Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non-Small Cell Lung Cancer Cells

Abstract

Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non-small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC.

Figure 1

Development of two ALK models resistant to alectinib and ceritinib conferring cross-resistance to crizotinib and all second-generation ALK inhibitors. (A and B) Second-generation ALK inhibitors resistant cells CHR H3122 (developed by exposing to alectinib) and LDKR H3122 (developed by exposing to ceritinib) were treated with alectinib or ceritinib for 4 days; cell viabilities were determined by WST assay. Resistant cells showed 10-fold lower drug sensitivity and were resistant to both second-generation ALK inhibitors. (C–E) Both resistant cell lines also showed different levels of resistance to the first-generation ALK inhibitor crizotinib and additional two novel ALK inhibitors, AP26113 and PF06463922. (Data were representative of three independent experiments)

Figure 2

Upregulation of activated p-ALK contributes to drug resistance. (A and B) Parental and resistant cells were treated with the indicated concentrations of drugs for 1 hour and then collected for protein isolation. Western blot was performed using the indicated primary antibodies. (C) Total RNA was isolated from cells and qRT-PCR was performed with ALK-specific primers according to manufacturer’s instructions. GAPDH primer was used for normalizing ALK mRNA expression levels (P < .001***, P < .0001****). (All experiments have been repeated three times)

Figure 3

RTKs alternative pathway activation contributes to resistance to second-generation ALK inhibitors. (A) Three hundred micrograms of proteins was isolated from cells and then used in the RTK array according to manufacturer’s instructions; compared with parental H3122 cells, CHR H3122 cells showed upregulation of phosphor-EGFR, and LDKR H3122 cells showed upregulation of phosphor-EGFR, phosphor-IGF-1R, and phosphor-HER3. (B) Total RNA was isolated from H3122, CHR H3122, and LDKR H3122 cells and analyzed by qRT-PCR, demonstrating overexpression of RTKs in the resistant cells (P < .001***, P < .0001****). (C and D) Cells were treated with indicated drugs for 1 hour; immunoblotting for RTKs in both resistant cell lines showed upregulation of phosphor-EGFR and phosphor-HER3 when compared with parental cells. (All experiments have been repeated three times)

Figure 4

Role of RTK ligands in the development of acquired resistance. (A) Total mRNA was isolated from cells and qRT-PCR was performed using specific primers for EGF, IGF, and NRG1. GAPDH was used as internal reference (P < .001***, P < .0001****). (B) Parental H3122 cells were seeded in 96-well plates overnight to allow for attachment and were incubated with stepwise increased concentrations of ALK inhibitors in the presence of indicated EGFR ligands (EGF and amphiregulin), HER3 ligand (NRG1), and IGF1R ligand (IGF) at a concentration of 100 ng/ml for 4 days. WST was performed to evaluate cell viabilities. (C–F) Parental H3122 cells were incubated with different concentrations of NRG1 and stepwise increased concentrations of ALK inhibitors for 4 days. WST assay was then performed.

Figure 5

The NRG1-HER3-EGFR axis is responsible for activated EGFR family–induced drug resistance. (A and B) Indicated concentrations of NRG1 and alectinib/ceritinib were added to cell culture media. Cells were incubated for 1 hour and lysed, and proteins were analyzed by Western blot with the indicated primary antibodies. (C) One thousand micrograms of protein was isolated for immunoprecipitation study; p-EGFR/p-HER3 was used as primary antibody to detect its expression in the HER3/EGFR pulled-down products. (D) Efficient knockdown of HER3 by three different siRNA constructs in H3122 cells. (E and F) H3122 cells and HER3 knockdown H3122 cells were incubated with stepwise increased concentrations of ALK inhibitors in the presence of 100 ng/ml of NRG1 for 4 days. WST was performed to evaluate cell viabilities.

Figure 6

Combination treatment to overcome resistance. (A) Both resistant cell lines CHR H3122 and LDKR H3122 were seeded in 96-well plates and cultured with indicated single drugs or 500 nM combination drugs for 4 days; WST assay was then performed. (B and C) Different concentrations of afatinib (from 31.25 nM to 500 nM) were combined with ALK inhibitors and added to cell culture media for 4 days. Viability was assessed by WST assay. (D and E) Proteins were collected after treatment with the indicated drugs for 2 hours, and immunoblotting was performed. Combination with afatinib induced downregulation of p-EGFR and its downstream signals.