American Association for Cancer Research
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Supplementary Figure from Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK–Positive Anaplastic Large Cell Lymphoma

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posted on 2023-04-03, 17:07 authored by M. Ceccon, L. Mologni, G. Giudici, R. Piazza, A. Pirola, D. Fontana, C. Gambacorti-Passerini

Supplementary Figure 1. Cell selection scheme. For every resistant cell lines all selection steps are shown. Numbers represent AP26113 concentration (nanomolar) added to the medium. For each step a new cell line was established and stored frozen. Cell lines resistant to the highest drug doses were preliminarily tested for AP26113 IC50 value.



ALK is a tyrosine kinase receptor involved in a broad range of solid and hematologic tumors. Among 70% to 80% of ALK+ anaplastic large cell lymphomas (ALCL) are caused by the aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late-stage and metastatic cases of lung cancer. However, patients frequently develop drug resistance to Crizotinib, mainly due to the appearance of point mutations located in the ALK kinase domain. Fortunately, other inhibitors are available and in clinical trial, suggesting the potential for second-line therapies to overcome Crizotinib resistance. This study focuses on the ongoing phase I/II trial small-molecule tyrosine kinase inhibitor (TKI) AP26113, by Ariad Pharmaceuticals, which targets both ALK and EGFR. Two NPM-ALK+ human cell lines, KARPAS-299 and SUP-M2, were grown in the presence of increasing concentrations of AP26113, and eight lines were selected that demonstrated resistance. All lines show IC50 values higher (130 to 1,000-fold) than the parental line. Mechanistically, KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, whereas SUP-M2–resistant cells harbor several point mutations spanning the entire ALK kinase domain. In particular, amino acid substitutions: L1196M, S1206C, the double F1174V+L1198F and L1122V+L1196M mutations were identified. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI-resistant cases.Implications: This work defines reliable ALCL model systems of AP26113 resistance and provides a valuable tool in the management of all cases of relapse upon NPM-ALK–targeted therapy. Mol Cancer Res; 13(4); 775–83. ©2014 AACR.

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