Multiple receptor tyrosine kinase activation related to ALK inhibitor resistance in lung cancer cells with ALK rearrangement

Abstract

The activation of alternative receptor tyrosine kinases (RTKs) is known to mediate resistance to ALK inhibitors. However, the role of multiple RTK activation in resistance has yet to be determined. Two crizotinib-resistant (H3122/CR-1 and H3122/CR-2) and one TAE684-resistant (H2228/TR) cell lines were established. Multi-RTK arrays and Western blots were performed to detect the activation of bypass signals. There were no secondary mutations in the sequencing. EGFR and MET were activated in H3122/CR-1 cells whereas EGFR and IGF1R were activated in H3122/CR-2 cells. Concomitant activation of MET did not contribute to resistance as crizotinib completely suppressed both p-MET and p-ALK in H3122/CR-1 cells, whose survival was not affected by crizotinib. However, combined inhibition of EGFR and ALK was effective in controlling this resistant cell line. In H3122/CR-2 cells, the inhibition of both ALK and IGF1R could effectively suppress cell growth, whereas simultaneous inhibition of ALK and EGFR brought about a less-effective suppression, indicating that IGF1R activation is the main resistance mechanism. H2228/TR cells showed activation of the HER family (EGFR, ErbB2, and ErbB3). Afatinib, a pan-HER inhibitor, was more potent in suppressing resistant cells than gefitinib when combined with crizotinib, which suggests that coactivation of ErbB2 and ErbB3 also contributes to resistance. Interestingly, all three resistant cell lines responded well to AUY922, which can inhibit ALK, EGFR, and IGF1R activity. Activation of multiple RTKs can occur during acquired resistance to ALK inhibitors, in which case the dominant or significant bypass signal should be identified to provide a more appropriate combination therapy.

Keywords: ALK; EGFR; IGF1R; lung cancer; resistance.

Figure 1. Development of acquired resistance to ALK inhibitors in the H3122 and H2228 cell lines

(A) Cells were treated with the indicated doses of crizotinib or TAE684 for 72 h, and cell viability was then determined using the MTT assay. (B) Cells were treated with the indicated doses of crizotinib or TAE684 for 6 h. Molecules associated with ALK signaling activity were detected using Western blot analysis. Densitometry values were determined relative to the control after normalization to the non-phosphorylated form of each protein. (C) Lentiviral constructs containing the negative control (NT) and ALK shRNAs were introduced into parental or resistant cells, and ALK suppression was confirmed by Western blot analysis. Cell viability was measured by cell counting. **P < 0.001 compared with negative control shRNAs.

Figure 2. Activation of other RTKs in acquired resistance to ALK inhibitors

(A) Cells were grown to confluence, and cell lysates were then prepared by protein extraction. 500 μg of the cell lysates were incubated on each membrane. A phospho-RTK array was performed as described in the Materials and Methods. (B) Lysates from each cell line were subjected to Western blot analysis. The indicated antibodies were used to evaluate the level of activated protein in Figure 2A.

Figure 3. Activation of EGFR and IGF1R in acquired resistance to ALK inhibitors

(A, B, and C) Cells were treated with a single drug or a combination of drugs, as indicated, for 72 h. Cell viability was measured using the MTT assay. (D, E, and F) Cells were treated with drugs as in the MTT assay. After 48 h, cells were harvested and subjected to Western blotting using the indicated antibodies. C, crizotinib; G, gefitinib; PHA, PHA 665752; BI, BI 836845; T, TAE684; A, apatinib.

Figure 4. Effect of EGF and IGF-1 on resistance to ALK inhibitors

H3122 (A) and H2228 (B) cells were treated with the indicated doses (μM) of crizotinib or TAE684 plus 100 ng/mL EGF or 100 ng/mL IGF-1. Cell viability was measured by cell counting. *P < 0.01, **P < 0.001, ***P < 0.0001 compared to crizotinib or TAE684 alone. CT, control; C, crizotinib; T, TAE684.

Figure 5. Response to an HSP90 inhibitor in H3122/CR and H2228/TR cell lines

(A) Cells were treated with the indicated concentrations of AUY922 for 72 h. Cell viability was measured using the MTT assay. (B) Cells were treated with the indicated concentrations of AUY922 for 48 h. Cell lysates were subjected to Western blotting using the indicated antibodies.