Article Figures & Data
Figures
- Figure 1.
Human cell lines surviving at different crizotinib concentrations were characterized. A, ALK expression at protein level was assessed by Western blot analysis and quantified by densitometry, after normalization on actin expression level. B, cell lines were cultured for 72 hours in the presence of different crizotinib concentrations. Then, proliferation rate was measured as 3H-thymidine incorporation level. Each curve derives from 2 independent experiments. Corresponding IC50 values are summarized in Table 2.
- Figure 2.
Human cell lines carrying dominant NPM-ALK mutations were characterized. A, dose–response curves obtained by 3H-thymidine incorporation assay after 72-hour treatment with crizotinib, AP26113, and NVP-TAE684. IC50 values related are summarized in Table 4. B, ALK and STAT3 phosphorylation status was assessed by Western blot analysis after 4-hour incubation with different concentrations of crizotinib, AP26113, or NVP-TAE684. C, apoptosis was quantified after 72-hour treatment with crizotinib, AP26113, and NVP-TAE684 using Annexin V–PI staining and analyzed by flow cytometry. D, after 72 hours of incubation with crizotinib, AP26113, and NVP-TAE684, cell viability was analyzed by MTS assay. K299CR06 viability was significantly reduced after AP26113 (P < 0.0001) and NVP-TAE684 (P = 0.0008) exposure but not in response to crizotinib, whereas SUPM2CR03 were not affected by any kind of treatment. Values obtained derive from at least 2 independent experiments.
- Figure 3.
Characterization of Ba/F3 cell lines stably transfected with WT or mutated human NPM-ALK. A, cell lines were cultured for 72 hours in the presence of crizotinib, AP26113, and NVP-TAE684 at different concentrations; 3H-thymidine incorporation was then measured. Curves derive from at least 2 independent experiments. Corresponding IC50 values are summarized in Table 5. B, ALK phosphorylation status in the presence of increasing doses of crizotinib, AP26113, and NVP-TAE684. Activation of the main ALK downstream effector STAT3 is also shown. ACTIN was used as a loading control.
- Figure 4.
Molecular modeling studies show the interaction between ALK domain bearing L1196Q and I1171N substitutions in combination with crizotinib and NVP-TAE684. A, the position of residues L1196 and I1171 is displayed in the catalytic domain of the human WT-ALK kinase bound to NVP-TAE684 (PDB 2XB7). The ligand is displayed as capped sticks and colored by atom type (carbon atoms in green). The protein is displayed as secondary structure. The residues are colored by atom type (carbon atoms in orange) and displayed as capped sticks. B, docking pose of crizotinib in the human WT-ALK kinase (PDB 2XP2). The ligand is displayed as capped sticks and colored by atom type (carbon atoms in green). The human L1196Q-ALK mutant model is displayed as secondary structure. The residue Q1196-R1 is colored by atom type (carbon atoms in orange) and displayed as space fill. C, docking pose of NVP-TAE684 in the human L1196Q-ALK mutant model. The ligand is displayed as capped sticks and colored by atom type. The protein is displayed as secondary structure. The residue Q1196-R2 is colored by atom type (carbon atoms in orange) and displayed as capped sticks. H-bonds are represented by black dashed lines, respectively, between the donor and the acceptor. D, the R-spine of the catalytic domain of the human WT-ALK kinase bound to NVP-TAE684 (PDB 2XB7) is shown. The protein is displayed as secondary structure. The R-spine residues are colored by atom type and displayed as space fill.
Tables
- Table 1.
ALK mRNA expression was measured by qRT-PCR and normalized on parental cell line expression level
Normalized ALK expression K299 1 K299CR01 1.7 K299CR03 2.68 K299CR06 4.19 K299CR1 4.58 SUP-M2 1 SUPM2CR02 3.76 SUPM2CR03 1.52 - Table 2.
IC50 values obtained by 3H-thymidine incorporation test for each crizotinib-resistant human cell line are summarized
Cell line IC50 (μmol/L) Normalized K299 0.023 1.0 K299CR01 0.27 11.7 K299CR03 0.45 19.6 K299CR06 0.948 41.2 K299CR1 0.726 31.6 SUP-M2 0.056 1.0 SUPM2CR01 0.26 4.6 SUPM2CR02 0.25 4.5 SUPM2CR03 0.388 6.9 NOTE: Each value derives from 2 independent experiments.
- Table 3.
All mutations identified, corresponding substitutions and relative frequencies are presented
Cell line Dose (μmol/L) Mutation Substitution Frequency (%) K299 0.1 μmol/L 4539T→A L1196Q 12.5 4485A→G N1178S 3 4596C→T P1215L 3 4936G→A M1328I 3 0.3 μmol/L 4539T→A L1196Q 83.8 4521T→C L1190P 2.7 0.6 μmol/L 4539T→A L1196Q 85.7 1 μmol/L 4539T→A L1196Q 58 SUP-M2 0.2 μmol/L 4464–65TC→AT I1171N 50 0.3 μmol/L 4464–65TC→AT I1171N 100 - Table 4.
IC50 values, expressed in μmol/L, obtained for K299CR06 and SUPM2CR03 cell lines and the same values normalized on parental cell line, are summarized
Crizotinib AP26113 NVP-TAE684 IC50 Norm IC50 Norm IC50 Norm K299 0.014 1.00 0.004 1.00 0.001 1.00 K299CR06 0.651 46.50 0.016 4.00 0.019 12.88 SUP-M2 0.057 1.00 0.014 1.00 0.005 1.00 SUPM2CR03 0.388 6.81 0.112 8.00 0.052 10.40 NOTE: All values shown derive from at least 2 independent experiments.
- Table 5.
IC50 values, expressed in μmol/L, obtained for each Ba/F3 cell line and relative therapeutic index (TI) calculated as IC50 parental line/IC50 mutant, are summarized
CRIZOTINIB AP26113 NVP-TAE684 IC50 Norm T.I. IC50 Norm T.I. IC50 Norm T.I. Ba/F3 1.261 36.090 1.000 0.820 78.021 1.000 0.760 127.389 1.000 Ba/F3 NA WT 0.035 1.000 36.090 0.011 1.000 78.021 0.006 1.000 127.389 Ba/F3 L1196Q 0.212 6.076 5.940 0.009 0.856 91.111 0.009 1.509 84.444 Ba/F3 I1171N 0.215 6.156 5.862 0.329 31.256 2.496 0.759 127.254 1.001 Ba/F3 L1196M 0.459 13.125 2.750 0.042 3.983 19.589 0.018 3.022 42.152 NOTE: IC50 values obtained derive from at least 2 independent experiments.
- Table 6.
Docking scores of crizotinib and NVP-TAE684 on human WT and L1196Q ALK
WT-ALK (PDB 2XP2) WT-ALK (PDB 2XB7) L1196Q-ALK (Q-R1) L1196Q-ALK (Q-R2) Crizotinib 7.16 - ND 6.73 NVP-TAE684 - 5.67 4.47 5.63 Abbreviation: ND, not defined.
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file - 39K
- Supplementary Figure 1 - PDF file - 2845K, Cell viability and absolute cell number during cell selection.
- Supplementary Figure 2 - PDF file - 2629K, ALK phosphorylation status of Crizotinib resistant human cell lines.
- Supplementary Figure 3 - PDF file - 590K, Chromatograms representative of the two major mutations found on KARPAS299 and SUP-M2 cell lines.
- Supplementary Figure 4 - PDF file - 177K, ALK phosphorylation status for K299 and K299CR06 at low Crizotinib doses.
- Supplementary Figure 5 - PDF file - 2602K, SUPM2CR03 cell line is resistant even at higher doses of AP26113 and NVP-TAE684 (100nM).
- Supplementary Figure 6 - PDF file - 9018K, Chromatograms show the presence of the correct point mutations in our site-directed mutagenized Ba/F3 NPM-ALK cell lines.
Volume 11, Issue 2
