Fibroblast Growth Factor Receptor Kinase Research Articles

Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ

The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.

Abstract A170: Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in bladder cancer harboring FGFR3-fusion or -mutation

Abstract Background: Fibroblast growth factor (FGF) / FGF receptor (FGFR) gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers. Recently, FGFR3 fusions (FGFR3-TACC3 and FGFR3-BAIAP2L1), which have potent oncogenic activity, have been discovered in bladder and lung cancer. FGFR kinase inhibitors are expected to be a targeted therapy for bladder cancer harboring FGFR3 gene alternations. A phase I clinical trial of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, is ongoing (NCT02038673). Method: We tested selectivity of ASP5878 among 128 kinases and sensitivity of ASP5878 on cell proliferation of bladder cancer cell lines. FGFR3 and ERK phosphorylation in FGFR3-dependent bladder cancer cell lines were evaluated with sandwich ELISA or Western blotting. In vivo antitumor effects of ASP5878 were examined in subcutaneously implanted bladder cancer cell lines in nude mice. Results: Among 128 kinases, only 9 kinases including wild-type FGFR1-4 and FGFR3/4 mutants were inhibited more than 50% by ASP5878 (200 nmol/L). The IC50 values of ASP5878 against FGFR1, 2, 3 and 4 kinases are 0.47, 0.60, 0.74 and 3.5 nmol/L, respectively. In addition, ASP5878 suppressed cell growth in several cancer cell lines harboring FGF / FGFR gene alternations. Among 25 bladder cancer cell lines, ASP5878 selectively inhibited cell proliferation of UM-UC-14 [FGFR3 (S249C) positive], RT-112 (FGFR3-TACC3 positive), RT-4 (FGFR3-TACC3 positive) and SW780 (FGFR3-BAIAP2L1 positive). FGFR3 and ERK phosphorylation in UM-UC-14 and RT-112 cell lines were inhibited by ASP5878 in a concentration-dependent manner. Furthermore, ASP5878 inhibited cell proliferation of gemcitabine-resistant RT-112 cells and adriamycin-resistant UM-UC-14 cells. Once-daily oral administration of ASP5878 induced tumor regression at 1 and 3 mg/kg in UM-UC-14 and RT-112 xenograft models, respectively, without body weight loss. Conclusions: These findings suggest that ASP5878 has the potential to be an oral targeted therapy against bladder cancer harboring FGFR3-TACC3 fusion or FGFR3 point mutation even after the acquisition of chemoresistance. Citation Format: Tomoyuki Suzuki, Aya Kikuchi, Taisuke Nakazawa, Masateru Iizuka, Ayako Nakayama, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu. Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in bladder cancer harboring FGFR3-fusion or -mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A170.

Structural analysis of the mechanism of phosphorylation of a critical autoregulatory tyrosine residue in FGFR1 kinase domain.

Receptor and nonreceptor tyrosine kinases are enzymes that play important roles in regulating signal transduction pathways in a variety of normal cellular process and in many pathological conditions. Ordered phosphorylation is required for receptor tyrosine kinase (RTK) activation, a process mediated by transient dimer formation of the kinase domains. This process is triggered by the tyrosine phosphorylation in the activation loop. Here, we report structural and biochemical analyses of the tyrosine kinase domain interaction of fibroblast growth factor receptor 1 (FGFR1) required for the initial phosphorylation step. On the basis of nuclear magnetic resonance (NMR) analysis and covalent cross-linking experiments, we propose a parallel symmetric dimer model where specific contacts are formed between the N-lobes and C-lobes, respectively, in the FGFR1 kinase domains. Moreover, assignment of the contact sites between two FGFR1 kinase domains are supported by a trans-phosphorylation assay and by mutational analyses. The present report shows the molecular mechanism underlying the control of trans-phosphorylation of a critical auto-regulatory site in FGF receptors' catalytic domain.

Abstract 3641: Identification of naphthyridines as potent inhibitors of fibroblast growth factor receptor kinase family

Abstract The fibroblast growth factor receptor (FGFR) tyrosine kinase family members, FGFR1, 2, 3 and 4, have roles in a variety of key cellular processes, including proliferation, migration, survival, and differentiation1. Aberrant activation of FGFRs through mutation, amplification, chromosomal translocation, and ligand up-regulation being strongly implicated in oncogenic signalling in many tumour types, has triggered efforts to identify selective FGFR inhibitors. As a result, several potent FGFR kinase inhibitors are currently being evaluated in clinical studies across many tumor types, including non-small cell lung, breast and bladder cancers. We have designed novel 1,5 and 1,7-naphthyridine derivatives that are potent kinase inhibitors of all FGFR family members in enzymatic and cellular systems. Initial hits were further optimized to increase potency and ADME properties leading to identification of a novel 1,5-naphthyridine-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. In vivo screening using an FGFR3-driven xenograft model revealed efficacious compounds that could be explored further as antitumoral agents. This report represents the first disclosure of the structure-activity relationship and synthesis pathway of novel naphthyridine chemical series displaying nanomolar affinity for FGFRs1, 2, 3 and 4. 1 Dieci M. V., Ardenos M., Andre F., Soria J.C. Cancer Discovery. 2013, 3(3) 264-279. Citation Format: Patrick R. Angibaud, Michel Obringer, Julien Marin, Matthieu Jeanty, Norbert Esser, Ron Gilissen, Peter King, Lieven Meerpoel, Olivier Querolle, David C. Rees, Bruno Roux, Gordon Saxty, Tinne Verhulst, Berthold Wroblowski, Christopher C. Murray, Jorge Vialard. Identification of naphthyridines as potent inhibitors of fibroblast growth factor receptor kinase family. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2015-3641

Abstract 4133: ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong activity on primary and metastatic tumor growth in FGFR-dependent and angiogenic cancer models

Abstract Genomic alterations in fibroblast growth factor receptors (FGFR) and upregulation of vascular endothelial growth factor receptors (VEGFR) are often found in the same cancer types such as gastric, lung and breast and these alterations correlate with patient survival and disease progression. In addition, both FGFR and VEGFR signaling promote tumor angiogenesis. Activation of FGFR signaling has also been described to function as a compensatory angiogenic signal following development of resistance to VEGF inhibition. ODM-203 is a novel and selective inhibitor of the FGFR and VEGFR families exhibiting equal potency to both receptors both in vitro and in vivo. In recombinant kinase assays, ODM-203 inhibits both the FGFR and VEGFR family kinases in the low nanomolar range (IC50 6-35 nM). In cells, ODM-203 suppresses FGFR and VEGFR signaling with similar potency. It also inhibits VEGF-induced endothelial cell (HUVEC) proliferation (IC50 33 nM) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50-150 nM). In vivo, ODM-203 shows strong anti-tumor activity in FGFR-dependent xenograft models and in angiogenic xenograft models with similar well tolerated doses. In addition ODM-203 inhibits metastatic tumor growth in angiogenic xenograft models. Lack of activity in a FGFR and VEGFR independent tumor models further supports the selective mode of action of ODM-203. Oral administration of ODM-203 was well tolerated with only FGFR or VEGFR inhibition related physiological events observed in vivo in rat and dog. Based on our findings, ODM-203 has a unique profile with equal potency for both FGFR and VEGFR kinase families which correlates with with strong activity on primary and metastatic tumor growth in FGFR-dependent and angiogenic tumor models. Therefore, a clinical trial with ODM-203 is ongoing in patients with solid tumors (NCT 02264418). Citation Format: Tim Holmström, Anu Moilanen, Mari Björkman, Tero Linnanen, Gerd Wohlfahrt, Stefan Karlsson, Riikka Oksala, Timo Korjamo, Susanta Samajdar, Srinivasan Rajagopalan, Shekar Chelur, Kishore Narayan, Raghuveer Ramachandra, Thomas Anthony, Samiulla DS, Murali Ramachandra, Pekka Kallio. ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong activity on primary and metastatic tumor growth in FGFR-dependent and angiogenic cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4133. doi:10.1158/1538-7445.AM2015-4133

Abstract 771: Preclinical characterization of the selective FGFR inhibitor INCB054828

Abstract Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. Genomic analyses of squamous cell lung, gastric and urothelial tumors have revealed recurrent genetic alterations in FGFR1, FGFR2 and FGFR3 genes, respectively. FGFR proteins contribute to the development of malignancies by promoting tumor cell proliferation, survival, and migration and supporting angiogenesis. Therefore targeting FGFR kinases may provide therapeutic benefit to patients with cancers that have genetic alterations in genes encoding components of the FGF-FGFR axis. INCB054828 is a potent inhibitor of FGFR1, FGFR2, and FGFR3 that has selective pharmacological activity against cancer cells with FGFR alterations. In vitro, INCB054828 potently inhibited the kinase activity of recombinant FGFR1, FGFR2 and FGFR3 enzymes and was highly selective against a panel of kinases including VEGFR2. In cellular assays, INCB054828 inhibited the autophosphorylation of FGFR proteins with low nanomolar IC50 values and blocked signal transduction by FGFR to downstream markers of pathway activation. Cancer cell lines that have genetic alterations in FGFR1, FGFR2 and FGFR3 were uniquely sensitive to growth inhibition by INCB054828, with IC50 values generally in the range of 3-50 nM, compared with cancer cell lines or normal cells without FGFR dependence (IC50 > 1500 nM). In vivo, once-daily oral administration of INCB054828 inhibited the growth of tumors that are dependent upon FGFR1, FGFR2 and FGFR3 activity at tolerated doses. Suppression of tumor growth was dose-dependent and correlated with pharmacodynamic inhibition of FGFR. Collectively, these preclinical studies demonstrate that INCB054828 potently and selectively inhibits models of FGFR-dependent cancers in vitro and in vivo, supporting the compound's clinical evaluation in patients harboring oncogenic FGFR activation. Citation Format: Phillip CC Liu, Liangxing Wu, Holly Koblish, Kevin Bowman, Yue Zhang, Ronald Klabe, Lynn Leffet, Darlise DiMatteo, Mark Rupar, Karen Gallagher, Michael Hansbury, Colin Zhang, Chunhong He, Paul Collier, Maryanne Covington, Richard Wynn, Swamy Yeleswaram, Kris Vaddi, Timothy Burn, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Preclinical characterization of the selective FGFR inhibitor INCB054828. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2015-771

A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication.

Influenza A virus causes annual epidemics and occasional pandemics in humans. Here, we investigated four members of the fibroblast growth factor receptor (FGFR) family; FGFR1 to 4, and examined their expression patterns in human lung epithelial cells A549 with influenza A virus infection. We identified a functional role of FGFR1 in influenza A/Puerto Rico/8/1934 (PR8) and A/Anhui/01/2005 (H5N1) virus replication. Our results showed that FGFR1 silencing by siRNA interference promoted influenza A/PR8 and H5N1 virus replication in A549 cells, while lentivirus-mediated exogenous FGFR1 expression significantly suppressed influenza A virus replication; however, FGFR4 did not have the same effects. Moreover, FGFR1 phosphorylation levels were downregulated in A549 cells by influenza A virus infection, while the repression of FGFR1 kinase using PD173074, a potent and selective FGFR1 inhibitor, could enhance virus replication. Furthermore, we found that FGFR1 inhibits influenza virus internalization, but not binding, during viral entry. These results suggested that FGFR1 specifically antagonizes influenza A virus replication, probably by blocking viral entry.

AZD-4547 exerts potent cytostatic and cytotoxic activities against fibroblast growth factor receptor (FGFR)-expressing colorectal cancer cells.

Colorectal cancer (CRC) causes significant mortalities worldwide. Fibroblast growth factor (FGF) receptor (FGFR) signaling is frequently dysregulated and/or constitutively activated in CRCs, contributing to cancer carcinogenesis and progression. Here, we studied the activity of AZD-4547, a novel and potent FGFR kinase inhibitor, on CRC cells. AZD-4547 inhibited CRC cell growth in vitro, and its activity correlated with the FGFR-1/2 expression level. AZD-4547 was cytotoxic and pro-apoptotic in FGFR-1/2-expressed CRC cell lines (NCI-H716 and HCT-116), but not in FGFR-1/2 null HT-29 cells. Further, AZD-4547 inhibited cell cycle progression and attenuated the activation of FGFR1-FGFR substrate 2 (FRS-2), ERK/mitogen-activated protein kinase (MAPK), and AKT/mammalian target of rapamycin (AKT/mTOR) signalings in NCI-H716 and HCT-116 cells. In vivo, AZD-4547 oral administration at effective doses inhibited NCI-H716 (high FGFR-1/2 expression) xenograft growth in nude mice. Phosphorylation of FGFR-1, AKT, and ERK1/2 in xenograft specimens was also inhibited by AZD-4547 administration. Thus, our preclinical studies strongly support possible clinical investigations of AZD-4547 for the treatment of CRCs harboring deregulated FGFR signalings.

Regulation of the Molecular Brake Region in FGFR2 Kinase

Unregulated activity of tyrosine kinases (TKS) is responsible for numerous developmental musculoskeletal diseases and cancers. The regulatory mechanisms of tks and how they are affected by point mutations are still in need of elucidation. Fibroblast growth factor receptor (FGFR) kinase is a TK whose regulatory element, the “molecular brake,” is thought to prevent constitutive activation of the receptor, despite not being near the kinase's active site. germline mutations in this “molecular brake” region in FGFR cause musculoskeletal disorders such as craniosynostosis and dwarfism, while somatic mutations can lead to uninhibited cell growth, causing an array of cancers. furthermore, it is thought that the majority of pathogenic activating mutations in protein kinases are in “molecular brake”-like regions. However, the mechanism through which the “molecular brake” of FGFR kinase is disengaged, allowing for signal transduction, is still unknown. using path-based molecular dynamics (MD) simulations, we have shown that the inward motion of the kinase's activation loop upon tyrosine phosphorylation is correlated with disengagement of the “molecular brake.” results of the string method in collective variables, which finds the minimum free energy path connecting the loop-out and loop-in states, indicate found that inward motion of loop occurs within 1 frame of “molecular brake” disengagement. Furthermore, umbrella sampling shows that in loop-out conformations, the disengaged state of the “molecular brake” is less stable, while in loop-in conformations, the disengaged state becomes more stable. Our results suggest that the “molecular brake” is allosterically controlled by the activation loop motion. An understanding of the “molecular brake” disengagement mechanism will provide avenues for targeted therapies to counteract pathologic over-activation of fgfr kinase leading to uncontrolled signal transduction and uninhibited cell growth.

Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases has been implicated in a wide variety of cancers. Despite a high level of sequence homology in the ATP-binding site, the majority of reported inhibitors are selective for the FGFR1-3 isoforms and display much reduced potency toward FGFR4, an exception being the Bcr-Abl inhibitor ponatinib. Here we present the crystal structure of the FGFR4 kinase domain and show that both FGFR1 and FGFR4 kinase domains in complex with ponatinib adopt a DFG-out activation loop conformation. Comparison with the structure of FGFR1 in complex with the candidate drug AZD4547, combined with kinetic characterization of the binding of ponatinib and AZD4547 to FGFR1 and FGFR4, sheds light on the observed differences in selectivity profiles and provides a rationale for developing FGFR4-selective inhibitors.

Abstract 5446: FPA144: A therapeutic antibody for treating patients with gastric cancers bearing FGFR2 gene amplification

Abstract A subset of patients with gastric cancer have an amplification of the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) gene. The amplification is most common in the diffuse type of gastric cancer and its presence correlates with poor patient prognosis. Although it has been reported that there is high expression of FGFR2 protein in patients with the amplification, it has been unknown which of the two major FGFR2 isoforms, FGFR2b or 2c, is expressed. In this study, we demonstrate, by both qPCR and immunohistochemistry using FGFR2b-selective antibodies, that it is the FGFR2b isoform, and not FGFR2c, that is overexpressed in gastric cancer tumors that contain the FGFR2 amplification. Five Prime Therapeutics has developed an FGFR2b-specific antibody, FPA144, to treat patients with gastric cancers bearing amplification of the FGFR2 gene. FPA144 is glycoengineered for enhanced antibody-dependent cell cytotoxicity (ADCC). FPA144 causes tumor growth inhibition by 72% to 100% in gastric cancer xenograft models with FGFR2 gene amplification that overexpress the FGFR2b protein isoform. In the SNU-16 model, FPA144 reduces the levels of FGFR2b protein expressed in the tumors by approximately 50%, and decreases both FGFR2b phosphorylation and phosphorylation of the downstream effector, FRS2. The anti-tumor effect of FPA144 is additive with the standard of care chemotherapy regimens of 5-fluoruracil/cisplatin and paclitaxel in the OCUM-2 gastric cancer xenograft model. Since FPA144, unlike small molecule FGFR tyrosine kinase inhibitors, blocks signaling by only the FGFR2b and not the other FGFRs, we expect a favorable toxicology profile, either alone or in combination with chemotherapy. In addition, by engaging immune effector cells for direct tumor cell killing, FPA144 has a second mechanism of action that small molecule FGFR tyrosine kinase inhibitors do not have. We anticipate starting clinical trials of FPA144 in the last quarter of 2014. Citation Format: Abigael T. Gemo, Amit M. Deshpande, Servando Palencia, David I. Bellovin, Thomas J. Brennan, Namrata S. Patil, Carol Huang, Gerrit Los, Kristen L. Pierce. FPA144: A therapeutic antibody for treating patients with gastric cancers bearing FGFR2 gene amplification. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2014-5446

Abstract 4425: Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells

Abstract Targeted therapy directed against ‘oncogene-addicted’ cancers can produce dramatic responses that are invariably of limited durability due to the emergence of subpopulations of drug-resistant cells. We found that many oncogene-addicted cancer cells treated with pathway-targeted drugs engage a positive feedback loop leading to activation of Stat3, which promotes cell survival, consequently limiting the overall drug response. This was observed in cancer cells driven by diverse mutationally-activated kinases, including EGFR, HER2, ALK, and MET, as well as mutant KRAS. We determined that inhibition of MEK signaling specifically promotes Stat3 engagement. MEK inhibition led to the autocrine-driven activation of Stat3 via the FGF receptor and JAK family kinases, and pharmacological inhibition of MEK together with JAK and FGFR effectively promoted tumor regression in vivo. These findings suggest that inhibition of a Stat3 feedback loop may enhance the response to a broad spectrum of drugs that target pathways of oncogene addiction. Citation Format: Ho-June Lee, Guanglei Zhuang, Yi Cao, Pan Du, Hyo-Jin Kim, Jeff Settleman. Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4425. doi:10.1158/1538-7445.AM2014-4425

Abstract LB-233: Unique physicochemical properties of the potent FGFR 1, 2, 3 and 4 inhibitor JNJ-42756493 contribute to prolonged target shutdown

Abstract The ability of drugs to penetrate into cells and tissues is a fundamentally important property that is essential for the efficacy of drugs targeting intracellular processes. Many weakly basic small molecule drugs are known to accumulate extensively in acidic sub-cellular compartments such as lysosomes, through a process called ion trapping that is dependent on the physico-chemical properties of a compound. ‘Lysosomal uptake’ has been shown to significantly influence the activity of many agents, including anticancer drugs, both positively and negatively. Here we describe the unique physico-chemical properties of JNJ-42756493, a potent inhibitor of fibroblast growth factor receptors (FGFRs) 1, 2, 3 and 4, currently undergoing clinical testing for the treatment of malignancies with FGFR pathway activating alterations. JNJ-42756493 has weakly basic and lipophilic characteristics, is highly permeable, and is fluorescent when exposed to UV light. The fluorescent property of JNJ-42756493 enabled rapid and convenient visualization of the drug (spatial and temporal localization) within the lysosomes of cultured cells exposed to JNJ-42756493. In order to evaluate the influence of this lysosomotropic property on the activity of JNJ-42756493, FGFR phosphorylation was monitored in a series of wash-out experiments performed in various cellular models with FGFR1 or FGFR2 amplifications. This comparative washout analysis demonstrated prolonged FGFR kinase inhibition by JNJ-42756493 in comparison to other FGFR inhibitors lacking lysosomotropic properties. This qualitative fluorescence-based data was complemented by accurate quantification of the amount of compound taken up by the cells through the use of radiolabelled JNJ-42756493. Our results unequivocally demonstrate that JNJ-42756493 accumulates at high concentrations within the lysosomal compartment of cells without undergoing metabolism. The continued, long lasting effects on the target FGF receptor kinase inhibition following drug washout, suggest a novel intrinsic slow release of the unmodified drug from these intracellular stores. The lysosomotropic property of JNJ-42756493 is proposed to be a major contributor to the efficient and persistent inhibition of FGFR phosphorylation leading to potent cellular and in vivo activity. Citation Format: Eleonora Jovcheva, Caroline Paulussen, Patrick Van Bergen, Yolanda Chong, Jorge Vialard, Laurence Mevellec, Patrick Angibaud, Timothy Perera. Unique physicochemical properties of the potent FGFR 1, 2, 3 and 4 inhibitor JNJ-42756493 contribute to prolonged target shutdown. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-233. doi:10.1158/1538-7445.AM2014-LB-233

Fibroblast and Epidermal Growth Factors Utilize Different Signaling Pathways to Induce Anchorage-independent Cell Transformation in JB6 Cl41 Mouse Skin Epidermal Cells.

Background:Extracellular stimulation of cells with growth factors such as epidermal growth factor (EGF) induces cell proliferation and cell transformation. Although fibroblast growth factor (FGF) is a well-known family member of growth factors and acts as a ligand of FGF receptor (FGFR), a receptor tyrosine kinase, in cytoplasmic membrane, the tumor promoter potential of FGF has not been clearly understood.Methods:The role of FGF as a tumor promoter was determined measuring its effects of cell proliferation and transformation by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and anchorage-independent cell transformation assays, respectively. The antibody specificity of phospho-RSK2 Tyr529 was determined by Western blotting using a purified FGFR kinase domain in vitro and the membrane fraction of JB6 Cl41 cells ex vivo. The signaling pathways mediated by FGF or EGF were determined by the comparisons of phosphorylation inhibitory efficacy using signaling inhibitors including kaempferol.Results:FGF acted as a tumor promoter. FGF induced cell proliferation by stimulation of G1/S cell cycle transition, and anchorage-independent cell transformation in JB6 Cl41 cells. FGF-induced FGFR phosphorylation was suppressed by kaempferol treatment in a dose dependent manner. Interestingly, FGF stimulation utilized a non-canonical signaling pathway to activate RSK2 and activating transcription factor (ATF)-1, which was not transduced by EGF stimulation. Importantly, kaempferol inhibited tyrosine phosphorylation of FGFR by FGF stimulation and nuclear accumulation of phospho-ATF-1 at Ser63. Moreover, although kaempferol, 4’-N-benzoyl staurosporine (PKC412), 2-(2’-amino-3’-methoxyphenyl)oxanaphthalen-4-one (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)buta-diene (U0126) inhibited EGF-induced anchorage-independent cell transformation in JB6 Cl41 cells, FGF-induced cell transformation in soft agar was only inhibited by PKC412 and kaempferol, but not by PD98059 and U0126.Conclusions:FGF acts as a tumor promoter and dual inhibition of kaempferol on the kinase activities of FGFR3 and RSK2 suppresses the FGF-induced neoplastic cell transformation through a non-canonical signaling pathway which is not utilized by EGF stimulation.

Structural Analysis of the Human Fibroblast Growth Factor Receptor 4 Kinase

The family of fibroblast growth factor receptors (FGFRs) plays an important and well-characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer, for example, breast cancer or rhabdomyosarcoma. Whereas FGFR1–FGFR3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study, we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type II inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4 inhibitors.

Drug Resistance via Feedback Activation of Stat3 in Oncogene-Addicted Cancer Cells

Pathway-targeted cancer drugs can produce dramatic responses that are invariably limited by the emergence of drug-resistant cells. We found that many drug-treated "oncogene-addicted" cancer cells engage a positive feedback loop leading to Stat3 activation, consequently promoting cell survival and limiting overall drug response. This was observed in cancer cells driven by diverse activated kinases, including EGFR, HER2, ALK, and MET, as well as mutant KRAS. Specifically, MEK inhibition led to autocrine activation of Stat3 via the FGF receptor and JAK kinases, and pharmacological inhibition of MEK together with JAK and FGFR enhanced tumor regression. These findings suggest that inhibition of a Stat3 feedback loop may augment the response to a broad spectrum of drugs that target pathways of oncogene addiction.

Increased MAP kinase inhibition enhances epiblast-specific gene expression in bovine blastocysts.

Mammalian blastocysts comprise three distinct lineages, namely, trophoblast, hypoblast, and epiblast, which develop into fetal placenta, extraembryonic yolk sac, and embryo proper, respectively. Pluripotent embryonic stem cells, capable of forming all adult cell types, can only be derived from the epiblast. In mouse and rat, this process is promoted by the double inhibition (2i) of mitogen-activated protein kinase kinase (MAP2K), which antagonizes FGF signaling, and glycogen synthase kinase 3 (GSK3), which stimulates the WNT pathway. We investigated variations of the 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro-fertilized embryos were cultured either in the presence of inhibitors of GSK3 (3 μM CHIR) and MAP2K (0.4 vs. 10 μM PD0325901, designated 2i and 2i+, respectively) or in 2i/2i+ with FGFR inhibitor (0.1 μM PD173074, designated 3i [2i and PD173074] and 3i+ [2i+ and PD173074]). Compared with 2i, both 2i+ and 3i+ potentiated the improvement in blastocyst morphology. Using an automated platform for multiplexed digital mRNA profiling, we simultaneously counted transcripts of 76 candidate genes in bovine blastocysts treated with multiple kinase inhibitors. We show that 2i+ medium specifically increased FGF4 and NANOG while reducing PDGFRalpha and SOX17 levels. The shift from a hypoblast to an epiblast gene expression signature was confirmed by quantitative PCR. A wide range of functionally related genes, including candidates involved in DNA methylation, were not significantly changed. This well-defined 2i+ effect was not observed after pharmacologically inhibiting FGF receptor or related MAP kinases (p38, JNK, and ERK5). In summary, our data suggest that increased MAP2K inhibition exerts its pluripotency-promoting effects through as yet unidentified signals.

Anti-fibroblast growth factor 23 antibody therapy.

The review is intended to provide an update on the expanding knowledge regarding diseases caused by the excess actions of fibroblast growth factor 23 (FGF23) and also on the new therapeutic measures for these diseases, with an emphasis on the anti-FGF23 antibody. FGF23 decreases serum phosphate and 1,25-dihydroxyvitamin D levels. After the cloning of FGF23, several hypophosphatemic diseases, including tumor-induced osteomalacia and X-linked hypophosphatemic rickets (XLHR), were shown to be caused by excess actions of FGF23. In addition, recent studies indicated that mutations in the family with sequence similarity 20, member C (FAM20C), HRAS and NRAS genes, also caused FGF23-related hypophosphatemic diseases. The inhibition of FGF23 production or activity is, theoretically, an ideal treatment for these hypophosphatemic diseases. The C-terminal fragment of FGF23, inhibitors of FGF receptor and extracellular signal-regulated kinase, and anti-FGF23 antibody were shown to inhibit FGF23 actions both in vitro and in vivo. A phase I clinical trial of anti-FGF23 antibody has shown that this antibody increases serum phosphate in patients with XLHR. These recent findings confirm that FGF23 has a pivotal role in phosphate metabolism. The inhibition of FGF23 production or activity is promising as a new therapy for FGF23-related hypophosphatemic diseases. Further studies are clearly necessary to establish the clinical utility and long-term safety of these measures.

Fibroblast growth factor receptors as therapeutic targets in neuroblastoma

To identify progression-driving molecular alterations in neuroblastoma, we used DNA deep-sequencing of 213 primary tumors, RNA deep-sequencing of 498 primary tumors, and microarray gene expression analysis of 709 tumors. While a heterogeneous mutation spectrum was observed in general, we identified putatively activating mutations in fibroblast growth factor receptor (FGFR) tyrosine kinase genes in several cases of high-risk neuroblastoma patients. In addition, gene expression data suggest that elevated FGFR expression in neuroblastoma is associated with a poor patient outcome, suggesting that FGFR may represent therapeutic targets in neuroblastoma. We are therefore investigating the effects of the FGFR inhibitors Dovitinib (TKI-258) and NVP-BGJ398, which have shown promising anticancer-activity in other malignancies, on growth characteristics of neuroblastoma cell lines. We observed that FGFR are highly expressed in the majority of the analysed neuroblastoma cell lines. Preliminary results of Dovitinib indicate that FGFR kinases may be suitable as a molecular target in neuroblastoma therapy.

Fibroblast growth factor signaling and inhibition in non‐small cell lung cancer and their role in squamous cell tumors

With the introduction of targeted agents primarily applicable to non-small cell lung cancer (NSCLC) of adenocarcinoma histology, there is a heightened unmet need in the squamous cell carcinoma population. Targeting the angiogenic fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway is among the strategies being explored in squamous NSCLC; these efforts are supported by growth-promoting effects of FGF signaling in preclinical studies (including interactions with other pathways) and observations suggesting that FGF/FGFR-related aberrations may be more common in squamous versus adenocarcinoma and other histologies. A number of different anti-FGF/FGFR approaches have shown promise in preclinical studies. Clinical trials of two multitargeted tyrosine kinase inhibitors are restricting enrollment to patients with squamous NSCLC: a phase I/II trial of nintedanib added to first-line gemcitabine/cisplatin and a phase II trial of ponatinib for previously treated advanced disease, with the latter requiring not only squamous disease but also a confirmed FGFR kinase amplification or mutation. There are several ongoing clinical trials of multitargeted agents in general NSCLC populations, including but not limited to patients with squamous disease. Other FGF/FGFR-targeted agents are in earlier clinical development. While results are awaited from these clinical investigations in squamous NSCLC and other disease settings, additional research is needed to elucidate the role of FGF/FGFR signaling in the biology of NSCLC of different histologies.