Translational Science Lorlatinib Treatment Elicits Multiple On- and Off-Target Mechanisms of Resistance in ALK-Driven Cancer Sara Redaelli 1 , Monica Ceccon 1 , Marina Zappa 1 , Geeta G. Sharma 1,2 , Cristina Mastini 1 , Mario Mauri 1 , Marion Nigoghossian 1,3 , Luca Massimino 1 , Nicoletta Cordani 1,4 , Francesca Farina 4 , Rocco Piazza 1,4 , Carlo Gambacorti-Passerini 1,2,4 , and Luca Mologni 1,2 Abstract Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non–small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interesting- ly, intracellular localization of NPM/ALKN 1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors con- firmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblasto- ma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorla- tinib treatment. Significance: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib. Cancer Res; 78(24); 6866–80. Ó2018 AACR. Introduction Activation of the anaplastic lymphoma kinase (ALK) is involved in the pathogenesis of different cancers, including ana- plastic large cell lymphoma (ALCL), non–small cell lung cancer (NSCLC) and neuroblastoma (1). ALK inhibitors (ALKi) were developed for specific treatment of ALK-positive patients (1, 2). Crizotinib demonstrated superior activity compared with chemo- therapy in NSCLC and showed exceptional response rates in refractory ALCL and inflammatory myofibroblastic tumor (IMT) patients (3–5). Unfortunately, the selection of drug-resistant clones has limited the long-term efficacy of crizotinib, especially in NSCLC (1, 6, 7). The knowledge of resistance mechanisms guided the quest for new drugs to overcome crizotinib failure. Several novel compounds were developed, by improving potency, selectivity, and brain penetration. Among these, lorlatinib (PF-06463922, a third-generation ALKi) showed activity against most drug-resistant mutants, including the highly refractory G1202R mutant (8–11). Indeed, ceritinib-resistant patient- derived cells carrying EML4/ALK mutations were shown to be sensitive to lorlatinib, while cells without ALK mutations were resistant (12), suggesting that resistance to this drug might arise from ALK-independent processes bypassing ALK dependency, as observed in a fraction of patients with NSCLC treated with other ALKi (6, 7, 13, 14). In such cases, drug combinations could provide effective therapeutic options (15, 16). On the other hand, compound mutations may also represent a big challenge, still poorly characterized. Indeed, a C1156Y/L1198F mutation was found in a patient relapsed on lorlatinib (17). Therefore, under- standing the mechanisms leading to tumor escape is a key to the development of better therapeutic choices. In this work, we investigated the spectrum of possible resistance mechanisms arising during lorlatinib treatment in ALK-dependent tumors. To this end, we kept ALCL, NSCLC, and neuroblastoma cells under selective pressure until drug-resistant clones evolved, in vitro and in vivo, from the original cell population. Materials and Methods Chemicals and cell lines Lorlatinib and crizotinib were provided by Pfizer. Ceritinib, erlotinib, afatinib, alectinib, and trametinib were purchased from 1 School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy. 2 European Research Initiative for ALK-Related Malignancies (ERIA), Cambridge, United Kingdom. 3 University Claude Bernard Lyon 1, Villeurbanne, France. 4 Hematology and Clinical Research Unit, San Gerardo Hospital, Monza, Italy. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Redaelli and M. Ceccon contributed equally to this article. Corresponding Author: Luca Mologni, School of Medicine and Surgery, Univer- sity of Milano-Bicocca, via Cadore 48, Monza 20900, Italy. Phone: 3902-6448- 8148; Fax: 3902-6448-8363; E-mail: luca.mologni@unimib.it doi: 10.1158/0008-5472.CAN-18-1867 Ó2018 American Association for Cancer Research. Cancer Research Cancer Res; 78(24) December 15, 2018 6866 on July 5, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst October 15, 2018; DOI: 10.1158/0008-5472.CAN-18-1867