FGFR1 Activation Research Articles

Abstract 5292: Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells

Abstract Obesity is a global epidemic with a predicted rate of 42% in the U.S.A by 2050. Epidemiologic studies show that obesity is a risk factor for developing cancer; however, the molecular mechanism has not been fully elucidated. Our published data demonstrate that fibroblast growth factor-2 (FGF2) released from fat cells (adipocytes) in the visceral adipose tissue (VAT) induces transformation/tumorigenicity in the skin and mammary epithelial cells. Specifically, FGF2 released from VAT stimulates epithelial cell growth in soft agar by inducing the proto-oncogene c-Myc. Growth in soft agar is a measure of transformation/tumorigenicity; neither transformation nor c-Myc induction in epithelial cells was reversible. c-Myc overexpression can initiate a process of genetic instability linked to tumor initiation. Our discovery of this novel direct path of VAT-stimulated tumorigenesis adds mechanistic insight to our earlier discovery that VAT secretions promote UVR-induced nonmelamoma skin cancer. The objective of our current study was to determine the mechanism by which FGF2 stimulates malignant transformation. We hypothesized that FGF2 from VAT induces c-Myc and subsequent genomic instability in epithelial cells, leading to increased carcinogenesis. To test hypothesis we generated a filtered conditioned-medium from the human VAT treated MCF-10A (mammary epithelial) and JB6 P+ (skin epithelial) cells and measured several downstream mediators of FGF2 and activation of FGFR-1 (FGF2 receptor). Following VAT treatment, epithelial cells demonstrated induced c-Myc protein expression along with ROS accumulation, elevated γ-H2AX foci, and increased micronucleus (MN) formation. We found that inhibition of c-Myc attenuated VAT-induced neoplastic transformation of MCF-10A and JB6 P+ cells, while constitutive activation of the c-Myc-induced spontaneous neoplastic transformation of JB6 P+ cells. Collectively, our data suggested FGF2 released from VAT interacts with FGFR-1 and activates c-Myc. The role of c-Myc in the formation of MN and DNA damage is under investigation. Determining the impact of excess VAT on cancer will lead to strategies to help prevent adiposity-associated cancers and identify individuals at risk for disease or individuals who may be susceptible to compounded genotoxicity due to DNA-damaging environmental exposures. Citation Format: Debrup Chakraborty, Vladislav Jdanov, Vanessa Benham, Blair Bullard, Jamie J. Bernard. Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5292.

PO-489 Tumour-specific oncogenic signalling activities define sensitivity to combinatorial MEK inhibition and stratify Kras-driven lung cancer

IntroductionWith the advent of molecular diagnostics and availability of targeted therapies, treatment of NSCLC has been improved, especially for tumours driven by mutant EGFR or ALK fusions. However, other frequently mutated genes, including KRAS, TP53 and STK11/LKB1, are still not targetable. Murine models of lung cancer that mimic human disease genomics and pathology can help refine diagnostic strategies, allowing discovery of new therapeutic options, identification of biomarkers and validation of the responses in vivo.Material and methodsPrimary cultures were derived from murine adenosquamous carcinoma (ASC) or adenocarcinoma (AC) tumours driven by mutant Kras and loss of Lkb1 (KL) or Tp53 (KP), and murine lung-derived healthy epithelia. Ex vivo drug sensitivity profiling, biomarker analysis and in vivo response validations were combined to identify NSCLC subtype-specific drug vulnerabilities. To identify biomarkers predictive of response, spatial analyses of murine and human tumours were conducted.Results and discussionsBy performing ex vivo functional screens, we identified histotype- and genotype-selective drug vulnerabilities. Inhibition of MEK, a downstream KRAS effector, caused a selective transient cytostasis in AC cells. However, long-term MEK inhibition was found to be ineffective in all cultures tested due to adaptive reactivation of MAPK and PI3K/AKT signalling. Resistance to MEK inhibitors was associated with activation of subtype-specific receptor tyrosine kinases: ERBB3 activation in KL;ASCs, adaptive ERBB2 and FGFR1 activation in KL;ACs, or adaptive FGFR1 activation in KP;ACs. Furthermore, while combined inhibition of MEK and ERBB kinases induced cell death in KL;ASC and KL;AC but not in KP;AC ex vivo culture, in vivo this combination showed cytotoxic response only in the squamous cell carcinoma (SCC) regions of KL;ASCs. This was in agreement with baseline ERBB activation occurring selectively in SCC tissue. Interestingly, activation of ERBB family receptors was detected in a significant proportion of human NSCLC tumours. Analysis of spatial signalling activities may therefore identify NSCLC patients that could benefit from combinatorial MEK and ERBB inhibition.ConclusionOur study validates the utility of NSCLC cultures to discover drug resistance mechanisms that can be overcome with subtype-specific drug combinations. Importantly, the combination of ex vivo drug response profiling and in situ spatial signalling shows that Kras-driven lung cancers stratify as functional subgroups.

Abstract LB-272: Discovery and characterization of a novel FGFR4 Inhibitor for the treatment of hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and has one of the fastest growing incidence among cancers. Aberrant FGF19-FGFR4 signaling occurs in 10-30% of HCC patients and drives tumor development and progression. Pan-FGFR inhibitors are associated with hyperphosphatemia in clinic due to inhibition of FGFR1. Therefore, selectively targeting FGFR4 may render a safer treatment approach with improved clinical benefits. Using advanced computation-aided structural analysis and medicinal chemistry design, we have discovered a novel and highly selective small molecular FGFR4 inhibitor, ABSK-011, demonstrating robust anti-tumor activity in hepatocellular carcinoma models. Method: ABSK-011 was evaluated in biochemical Caliper assay and Celltiter-Glo assay for its inhibition on FGFR4 enzymatic and cellular activities. Its selectivity against other FGFR family members was analyzed through related biochemical, cellular, and KinomeScan profiling assays. The pharmacokinetics (PK) profile of ABSK-011 was characterized across various species of animals including mouse, rat and dog. Efficacy studies were conducted in two HCC xenograft models, and preliminary toxicity profile was also evaluated. Results: ABSK-011 inhibits FGFR4 with IC50 < 10 nM in biochemical assay and exhibits at least 50 fold selectivity against other FGFR kinases. The strong selectivity arises from the ability of ABSK-011 to irreversibly modify Cys552, a residue within the active site of FGFR4 that is not conserved among other FGFR kinases. ABSK-011 also shows greater than 800-fold selectivity against other RTK members. In HCC cells with autocrine production of FGF19, ABSK-011 inhibits the auto-phosphorylation of FGFR4 and blocks signal transduction from FGFR4 to downstream pathway activation. HCC cell lines harboring amplification and overexpression of FGF19 are uniquely sensitive to growth inhibition by ABSK-011 (EC50 < 50 nM) compared with cancer cell lines with FGFR1 dependence (EC50 > 2000 nM), confirming selectivity for FGFR4 over FGFR1. In vivo PK study demonstrated high exposure and superior DMPK profile of ABSK-011 in mouse, rat and dog. In preclinical in vivo studies, oral administration of ABSK-011 strongly inhibits the growth and induces regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity. Suppression of tumor growth is dose dependent and well correlated with pharmacodynamic inhibition of FGFR4 signaling. Safety studies showed that ABSK-011 has no inhibition on CYP family members, hERG, etc. Conclusion: ABSK-011, presented here by Abbisko Therapeutics, is a highly potent, selective, and oral-available small molecular FGFR4 inhibitor. Its superior profile supports fast-track preclinical development. Citation Format: Zhui Chen. Discovery and characterization of a novel FGFR4 Inhibitor for the treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-272.

Abstract 4819: High-throughput screening of glioma stem-like cells (GSCs) identifies synergistic therapeutic combination of FGFR inhibitor and CDK4/6 inhibitor

Abstract High-grade gliomas are challenging tumors to treat. The lack of efficacy of single-agent therapy emphasizes the importance of developing combination strategies to overcome acquired resistance. Quantitative high-throughput screening (HTS) of 2 and 3 drug combinations at 5 concentrations (maximal concentration of 1 uM) of each agent were evaluated in a panel of GSCs to identify potentially novel combination therapy. Genomic, proteiomic (reverse protein lysate array, RPPA) and GSC subtype were correlated with drug IC50 to find biomarkers of drug sensitivity. GSCs were classified as sensitive (IC50 ≤ 1µM) or resistant (IC50 >1µM) for single-agent screening. The Bliss independence model and combination index (CI) criteria were used to quantify the synergy of compound combinations. Evaluation of drug combination data for synergistic combination identified FGFR inhibitor LY2874455 and CDK4/6 inhibitor LY2835219 as the most effective combinations for multiple drug sets. Abnormal activation of FGFR and alterations in the CDKN2A-CDK4/6-Retinoblastoma 1 (RB1) pathway have been implicated in glioblastoma pathogenesis. We further investigated the in vitro combination efficacy of FGFR inhibitor and CDK4/6 inhibitor. Compared to single agent, combination of FGFR inhibitor and CDK4/6 inhibitor induced prominent cleaved poly ADP ribose polymerase (c-PARP), an index of apoptosis, which was further confirmed by Annexin V staining. Combination therapy significantly inhibited p-RB, p-STAT3, p-Akt, p-Erk and p-c-Jun expression compared to single agent. Genomic data demonstrated that high PDGFRα expression and low methylation correlated to sensitivity to the FGFR and CDK4/6 inhibitor combination. These findings were validated by demonstrating that this synergistic sensitivity was decreased by knocking out PDGFRα by CRISP/Cas9 system with the combination index (CI) at IC50 increased from 0.964 to 2.247. In addition, Western blotting showed that expression of PDGFRα and Notch1 was significantly inhibited by combination treatment. However, this combination failed to show any survival and tumor inhibition benefits in intracranial xenograft mouse models. Citation Format: Jianwen Dong, Emmanuel Martinez-Ledesma, Nghi Nguyen, Shaofang Wu, Yuji Piao, Ningyi Tiao, Soon Young Park, David Brunell, Clifford Stephan, Roel Verhaak, Erik Sulman, Veerakumar Balasubramaniyan, John F. de Groot. High-throughput screening of glioma stem-like cells (GSCs) identifies synergistic therapeutic combination of FGFR inhibitor and CDK4/6 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4819.

Abstract 899: Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells

Abstract Because about 10% of gastric cancers show amplification of FGFR2, inhibition of FGFR2 activation has been regarded as one of therapeutic targets. AZD4547, a selective inhibitor of the FGFR1-3 tyrosine kinases, was developed to inhibit FGFR signaling; however, its efficacy is limited by emergency of the acquired resistance. We tried to clarify a resistance mechanism against the FGFR inhibitor. The cell line resistant against AZD4547 was established using SNU-16 (SNU16R), an FGFR2-amplified gastric cancer cell line, by culturing with increasing concentration of AZD4547. The expression levels of FGFR or phosphorylated FGFR (pFGFR), EphB3 and downstream signaling molecules, proteins involved in epithelial-mesenchymal transition (EMT) and apoptosis, were determined by Western blot. Cell viability was measured with EZ-Cytox kit reagent (DoGen, Seoul, Korea). Relative level of tyrosine phosphorylation of receptor tyrosine kinases (RTKs) was evaluated with Proteome ProfilerTM array by human phosphor-RTK kit (R&D Systems). Apoptosis was determined by Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea). Cell cycle assay was performed using Beckman Coulter flow cytometer and WinMDI for determination of cell numbers in G1, S, G2/M phases. Here we show the following: 1) establishment of a gastric cancer cell line (SNU16) resistant to AZD4547 (SNU16R); 2) expression of EphB3 receptor in SNU16R and induction of apoptosis and cell cycle arrest as well as inhibition of cell proliferation and suppressed expression of EMT markers under the treatment EphB3 inhibitor in SNU16R; 3) demonstration of mTOR as a downstream signaling cascade molecule of EphB3 by showing increased expression of phosphorylated (p-) mTOR and its downstream molecules in SNU16R and suppressed expression of p-mTOR by the EphB3 inhibitor. Cell cycle arrest, inhibition of cell proliferation and suppressed expression of p-mTOR under treatment with everolimus were also shown. We concluded that phosphorylation of mTOR through activation of EphB3 receptor is one of the mechanisms that confer resistance to AZD 4547, a selective FGFR inhibitor, in gastric cancer cells. Citation Format: Suk-young Lee, YooJin Na, YoonA Jung, JungLim Kim, Sang Cheul Oh, Dae-Hee Lee. Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 899.

Abstract 5079: Role of syntaxin-1A, a synaptic-related protein, in HER2-enriched and luminal B breast cancer progression

Abstract Breast cancer (BC) is the most prevalent cancer in women. It can be classified into 4 subtypes according to their gene expression signature, each one characterized by its aggressiveness and treatment approach. Mortality has declined as a result of earlier diagnoses and the availability of adjuvant therapies. However, a significant number of patients do not respond to these therapies or develop resistance after an initial response. The therapies currently available fail at curing or preventing BC tumor progression and metastasis. Therefore, there is an urgent need to develop novel therapeutic tools to improve BCs patients´ survival rates. The interaction of cancer cells with the stroma has been well established and has led to exciting new therapies. However, the presence and relevance of nerve axons and their secreted neurotransmitters and neuropeptides in the tumor microenvironment has been acknowledged only very recently. We have identified 6 nervous system related genes differentially expressed in BC subtypes. Among them, Syntaxin-1A (STX1A) is up-regulated in luminal B and HER2-enriched tumors, and its expression significantly correlates with a shorter overall survival and metastasis free survival. Therefore, we hypothesized that STX1A regulates luminal B and HER2 enriched tumors progression and metastasis development. STX1A belongs to the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins superfamily that regulate membrane fusion events, including exocytosis and neurotransmission, intracellular trafficking, and cell proliferation. To test its role in breast cancer we have characterized STX1A pathway in a panel of BC cells. Our results showed that STX1A is overexpressed in luminal B and HER2-enriched cell lines at protein and mRNA level, as we have seen in patient's database. Moreover we have analyzed STX1A expression in cell lines that were derived either from lung or brain metastases respectively and we have discovered that the cells that have migrated to the brain or to the lung have increased STX1A expression in comparison to the parentals. Moreover, downregulation of STX1A using siRNA results in inhibition of epithelial to mesenchymal markers, such as Twist-1, indicating a possible role of STX1A in tumor cell migration and metastasis. Furthermore, downregulation of STX1A also represses FGFR2 activation, suggesting that STX1A might regulate tyrosine kinase receptors membrane trafficking. In conclusion we propose STXIA and SNARE proteins as potential biomarkers and targets for novel BC therapy. Citation Format: Aleix Noguera-Castells, Leire Recalde-Percaz, Núria Moragas, Patricia Fernández-Nogueira, Gemma Fuster, Anna López-Plana, Patricia Jauregui, Pere Gascón, Paloma Bragado, Mario Mancino. Role of syntaxin-1A, a synaptic-related protein, in HER2-enriched and luminal B breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5079.

NCAM2 Fibronectin type-III domains form a rigid structure that binds and activates the Fibroblast Growth Factor Receptor

NCAM1 and NCAM2 have ectodomains consisting of 5 Ig domains followed by 2 membrane-proximal FnIII domains. In this study we investigate and compare the structures and functions of these FnIII domains. The NCAM1 and -2 FnIII2 domains both contain a Walker A motif. In NCAM1 binding of ATP to this motif interferes with NCAM1 binding to FGFR. We obtained a structural model of the NCAM2 FnIII2 domain by NMR spectroscopy, and by titration with an ATP analogue we show that the NCAM2 Walker A motif does not bind ATP. Small angle X-ray scattering (SAXS) data revealed that the NCAM2 FnIII1-2 double domain exhibits a very low degree of flexibility. Moreover, recombinant NCAM2 FnIII domains bind FGFR in vitro, and the FnIII1-2 double domain induces neurite outgrowth in a concentration-dependent manner through activation of FGFR. Several synthetic NCAM1-derived peptides induce neurite outgrowth via FGFR. Only 2 of 5 peptides derived from similar regions in NCAM2 induce neurite outgrowth, but the most potent of these peptides stimulates neurite outgrowth through FGFR-dependent activation of the Ras-MAPK pathway. These results reveal that the NCAM2 FnIII domains form a rigid structure that binds and activates FGFR in a manner related to, but different from NCAM1.

Fibroblast growth factor 23 and Klotho contribute to airway inflammation.

Circulating levels of fibroblast growth factor (FGF)23 are associated with systemic inflammation and increased mortality in chronic kidney disease. α-Klotho, a co-receptor for FGF23, is downregulated in chronic obstructive pulmonary disease (COPD). However, whether FGF23 and Klotho-mediated FGF receptor (FGFR) activation delineates a pathophysiological mechanism in COPD remains unclear. We hypothesised that FGF23 can potentiate airway inflammation via Klotho-independent FGFR4 activation.FGF23 and its effect were studied using plasma and transbronchial biopsies from COPD and control patients, and primary human bronchial epithelial cells isolated from COPD patients as well as a murine COPD model.Plasma FGF23 levels were significantly elevated in COPD patients. Exposure of airway epithelial cells to cigarette smoke and FGF23 led to a significant increase in interleukin-1β release via Klotho-independent FGFR4-mediated activation of phospholipase Cγ/nuclear factor of activated T-cells signalling. In addition, Klotho knockout mice developed COPD and showed airway inflammation and elevated FGFR4 expression in their lungs, whereas overexpression of Klotho led to an attenuation of airway inflammation.Cigarette smoke induces airway inflammation by downregulation of Klotho and activation of FGFR4 in the airway epithelium in COPD. Inhibition of FGF23 or FGFR4 might serve as a novel anti-inflammatory strategy in COPD.

Protein Tyrosine Phosphatase Receptor Type G (PTPRG) Controls Fibroblast Growth Factor Receptor (FGFR) 1 Activity and Influences Sensitivity to FGFR Kinase Inhibitors

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.

FGFR1 fusion kinase regulation of MYC expression drives development of stem cell leukemia/lymphoma syndrome.

Oncogenic transformation of hematopoietic stem cells by chimeric fusion kinases causing constitutive activation of FGFR1 leads to a stem cell leukemia/lymphoma (SCLL) syndrome, accompanied by widespread dysregulation of gene activity. We now show that FGFR1 activation is associated with upregulation of MYC and pharmacological suppression of FGFR1 activation leads to downregulation of MYC and suppression of MYC target genes. Luciferase reporter assays demonstrate FGFR1 can directly regulate MYC expression and this effect is enhanced in the presence of chimeric FGFR1 kinases. In SCLL cells, a truncated form of FGFR1 is generated by granzyme B cleavage of the chimeric kinases, producing a nucleus-restricted derivative that can bind MYC regulatory regions. Mutation of the granzyme B cleavage site prevents relocation to the nucleus but does not suppress MYC activation, suggesting additional mechanisms of MYC activation in the presence of cytoplasm-restricted chimeric kinases. We show one of these mechanisms involves activating cytoplasmic STAT5, which upregulates MYC independent of the truncated FGFR1 kinase. Targeting MYC function using shRNA knockdown and 10054-F8 in SCLL cells leads to inhibition of cell proliferation and synergizes with the BGJ398 FGFR1 inhibitor, suggesting a combination therapy that could be used in the treatment of SCLL.

Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3–RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin β3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3–RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

An FGFR3/MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers.

FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3. Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo. A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti‐FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation.

Abstract P5-05-03: Obesity drives breast cancer progression through estrogen dependent and independent mechanisms

Abstract Background: More than 200,000 women are diagnosed with breast cancer each year and 75% develop estrogen receptor positive (ER+) tumors. Obesity is an independent risk factor for the development of ER+ breast cancer, particularly after menopause, and affects 40% of US women. Obese women are more likely to be diagnosed with advanced tumors, lymph node involvement, and less likely to respond to endocrine therapy versus lean women. Mechanisms underlying the increased risk and worse prognosis of obese women are poorly understood. The dogma in the field states that estrogen production is the main contributor to obesity-associated ER+ breast cancer. We show that this is not always the case. Given the epidemic proportions of obesity in the US, we need better pre-clinical models that will inform focused clinical trials and interventions for patients. In this study, we describe a novel mouse model of obesity and ER+ breast cancer patient-derived xenografts (PDX). Our studies highlight the heterogeneity of responses within the ER+ breast cancer subtype to the obese environment and implicate both estrogen-dependent and independent mechanisms of obesity-associated tumor progression. Methods: ER+, FGFR1-amplified or non-amplified human tumors were established in ovariectomized lean and obese mice in the presence of high or low estradiol (E2). To simulate the hormonal environment of women on aromatase inhibitors, E2 was removed from half of the mice in each adiposity group and the study was terminated 3 weeks later. Weight gain, body fat percentage, and adipose tissue as well as tumor characteristics were analyzed. Results: Prior to EWD, obese mice were heavier and had higher body fat percentage than lean mice and also displayed a phenotype of metabolic dysfunction. This trend was accelerated after EWD, with obese mice gaining more weight due to body fat accumulation. Tumors responded in one of two ways: Regardless of FGFR1 amplification, obesity promoted ER+ tumor growth in the presence of low (postmenopausal), but not high (premenopausal) E2. In the presence of low E2, tumor PR levels were higher in obese compared to lean mice, suggesting hyperactive ER signaling. In FGFR1-amplified tumors, obesity promoted tumor growth after EWD. In addition, EWD induced excess fat deposition in visceral depots in both lean and obese mice; however, obese mice also gained fat in mammary adipose depots. Mammary fat pad mass and rate of post-EWD weight gain directly correlated with adipose FGF1 levels. Tumors from obese mice had higher levels of phosphorylated FGFR1, without changes in total FGFR1, compared to lean mice. Conclusions: Utilizing a unique PDX model system, we show that obesity promotes tumor progression in the presence of low E2, and also after EWD, and identify growth factor receptor signaling as a mediator of these phenotypes. The activation of FGFR1 may underlie increased breast cancer risk and recurrence observed in obese, postmenopausal women. Citation Format: Wellberg EA, Johnson SJ, Jacobsen BM, Anderson SM, Sartorius CA, MacLean PS, Kabos P. Obesity drives breast cancer progression through estrogen dependent and independent mechanisms [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-03.

Anlotinib inhibits angiogenesis via suppressing the activation of VEGFR2, PDGFRβ and FGFR1

Tumor cells recruit vascular endothelial cells and circulating endothelial progenitor cells to form new vessels to support their own growth and metastasis. VEGF, PDGF-BB and FGF-2 are three major pro-angiogenic factors and applied to promote angiogenesis. In this research, we demonstrated that anlotinib, a potent multi-tyrosine kinases inhibitor (TKI), showed a significant inhibitory effect on VEGF/PDGF-BB/FGF-2-induced angiogenesis in vitro and in vivo. Wound healing assay, chamber directional migration assay and tube formation assay indicated that anlotinib inhibited VEGF/PDGF-BB/FGF-2-induced cell migration and formation of capillary-like tubes in endothelial cells. Furthermore, anlotinib suppressed blood vessels sprout and microvessel density in rat aortic ring assay and chicken chorioallantoic membrane (CAM) assay. Importantly, according to our study, the anti-angiogenic effect of anlotinib is superior to sunitinib, sorafenib and nintedanib, which are three main anti-angiogenesis drugs in clinic. Mechanistically, anlotinib inhibits the activation of VEGFR2, PDGFRβ and FGFR1 as well their common downstream ERK signaling. Therefore, anlotinib is a potential agent to inhibit angiogenesis and be applied to tumor therapy.

Fibroblast growth factor type 1 receptor stimulation of T-type Ca2+ channels in sensory neurons requires the phosphatidylinositol 3-kinase and protein kinase A pathways, independently of Akt

Fibroblast growth factor-23 (FGF-23) secreted by osteocytes is known as a circulating factor that is essential for phosphate homeostasis. Recent studies have implicated FGF-23 in the nociceptive signalling of peripheral sensory neurons. However, the relevant mechanisms underlying this effect are not known. In this study, we determine the role of FGF-23 in regulating T-type Ca2+ channels (T-type channels) in small-diameter dorsal root ganglion (DRG) neurons in mice. Our results show that FGF-23 increases T-type channel currents in a concentration-dependent manner. This FGF-23-induced response was dependent on FGF type 1 receptor (FGFR1) and was accompanied by a depolarizing shift in the steady-state inactivation curve. Pretreatment of neurons with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 prevented the FGF-23-mediated T-type channel response. Analysis of phospho-Akt (p-Akt) revealed that FGF-23 significantly activated Akt, but Akt inhibition did not affect the FGF-23-induced T-type channel current increase. The cell-permeable protein kinase A (PKA) inhibitor KT-5720 pretreatment and intracellular application of PKI 6–22 both abolished the stimulatory effects of FGF-23 on T-type channels, but inhibition of PKC had no effect. In summary, these findings indicate that FGF-23 stimulates T-type channel activity via activation of FGFR1, which is coupled to the PI3K-dependent PKA signalling cascade in small DRG neurons.

A role for FGFR1 in paediatric gliomas

Paediatric high-grade gliomas (pHGGs) are highly invasive tumours associated with extremely poor prognosis. There is urgent clinical need to develop novel therapeutic strategies that can target the process of tumour migration and invasion. Fibroblast growth factor receptors (FGFRs) are transmembrane receptor tyrosine kinases associated with glioma tumourigenesis that play a role in regulating cell migration and invasion. An increased frequency of FGFR1 mutations has recently been described in paediatric gliomas (Zhang et al. 2013). Here, the role of FGFR1 in paediatric gliomas was investigated. FGFR1 levels (phosphorylated/unphosphorylated) and cellular localisation were determined by immunofluorescence in two pHGG cell lines (KNS42 and SF188) and five short-term cultures derived from pilocytic astrocytomas (grade 1). FGFR1 activity was stimulated by addition of FGF2. The activity of three FGFR1 inhibitors on cell migration after stimulation was assessed by live cell imaging and in a 3D spheroid invasion assay. FGFR1 levels in low and high-grade paediatric and adult tumour types were investigated in a commercial tissue microarray (TMA). FGFR1 was found to be expressed among all cell lines examined by immunofluorescence. Phosphorylated (activated) FGFR1 was elevated in some cell lines after stimulation with FGF2. Live cell imaging of inhibitor-treated cells and spheroid invasion assays suggest pro-migratory effects of FGFR1 inhibition in low-grade versus anti-migratory in high-grade tumours. High FGFR1 expression in astrocytomas from patients aged under 18 compared to the 18–60 age group (χ2 = 11.578, p = 0.0031) was determined in the TMA sample set. There was a significant association of high FGFR1 levels with grade 2 and 3 astrocytomas. We report the presence of FGFR1 among low and high-grade paediatric gliomas and a role in cell migration after stimulation with ligand. Treatment with FGFR1 inhibitors may elicit a different effect on cell migration depending on tumour grade.

Novel therapeutic strategy for cervical cancer harboring FGFR3-TACC3 fusions

We previously found that therapeutic targetable fusions are detected across various cancers. To identify therapeutic targetable fusion in uterine cervical cancer, for which no effective gene targeted therapy has yet been clinically applied, we analyzed RNA sequencing data from 306 cervical cancer samples. We detected 445 high confidence fusion transcripts and identified four samples that harbored FGFR3-TACC3 fusion as an attractive therapeutic target. The frequency of FGFR3-TACC3-fusion-positive cervical cancer is also 1.9% (2/103) in an independent cohort. Continuous expression of the FGFR3-TACC3 fusion transcript and protein induced anchorage-independent growth in the cervical epithelial cell line established from the ectocervix (Ect1/E6E7) but not in that from endocervix (End1/E6E7). Injection of FGFR3-TACC3 fusion-transfected-Ect1/E6E7 cells subcutaneously into NOG mice generated squamous cell carcinoma xenograft tumors, suggesting the association between FGFR3-TACC3 fusion and squamous cell carcinogenesis. Transfection of a FGFR3-TACC3 fusion transcript into four cervical cancer cell lines (SiHa, ME180, HeLa, and Ca Ski) induced activation of the MAPK pathway and enhancement of cell proliferation. Transcriptome analysis of the FGFR3-TACC3 fusion-transfected cell lines revealed that an IL8-triggered inflammatory response was increased, via activation of FGFR3–MAPK signaling. Continuous expression of FGFR3-TACC3 fusion led to activation of the PI3K–AKT pathway only in the two cell lines that harbored PIK3CA mutations. Sensitivity to the FGFR inhibitor, BGJ398, was found to depend on PIK3CA mutation status. Dual inhibition of both FGFR and AKT showed an obvious synergistic effect in cell lines that harbor mutant PIK3CA. Additionally, TACC3 inhibitor, KHS101, suppressed FGFR3-TACC3 fusion protein expression and showed antitumor effect against FGFR3-TACC3 fusion-transfected cell lines. FGFR3-TACC3 fusion-positive cancer has frequent genetic alterations of the PI3K/AKT pathway and selection of appropriate treatment based on PI3K/AKT pathway status should be required.

Activating FGFR1 Mutations in Sporadic Pheochromocytomas

IntroductionPheochromocytomas are neuroendocrine tumors of the adrenal glands. Up to 40% of the cases are caused by germline mutations in one of at least 15 susceptibility genes, making them the human neoplasms with the highest degree of heritability. Recurrent somatic alterations are found in about 50% of the more common sporadic tumors with NF1 being the most common mutated gene (20–25%). In many sporadic tumors, however, a genetic explanation is still lacking.Materials and methodsWe investigated the genomic landscape of sporadic pheochromocytomas with whole-exome sequencing of 16 paired tumor and normal DNA samples and extended confirmation analysis in 2 additional cohorts comprising a total of 80 sporadic pheochromocytomas.ResultsWe discovered on average 33 non-silent somatic variants per tumor. One of the recurrently mutated genes was FGFR1, encoding the fibroblast growth factor receptor 1, which was recently revealed as an oncogene in pediatric brain tumors. Including a subsequent analysis of a larger cohort, activating FGFR1 mutations were detected in three of 80 sporadic pheochromocytomas (3.8%). Gene expression microarray profiling showed that these tumors clustered with NF1-, RET,- and HRAS-mutated pheochromocytomas, indicating activation of the MAPK and PI3K-AKT signal transduction pathways.ConclusionBesides RET and HRAS, FGFR1 is only the third protooncogene found to be recurrently mutated in pheochromocytomas. The results advance our biological understanding of pheochromocytoma and suggest that somatic FGFR1 activation is an important event in a subset of sporadic pheochromocytomas.

Klotho Inhibits Interleukin-8 Secretion from Cystic Fibrosis Airway Epithelia

Chronic inflammation is a hallmark of cystic fibrosis (CF) and associated with increased production of transforming growth factor (TGF) β and interleukin (IL)-8. α-klotho (KL), a transmembrane or soluble protein, functions as a co-receptor for Fibroblast Growth Factor (FGF) 23, a known pro-inflammatory, prognostic marker in chronic kidney disease. KL is downregulated in airways from COPD patients. We hypothesized that both KL and FGF23 signaling modulate TGF β-induced IL-8 secretion in CF bronchial epithelia. Thus, FGF23 and soluble KL levels were measured in plasma from 48 CF patients and in primary CF bronchial epithelial cells (CF-HBEC). CF patients showed increased FGF23 plasma levels, but KL levels were not different. In CF-HBEC, TGF-β increased KL secretion and upregulated FGF receptor (FGFR) 1. Despite increases in KL, TGF-β also increased IL-8 secretion via activation of FGFR1 and Smad 3 signaling. However, KL excess via overexpression or supplementation decreased IL-8 secretion by inhibiting Smad 3 phosphorylation. Here, we identify a novel signaling pathway contributing to IL-8 secretion in the CF bronchial epithelium with KL functioning as an endocrine and local anti-inflammatory mediator that antagonizes pro-inflammatory actions of FGF23 and TGF-β.

TRPA1\u2013FGFR2 binding event is a regulatory oncogenic driver modulated by miRNA-142-3p

Recent evidence suggests that the ion channel TRPA1 is implicated in lung adenocarcinoma (LUAD), where its role and mechanism of action remain unknown. We have previously established that the membrane receptor FGFR2 drives LUAD progression through aberrant protein–protein interactions mediated via its C-terminal proline-rich motif. Here we report that the N-terminal ankyrin repeats of TRPA1 directly bind to the C-terminal proline-rich motif of FGFR2 inducing the constitutive activation of the receptor, thereby prompting LUAD progression and metastasis. Furthermore, we show that upon metastasis to the brain, TRPA1 gets depleted, an effect triggered by the transfer of TRPA1-targeting exosomal microRNA (miRNA-142-3p) from brain astrocytes to cancer cells. This downregulation, in turn, inhibits TRPA1-mediated activation of FGFR2, hindering the metastatic process. Our study reveals a direct binding event and characterizes the role of TRPA1 ankyrin repeats in regulating FGFR2-driven oncogenic process; a mechanism that is hindered by miRNA-142-3p.