Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors

Abstract

Aberrant forms of the anaplastic lymphoma kinase (ALK) have been implicated in the pathogenesis of multiple human cancers, where ALK represents a rational therapeutic target in these settings. In this study, we report the identification and biological characterization of X-376 and X-396, two potent and highly specific ALK small molecule tyrosine kinase inhibitors (TKIs). In Ambit kinome screens, cell growth inhibition studies, and surrogate kinase assays, X-376 and X-396 were more potent inhibitors of ALK but less potent inhibitors of MET compared to PF-02341066 (PF-1066), an ALK/MET dual TKI currently in clinical trials. Both X-376 and X-396 displayed potent antitumor activity in vivo with favorable pharmacokinetic and toxicity profiles. Similar levels of drug sensitivity were displayed by the three most common ALK fusion proteins in lung cancer (EML4-ALK variants E13;A20, E20;A20, and E6b;A20) as well as a KIF5B-ALK fusion protein. Moreover, X-396 could potently inhibit ALK kinases engineered with two point mutations associated with acquired resistance to PF-1066, L1196M, and C1156Y, when engineered into an E13;A20 fusion variant. Finally, X-396 displayed synergistic growth inhibitory activity when combined with the mTOR inhibitor rapamycin. Our findings offer preclinical proof-of-concept for use of these novel agents to improve therapeutic outcomes of patients with mutant ALK-driven malignancies.

©2011 AACR.

Figures

Figure 1. Structure and specificity of ALK inhibitors

(A) Chemical structure of PF-1066 compared to X-376. (B) Kinase dendrograms for PF-1066 and X-376. Note that racemic mixtures were used in these specific assays. The size of the red circle is proportional to the degree of inhibition as depicted in the figure legend. Numerical data as well as IC50 values for X-376 against specific kinases are listed in Supplemental Table 1. (C) A model of X-376 in the ATP binding site of the ALK kinase domain (only residues within 5Å of X-376 are shown). (D) Rotated view highlighting hydrogen bonds between X-376 and amide peptides of hinge residues (E1197 and M1199). In addition to hydrogen bonds A and B, which are also present in the PF-1066/ALK complex, X-376 can form two more hydrogen bonds, C and D, with the receptor, thereby further increasing its binding affinity.

Figure 1. Structure and specificity of ALK inhibitors

(A) Chemical structure of PF-1066 compared to X-376. (B) Kinase dendrograms for PF-1066 and X-376. Note that racemic mixtures were used in these specific assays. The size of the red circle is proportional to the degree of inhibition as depicted in the figure legend. Numerical data as well as IC50 values for X-376 against specific kinases are listed in Supplemental Table 1. (C) A model of X-376 in the ATP binding site of the ALK kinase domain (only residues within 5Å of X-376 are shown). (D) Rotated view highlighting hydrogen bonds between X-376 and amide peptides of hinge residues (E1197 and M1199). In addition to hydrogen bonds A and B, which are also present in the PF-1066/ALK complex, X-376 can form two more hydrogen bonds, C and D, with the receptor, thereby further increasing its binding affinity.

Figure 2. Potency of X-376 and X-396 in ALK mutant cell lines

(A-D) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion, H2228 lung cancer cells harboring the EML4-ALK E6a/b;A20 fusion, SUDHL-1 lymphoma cells containing NPM-ALK fusion, and SY5Y neuroblastoma cells with an activating mutation within the ALK kinase domain (ALK F1174L) were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details. (E) Apoptosis is induced by X-376 treatment. H3122 cells were treated with increasing concentrations of X-376 for 72h. Cells were stained with annexin V (AV) and propidium iodide (PI) and counted on a FACSCanto II machine. Viable cells are defined as the AV/PI double negative population. Apoptotic cells are defined as the sum of AV positive, PI negative plus AV/PI double positive cell populations. (F) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20. At 48 hours post transfection, the cells were treated with increasing amounts of the ALK TKIs for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (G) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion were treated with increasing amounts of ALK TKIs for 1 hour as indicated. Lysates were subjected to immunoblotting with the specified antibodies. The asterisks (*) in the ALK and phospho-AKT blot indicate an empty lane (no lysate loaded) on the gel.

Figure 2. Potency of X-376 and X-396 in ALK mutant cell lines

(A-D) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion, H2228 lung cancer cells harboring the EML4-ALK E6a/b;A20 fusion, SUDHL-1 lymphoma cells containing NPM-ALK fusion, and SY5Y neuroblastoma cells with an activating mutation within the ALK kinase domain (ALK F1174L) were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details. (E) Apoptosis is induced by X-376 treatment. H3122 cells were treated with increasing concentrations of X-376 for 72h. Cells were stained with annexin V (AV) and propidium iodide (PI) and counted on a FACSCanto II machine. Viable cells are defined as the AV/PI double negative population. Apoptotic cells are defined as the sum of AV positive, PI negative plus AV/PI double positive cell populations. (F) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20. At 48 hours post transfection, the cells were treated with increasing amounts of the ALK TKIs for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (G) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion were treated with increasing amounts of ALK TKIs for 1 hour as indicated. Lysates were subjected to immunoblotting with the specified antibodies. The asterisks (*) in the ALK and phospho-AKT blot indicate an empty lane (no lysate loaded) on the gel.

Figure 2. Potency of X-376 and X-396 in ALK mutant cell lines

(A-D) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion, H2228 lung cancer cells harboring the EML4-ALK E6a/b;A20 fusion, SUDHL-1 lymphoma cells containing NPM-ALK fusion, and SY5Y neuroblastoma cells with an activating mutation within the ALK kinase domain (ALK F1174L) were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details. (E) Apoptosis is induced by X-376 treatment. H3122 cells were treated with increasing concentrations of X-376 for 72h. Cells were stained with annexin V (AV) and propidium iodide (PI) and counted on a FACSCanto II machine. Viable cells are defined as the AV/PI double negative population. Apoptotic cells are defined as the sum of AV positive, PI negative plus AV/PI double positive cell populations. (F) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20. At 48 hours post transfection, the cells were treated with increasing amounts of the ALK TKIs for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (G) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion were treated with increasing amounts of ALK TKIs for 1 hour as indicated. Lysates were subjected to immunoblotting with the specified antibodies. The asterisks (*) in the ALK and phospho-AKT blot indicate an empty lane (no lysate loaded) on the gel.

Figure 3. X-376 and X-396 effectively inhibit the growth of H3122 in vivo

Athymic nu/nu female mice were injected subcutaneously with H3122 lung cancer cells harboring the EML4-ALK E13;A20 fusion. When tumors reached an average volume of 290mm3, mice were treated with X-376, X-396, or vehicle alone by oral gavage (n=6 per group). Tumor volumes (A) and mouse weights (B) were assessed every 2-3 days.

Figure 4. Effects of ALK TKIs against various ALK fusion variants and the EML4-ALK L1196M and C1156Y point mutations

(A) 293 cells were transiently transfected with expression plasmids encoding various ALK fusions. At 48 hours post transfection, the cells were treated with increasing amounts of X-376 for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (B-C) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20 WT alongside 3Flag-EML4-ALK E13;A20 L1196M (B) or 3Flag-EML4-ALK E13;A20 C1156Y (C). At 48 hours post transfection, the cells were treated with increasing amounts of the indicated ALK TKI for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. The exposure of the pALK blot was selected to highlight the difference in baseline phosphorylation between wild-type and mutants. (D) Western blot demonstrating EML4-ALK E13;A20 WT, L1196M, and C1156Y expression in Ba/F3 cell lines. (E) Ba/F3 cells expressing EML4-ALK E13;A20 WT, L1196M, or C1156Y were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details.

Figure 4. Effects of ALK TKIs against various ALK fusion variants and the EML4-ALK L1196M and C1156Y point mutations

(A) 293 cells were transiently transfected with expression plasmids encoding various ALK fusions. At 48 hours post transfection, the cells were treated with increasing amounts of X-376 for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (B-C) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20 WT alongside 3Flag-EML4-ALK E13;A20 L1196M (B) or 3Flag-EML4-ALK E13;A20 C1156Y (C). At 48 hours post transfection, the cells were treated with increasing amounts of the indicated ALK TKI for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. The exposure of the pALK blot was selected to highlight the difference in baseline phosphorylation between wild-type and mutants. (D) Western blot demonstrating EML4-ALK E13;A20 WT, L1196M, and C1156Y expression in Ba/F3 cell lines. (E) Ba/F3 cells expressing EML4-ALK E13;A20 WT, L1196M, or C1156Y were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details.

Figure 4. Effects of ALK TKIs against various ALK fusion variants and the EML4-ALK L1196M and C1156Y point mutations

(A) 293 cells were transiently transfected with expression plasmids encoding various ALK fusions. At 48 hours post transfection, the cells were treated with increasing amounts of X-376 for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (B-C) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20 WT alongside 3Flag-EML4-ALK E13;A20 L1196M (B) or 3Flag-EML4-ALK E13;A20 C1156Y (C). At 48 hours post transfection, the cells were treated with increasing amounts of the indicated ALK TKI for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. The exposure of the pALK blot was selected to highlight the difference in baseline phosphorylation between wild-type and mutants. (D) Western blot demonstrating EML4-ALK E13;A20 WT, L1196M, and C1156Y expression in Ba/F3 cell lines. (E) Ba/F3 cells expressing EML4-ALK E13;A20 WT, L1196M, or C1156Y were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details.

Figure 5. The mTOR inhibitor, rapamycin, is synergistic with X-396

H3122 (A) and H2228 (B) were treated with rapamcyin alone, X-396 alone, rapamycin plus X-396 for 72 hours. Cell titer blue assays were performed to assess proliferation as per methods. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. p values were calculated using the Wilcoxon rank-sum test. (C) H3122 cells were treated with rapamcyin alone, X-396 alone, or rapamycin plus X-396 at the indicated concentrations for 1 hour. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins.