective monovalent antagonist of FGFR1 showing antitumor activity in preclinical lung cancer models

Abstract Despite the considerable progress made with immunotherapies and targeted therapies, lung cancer remains a devastating disease with a high medical need for more effective and safer therapeutic options. Targeted therapies have delivered meaningful benefit to subsets of lung adenocarcinoma patients harboring specific oncogenic driver alterations but have not yet been extensively explored in other lung cancer subtypes. In the case of lung squamous carcinoma and small-cell lung cancer, amplification and overexpression of FGFR1 have been described as potential oncogenic drivers.Several small molecule tyrosine kinase inhibitors of the FGFR family are in clinical development but initial reports have suggested limited therapeutic benefit of these agents when applied as monotherapy in FGFR1-amplified lung cancer patients. To overcome toxicities associated with pan-FGFR inhibition, such as dysregulation of FGF23-mediated phosphate homeostasis, M6123 was developed as an FGFR1-selective biological antagonist. An immunization campaign in OmniRats® led to the identification of the fully human, potent and highly selective FGFR1 antibody A08 which was converted into the monovalent FGFR1 antagonist M6123 based on the proprietary SEED platform. Furthermore, M6123 was engineered for enhanced ADCC effector function. In contrast to bivalent FGFR1 antibodies, M6123 was well tolerated in mice, with no signs of body weight loss. Of note, M6123 did not interfere with hormonal FGF23 signaling in mice, a severe side effect of FGFR kinase inhibitors. Concentration-dependent inhibition of FGFR1 auto-phosphorylation was demonstrated in FGFR1-overexpressing lung cancer cells both in vitro and in vivo. Moreover, M6123 monotherapy had strong antitumor activity in specific FGFR1-expressing cell line- and patient-derived xenograft models, whilst combination with chemotherapy significantly enhanced efficacy. In summary, M6123 is a unique and clearly differentiated selective FGFR1 antagonist which warrants further testing in preclinical models to refine a predictive biomarker strategy for selection of potentially sensitive lung cancer patients. Citation Format: Christina Esdar, Christel Iffland, Astrid Zimmermann, Angela Lim, Michael Sanderson, Andree Blaukat. M6123 is an ADCC-enhanced and selective monovalent antagonist of FGFR1 showing antitumor activity in preclinical lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4853.

Abstract 3080: Activity of pan-FGFR inhibitor rogaratinib and PI3K inhibitor copanlisib in preclinical urothelial bladder cancer models

Abstract Rogaratinib is a potent small molecule pan-FGFR inhibitor that leads to downregulation of MAPK and PI3K signaling (1). In a recent Phase I study rogaratinib demonstrated higher ORR in locally advanced or metastatic urothelial bladder cancer (UBC) with FGFR mRNA overexpression in patients with PIK3CA and RASwildtype than in those harboring PIK3CA or RAS mutations (2). In UBC, about 25% of tumors contain mutated PIK3CA of which 75% represent hot spot mutations E545K or E542K (3). Copanlisib is a potent pan class I PI3K inhibitor with predominant activity against the α and δ isoforms being approved for treatment of relapsed follicular lymphoma (4). We evaluated the activity of rogaratinib in cellular and in vivo efficacy studies in bladder cancer models in monotherapy and in combination with copanlisib. Cell proliferation and viability in response to rogaratinib, copanlisib or the combination of both was studied in rogaratinib-sensitive UBC cell lines like RT112 as well as in a recombinant RT112-PIK3CAE545K cell line. Rogaratinib potently inhibited cell proliferation with IC50 values in the double nM range. Copanlisib’s potency varied between 20 and 900 nM. In combination, rogaratinib and copanlisib increased cell death and were synergistic as shown by their proliferation combination indices for the absolute IC50 and IC80. Expression of the PIK3CAE545K hot spot mutant in RT112 cells decreased the anti-proliferative efficacy of rogaratinib but did not change the sensitivity to copanlisib. In vivo, rogaratinib led to tumor regression in the FGFR1-expressing JMSU1 xenograft and strongly inhibited tumor growth in the FGFR3-driven RT112 model. Copanlisib did not show significant inhibition of tumor growth in monotherapy in either model. In the JMSU1 model the combination was not superior to rogaratinib alone at maximal tolerated doses. In the RT112 as well as in the RT112-PIK3CAE545K model, the combination significantly improved anti-tumor activity. In a PDX model with FGFR3 overexpression and a PIK3CAH1047R hot spot mutation rogaratinib did not inhibit tumor growth significantly while copanlisib displayed significant anti-tumor effects. The combination of both drugs reduced tumor growth compared to either monotherapy group. In conclusion, in UBC tumor models overexpressing FGFR with differing sensitivities to rogaratinib, its anti-tumor activity could be further enhanced when combined with copanlisib and suggests that PIK3CA mutations may play a role in reduced sensitivity of UBC to FGFR inhibition. These promising results warrant further development of rogaratinib in monotherapy and in combination. Clinical trials with rogaratinib are currently recruiting (NCT03517956, NCT03410693, NCT03473756).

Abstract 314: Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer

Abstract In estrogen-receptor positive (ER+) breast cancer, therapies that target the ER have led to reductions in recurrence and mortality. However, in the metastatic setting, resistance to ER-targeted therapies is near universal. Various resistance mechanisms have been proposed, including ESR1 activating mutations and upregulation of alternative signal transduction pathways, yet clinically relevant resistance mechanisms have not been clearly defined. We conducted a genome-scale gain-of-function screen in ER+ breast cancer cell lines, spanning 17,255 overexpressed open reading frames (ORFs), to investigate genes whose overexpression is sufficient to confer resistance to selective estrogen receptor degraders (SERDs). Among several validated resistance genes, the fibroblast growth factor receptor (FGF/FGFR) pathway was among the top pathways, with several FGF ligands scoring as top resistance genes in this screen. In parallel, we performed whole exome sequencing (WES) in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-targeted therapy. The FGFR pathway was altered in 40% (24 of 60) of the post-resistance biopsies; alterations included FGFR1 amplifications (9 patients), FGFR2 amplifications and activating mutations (4 patients), and FGF3 amplifications (17 patients). In 12 out of the 24 cases (5 FGFR1, 4 FGFR2, 3 FGF3), the FGF/FGFR pathway alterations were present in the resistant tumors and not detected in the pre-treatment tumors, suggesting that these alterations were acquired under the selective pressure of ER-directed therapy. Overexpression of wildtype FGFR1, FGFR2, FGF3 in ER+ breast cancer cells (T47D and MCF7) led to resistance to fulvestrant as well as the combination of fulvestrant and the CDK4/6 inhibitor palbociclib. This resistance phenotype was reversed by the FGFR inhibitor PD173074, and, to a lesser extent, the MEK inhibitor trametinib and mTOR inhibitor everolimus, suggesting several potential treatment strategies. The three FGFR2 mutations (M538I, N550K and K660N) identified in these patients induced hyperactive FGFR signaling and conferred resistance to fulvestrant, which was abrogated by the irreversible FGFR inhibitors FIIN-2 and FIIN-3. Together, these results offer new insights into endocrine resistance and suggest new clinical approaches to overcome or preempt therapeutic resistance. Citation Format: Pingping Mao, Ofir Cohen, Kailey Kowalski, Justin Kusiel, Jorge Buendia, Pedro Exman, Seth A. Wander, Adrienne G. Waks, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 314.

Abstract 3116: Targeting fibroblast growth factor receptors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two main histopathological subtypes: embryonal RMS (eRMS), characterized by different genomic changes or alveolar RMS (aRMS), driven by the oncogenic fusion protein PAX3-FOXO1. The significant toxicity associated with conventional chemotherapies represents a major complication in pediatric oncology. To improve current therapies, we adopted two different strategies targeting fibroblast growth factor receptors (FGFR) in RMS. FGFR1-4 are a family of transmembrane receptor tyrosine kinases. Their activation upon binding of fibroblast growth factors (FGF) triggers pro-survival and proliferative signals. Our goal is to deliver drugs specifically to the tumor site by taking advantage of FGFR4 overexpression in RMS. To this end, we will covalently link FGFR4 specific nanobodies to the surface of liposomal vincristine in order to actively target RMS cells. We have established the optimal conditions to formulate liposomes loaded with vincristine. Following nanobody phage display selection on recombinant FGFR4 we focused on the top scoring candidates. Flow cytometry analysis on FGFR4-expressing versus FGFR4 knock-out RMS cell lines showed receptor-specific binding of three nanobodies. In activation assays with the FGFR4 specific growth factor FGF19, we demonstrated that the three binding candidates also blocked downstream ERK activation in RMS cells. We will now assess their theranostic potential on drug-loaded and fluorescently labeled nanovesicles on RMS tumor cells in vitro and in xenografts in vivo. Surprisingly, we observed change in cell morphology followed by cell death upon exposure to FGF2 in a subset of cultured cells established from eRMS patient-derived xenografts. Inappropriate expression of FGFRs and FGF signaling is implicated in tumor progression and therefore our findings appear contradictory. Dose-response experiments have shown that FGFR inhibition with small molecule inhibitors completely rescued FGF2 toxicity. In contrast, however, we detected high expression levels of FGFR1, 2 and 4 as well as activating mutations of FGFR4 in FGF2-sensitive eRMS cells. Therefore, our results are of upmost clinical relevance since genetically-based drug selection could lead to an inappropriate treatment inducing tumor promoting conditions. Hence, our second goal is to further unravel the molecular mechanism underlying the toxic effect of FGF-2 in a subset of eRMS tumors to avoid potentially harmful treatments. In summary, we have identified FGFR4 specific nanobodies that bind to the receptor and block downstream signaling in RMS cells. Active drug delivery of liposomal vincristine to the tumor site has the potential to enhance the therapeutic impact and decrease side effects. Moreover, we discovered a toxic effect of FGF2 in a subgroup of eRMS patient derived xenograft cells which might open new avenues for treatment. Citation Format: Nagjie Alijaj, Sandrine Moutel, Maxim Gray, Maurizio Roveri, Gabriele Manzella, Marco Wachtel, Franck Perez, Beat Schäfer, Michele Bernasconi. Targeting fibroblast growth factor receptors in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3116.

Fibroblast growth factor receptor promotes progression of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer striking 200 000 Americans annually. Further, in organ transplant patients, there is a 65- to 100-fold increased incidence of cSCC compared to the general population. Excision of cSCC of the head and neck results in significant facial disfigurement. Therefore, increased understanding of the mechanisms involved in the pathogeneses of cSCC could identify means to prevent, inhibit, and reverse this process. In our previous studies, inhibition of fibroblast growth factor receptor (FGFR) significantly decreased ultraviolet B-induced epidermal hyperplasia and hyperproliferation in SKH-1 mice, suggesting an important role for FGFR signaling in skin cancer development. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR in cSCC cells and normal epidermal keratinocytes revealed protein overexpression and increased FGFR2 activation in cSCC cells compared to normal keratinocytes. Further, tumor cell-specific overexpression of FGFR2 was detected in human cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Pretreatment with the pan-FGFR inhibitor; AZD4547 significantly decreased cSCC cell-cycle traverse, proliferation, migration, and motility. Interestingly, AZD4547 also significantly downregulated mammalian target of rapamycin complex 1 and AKT activation in cSCC cells, suggesting an important role of these signaling pathways in FGFR-mediated effects. To further bolster the in vitro studies, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with SCC12A tumor xenografts treated with AZD4547 (15 mg/kg/bw, twice weekly oral gavage) exhibited significantly decreased tumor volume compared to the vehicle-only treatment group. The current studies provide mechanistic evidence for the role of FGFR and selectively FGFR2 in the early progression of cSCC and identifies FGFR as a putative therapeutic target in the treatment of skin cancer.

FGFR4 overexpression and hotspot mutations in metastatic ER+ breast cancer are enriched in the lobular subtype

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.

Neferine inhibits MDA-MB-231 cells growth and metastasis by regulating miR-374a/FGFR-2

BackgroundNeferine (NEF) is a major bisbenzylisoquinline alkaloid mainly exists in the seed embryo of Nelumbo nucifera (Gaertn.) that possesses anti-tumor effects. Our study designed to check the effect of NEF on breast cancer MDA-MB-231 cells and further explore the potential mechanism. MethodsMDA-MB-231 cells were administrated with various dosages of NEF for 24 h after which cell viability was measured. The effects of NEF on cell proliferation, apoptosis, migration and invasion were assessed by BrdU staining, flow cytometry assay and Transwell assay. Western blot was utilized to assess the accumulation of proteins related with proliferation, apoptosis, metastasis, PI3K/AKT and MEK/ERK pathways. ResultsViability was efficiently reduced by NEF in a dose-dependent manner. NEF (8 μM) significantly suppressed cell proliferation, migration and invasion but enhanced apoptosis in MDA-MB-213 cells. Interestingly, NEF suppressed miR-374a expression and miR-374a mediated the inhibitory effect of NEF. Moreover, miR-374a positively regulated FGFR-2 expression and FGFR-2 overexpression impeded the effect of NEF on MDA-MB-213 cells. FGFR-2 overexpression abolished the suppressive effect of NEF on PI3K/AKT and MEK/ERK pathways. ConclusionWe found that NEF possessed the anti-growth and anti-metastasis effect on MDA-MB-231 cells through regulating miR-374a/FGFR-2, which might provide new insight for breast cancer management.

Zebrafish FGFR3 is a negative regulator of RLR pathway to decrease IFN expression

Fibroblast growth factor receptor (FGFR) 3 is one of the four distinct membrane-spanning tyrosine kinases required for proper skeletal development. In fish, the role of FGFR3 is still unclear. In this article, we reveal that zebrafish FGFR3 is a negative regulator of interferon (IFN) production in the innate immune response by suppressing the activity of TANK-binding kinase 1 (TBK1) in the process of virus infection. qPCR experiments demonstrate that the transcriptional level of cellular FGFR3 was upregulated by infection with spring viremia of carp virus (SVCV), indicating that FGFR3 might be involved in the process of host cell response to viral infection. Then, overexpression of FGFR3 significantly impeded the IFN promoter activity induced by a stimulator. In addition, the capabilities of a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system to activate IFN promoter were decreased during the overexpression of FGFR3. Subsequently, FGFR3 decreased the phosphorylation of interferon regulatory factor 3 (IRF3) and mediator of IRF3 activation (MITA) by TBK1. These findings suggest that zebrafish FGFR3 is a negative regulator of IFN by attenuating the kinase activity of TBK1, leading to the suppression of IFN expression.