P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

Abstract

The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%-5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression.

Keywords: (sh)RNA, small hairpin; ABC, adenosine triphosphate (ATP)-binding cassette; ALK; ALK, anaplastic lymphoma kinase; ATP, adenosine triphosphate; BAC, bronchioloalveolar carcinoma; BBB, blood–brain barrier; BCRP, breast cancer resistance protein; CAF, cyclophosphamide, doxorubicin, and fluorouracil; CSCs, cancer stem/initiating cells; CT, computed tomography; Ceritinib; Crizotinib; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; FISH, fluorescence in situ hybridization; IC50, half-maximal inhibitory concentration; IHC, immunohistochemical; IRB, institutional review board; K562/VCR, K562-derived vincristine-resistant; LCNEC, large cell neuroendocrine carcinoma; MRP1, multidrug Resistance-associated Protein 1; ORR, overall response rate; OS, overall survival; P-glycoprotein; P-gp, P-glycoprotein; PFS, progression-free survival; ROS1, v-ros avian ur2 sarcoma virus oncogene homolog 1; RPMI, Roswell Park Memorial Institute; Resistance; SP, side population; TKI, tyrosine kinase inhibitor; TNM, tumor-node-metastasis; Tyrosine kinase.

Fig. 1

Response to ceritinib and obtained tumor samples from ALK-rearranged NSLCL patient. (A) An axial CT scan of the chest showing the patient's tumor burden prior to ceritinib treatment (left), after response to ceritinib treatment (center), and at the time of disease progression while on ceritinib (right). (B) X-ray and CT scan of the chest showing accumulation of pleural effusion at the time of disease progression on ceritinib. The picture was taken 4 days before thoracentesis from which the JFCR013-2 cell line was established. (C) Treatment history of JFCR013 and time line of the treatment, examination, and sample acquisition. (D) A summary of all obtained specimens and genetic alteration of ALK analysis by Sanger sequencing of the EML4-ALK and ALK kinase domain and deep sequencing of the ALK kinase domain (Illumina TruSeq amplicon panel). (E) Break-apart FISH analysis with a 5′-ALK probe (red) and a 3′-ALK probe (green) of a formalin-fixed paraffin-embedded specimen from the patient. ALK rearrangement was indicated by the presence of individually isolated red 3′-ALK green probes. Nuclei were stained with 4′,6-diamidino-2-phenylindole.

Fig. 2

Ceritinib-refractory patient-derived cells were resistant to ceritinib and crizotinib but not to alectinib. (A–C) JFCR013-2 or H3122 cells were treated with the indicated concentrations of crizotinib, ceritinib, alectinib, or TAE684 for 72 h. After incubation, cell viability was measured using the CellTiter-Glo assay. Calculated IC50 values are shown in the table (C). (D–G) JFCR-013-2 and H3122 cells were treated with the indicated concentration of ceritinib (D), crizotinib (E), alectinib (F), or TAE684 (G) for 3 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies.

Fig. 3

P-gp was highly expressed in JFCR-013 cells. (A) P-gp expression in the indicated cell lines was analyzed by immunohistochemistry. (B) P-gp, ALK or β-actin protein expression analyzed by immunoblotting. (C) Cell surface P-gp expression analyzed by flow cytometry after incubation of each cell line with P-gp monoclonal antibody (blue) or control IgG (red). (D) ABCB1 or BCRP (ABCG2) mRNA expression levels analyzed by quantitative RT-PCR. Relative mRNA expression was calculated using β-actin (ACTB) mRNA expression as reference. ND (not detected) means no PCR amplicon of ABCB1 or ABCG2 was detected.

Fig. 4

P-gp overexpression was observed only in the tumor after ceritinib treatment. (A) IHC analysis of P-gp or ALK protein expression in JFCR-013-pre and ceritinib-resistant JFCR013-2 and JFCR013-6-1 (autopsy) specimens. (B) P-gp protein expression in the indicated cell lines and in autopsy specimens analyzed by immunoblotting. (C) ABCB1 mRNA expression level in autopsy specimens was analyzed by quantitative RT-PCR. In particular, JFCR013-6-4 reflects normal lung tissue. Relative mRNA expression was calculated using β-actin (ACTB) mRNA expression as a reference.

Fig. 5

P-gp overexpression confers ceritinib and crizotinib resistance. (A) P-gp protein expression in the indicated shRNA-induced cells analyzed by immunoblotting. (B) JFCR013-2- and shABCB1-induced JFCR013-2 cells treated with serially diluted concentrations of ceritinib or crizotinib for 72 h. After incubation, cell viability was measured by CellTiter-Glo Assay. Calculated IC₅₀ values are shown in the table. (C) P-gp and β-actin protein expression in H3122 and ABCB1-overexpressing H3122 cells analyzed by immunoblotting. (D, E) H3122 or ABCB1-overexpressing H3122 cells treated with the indicated concentrations of ceritinib (left) or crizotinib (right) for 72 h. After incubation, cell viability was measured using the CellTiter-Glo assay. Calculated IC50 values are shown in (E).

Fig. 6

Ceritinib is a P-gp substrate and P-gp inhibition reduces drug export. (A) JFCR013-2 cells and shABCB1-(#86) induced JFCR013-2 cells incubated with ¹⁴C-labeled ceritinib (100 nM) plus verapamil (10 μM) or MS209 (5 μM) at 37 °C. At the indicated time points, incorporated ¹⁴C-ceritinib was measured using a liquid scintillation counter. (B) K562 and P-gp-overexpressing K562/VCR cells incubated with ¹⁴C-labeled ceritinib (100 nM) plus verapamil (10 μM) or MS209 (5 μM) for 8 h at 37 °C. After washing the cells, incorporated ¹⁴C-ceritinib was measured using a liquid scintillation counter.

Fig. 7

P-gp inhibitor treatment sensitized the cells to ceritinib in vitro and in vivo. (A) JFCR-013-2 cells treated with the indicated concentrations of ceritinib plus verapamil (10 μM), MS209 (5 μM), or FTC (5 μM) for 72 h. After incubation, cell viability was measured using the CellTiter-Glo assay. Calculated IC50 values are shown in the lower table. (B, C) JFCR-013-2 cells treated with the indicated concentrations of ceritinib with or without P-gp inhibitor MS209 (5 μM) or verapamil (10 μM) for 3 h (B) or for 1–48 h (C). Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D, E) JFCR013-2 cells were subcutaneously injected into nude mice. After the tumor achieved a size of approximately 100 mm³, the mice were randomized by tumor size and daily treatment with 50 mg/kg of ceritinib with or without 200 mg/kg of MS209 was started. Tumor volumes were measured as follows: 0.5 × length × width × width. Tumor sizes relative to day 0 are shown in (D), and changes in body weight are shown in (E).

Fig. 8

P-gp overexpression in ALK-rearranged NSCLC clinical specimens. (A) ALK mutation status and P-gp expression status in an ALK-TKI-resistant tumor before and after ALK-TKI treatment. (B, E) IHC analysis of P-gp protein expression in pre-alectinib- and post-ceritinib-treated JFCR025 specimens (B) and pre-crizotinib-, post-crizotinib-/pre-ceritinib-, or post-ceritinib-treated MGH015 specimens (E). (C) H3122 and ceritinib-resistant patient-derived JFCR025 cells were analyzed by immunoblotting with the indicated antibodies. (D) JFCR025 cells were treated with the indicated concentrations of ceritinib, crizotinib, alectinib or PF-06463922 with or without MS209 (5 μM) for 72 h. After incubation, cell viability was measured using the CellTiter-Glo assay.