Fibroblast growth factor receptor 3 overexpression mediates ALK inhibitor resistance in ALK-rearranged non-small cell lung cancer

Abstract

The rearrangement of anaplastic lymphoma kinase (ALK) occurs in 3%-5% of patients with non-small cell lung cancer (NSCLC) and confers sensitivity to ALK-tyrosine kinase inhibitors (TKIs). For the treatment of patients with ALK-rearranged NSCLC, various additional ALK-TKIs have been developed. Ceritinib is a second-generation ALK-TKI and has shown great efficacy in the treatment of patients with both newly diagnosed and crizotinib (a first-generation ALK-TKI)-refractory ALK-rearranged NSCLC. However, tumors can also develop ceritinib resistance. This may result from secondary ALK mutations, but other mechanisms responsible for this have not been fully elucidated. In this study, we explored the mechanisms of ceritinib resistance by establishing ceritinib-resistant, echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive H3122 cells and ceritinib-resistant patient-derived cells. We identified a mechanism of ceritinib resistance induced by bypass signals that is mediated by the overexpression and activation of fibroblast growth factor receptor 3 (FGFR3). FGFR3 knockdown by small hairpin RNA or treatment with FGFR inhibitors was found to resensitize the resistant cells to ceritinib in vitro and in vivo. FGFR ligands from either human serum or fetal bovine serum were able to activate FGFR3 and induce ceritinib resistance. A detailed analysis of ceritinib-resistant patient-derived specimens confirmed that tyrosine-protein kinase Met (cMET) amplification induces ceritinib resistance. Amplified cMET counteractivated EGFR and/or Her3 and induced ceritinib resistance. These results reveal multiple ceritinib resistance mechanisms and suggest that ceritinib resistance might be overcome by identifying precise resistance mechanisms.

Keywords: ALK kinase; FGFR3; ceritinib; drug resistance; lung cancer.

FIGURE 1

Anaplastic lymphoma kinase (ALK) amplification in the H3122 LR1 and LR5 and ALK L1198F mutation in the H3122 LR2 conferred intermediate and high resistance to ceritinib, respectively. A, C, G, The H3122 and H3122 LR cells were treated with the indicated concentrations of ALK inhibitors for 72 h. Calculated IC50 values are shown in (D). B, The ceritinib (LDK378)‐resistant H3122 cells were established by treating the parental H3122 (H3122 pt) cells with increasing concentrations of ceritinib over 6 months. E, The H3122 and H3122 LR1 and LR5 cells were treated with the indicated concentrations of ceritinib or crizotinib for 6 h. After incubation, the cells were lysed and analyzed by immunoblotting. F, Genomic DNA was extracted from five cell lines, and each relative ALK gene copy number was analyzed by qRT‐PCR. H, The Ba/F3 expressing EML4‐ALK‐WT or ‐L1198F cells were treated with the indicated concentrations of ceritinib for 4 h. After incubation, the cells were lysed and analyzed by immunoblotting.

FIGURE 2

Ceritinib resistance was induced by ligand‐dependent activation of FGFR3 in the H3122 LR3 cells. A, C, The H3122 and H3122 LR3 cells were treated with various concentrations of ceritinib with or without erlotinib (1 μM), cabozantinib (1 μM), or infigratinib (100 nM) for 72 h. B, The phosphorylation levels of 49 RTKs in the H3122 and H3122 LR3 cells were measured using an RTK array with cell lysates treated with or without 1 μM of ceritinib for 8 h. D, The H3122 LR3 cells were treated with ceritinib (1 μM) with or without infigratinib (100 nM) for 1–48 h. After incubation, the cells were lysed and analyzed by immunoblotting.

FIGURE 3

FGFR3 knockdown in H3122 LR3 cells restored sensitivity to ceritinib. A, Quantitative real‐time PCR was used to measure FGFR (FGFR1‐4) mRNA expression levels in the H3122 and H3122 LR3 cells. The H3122 LR3 mRNA expression levels (FGFRs/GAPDH) relative to those in the H3122 parental cells are shown in the bar graph. B, FGFR3 protein expression in the indicated shRNA‐infected H3122 LR3 cells was analyzed by immunoblotting. C, H3122 LR3 and shFGFR3 H3122 LR3 cells were treated with the indicated concentrations of ceritinib for 72 h. D, The H3122 LR3 and shFGFR3 H3122 LR3 cells were treated with 1 μM ceritinib for the indicated amount of time (0–2 h). After incubation, the cells were lysed and analyzed by immunoblotting. E, H3122 LR3 control cells and H3122 LR3‐sh374 cells were plated in RPMI‐1640 containing the indicated concentrations of human AB serum (Mediatech). Beginning the next day, they were cultured with 300 nM ceritinib for 0–96 h.

FIGURE 4

Combined treatment with alectinib and the FGFR inhibitor, zoligratinib, effectively resensitized H3122 LR3 cells to ceritinib in vivo. A, The H3122 LR3 cells were treated with various concentrations of alectinib with or without 1 μM of zoligratinib for 72 h. B, Schematic representation of the treatment schedule. After the tumors reached sizes of approximately 150 mm³, the mice were randomized by tumor size, and daily treatment with 50 mg/kg of alectinib with or without 50 mg/kg of zoligratinib was initiated. The tumor volumes were measured as 0.5 × length × width × height. The mean tumor volumes are shown in (C). *p < 0.05.

FIGURE 5

New ceritinib resistance mechanisms and therapeutic strategies to overcome them were identified. A diagrammatic representation of the identified ceritinib resistance mechanisms mediated by anaplastic lymphoma kinase (ALK) mutation (left) and bypass pathway activation resulting from FGFR3 or cMET overexpression.