Ceritinib

PC9 cells were subcutaneously injected into 4- to 6-week-old NOD-scid IL2Rg^null (NSG) recipient mice, produced from in-house breeding colony, with breeders purchased from Jackson Laboratories. Each animal received two contralateral injections, containing 10⁶ tumor cells, suspended in 100 μL of 1:1 mix of RPMI/BME type 3 (R&D Systems). Three weeks after injections, animals were randomized into treatment and control groups, and subjected to daily oral gavage with 2 mg/kg osimertinib (ChemieTek, #CT-A9291), 25 mg/kg ceritinib (ChemieTek, #CT-LDK378), combination of the two drugs, or vehicle control (0.5% methyl cellulose/0.5% Tween 80). Tumor diameters, measured by electronic calipers, and animal weights were measured weekly. For tumor volume calculations, spherical shape of tumors was assumed. After four weeks of treatment, animals were euthanized, and tumors were weighted. The results were reproduced in two independent experiments, in both males and females. Xenograft studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the H. Lee Moffitt Cancer Center. Animals were maintained under AAALAC-accredited specific pathogen-free housing vivarium and care and veterinary supervision following the standard guidelines for temperature and humidity, with 12-hour/12-hour light/dark cycle.

Stably transduced Ba/F3 cells were cultured in RPMI medium supplemented with IL-3 (1 ng ml⁻¹). For the cell proliferation assay, Ba/F3 cells were transferred into IL-3 depleted RPMI medium, and cell growth was quantified in quadruplicates every 2–4 days by a luminescence assay (Promega). For cell viability assays and ALK inhibitor–dose-response curves, 2,000 Ba/F3 cells were plated in triplicates in 96-well plates with increasing concentrations of the ALK inhibitors crizotinib (LC laboratories), TAE-684 (ChemieTek), or ceritinib (ChemieTek) as indicated. All drugs were re-suspended in DMSO. The cell viability was assessed after 72 h by a luminescence assay (Promega). Results were normalized to cell growth in medium containing an equivalent concentration of DMSO. The inhibition curve was determined with GraphPad Prism 6.0 software using the ‘log(inhibitor) vs response – variable slope’ nonlinear regression model. For immunoblots, 10 million Ba/F3 cells were harvested after 2 h treatment with crizotinib, washed in ice-cold PBS, and lysed in RIPA buffer. All assays were independently performed at least twice and a representative experiment is shown.