Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M ‘gate-keeper’ mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(±)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

Purpose: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. Results: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC₅₀ values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. Clin Cancer Res; 20(12); 3299–309. ©2014 AACR.

Purpose: We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design: FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and epidermal growth factor receptor (EGFR) dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results: FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed, whereas only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and extracellular signal-regulated kinase signaling in three HNSCC cell lines, whereas three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR tyrosine kinase inhibitors (TKI) with no contribution from FGFR pathways. Conclusions: FGFR signaling was dominant or codominant with EGFR in six HNSCC lines, whereas three lines exhibited little or no role for FGFRs and were highly EGFR dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. Clin Cancer Res; 17(15); 5016–25. ©2011 AACR.

The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non–small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. Cancer Res; 75(20); 4398–406. ©2015 AACR.