Cell line H3122 H2228 H838 H23 ND, not determined. ALK status EML4-ALK v1 EML4-ALK v3a ⁄ 3b ALK negative ALK negative Table 1: Inhibitory activity of crizotinib and TAE684 on ALK-posi- tive and ALK-negative NSCLC lines 100 mg ⁄ kg of crizotinib for 21 days reduced tumor growth in a dose-dependent manner, with 14% tumor regression observed as the best response to treatment (Figure 1B). To identify kinase domain mutants resistant to crizotinib, we first created a Ba ⁄ F3 cell line expressing native (unmutated) EML4-ALK variant 1. This cell line was inhibited by crizotinib with an IC50 of 132 nM, representing a selectivity differential of ninefold over parental Ba ⁄ F3 cells (Table 2). These assays guided us to use a crizotinib concentration range in our mutagenesis screens of 250– 2000 nM. Ba ⁄ F3 cells expressing native EML4-ALK were exposed to the DNA-modifying agent ENU, cultured in 96-well plates in the presence of crizotinib dilutions and monitored for cell growth. Growth was observed in all wells containing 250 nM crizotinib. Approximately, 60% of wells at 500 nM crizotinib showed out- growth. At higher concentrations (720, 1000, and 1440 nM), cell growth was observed in progressively fewer wells, with the only concentration showing no outgrowth being 2000 nM. Sequencing identified a total of 422 mutations representing amino acid exchanges at 16 different sites (Figure 2A). The spectrum of mutations was narrowed with increasing crizotinib concentrations, in terms of both the sites modified and the number of alternative amino acids identified at each position. Mutations at 15 different Crizotinib-Resistant EML4-ALK Mutations Crizotinib Cell growth (GI50, nM) 62 € 18 121 € 61 1307 € 270 1773 € 743 p-ALK (IC50, nM) 43 € 37 55 € 4 ND ND TAE684 Cell growth (GI50, nM) 1.5 € 0.6 3.8 € 2 406 € 217 618 € 283 p-ALK (IC50, nM) 1.0 3.7 ND ND sites were detected at 500 nM crizotinib, eight sites at 720 nM, six sites at 1000 nM, and two sites at 1440 nM. The mutated residues identified at the highest crizotinib concentrations in our screen were C1156, I1171, F1174, L1196, S1206, and G1269. Similar results were obtained in two additional experiments. Interestingly, one of the residues most frequently mutated in our screen, F1174, is also one of the most frequently identified posi- tions for activating mutations in neuroblastoma (13). The same is true for the residue R1275 of ALK; yet, mutations at this site were not recovered in our screen. Consistent with this, we found that the introduction of R1275Q into EML4-ALK had no negative impact on sensitivity to crizotinib (IC50 47 € 8 nM). We selected the ten most frequently identified mutants, each at a different residue, for further analysis (Table 2). As expected, the IC50 values for viability of Ba ⁄ F3 cells expressing these mutants were all above that for Ba ⁄ F3 cells expressing native EML4-ALK, with IC50s ranging from 231 to 981 nM. The three most resistant mutants, L1196M, S1206R, and G1269S, all had IC50s within two- fold of parental, ALK-negative, Ba ⁄ F3 cells (IC50 1176 nM). There was no evidence that the mutations increased the basal activity of ALK as assessed by levels of p-ALK and downstream signaling pro- teins (Figure 2C). However, the ability of crizotinib to inhibit ALK Table 2: Inhibitory activity of crizotinib and TAE684 on Ba ⁄ F3 cells expressing EML4-ALK mutants Ba ⁄ F3 line Parentalb Native EML4-ALK T1151K L1152V C1156Y I1171T F1174C L1196M S1206R E1210K F1245C G1269S Crizotinib Viability (IC50s, nM) ENU clonea 1176 € 282 132 € 45 231 € 106 237 € 25 489 € 48 393 € 134 479 € 40 981 € 113 681 € 176 318 € 143 425 € 100 953 € 213 Reintroduced N ⁄ A N ⁄ A 152 € 48 162 € 89 ND 413 € 62 319 € 90 1215 € 708 728 € 362 297 € 92 269 € 194 1196 € 649 p-ALK (IC50, nM) ENU clone N ⁄ A 102 € 70 ND ND 408 € 67 ND 165 € 112 1162 € 209 356 € 173 ND ND 1366 € 52 TAE684 Viability (IC50, nM) ENU clone 1283 € 348 8 € 1 ND ND 37 € 5 ND 40 € 2 20 € 3 80 € 9 ND ND 33 € 9 aENU clone: Ba ⁄ F3 cells obtained from the crizotinib mutagenesis screen. bGrown in the presence of 10 ng ⁄ mL IL-3. ND, not determined; N ⁄ A, not applicable; ALK, anaplastic lymphoma kinase. Chem Biol Drug Des 2011; 78: 999–1005 p-ALK (IC50, nM) ENU clone N ⁄ A 5 € 3.5 ND ND 11 € 6 ND 6.7 € 2.5 3.3 € 0.5 16 € 11 ND ND 35 € 1 1001