Zhang et al. ing that crizotinib inactivity in the mutant ALK efficacy studies is because of the inadequate target inhibition. TAE684 is a previously described ALK inhibitor (11) that we have con- firmed to be substantially more potent and selective than crizotinib in ALK-driven NSCLC models (Table 1). TAE684 inhibited the viability of Ba ⁄ F3 cells expressing native EML4-ALK (IC50 8 nM) or the five mutants that conferred the greatest resistance to crizotinib (IC50s of 20–80 nM) all with substantial selectivity over parental, ALK-negative Ba ⁄ F3 cells (IC50 1283 nM; Table 2). Potent inhibition of p-ALK and downstream signaling was also observed (Table 2; Figure S2). Discussion In this study, we have used an accelerated mutagenesis strategy to identify an extensive set of mutations in ALK that can confer resistance to crizotinib. Alterations at 16 different amino acids were observed, with three of them, L1196M, S1206R and G1269S, rendering cells completely insensitive in mouse xenograft studies. Interestingly, use of an alternative approach, in which an ALK-posi- tive NSCLC cell line is exposed to increasing doses of crizotinib, led to the identification of one mutation, L1196M, that could confer resistance to crizotinib (15). Our results confirm that kinase domain mutations are a potential mechanism for acquired resistance to criz- otinib and identify a novel, sizable panel of specific candidate mutations for correlation with clinical studies. An important factor in the resistance susceptibility of crizotinib appears to be its relatively narrow window of activity against ALK- positive versus ALK-negative cell lines: a differential of approxi- mately 10- to 20-fold in our studies (Tables 1 and 2). This means that even modest potency reductions linked to single mutations may abrogate the selective activity of the compound. Ultimately, the range of ALK mutations observed clinically will depend on phar- macologic considerations, such as drug exposure and target inhibi- tion levels in patients. By analogy with CML (16), however, more potent ALK inhibitors should be able to overcome crizotinib-resistant mutants. Indeed, we show that a more potent and selective ALK inhibitor, TAE684, maintains substantial activity against the muta- tions that confer the greatest resistance to crizotinib, with all mutants inhibited with at least 15-fold selectivity over ALK-negative cells. Recently, three additional ALK inhibitors, AP26113 (15), CH5424802 (17), and X-396 (18), have also be shown to be capable of inhibiting the L1196M variant of ALK in preclinical studies. Con- sistent with our observations regarding TAE684, each of these com- pounds has also been shown to be a more potent and selective inhibitor of ALK than crizotinib. Most of the mutations can be rationalized based on structural anal- ysis (Figure 2B). The L1196M gatekeeper mutation likely sterically impedes crizotinib binding. S1206, located near the ribose binding pocket of ATP, makes a contact with crizotinib, in the docked model, that would be eliminated by the S1206R mutation. Finally, G1269 (with D1268) forms a small hydrophobic pocket that binds the 3-flu- oro-2,6-dichlorophenyl group of crizotinib. This interaction would be disrupted by the G1269S mutation. Other mutated residues likely stabilize the conformation of the crizotinib contact residues, includ- 1004 ing V1180 and R1181 (in contact with L1196); E1210 (in contact with S1206); and D1268, F1174, F1245, I1171, Y1278, and E1241 (in direct or indirect contact with G1269). The three residues in group 4 (T1151, L1152, and C1156) do not make direct contacts with criz- otinib, but likely have indirect conformational roles. TAE684, on the other hand, has limited molecular contact interactions with the gatekeeper residue L1196 as well as with G1269 of the DFG motif, according to the recently published crystal structure (19), and is thus less susceptible to these two mutations. However, TAE684 is quite sensitive to the S1206R mutation. Analysis of the crystal structure indicates that the mutated arginine 1206 is likely to form a stabilized side chain conformation by interacting with its neigh- boring two acidic residues (D1203 and E1210), and such a confor- mation may be incompatible with the optimized binding pose of TAE684 in the ALK protein. Several isolated mutations were at positions where activating muta- tions have previously been identified in ALK-expressing neuroblas- toma (F1174, T1151, I1171, F1245, and Y1278) (13,20,21). In particular, F1174 is one of the most frequently mutated residues in neuroblastoma, and the mutations of F1174 to Cys, Val, Ile, and Leu were observed in our screen. F1174 is at the loop C-terminal to the alpha-helix C and forms a hydrophobic patch with neighboring resi- dues including F1241 of the DFG motif. F1174L may therefore stabi- lize an active conformation that is both more oncogenic and less favored for crizotinib binding. This screen has several potential limitations. Ba ⁄ F3 cells in vitro are unlikely to faithfully recapitulate the cellular context of ALK-dri- ven primary human tumors. Additionally, mutation-focused screens do not probe alternative resistance mechanisms, such as gene amplification or upregulation of parallel signaling pathways. Never- theless, such screens have proved highly predictive with other kin- ases (10). Most importantly, the clinical relevance of our findings is supported by the recent identification, after completion of our study, of the L1196M and C1156Y mutations from a patient with NSCLC with acquired resistance to crizotinib (22) and a separate report identifying the F1174L mutation in an IMT patient with similar acquired resistance (23). Our studies suggest multiple additional mutations that have the potential to confer resistance to crizotinib in patients and provide guidance for the rational design and optimi- zation of potent and selective second generation drugs that may be able to overcome ALK-based mechanisms of resistance. Acknowledgments All authors are employees and shareholders of ARIAD Pharmaceuti- cals, Inc. References 1. Morris S.W., Kirstein M.N., Valentine M.B., Dittmer K.G., Shapiro D.N., Saltman D.L., Look A.T. (1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lym- phoma. Science;263:1281–1284. Chem Biol Drug Des 2011; 78: 999–1005