The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer

Abstract

Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation of the next-generation ALK TKI, ceritinib (LDK378), in the setting of crizotinib resistance. An interrogation of in vitro and in vivo models of acquired resistance to crizotinib, including cell lines established from biopsies of patients with crizotinib-resistant NSCLC, revealed that ceritinib potently overcomes crizotinib-resistant mutations. In particular, ceritinib effectively inhibits ALK harboring L1196M, G1269A, I1171T, and S1206Y mutations, and a cocrystal structure of ceritinib bound to ALK provides structural bases for this increased potency. However, we observed that ceritinib did not overcome two crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these mutations was identified in 5 of 11 biopsies from patients with acquired resistance to ceritinib. Altogether, our results demonstrate that ceritinib can overcome crizotinib resistance, consistent with clinical data showing marked efficacy of ceritinib in patients with crizotinib-resistant disease.

Significance: The second-generation ALK inhibitor ceritinib can overcome several crizotinib-resistant mutations and is potent against several in vitro and in vivo laboratory models of acquired resistance to crizotinib. These findings provide the molecular basis for the marked clinical activity of ceritinib in patients with ALK-positive NSCLC with crizotinib-resistant disease. Cancer Discov; 4(6); 662-73. ©2014 AACR. See related commentary by Ramalingam and Khuri, p. 634 This article is highlighted in the In This Issue feature, p. 621.

Figure 1. Ceritinib is a potent ALK inhibitor on crizotinib naïve models

A-B) Cell survival assay of H3122 (A) and H2228 (B) cells treated with the indicated doses of crizotinib or ceritinib for 72 hours. The cell survival was assayed by Cell-Titer-Glo. C-D) H3122 (C) and H2228 (D) cells were treated with the indicated concentrations of crizotinib or ceritinib for 6 hours. Lysates were probed with antibodies directed against the specified proteins. E) SCID beige bearing H2228 cells were administered Crizotinib or ceritinib orally once daily for 14 days. The arrow indicated when treatments were stopped and tumor growth was monitored in animals up to 4 months. Tumor volumes are presented as mean +- SD (n=8).

Figure 2. EML4-ALK L1196M and G1269A mutations are sensitive to ceritinib in vitro

A) GI50 values of cell survival assay for crizotinib or ceritinib in cell lines harboring L1196M or G1269A crizotinib resistant-mutations. B-D) H3122 CR1 (B), MGH021-4 (C) and MGH045 (D) cells were treated with the indicated concentrations of crizotinib or ceritinib for 6 hours. Lysates were probed with antibodies directed against the indicated proteins. E) Nude mice bearing MGH045 cell line were treated with 100 mg/kg crizotinib or ceritinib 25 mg/kg. Tumor volumes are presented as mean +- SD (n=6).

Figure 3. Ceritinib is active on ALK wild-type crizotinib resistant cell line

A) Abdominal computed tomography (CT) images of patient MGH051 prior to treatment with crizotinib and after 11 weeks of crizotinib. Several new hepatic metastases (yellow arrows) were detectable after crizotinib treatment consistent with disease progression. A repeat biopsy of a hepatic metastasis was performed within 2 weeks of crizotinib discontinuation. B) MGH051 cells were treated with the indicated doses of crizotinib or ceritinib for 7 days. After the incubation, the cell survival was assayed by Cell-Titer-Glo. C) MGH051 cells were treated with the indicated concentrations of crizotinib or ceritinib for 24 hours. Lysates were probed with antibodies directed against the indicated proteins.

Figure 4. Ba/F3 models of ALK-crizotinib resistant mutations

A) IC50 of ceritinib across different Ba/F3 cell lines expressing wild-type or mutated ALK TK and including parental, IL-3–dependent Ba/F3 cells are shown. B-C) ALK resistant mutations mapped onto ALK/ceritinib (PDB 4MKC) (B) and ALK/crizotinib (PDB 2XP2) (C) co-crystal structures. Beta strand secondary structural elements of N-terminal lobe and the αC-helix of the N-terminal lobe are shown in orange and purple, respectively. Helical structural elements of the C-terminal lobe are shown in blue. Residues of the activation loop (A-loop) and catalytic loop are shown in red and orange, respectively. Residues involved in resistant mutations are depicted as green spheres. Inhibitor molecules are depicted as stick representations with carbons colored yellow and cyan for crizotinib and ceritinib, respectively. Nitrogens are colored dark blue, oxygens colored red, and chlorine green for both inhibitors. Fluoride is colored white (crizotinib) and sulfur atoms are colored yellow (ceritinib). Transparent surfaces for the inhibitors are displayed. Zoomed in view boxes for G1269 and L1196 residues are shown. Figures were rendered with MacPymol (The PyMOL Molecular Graphics System, Version 1.4 Schrödinger, LLC).

Figure 5. EML4-ALK C1156Y, I1171T, G1202R mutations sensitivity to ceritinib

A-D) SCID beige mice bearing H2228 crizotinib resistant tumors EML4-ALK wild-type (A), I1171T (B), C1156Y (C) or G1202R (D) were treated with 100 mg/kg crizotinib or 50 mg/kg ceritinib, once daily for 12 to 22 days. Tumor volumes are presented as mean +- SD (n=5-8).

Figure 6. Ceritinib resistant tumors acquired mutations at positions G1202 or F1174

A) ALK mutational status in ceritinib resistant patient tumors before and after ceritinib treatment. B) Thoracic computed tomography (CT) images of patient MGH011 during crizotinib or ceritinib treatments. Sites of biopsies (red arrows) revealed the presence of different ALK secondary mutations throughout the treatments. Tumor growth observed during ceritinib treatment is consistent with disease progression.