FGFR Signaling as a Candidate Therapeutic Target for Cancers Resistant to Carbon Ion Radiotherapy

Abstract

Radiotherapy is an essential component of cancer therapy. Carbon ion radiotherapy (CIRT) promises to improve outcomes compared with standard of care in many cancers. Nevertheless, clinicians often observe in-field recurrence after CIRT. This indicates the presence of a subset of cancers that harbor intrinsic resistance to CIRT. Thus, the development of methods to identify and sensitize CIRT-resistant cancers is needed. To address this issue, we analyzed a unique donor-matched pair of clinical specimens: a treatment-naïve tumor, and the tumor that recurred locally after CIRT in the same patient. Exon sequencing of 409 cancer-related genes identified enrichment of somatic mutations in FGFR3 and FGFR4 in the recurrent tumor compared with the treatment-naïve tumor, indicating a pivotal role for FGFR signaling in cancer cell survival through CIRT. Inhibition of FGFR using the clinically available pan-FGFR inhibitor LY2874455 sensitized multiple cancer cell lines to carbon ions at 3 Gy (RBE: relative biological effectiveness), the daily dose prescribed to the patient. The sensitizer enhancement ratio was 1.66 ± 0.17, 1.27 ± 0.09, and 1.20 ± 0.18 in A549, H1299, and H1703 cells, respectively. Our data indicate the potential usefulness of the analytical pipeline employed in this pilot study to identify targetable mutations associated with resistance to CIRT, and of LY21874455 as a sensitizer for CIRT-resistant cancers. The results warrant validation in larger cohorts.

Keywords: FGFR; LY2874455; carbon ion radiotherapy; next-generation sequencer; radiosensitization; somatic mutations; uterine cervical cancer.

Figure 1

Quality assurance of next-generation sequencing data. (A) Verification of mutations in PIK3CA (c.1633G>A) by Sanger sequencing. Red arrows show variant. (B) Lego plots showing mutational patterns in a three-base context. The identified somatic single-nucleotide variants are grouped based on base substitution pattern and the neighboring bases.

Figure 2

Enrichment analysis for somatic non-synonymous mutations comparing T2 with T1. (A) Subtraction of VF_T1 from VF_T2. The genes showing subtraction values >10% are listed. (B) Sum of gene ontology (GO) enrichment scores for the genes listed in A (see Section 4.6 for the calculation of GO enrichment scores).

Figure 3

Concentration-dependent effect of LY2874455 (LY). (A) Immunoblots showing suppression of phosphorylation of extracellular signal-regulated kinase (ERK). Cells were exposed to LY for 1 h before collection. pERK, phosphorylated ERK. (B) Quantitation of immunoblots shown in A. The ratio of pERK to total ERK is shown relative to untreated controls and normalized to GAPDH. (C) Clonogenic survival of cells treated by LY28744554 alone. Dashed line indicates 40 nM.

Figure 4

Sensitizing effect of LY2874455 on carbon ions as assessed by clonogenic assays. Cells were exposed to LY2874455 (LY, 40 nM) for 1 h and irradiated with carbon ions for 3 Gy (RBE).