Abstract
Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non–small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib)-resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy.
Highlights
The anaplastic lymphoma kinase (ALK) fusion gene is found in about 3-5% of patients with non-small cell lung cancer (NSCLC) [1, 2]
We established three types of ALK-TKIresistant H2228 non–small cell lung cancer (NSCLC) cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelialmesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations
Because the low expression of E-cadherin suggested the occurrence of an epithelial-mesenchymal transition (EMT) phenotype [17], we evaluated the expression of other EMT-related genes: FN1, ZEB1, VIM encoding vimentin, and AXL were overexpressed in anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKI)-resistant cells (Figure 2A)
Summary
The anaplastic lymphoma kinase (ALK) fusion gene is found in about 3-5% of patients with non-small cell lung cancer (NSCLC) [1, 2]. Two major mechanisms of resistance to crizotinib in ALK-positive NSCLC patients have been previously described. ALK-positive NSCLC tumors showed multiple gatekeeper mutations, including 1151Tins, L1152R, C1156Y, F1174L, L1196M, G1202R, S1206Y and G1269A, after treatment with crizotinib. Another crizotinib-resistant mechanism is the activation of alternative survival signaling pathways, including EGFR activation, KIT activation having a KRAS mutation and IGF-1R activation [6, 7, 9,10,11]. The activation of bypass pathways has been found to be a mechanism of resistance to alectinib and ceritinib [12,13,14] Alternative signaling activation, such as MET against crizotinib, RET against alectinib and IGF-1R and INSR against ceritinib, has been reported [10, 15, 16]. The development of drug resistance in NSCLC patients with ALK is a major challenge that needs to be overcome
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