Zhang et al. Unambiguous structural assignments were made by routine spectro- scopic methods including NMR, LC-MS, and CHN analyses. In vitro cell growth, viability, and signaling Cells were treated with crizotinib, TAE684, or vehicle (DMSO) for 72 h. The effect on NSCLC growth was assessed using CyQuant (Invitrogen). The concentration causing 50% growth inhibition (GI50) was deter- mined by subtracting the cell count at time zero and plotting relative to vehicle-treated cells. The effect on Ba ⁄ F3 cell viability (IC50) was assessed using CellTiter-96 AQueous One (Promega, Madison, WI, USA) and plotting viable cells relative to vehicle-treated cells. Cell lysates prepared after 2-h treatment with compound were ana- lyzed by immunoblotting, using antibodies against p-ALKY1604, total- ALK, p-STAT3Y705, p-AKTS473, p-ERK1 ⁄ 2T202 ⁄ Y204, p-S6PT240 ⁄ 244, or by PathScan Sandwich ELISA against p-ALKY1604 and total-ALK (Cell Signaling, Danvers, MA, USA). EML4-ALK cloning and generation of cell lines The native EML4-ALK variant 1 gene (Genbank: BAF73611.1; BlueSky Biotech, Worcester, MA, USA) was cloned into the retroviral vector pMSCV-Neo (Clontech, Mountain View, CA, USA), which was intro- duced into Ba ⁄ F3 cells by retroviral transduction. After the selection, cells were grown in the absence of IL-3. Plasmids encoding specific EML4-ALK mutations were generated with the QuikChange site- directed mutagenesis kit (Stratagene, Santa Clara, CA, USA). In vitro mutagenesis screen Ba ⁄ F3 cells expressing native EML4-ALK were treated overnight with 100 lg ⁄ mL N-ethyl-N-nitrosourea (ENU; Sigma Aldrich, St. Louis, MO, USA) and then distributed into 96-well plates containing 250, 500, 720, 1000, 1440, or 2000 nM crizotinib. The cells were grown in standard growth medium, without IL-3, for 5 weeks. Cells from the wells containing substantial outgrowth were expanded under the original selection conditions, genomic DNA extracted, and the ALK kinase region sequenced by Taq DyeDeoxy Terminator Cycle Sequencing (Applied Biosystems, Carlsbad, CA, USA). Model of crizotinib bound to ALK A homology model of ALK was built based on the crystal structure of activated insulin kinase (PDB code: 1ir3) using PRIME (v2.0; Schro- dinger). Crizotinib was docked into ALK using GLIDE SP (v5.0; Schro- dinger) with postdocking minimization and the top-scoring pose chosen for further analysis. Subcutaneous tumor models H3122 or Ba ⁄ F3 cells expressing EML4-ALK were implanted into the right flank of female Severe Combined Immunodeficiency (SCID) Beige mice. Crizotinib or vehicle (water) was administered once daily by oral gavage and mean tumor volume (L · W 2 · 0.5) calculated for each group. Tumor growth inhibition (TGI) or regression (TR) was calculated as follows: TGI = (1 ) DT ⁄ DC) · 100 was used when DT > 0, where DT and DC represent the mean tumor volume 1000 changes in treatment and control groups, respectively. When DT < 0, the formula TR = (DT ⁄ Ti) · 100 was used where Ti is the mean tumor volume for the group at the start of treatment. Tumor mea- surement data were analyzed with a one-way ANOVA test (GRAPHPAD PRISM, La Jolla, CA, USA). Statistical significance was determined using Dunnett's test. P-ALK levels were measured in homogenized tumors by ELISA. Crizotinib concentrations in plasma were deter- mined by LC ⁄ MS ⁄ MS. Results To understand the potential impact of resistant mutations on crizoti- nib efficacy, we first characterized its activity in in vitro and in vivo models of NSCLC. In H3122 cells, which express EML4-ALK variant 1, crizotinib inhibited ALK phosphorylation (p-ALK) with an IC50 of 43 nM and cell growth with a GI50 of 62 nM (Figure 1A and Table 1). This was accompanied by inhibition of p-ERK and p-S6P, although with minimal effects on STAT3 phosphorylation. Similar results were obtained with H2228 cells, which express EML4-ALK variant 3 (12). By contrast, IC50 values for two ALK-negative NSCLC cell lines were >1000 nM (Table 1). These data establish that crizotinib differentially inhibits the growth of EML4-ALK NSCLC cell lines relative to ALK- negative cells with approximately 10- to 20-fold selectivity. We also characterized the activity of crizotinib in a mouse H3122 xenograft model. Once daily oral administration of 25, 50, or A B Figure 1: Crizotinib inhibits signaling and proliferation in EML4- ALK-dependent NSCLC cell lines. (A) immunoblot analysis of p-ALK and downstream signaling in H3122 cells treated with crizotinib. (B) In vivo efficacy of crizotinib in an H3122 subcutaneous xenograft model. Mean tumor volume €SEM is plotted. Chem Biol Drug Des 2011; 78: 999–1005