Crizotinib-Resistant EML4-ALK Mutations Figure 2: Spectrum of EML4-ALK mutations recovered in a mutagenesis screen. (A) resistant clones recovered from ENU-treated Ba ⁄ F3 cells expressing native EML4-ALK cultured with indicated concentrations of crizotinib. Each bar represents the relative percentage of the indi- cated EML4-ALK kinase domain mutant among recovered clones. The percentage of wells surveyed that contained outgrowth is indicated. (B) position of mutations in a model of crizotinib (shown in yellow) bound to the ALK kinase domain. (C) immunoblot analysis of p-ALK and down- stream signaling in Ba ⁄ F3 cells carrying native and mutant variants of EML4-ALK. phosphorylation was substantially reduced in all mutants tested, with IC50s >1000 nM for L1196M and G1269S (Table 2). A similar impairment was seen on the ability of crizotinib to inhibit down- stream signaling in cells expressing the L1196M mutant (Fig- ure S2A). We verified that crizotinib resistance was solely dependent on the mutations by analyzing Ba ⁄ F3 cell lines express- ing mutant EML4-ALK fusions generated by site-directed mutagene- sis and observing similar results (Table 2, 'Reintroduced'). The 16 mutations are located around the kinase active site and can be categorized into five groups, involved in either direct or indirect contacts with crizotinib. As shown in Figure 2B, group 1 is located around the gatekeeper position, group 2 is located around the kinase hinge region, group 3 is located near the ribose binding pocket, group 4 is located at the loop of the N-terminus of a-helix C, and group 5 is located in the vicinity of the kinase DFG (Asp- Phe-Gly) motif of the activation loop. The three mutations that con- ferred the strongest resistance were the L1196M gatekeeper resi- due, S1206R at the solvent front, and G1269S near the DFG motif. We characterized the sensitivity of these three mutants in mouse xenograft studies (Figure 3A). Ba ⁄ F3 cells expressing native EML4- ALK grew robustly as subcutaneous xenografts in SCID mice. Daily oral treatment of these mice with crizotinib at 100 mg ⁄ kg induced a modest tumor growth inhibition of 33%, which was not statisti- cally significant (p > 0.05), and 200 mg ⁄ kg [the highest reported daily oral dose for crizotinib in mice (14)] induced complete regres- sions by 12 days of treatment. However, analogous Ba ⁄ F3 xeno- grafts expressing L1196M, S1206R, or G1269S mutants were completely insensitive to these doses, with no statistically signifi- cant changes in tumor growth rate. In pharmacodynamic studies (Figure 3B), xenografts expressing native EML4-ALK exhibited a 60–70% inhibition in p-ALK levels at 6 h postdose, with more pronounced inhibition at 24 h. By contrast, p-ALK levels were reduced by approximately 25–35% at 6 h in tumors expressing L1196M or S1206R, with a partial recovery at 24 h. There was no significant inhibition in tumors expressing the G1269S mutation. Drug exposure was similar in all models, confirm- A B Figure 3: Crizotinib is not efficacious in mouse Ba ⁄ F3 xenograft models expressing EML4-ALK mutants. (A) In vivo efficacy of crizotinib in EML4-ALK-driven Ba ⁄ F3 models. Mean tumor volume €SEM was plotted. (B) PK ⁄ PD analysis after treating tumor-bearing mice with a single dose of vehicle (V, for 6 h) or 200 mg ⁄ kg crizotinib. Chem Biol Drug Des 2011; 78: 999–1005 1003