Alectinib

NCI-H3122 cells were provided by Pasi A. Jänne (DanaFarber Cancer Institute, Boston, MA). H3122 cells were maintained in RPMI 1640 medium with gentamicin (Gibco, Grand Island, NY) and supplemented with 10% fetal bovine serum (Gibco). The cell line was incubated at 37°C in a humidified atmosphere of 5% carbon dioxide. Crizotinib and alectinib were purchased from Selleck Chemicals (Houston, TX) and dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) for experiments. Ceritinib was obtained from Active Biochem (Maplewood, NJ). ALK inhibitor-resistant cell lines (CR1, LR1, and CH1) were established by exposing parental H3122 cells to Crizotinib, Ceritinib (LDK378), and alectinib (CH-5424802), respectively, at doses of 100 nM to 1 μM. Double-resistant cell lines (CR1LR1 and CR1CH1) were generated by exposing the Crizotinib-resistant cell line CR1 to Ceritinib and alectinib, respectively. The subclonal resistant cell lines exhibited ≥ 5-fold greater IC₅₀ values for ALK inhibitors than parental cells, as determined using the cell viability assay, and this phenotype was stable for at least 6 months without ALK inhibitor treatment.

Karpas-299 cells were plated 24 hours before alectinib (Selleckchem) treatment at 0.05 × 10⁶ cells/ml. alectinib dose-escalation studies (0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 nM) were performed in triplicate and terminated when untreated control cultures reached three population doublings. Given that 10 nM alectinib was the maximum concentration at which Karpas-299 proliferation was not inhibited, cells were treated with 10 nM alectinib and an escalating dose of rapamycin (0.1, 1, 10, 100, and 100 ng/ml) in triplicate and terminated when 10 nM alectinib alone control cultures reached three population doublings. Cell counts and viability were determined using a Vi-CELL Cell Viability Analyzer using trypan blue exclusion.