Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer

Abstract

Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

Figures

Figure 1. FGFR1 is amplified in squamous-cell lung cancer

(A) Left panel: Significant (14) (FDR-value; x-axis) amplifications across all chromosomes (y-axis) in squamous-cell lung cancer (SQLC; n=155) as assessed by GISTIC. Right panel: Copy-number alterations (blue=deletion; white=copy number-neutral; red=amplification) at chromosome 8 (y-axis) across all SQLC samples (x-axis). Samples are ordered according to focal amplification of FGFR1. (B) Significant (G-score; y-axis) copy number changes in adenocarcinoma (AC; n=77), (black line) and SQLC (red dotted line) at chromosome 8. The q-value for the presence of 8p12 amplification is 8.82*10−28 for squamous-cell lung cancer and greater than 0.25 for adenocarcinoma. The chromosomal positions of FGFR1 (8p12) and MYC are highlighted (black arrows) (C) Frequency of FGFR1 amplification (% of samples ≥ copy number 4; y-axis) in non-SQLC lung cancer from a published dataset (14), AC and SQLC. P-values indicate statistical significance. (D) FISH analysis (green = control; red = FGFR1) of 153 SQLC samples (FGFR1-HA: copy number >9; FGFR1-LA: copy number >2 <9; FGFR1-N: copy number 2). Presented are example images from the three different FGFR1 amplification groups.

Figure 2. FGFR1 amplification and sensitivity to FGFR inhibition

(A) GI50-values (y-axis) of PD173074 across 83 lung cancer cell lines (x-axis). FGFR1-amplified (copy number≥4) cell lines are marked with asterisks. (B) Copy number alterations (x-axis, blue=deletion; white= copy number 2; red= amplification) on chromosome 8 with a zoom in on 8p12 (FGFR1 locus is highlighted) across all cell lines (y-axis). (C) Induction of apoptosis (difference between PD173074 at 1µM and DMSO control after 72h; y-axis) across 24 cell lines (x-axis; asterisks denote FGFR1 amplification copy number≥4) as measured by flowcytometry (after Annexin V/PI staining). (D) FGFR1-amplified cell lines were plated in soft agar and treated either with DMSO (control) or decreasing concentrations of PD173074. (E) Phosphorylation of FGFR and of downstream molecules in FGFR1-amplified (H1581, H520) and in FGFR1 wildtype (EGFR-mutant) cells (HCC827) after treatment with PD173074 as assessed by immunoblotting.

Figure 3. FGFR1-amplified cells are dependent on FGFR1 in vitro and in vivo

(A) Left panel: Viability (PD173074 treatment as compared to DMSO control) of FGFR1-amplified cells expressing wildtype or mutant (V561M) FGFR1 treated with PD173074 (0.5µM white bars; 1.0µM grey bars). Right panel: phosphorylation of FGFR in the FGFR1V561M and FGFR1wt cells detected by immunoblotting. (B) Upper panel: Viability (PD173074 treatment as compared to DMSO control; y-axis) of H1581 cells after transduction with control shRNA or shRNA targeting FGFR1. Right panel: Silencing of FGFR1 in H1581 cells was confirmed by immunoblotting. (C) In mice engrafted with H1581 cells either treated with vehicle or PD173074 (dosage as indicated; y-axis), tumor volume was measured over time (x-axis).