Fibroblast Growth Factor Receptor Family Research Articles

Targeted disruption of the intracellular domain of receptor FgfrL1 in mice.

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1.

A novel fibroblast growth factor receptor family member promotes neuronal outgrowth and synaptic plasticity in aplysia.

Fibroblast Growth Factor (FGF) Receptors (FGFRs) regulate essential biological processes, including embryogenesis, angiogenesis, cellular growth and memory-related long-term synaptic plasticity. Whereas canonical FGFRs depend exclusively on extracellular Immunoglobulin (Ig)-like domains for ligand binding, other receptor types, including members of the tropomyosin-receptor-kinase (Trk) family, use either Ig-like or Leucine-Rich Repeat (LRR) motifs, or both. Little is known, however, about the evolutionary events leading to the differential incorporation of LRR domains into Ig-containing tyrosine kinase receptors. Moreover, although FGFRs have been identified in many vertebrate species, few reports describe their existence in invertebrates. Information about the biological relevance of invertebrate FGFRs and evolutionary divergences between them and their vertebrate counterparts is therefore limited. Here, we characterized ApLRRTK, a neuronal cell-surface protein recently identified in Aplysia. We unveiled ApLRRTK as the first member of the FGFRs family deprived of Ig-like domains that instead contains extracellular LRR domains. We describe that ApLRRTK exhibits properties typical of canonical vertebrate FGFRs, including promotion of FGF activity, enhancement of neuritic outgrowth and signaling via MAPK and the transcription factor CREB. ApLRRTK also enhanced the synaptic efficiency of neurons known to mediate in vivo memory-related defensive behaviors. These data reveal a novel molecular regulator of neuronal function in invertebrates, provide the first evolutionary linkage between LRR proteins and FGFRs and unveil an unprecedented mechanism of FGFR gene diversification in primeval central nervous systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-014-1803-2) contains supplementary material, which is available to authorized users.

Ligand-associated ERBB2/3 activation confers acquired resistance to FGFR inhibition in FGFR3-dependent cancer cells

Somatic alterations of Fibroblast Growth Factor Receptors (FGFRs) have been described in a wide range of malignancies. A number of anti-FGFR therapies are currently under investigation in clinical trials for subjects with FGFR gene amplifications, mutations and translocations. Here, we develop cell line models of acquired resistance to FGFR inhibition by exposure of cell lines harboring FGFR3 gene amplification and translocation to the selective FGFR inhibitor BGJ398 and multi-targeted FGFR inhibitor ponatinib. We show that the acquisition of resistance is rapid, reversible and characterized by an epithelial to mesenchymal transition (EMT) and a switch from dependency on FGFR3 to ERBB family members. Acquired resistance was associated with demonstrable changes in gene expression including increased production of ERBB2/3 ligands which were sufficient to drive resistance in the setting of FGFR3 dependency but not dependency on other FGFR family members. These data support the concept that activation of ERBB family members is sufficient to bypass dependency on FGFR3 and suggest that concurrent inhibition of these two pathways may be desirable when targeting FGFR3 dependent cancers.

Essential role of AKT in tumor cells addicted to FGFR

Tumor cells with genetic amplifications or mutations in the fibroblast growth factor receptor (FGFR) family are often addicted to FGFR and heavily dependent on its signaling to survive. Although it is critical to understand which signaling pathway downstream of FGFR plays an essential role to guide the research and development of FGFR inhibitors, it has remained unclear partly because the tool compounds used in the literature also hit many other kinases, making the results difficult to interpret. With the development of a potent FGFR-specific inhibitor, BGJ398, we are now able to dissect various pathways with low drug concentrations to minimize multiple-target effects. Importantly, here, we show that inhibition of FGFR signaling by BGJ398 leads to only transient inhibition of ERK1/2 phosphorylation, whereas the inhibitory effect on AKT phosphorylation is sustainable, indicating that AKT, not ERK as commonly believed, serves as an appropriate pharmacodynamic biomarker for BGJ398. Although AKT inhibition by a pan-PI3K inhibitor alone has almost no effect on cell growth, heterologous expression of myr-AKT, an active form of AKT, rescues BGJ398-mediated suppression of tumor cell proliferation. These results indicate that AKT is an essential component downstream of FGFR. Finally, combination of the FGFR inhibitor BGJ398 with rapamycin significantly inhibits AKT phosphorylation and enhances their antiproliferative effects in FGFR-addicted cells, suggesting an effective combination strategy for clinical development of FGFR inhibitors.

Emergence of FGFR family gene fusions as therapeutic targets in a wide spectrum of solid tumours

The emergence of fibroblast growth factor receptor (FGFR) family fusions across diverse cancers has brought attention to FGFR-derived cancer therapies. The discovery of the first recurrent FGFR fusion in glioblastoma was followed by discoveries of FGFR fusions in bladder, lung, breast, thyroid, oral, and prostate cancers. Drug targeting of FGFR fusions has shown promising results and should soon be translating into clinical trials. FGFR fusions form as a result of various mechanisms – predominantly deletion for FGFR1, translocation for FGFR2, and tandem duplication for FGFR3. The ability to exploit the unique targetability of FGFR fusions proves that FGFR-derived therapies could have a promising future in cancer therapeutics. Drug targeting of fusion genes has proven to be an extremely effective therapeutic approach for cancers such as the recurrent BCR–ABL1 fusion in chronic myeloid leukaemia. The recent discovery of recurrent FGFR family fusions in several cancer types has brought to attention the unique therapeutic potential for FGFR-positive patients. Understanding the diverse mechanisms of FGFR fusion formation and their oncogenic potential will shed light on the impact of FGFR-derived therapy in the future.

A new chemical probe for quantitative proteomic profiling of fibroblast growth factor receptor and its inhibitors

Recent advances in mass spectrometry-based chemical proteomics allow unbiased analysis of drug-target interactions under close to physiological conditions. In this study, we designed and synthesized two small molecule probes targeting fibroblast growth factor receptors (FGFRs) and applied them to evaluate the selectivity profiles of the FGFR inhibitors Dovitinib and Orantinib. Probe F2 was capable of enriching all members of the FGF receptor family as well as other kinases involved in cancer such as KDR, FLT4 and RET from lysates of cancer cells or human placenta tissue. In combination with the established Kinobeads™ approach, probe F2 facilitated the identification of the target spectrum of the two inhibitors confirming many of the previously identified (off-) targets such as AURKA, FLT4-VEGFR3, IKBKE and PDGFRβ. The newly synthesized probe enlarges the arsenal of chemical proteomic tools for the expression profiling of kinases and selectivity profiling of their inhibitors. It will also be useful in applications aiming at a better understanding of a drug's cellular mechanisms of action as well as highlighting potential beneficial or adverse side effects. The synthesis of a new chemical affinity probe targeting FGF-receptors and many other kinases improved the general scope of drug selectivity profiling by chemical proteomics. The application of the developed chemical tool identified most of the known targets for the advanced clinical kinase inhibitors Dovitinib and Orantinib thus exemplify the practical utility of the developed probe and the results obtained shed further light on how these drugs exert their anti-cancer activity in cells. More generally speaking, the significance of the work is that the molecular tools presented here extend the application scope of kinobeads based kinase profiling to FGFR/VEGFR/PDGFR families, which thus may be generically employed for selectivity profiling of kinase inhibitors using chemical proteomics. The overall aim of such studies is to improve our understanding of how target as well as off-target profiles can be used to assess or predict the therapeutic efficacy of a drug.

Evidence that the novel receptor FGFRL1 signals indirectly via FGFR1

Fibroblast growth factor (FGF) receptor-like protein 1 (FGFRL1) is a recently discovered member of the FGF receptor (FGFR) family. Similar to the classical FGFRs, it contains three extracellular immunoglobulin-like domains and interacts with FGF ligands. However, in contrast to the classical receptors, it does not contain any intracellular tyrosine kinase domain and consequently cannot signal by transphosphorylation. In mouse kidneys, FgfrL1 is expressed primarily at embryonic stages E14–E15 in regions where nascent nephrons develop. In this study, we used whole-mount in situ hybridization to show the spatial pattern of five different Fgfrs in the developing mouse kidney. We compared the expression pattern of FgfrL1 with that of other Fgfrs. The expression pattern of FgfrL1 closely resembled that of Fgfr1, but clearly differed from that of Fgfr2–Fgfr4. It is therefore conceivable that FgfrL1 signals indirectly via Fgfr1. The mechanisms by which FgfrL1 affects the activity of Fgfr1 remain to be elucidated.

Functional roles of fibroblast growth factor receptors (FGFRs) signaling in human cancers.

The fibroblast growth factor receptors (FGFRs) regulate important biological processes including cell proliferation and differentiation during development and tissue repair. Over the past decades, numerous pathological conditions and developmental syndromes have emerged as a consequence of deregulation in the FGFRs signaling network. This review aims to provide an overview of FGFR family, their complex signaling pathways in tumorigenesis, and the current development and application of therapeutics targeting the FGFRs signaling for treatment of refractory human cancers.

Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

Novel Binding Partners and Differentially Regulated Phosphorylation Sites Clarify Eps8 as a Multi-Functional Adaptor

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.

Abstract 2083: Ponatinib potently inhibits the activity of mutant variants of FGFR commonly found in endometrial, lung and other cancers.

Abstract Introduction: Members of the fibroblast growth factor receptor family (FGFR1-4) are mutationally-activated in a number of tumor types. A broad array of mutations have been observed that likely activate through different mechanisms, given the disparate functions of the domains they are found in: the ligand binding (LB) domain, the extracellular (EC) domain that effects receptor dimerization, the transmembrane (TM) domain, and the kinase domain (KD). FGFR2 is mutated in 10% of endometrial cancers, with 90% of all mutations found at six specific codons. In addition, mutations have been found in 12% of lung squamous cell carcinomas (SCCs; FGFR1-4), 50% of bladder cancers (FGFR3), and in glioblastoma multiforme (GBM; FGFR1). Ponatinib (AP24534) is an oral multi-targeted kinase inhibitor being developed for the treatment of CML or Ph+ ALL. Previously, ponatinib was shown to also be a potent inhibitor of all 4 FGFRs, inhibiting viability of Ba/F3 cells dependent on activated variants of native FGFR1-4 (via fusion to a TEL dimerization domain) with IC50s of 14-47 nM. Similar potency was observed in HEK cells transiently transfected to overexpress full-length FGFR1-3, as measured by inhibition of FGFR phosphorylation (IC50s 15-35 nM). Here, the HEK cell system was used to test the ability of ponatinib to inhibit a broad panel of naturally-occurring mutant variants of FGFR1-3, with a primary focus on mutant variants of FGFR2 that have been observed in endometrial and/or SCCs. Results: Of 12 FGFR2 mutants tested, ponatinib inhibited the activity of 10 with high potency and 2 with moderate potency. The 10 mutants inhibited most potently (IC50s < 45 nM) included the 2 mutants that together account for over half of those observed in endometrial cancer (S252W and N550K) and included mutants located in all 4 functional domains: the LB domain (S252W and P253R), the EC domain (Y376C and W290C), the TM domain (C383R), and the KD (I548V, N550K, G584W, K660N and R738K). The 2 FGFR2 mutants inhibited with moderate potency (IC50: 113-152 nM) were N550H and K660E. Select mutant versions of FGFR1 and 3 were also examined. Ponatinib had moderate potency against the FGFR3-S249C EC domain mutant (IC50 128 nM) commonly found in bladder cancer, and did not inhibit the less common K652E KD mutant. Ponatinib potently inhibited the FGFR1-N546K KD mutant found in GBM (IC50 18 nM). Importantly, in patients dosed once daily at 45 mg with ponatinib, peak and trough concentrations are 145 nM and 64 nM, respectively. These exposure levels exceed the IC50s for most mutants tested here. Conclusion: Ponatinib potently inhibited the activity of a broad spectrum of FGFR mutant variants, including FGFR2 mutants observed in endometrial and SCCs. Most of the mutant variants were inhibited at concentrations shown to be achievable in the clinic. These results provide strong support for clinical evaluation of ponatinib in FGFR-driven cancers. Citation Format: Alexa B. Schrock, Joseph M. Gozgit, Tim Clackson, Victor M. Rivera. Ponatinib potently inhibits the activity of mutant variants of FGFR commonly found in endometrial, lung and other cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2013-2083

A Receptor Tyrosine Kinase Network Composed of Fibroblast Growth Factor Receptors, Epidermal Growth Factor Receptor, v-erb-b2 Erythroblastic Leukemia Viral Oncogene Homolog 2, and Hepatocyte Growth Factor Receptor Drives Growth and Survival of Head and Neck Squamous Carcinoma Cell Lines

Our laboratory has previously shown that some gefitinib-insensitive head and neck squamous cell carcinoma (HNSCC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. Herein, we deployed a whole-genome loss-of-function screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ8010, in HNSCC cell lines. Three HNSCC cell lines expressing a genome-wide small hairpin RNA (shRNA) library were treated with AZ8010 and the abundance of shRNA sequences was assessed by deep sequencing. Under-represented shRNAs in treated cells are expected to target genes important for survival with AZ8010 treatment. Synthetic lethal hits were validated with specific inhibitors and independent shRNAs. We found that multiple alternate receptors provided protection from FGFR inhibition, including receptor tyrosine kinases (RTKs), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), and hepatocyte growth factor receptor (MET). We showed that specific knockdown of either ERBB2 or MET in combination with FGFR inhibition led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ERBB family members or MET. Moreover, the triple combination of FGFR, MET, and ERBB family inhibitors showed the largest inhibition of growth and induction of apoptosis compared with the double combinations. These results reveal a role for alternate RTKs in maintaining progrowth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR, ERBB family members, and MET.

Abstract P4-06-08: Clinical utility of functional analysis of FGFR kinase family for selecting patients who may benefit from FGFR inhibitors

Abstract Background: Fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) are currently under investigation as therapeutic targets for the treatment of breast cancer. Members of this kinase family have been associated with breast cancer progression and resistance to current therapeutics. FGFR1 amplifications range from 7.5–17% in breast cancers and are associated with a shorter overall survival. FGFR1 amplified cell lines drive activation of AKT, ERK, and RSK and are highly sensitive to FGFR1 inhibition, suggesting an oncogenic addiction to FGFR1. FGFR2 is activated in lapatinib-resistant, HER2-positive cells. Likewise, FGFR3 expression levels are elevated in tamoxifen-resistant, ER positive patients. FGFR 4 overexpression correlated with poor response to chemotherapy due to activation of MAPK and increase in B-cell lymphoma extra large (BCL-XL) levels. Using an immuno-microarray system, this study seeks to determine FGFR signal transduction pathway modulation post FGFR inhibitor treatment in breast cancer cell lines expressing varying levels of FGFR1, 2, 3 or 4. Method: FGFR 1, 2, 3, and 4 amplified cell lines (MDA-MB-134VI (KRAS), SNU16, RT112, and MDA-MB-453) were treated with varying dosages (1, 10, 100, and 1000nM) of several FGFR inhibitors (AZD4547 (pan FGFR), PD173074 (FGFR1,3) and Ponatinib-AP24534 (FGFR1)) either in presence or absence of corresponding FGF (FGF1, FGF7, FGF9, and FGF19) stimulation. Expression and activation of FGFR 1, 2, 3, and 4 and downstream signaling proteins FRS2, AKT, ERK, MEK, and RSK were measured utilizing a proximity based immuno-microarray. Results: FGFR1, 2, 3, 4 protein expression was detected in each of the corresponding four FGFR1, 2, 3, 4 amplified cell lines. Total protein levels remained unchanged with or without FGFs stimulation. Levels of FGFR1 phosphorylation increased 5 folds in MDA-MB-134VI stimulated with FGF1. FGFR2 and FGFR3 activation increased by 2–3 folds in SNU16 and RT112 stimulated with FGF7, and FGF9 respectively. FGFR4 activation levels in MDA-MB-453 remained unchanged due to Y367C mutation. ERK was activated in both FGF1 stimulated and unstimulated group in MDA-MB134 due to the KRAS mutation. RSK activation increased in both SNU16 and RT112 when stimulated with FGF7, and FGF9 respectively. FGFR inhibitors treated data analysis is currently in progress and will be presented. Conclusion: We have demonstrated utility of specific FGFR analysis using well defined model systems. FGFR- profiling should be considered during the clinical work-up in order to select patients who may benefit from regimen containing specific FGFR inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-08.

FGFR Genetic Alterations Predict for Sensitivity to NVP-BGJ398, a Selective Pan-FGFR Inhibitor

Patient stratification biomarkers that enable the translation of cancer genetic knowledge into clinical use are essential for the successful and rapid development of emerging targeted anticancer therapeutics. Here, we describe the identification of patient stratification biomarkers for NVP-BGJ398, a novel and selective fibroblast growth factor receptor (FGFR) inhibitor. By intersecting genome-wide gene expression and genomic alteration data with cell line-sensitivity data across an annotated collection of cancer cell lines called the Cancer Cell Line Encyclopedia, we show that genetic alterations for FGFR family members predict for sensitivity to NVP-BGJ398. For the first time, we report oncogenic FGFR1 amplification in osteosarcoma as a potential patient selection biomarker. Furthermore, we show that cancer cell lines harboring FGF19 copy number gain at the 11q13 amplicon are sensitive to NVP-BGJ398 only when concomitant expression of β-klotho occurs. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in selected patients with cancer harboring tumors with the identified predictors of sensitivity. The success of a personalized medicine approach using targeted therapies ultimately depends on being able to identify the patients who will benefit the most from any given drug. To this end, we have integrated the molecular profiles for more than 500 cancer cell lines with sensitivity data for the novel anticancer drug NVP-BGJ398 and showed that FGFR genetic alterations are the most significant predictors for sensitivity. This work has ultimately endorsed the incorporation of specific patient selection biomakers in the clinical trials for NVP-BGJ398.

Fibroblast Growth Factor Receptor 2: Expression, Roles, and Potential As a Novel Molecular Target for Colorectal Cancer

The fibroblast growth factor receptor (FGFR) family consists of four members, named FGFR1, 2, 3, and 4. All 4 FGFRs and their ligands, fibroblast growth factors (FGFs), are expressed in colorectal cancer (CRC). Recent studies have shown that FGFR2 plays important roles in cancer progression; therefore, it is of great interest as a novel target for cancers. Expression of FGFR2 regulates migration, invasion, and growth in CRC. Expression of the FGFR2 isoform FGFR2 IIIb was associated with well-differentiated histological types, and its specific ligand, FGF7, enhanced angiogenesis and adhesion to type-IV collagen via FGFR2 IIIb in CRC. FGFR2 IIIc is detected in CRC, but its roles have not been well elucidated. Interactions between FGFR2 IIIb and IIIc and FGFs may play important roles in CRC via autocrine and/or paracrine signaling. Several kinds of molecular-targeting agents against FGFR2 have been developed; however, it is not clear how a cancer treatment can most effectively inhibit FGFR2 IIIb or FGFR2 IIIc, or both isoforms. The aim of this paper is to summarize the roles of FGFR2 and its isoforms in CRC and clarify whether they are potent therapeutic targets for CRC.

SOX2 and FGFR1 gene copy number in surgically resected non-small cell lung cancer (NSCLC).

7061 Background: SOX2 is a member of the SRY-related HMG-box family of transcription factors and has been shown to be frequently amplified and overexpressed in squamous cell lung cancer, with conflicting results regarding its prognostic relevance. Similarly, FGFR1, a transmembrane tyrosine kinase receptor belonging to the fibroblast growth factor receptor family, has been recently reported to be amplified in squamous cell lung carcinomas, suggesting a potential role for FGFR1 as a therapeutic target in NSCLC. Aim of the present study is to evaluate SOX2 and FGFR1 gene copy number in surgically resected NSCLCs, to investigate their prognostic relevance and their association with clinico-pathological characteristics. Methods: SOX2 and FGFR1 gene copy number was assessed by fluorescence in situ hybridization (FISH) in tissue microarray cores from 447 surgically resected NSCLCs. Each patient was given a score ranging from 1 to 6 according to increasing mean copy number per cell of each gene, with 6 indicating true gene amplification. Results: SOX2 and FGFR1 FISH was successfully performed in 445 patients (pts), which were grouped as + (score 5-6) and - (score 1-4). Using this scoring system 105 (23.6%) pts tested SOX2+, while 74 (16.6%) pts resulted FGFR1+. True gene amplification for SOX2 and FGFR1 was observed in 19 (4.3%) and 37 (8.3%) cases, respectively. SOX2+ and FGFR1+ status was significantly associated with squamous histology (p<.001). Additionally, SOX2+ pts had a significantly higher chance of being former/current smokers, male and FGFR1+. FGFR1 gene status had no prognostic impact in the whole population and in the squamous cell carcinoma subgroup. Conversely, SOX2+ pts had significantly longer overall survival compared with SOX2- pts (HR 0.68, p=.020). When restricting survival analysis to squamous cell histology, stage I-II SOX2+ pts had a significant survival advantage compared with SOX2- group (HR 0.38, p=.006), while no difference was observed in stage III-IV pts. Conclusions: Increased SOX2 and FGFR1 gene copy number is a common event in lung cancer pts with squamous cell histology. SOX2 gene gain is a favorable prognostic factor in surgically resected pts, particularly in early stage squamous cell cancers.

Abstract B35: Alternate receptor tyrosine kinases provide protection from FGFR inhibition in HNSCC cells as identified by genome-wide shRNA screening

Abstract The efficacy of many biologically targeted cancer therapies is limited by intrinsic and acquired resistance. Head and neck squamous carcinoma (HNSCC) patients show only a 4–10% response to the epidermal growth factor receptor (EGFR) inhibitors, gefitinib and erlotinib, despite EGFR over-expression in over 90% of tumors. Our lab has previously shown that gefitinib insensitive non-small cell lung cancer (NSCLC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. RNAi-based loss-of-function screens are an important tool to identify synthetic lethal interactions between the inhibition of two genes in order to efficiently identify functional survival mechanisms and potent drug combinations. We deployed a whole genome screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ12908010, in HNSCC cell lines. We found that multiple alternate receptors provided protection from FGFR inhibition in these cell lines, including the receptor tyrosine kinases (RTKs) epidermal growth factor receptor 2 (ErbB2) and hepatocyte growth factor receptor (Met). We validated the results of the screen by showing that specific knockdown of either ErbB2 or Met in combination with FGFR inhibitor treatment led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ErbB2 or Met. The combination therapies did not lead to greater inhibition of growth in the cell lines where ErbB2 and Met were not identified as synthetic lethal with FGFR. We extended the study to find that the combination of knockdown of either ErbB2 or Met with FGFR inhibition decreased the growth of additional cell lines, indicating that the combination therapies could be broadly applicable. These results reveal a role for alternate RTKs in maintaining pro-growth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR and either ErbB family members or Met.

Species‐specific biological effects of FGF‐2 in articular cartilage: Implication for distinct roles within the FGF receptor family

Existing literature demonstrates that fibroblast growth factor-2 (FGF-2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF-2 plays a catabolic and anti-anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF-2 has been identified as an anabolic mediator as ablation of the FGF-2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species-specific differences in FGF-2-mediated biological effects. In this study, we provide a mechanistic understanding by which FGF-2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species-specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra-articular injection of FGF-2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF-2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF-2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future.

Abstract 3662: FGFR2 gene amplification and overexpression frequently occur in squamous cell carcinomas of the lung

Abstract Introduction: To date, only a few genetic abnormalities have been reported to be involved in tumorigenesis of squamous cell carcinomas (SqCCs) of the lung. Recently, fibroblast growth factor receptor (FGFR) 1 gene amplification was identified as a potential target for treatment in approximately 20% of SqCCs. In order to identify the clinical significance of FGFR family (FGFR1, FGFR2, FGFR3, and FGFR4) in SqCC, we performed a clinical study on the FGFR family in patients with non-small cell lung cancers (NSCLCs). Methods: Consecutive 138 NSCLC patients were studied. Gene expressions of FGFR family were evaluated by quantitative real-time RT-PCR. Intratumoral protein expressions of FGFR1, FGFR2, and Ki-67 were evaluated by immunohistochemistry. Gene amplifications of FGFR1 and FGFR2 were investigated by FISH. In addition, mutations of EGFR (exon 19-22) and KRAS (exon 1) were also investigated using either PCR-SSCP or PCR-RFLP, confirmed by direct sequencing. Results: Among 138 NSCLCs, 91 tumors were adenocarcinoma, 37 tumors were SqCCs, and 10 tumors were other types. Regarding FGFR gene expressions in NSCLCs, The FGFR2 gene expression was significantly higher in NSCLCs with both wild-type EGFR and wild-type KRAS than in NSCLCs with either mutant EGFR or mutant KRAS (P = 0.0012). FGFR2 gene expression was significantly higher in SqCCs than in adenocarcinomas (P < 0.0001). Furthermore, the FGFR2 gene expression was the highest among the gene expressions of FGFR family in SqCC. In contrast, no significant difference was observed in FGFR1 gene expression in relation to tumor histology. Regarding FGFR protein expressions in NSCLCs, the intratumoral FGFR2 protein expression significantly correlated with the FGFR2 gene expression (P < 0.0001, r = 0.5492), and the intratumoral FGFR2 protein expression was also significantly higher in SqCCs than in adenocarcinomas (P = 0.0008). Furthermore, the FGFR2 protein expression significantly correlated with Ki-67 proliferation index (P < 0.0001, r = 0.4677). In FISH analysis, FGFR1 gene amplification was detected in 34.3% (12/35) of SqCCs, and FGFR2 gene amplification was detected in 22.9% (8/35) of SqCCs. Furthermore, the Ki-67 proliferation index was significantly higher in SqCCs with FGFR2 gene amplification than in SqCCs without FGFR2 gene amplification (P = 0.0069). Conclusions: FGFR2 gene amplification and overexpression frequently occur and correlate with the tumor proliferation in SqCCs of the lung. FGFR2 could be a potential molecular target for the treatment of SqCCs of the lung as well as FGFR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3662. doi:1538-7445.AM2012-3662

Abstract 3858: Gains in FGF19 are predictive of response to the fibroblast growth factor receptor (FGFR) small molecule tyrosine kinase inhibitor BGJ 398 in vitro

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer death world-wide. Sorafenib, a multi-targeted small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor and other kinases is the only approved systemic agent in advanced HCC. Despite multiple studies defining the molecular heterogeneity of HCC, clinical trials of new agents do not pre-select for predictive markers. The fibroblast growth factor (FGF) receptor consists of 4 receptor tyrosine kinases and over 20 ligands. Differential expression and activation of these receptors and ligands have been implicated in oncogenesis and the pathogenesis of HCC (Sawey 2011). BGJ398 is a selective small molecule tyrosine kinase inhibitor of the FGFR family of receptors 1-4. Given the molecular diversity of HCC, we hypothesized that there would be a subset of HCC that would be more dependent on FGFR inhibition and used cell line models to identify predictive markers of response to BGK 398. Methods: 20 human HCC cell lines were used. In vitro dose response curves were generated using direct cell count assays over 6 days. IC 50 values were calculated from the dose response curves. Genomic data on the cell lines was derived using the Agilent 105K CGH platform. These data were analyzed in the context of the sensitivity data to BGJ 398. Western blot analysis was performed for effects of BGJ398 on phospho-FRS2Δ (tyr-196), a downstream signaling molecule for FGFR activation. Flow cytometry was performed for effects of BGJ 398 on the cell cycle and apoptosis. Results: There was differential sensitivity to BGJ 398 across the HCC cell lines. 3 cell lines were identified as having IC 50 values less than 1 μM versus the remaining cell lines which were all >1 uM. When analyzed in the context of the CGH data, all 3 cell lines had gains in FGF 19 of log2 ratio of 0.9 to 2.6. BGJ inhibited FRS2α activation in all cell lines tested. Flow cytometry revealed the BGJ induced cell cycle arrest in sensitive lines without significant apoptosis. Conclusions: Gains in FGF19 are predictive for growth inhibitory effects of BGJ398 in human HCC cell lines in vitro and provide rationale for patient selection in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3858. doi:1538-7445.AM2012-3858