AXL confers intrinsic resistance to osimertinib and advances the emergence of tolerant cells

Abstract

A novel EGFR-tyrosine kinase inhibitor (TKI), osimertinib, has marked efficacy in patients with EGFR-mutated lung cancer. However, some patients show intrinsic resistance and an insufficient response to osimertinib. This study showed that osimertinib stimulated AXL by inhibiting a negative feedback loop. Activated AXL was associated with EGFR and HER3 in maintaining cell survival and inducing the emergence of cells tolerant to osimertinib. AXL inhibition reduced the viability of EGFR-mutated lung cancer cells overexpressing AXL that were exposed to osimertinib. The addition of an AXL inhibitor during either the initial or tolerant phases reduced tumor size and delayed tumor re-growth compared to osimertinib alone. AXL was highly expressed in clinical specimens of EGFR-mutated lung cancers and its high expression was associated with a low response rate to EGFR-TKI. These results indicated pivotal roles for AXL and its inhibition in the intrinsic resistance to osimertinib and the emergence of osimertinib-tolerant cells.

Fig. 1

Osimertinib activated AXL in EGFR-mutated NSCLC cells in vitro. a Human tyrosine kinase phosphorylation array analysis of PC-9 cells in the presence or absence of osimertinib (100 nmol/L) for 72 h. b PC-9 cells were treated with osimertinib (100 nmol/L), lysed, and the indicated proteins detected by western blotting. c Nonspecific siRNA control, specific siRNA for AXL, HER3 or MET introduced into the indicated cells. After 24 h, the cells were incubated with or without osimertinib (100 nmol/L) for 72 h and cell viability was determined using MTT assays. *P < 0.05 for comparisons of osimertinib-treated cells with parental cells treated with nonspecific control siRNA. Comparisons by paired Student’s t tests. d PC-9 cells were treated for 72 h with the indicated siRNAs, or combinations of the indicated siRNAs and cell viability was determined using MTT assays. *P < 0.05 compared with the respective parental cells. Comparisons by paired Student’s t tests. e The indicated siRNAs were introduced into PC-9 cells. After 24 h, the cells were incubated with or without osimertinib (100 nmol/L) for 72 h and lysed, and the indicated proteins detected by western blotting. f Cell lines were treated with or without osimertinib (100 nmol/L) for 72 h. The cells were lysed and the indicated proteins were detected by western blotting with immunoprecipitation of the indicated proteins

Fig. 2

AXL protein expression inversely correlated with susceptibility to EGFR-TKIs. a The EGFR-mutated NSCLC cell lines PC-9, PC-9GXR, PC-9KGR, H1650, H1975, HCC4011, HCC827, H3255, and HCC4006 were lysed and the indicated proteins were detected by western blotting. b The IC50 values for the EGFR-TKIs gefitinib and osimertinib in EGFR-mutated NSCLC cells. The IC50 values for both gefitinib and osimertinib were significantly higher in cells expressing high levels of AXL compared to cells expressing low levels of AXL. P values were calculated using the Mann Whitney U test. c Correlation between the cytoplasmic AXL protein expression levels determined immunohistochemically and the response to treatment with EGFR-TKIs in EGFR-mutated NSCLC specimens from 46 patients. d Correlation between the expression levels of the cytoplasmic AXL protein, determined immunohistochemically, and response to treatment with osimertinib in EGFR-mutated NSCLC specimens from 11 patients

Fig. 3

AXL knockdown sensitized EGFR-mutated NSCLC cells to osimertinib in vitro. a Nonspecific siRNA control or AXL-specific siRNAs (#1 and #2) were introduced into PC-9, PC-9GXR, HCC4011, HCC827, and H3255 cells. After 24 h, the cells were incubated with osimertinib (100 nmol/L) or gefitinib (100 nmol/L) for 72 h and the cell growth was determined using MTT assays. The percentage of growth is shown relative to the growth of untreated control cells. b Quantitative determination of the inhibition of cell viability of high-AXL-expressing and low-AXL-expressing EGFR-mutant cells transfected with nonspecific siRNA control or AXL-specific siRNAs after treatment with osimertinib or gefitinib. Paired Student’s t tests were used for comparisons. c Nonspecific siRNA control or AXL-specific siRNAs were introduced into PC-9, PC-9GXR, and HCC4011 cells. After 24 h, the cells were incubated with osimertinib (100 nmol/L) for 1 h. The cells were lysed and the indicated proteins were detected by western blotting

Fig. 4

AXL inhibitor sensitized AXL-high EGFR-mutant NSCLC cells to osimertinib. a PC-9, PC-9GXR, HCC4011, and HCC827 cells were incubated with osimertinib in the presence or absence of AXL inhibitor NPS1034 (1 μmol/L) for 72 h and the cell viability was determined using MTT assays. Data are representative of three independent experiments that produced similar results. b, c The indicated cells were incubated with osimertinib (100 nmol/L) with or without NPS1034 (1 μmol/L) for 1 h (b) and 72 h (c). The cells were lysed and the indicated proteins were detected by western blotting

Fig. 5

AXL inhibitor prevented the emergence of drug-tolerant cells to osimertinib. a Drug-tolerant (DT) cells previously treated with 3 μmol/L osimertinib for 9 days were treated with the indicated concentrations of osimertinib for 72 h and their viability was assessed using MTT assays. b PC-9 parental cells and DT cells generated by treatment with 1 or 3 μmol/L osimertinib were lysed and the indicated proteins were detected by western blotting. c PC-9 parental cells and DT cells generated by treatment with 3 μmol/L osimertinib for 9 days were lysed and the indicated proteins were detected by western blotting with immunoprecipitation of the indicated proteins. d PC-9, HCC4011 parental cells, and DT cells generated by treatment with 3 μmol/L osimertinib were treated with 1 μmol/L of NPS1034, OSI906, or a combination of these agents for 72 h and the cell viability was assessed using MTT assays. e The indicated cells were incubated with osimertinib (3 μmol/L) with or without NPS1034 (1 μmol/L) for 1 h. The cells were lysed and the indicated proteins were detected by western blotting. f Cells were treated with DMSO, 100 nmol/L osimertinib, 1 μmol/L NPS1034, or a combination of 100 nmol/L osimertinib and 1 μmol/L NPS1034 for 3 weeks with the drugs replenished every 72 h. The plates were stained with crystal violet and visually examined. A plate representative of three independent experiments is shown

Fig. 6

AXL inhibition shrank PC-9 tumors treated with osimertinib in vivo. Stable PC-9 cell lines were generated by the introduction of short hairpin RNAs (shRNA) that mediated inhibition of AXL expression (#37 and #38) and control nontargeting (SCR) shRNA. a The cells were lysed and the indicated proteins were detected by western blotting. b Cells were incubated for the indicated times and their viability assessed using MTT assays. c Cells were incubated with gefitinib or osimertinib at the indicated concentrations for 72 h and the cell viability assessed using MTT assays. d Following the subcutaneous injection of the indicated cells into nude mice, vehicle (control) or osimertinib (2.5 or 5 mg/kg) were administered. Tumor volumes were determined and the results are plotted over time from the start of treatment (mean ± SEM). e Western blotting analysis of the presence of the indicated proteins from the harvested tumors as described for (d). f Quantification of proliferating cells, as determined by their Ki-67-positive proliferation index (percentage of Ki-67-positive cells) as described in the Methods. Columns, mean of five evaluated areas; bars, SD. Comparisons by paired Student’s t tests. g Representative images of PC-9 xenografts containing the indicated shRNAs following immunohistochemical staining with antibodies specific for human Ki-67. Bar, 100 μm

Fig. 7

AXL inhibitor with osimertinib inhibited growth of AXL-high tumors in vivo. a PC-9 cell-line-derived xenograft (CDX) tumors were treated with vehicle (control), NPS1034 50 mg/kg, osimertinib 5 mg/kg, or NPS1034 50 mg/kg plus osimertinib 5 mg/kg (n = 6 each). Tumor volumes were measured over time from the start of treatment and the results are shown (mean ± SEM). b PC-9 CDX tumors were treated with osimertinib (5 mg/kg) for 8 days followed by the continuous administration of 5 mg/kg osimertinib or in combination with 50 mg/kg NPS1034 (n = 7 each) administered. The results of tumor volume are plotted (mean ± SEM). c The indicated cells and patient-derived xenograft (PDX) tumors (TM00784) were lysed and the indicated proteins were detected by western blotting. d PDX tumors (TM00784) were untreated (control), treated with 50 mg/kg NPS1034, 5 mg/kg osimertinib, or a combination of 50 mg/kg NPS1034 and 5 mg/kg osimertinib (n = 4 each). The results of tumor volume are plotted (mean ± SEM). e PDX tumors (TM00784) were treated with 5 mg/kg osimertinib for 7 days followed by continuous administration of 5 mg/kg osimertinib or in combination with 50 mg/kg NPS1034 (n = 4 each). The results of tumor volume are plotted (mean ± SE)