Chem Biol Drug Des 2011; 78: 999–1005 Research Letter ª 2011 John Wiley & Sons A/S doi: 10.1111/j.1747-0285.2011.01239.x Crizotinib-Resistant Mutants of EML4-ALK Identiﬁed Through an Accelerated Mutagenesis Screen Sen Zhang, Frank Wang, Jeffrey Keats, Xiaotian Zhu, Yaoyu Ning, Scott D. Wardwell, Lauren Moran, Qurish K. Mohemmad, Rana Anjum, Yihan Wang, Narayana I. Narasimhan, David Dalgarno, William C. Shakespeare, Juan J. Miret, Tim Clackson and Victor M. Rivera* ARIAD Pharmaceuticals, Inc. Cambridge, MA 02139, USA *Corresponding author: Victor M. Rivera, victor.rivera@ariad.com Activating gene rearrangements of anaplastic lym- phoma kinase (ALK) have been identiﬁed as driver mutations in non-small-cell lung cancer, inﬂamma- tory myoﬁbroblastic tumors, and other cancers. Crizotinib, a dual MET ⁄ ALK inhibitor, has demon- strated promising clinical activity in patients with non-small-cell lung cancer and inﬂammatory myo- ﬁbroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK muta- tions that can confer resistance to crizotinib. Muta- tions at 16 residues were identiﬁed, structurally clustered into ﬁve regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identiﬁed in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively mod- est potency in ALK-positive non-small-cell lung can- cer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identiﬁes multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib’s narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. Key words: crizotinib, EML4-ALK, ENU mutagenesis, gatekeeper, resistance Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#Online Open_Terms Received 26 July 2011, revised 29 August 2011 and accepted for publi- cation 3 September 2011 Chromosomal translocations of anaplastic lymphoma kinase (ALK), originally identified in anaplastic large cell lymphoma (1), have now been found in multiple tumor types, including inflammatory myofib- roblastic tumors (IMT), and in 3–7% of non-small-cell lung cancers (NSCLC) (2,3). Activating mutations and ALK gene amplifications have also been detected in neuroblastomas (4). Preclinical studies demonstrate that ALK inhibition induces apoptosis and tumor regression in models of ALK-expressing tumors, identifying ALK as a driver mutation and underscoring its potential as a therapeutic target (5). Recently reported data from a phase 1 trial of crizotinib (PF-02341066), a dual MET ⁄ ALK inhibitor (6) in ALK-positive patients with NSCLC, revealed significant clinical efficacy (7). Combined with a response in a patient with ALK-positive IMT (8), these data pro- vide clinical validation of ALK as a target and proof-of-concept for the targeted use of ALK inhibitors in ALK-driven tumors. Treatment for tumors expressing driver kinases with targeted inhibi- tors commonly leads to acquired resistance because of point muta- tions in the kinase domain. In vitro accelerated mutagenesis screens are powerful methods for identifying such mutations (9,10) and have successfully predicted and recapitulated the spectra of mutations observed clinically – for example, following the treatment for chronic myeloid leukemia (CML) with different BCR-ABL inhibi- tors (10). In this study, we conducted a mutagenesis screen to iden- tify the potential resistance mechanisms to crizotinib in ALK-driven tumors and determined whether a more potent ALK inhibitor, TAE684 (11), could overcome resistance. Methods and Materials Cell lines and reagents H2228, H838, and H23 NSCLC lines were obtained from the Ameri- can Type Culture Collection (ATCC) and Ba ⁄ F3 cells from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). ATCC cell lines were authenticated by ATCC's routine Cell Biology Program and were used within 6 months of receipt. Ba ⁄ F3 cells were used within 6 months of receipt from DSMZ that authenticates human cell lines by routine multiparameter methods prior to accession. H3122 cells were obtained from NCI with no further authentication made. Crizotinib and TAE684 (Figure S1) were synthesized at ARIAD Pharmaceuticals. 999