Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma

Daria Zdzalik,Anna Bujak,Monika Lamparska-Przybysz,Karolina Dzwonek,Barbara Dymek,Pawel Gunerka,Paulina Grygielewicz,Maciej Wieczorek

doi:10.1007/s00432-014-1589-3

Abstract

PurposeCrizotinib, the first FDA-approved ALK inhibitor, showed significant antitumor activity in young patients with anaplastic large-cell lymphoma (ALCL) frequently displaying ALK rearrangement. However, long-term therapeutic benefits of crizotinib are limited due to development of drug resistance. CH5424802—more potent and selective ALK inhibitor—comprises a good candidate for second-line treatment in crizotinib-relapsed patients. The aim of this study was to determine possible mechanisms of resistance to ALK inhibitors that can appear in ALCL patients.MethodsALK+ ALCL cell lines resistant to crizotinib (Karpas299CR) and to CH5424802 (Karpas299CHR) were established by long-term exposure of Karpas299 cells to these inhibitors. Next, alterations in their sensitivity to ALK, HSP90 and mTOR inhibitors were investigated by cell viability and BrdU incorporation assays and immunoblot analysis.ResultscDNA sequencing of ALK kinase domain revealed activating mutations—I1171T in Karpas299CR and F1174C in Karpas299CHR. The resistant cells displayed diminished sensitivity to structurally unrelated ALK inhibitors—crizotinib, CH5424802 and TAE684. Nevertheless, CH5424802 and TAE684 were still more potent against the resistant cells than crizotinib. Moreover, Karpas299CR and Karpas299CHR cells remained sensitive to HSP90 or mTOR inhibitors.ConclusionsResistance mediated by activating mutations in ALK kinase domain may emerge in ALCL patients during ALK inhibitors treatment. However, more potent second-generation ALK inhibitors, HSP90 or mTOR inhibitors may represent an effective therapy for relapsed ALK+ ALCL patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00432-014-1589-3) contains supplementary material, which is available to authorized users.

Highlights

Anaplastic lymphoma kinase (ALK) was initially discovered in anaplastic large-cell lymphoma (ALCL) as a component of fusion protein NPM-ALK formed as a result of the t(2;5)(p23;q35) chromosomal translocation (Morris et al 1994; Shiota et al 1995)
We found that I1171T and F1174C mutations in ALK kinase domain (KD) emerge as a mechanism of acquired resistance to crizotinib and CH5424802, respectively
To establish NPM-ALK+ ALCL Karpas299 cell lines resistant to crizotinib and CH5424802, two structurally unrelated ALK kinase inhibitors, Karpas299 cells were cultured with increasing concentrations of inhibitors for about 4 months

Summary

Introduction

Anaplastic lymphoma kinase (ALK) was initially discovered in anaplastic large-cell lymphoma (ALCL) as a component of fusion protein NPM-ALK formed as a result of the t(2;5)(p23;q35) chromosomal translocation (Morris et al 1994; Shiota et al 1995). The discovery of EML4ALK fusion in 2007 as oncogenic driver in non-small-cell lung cancer (NSCLC) and the identification of activating mutations in ALK gene in neuroblastoma made ALK one of the most extensively studied targets in the field of kinase inhibitor drug development (Chen et al 2008; George et al 2008; Janoueix-Lerosey et al 2008; Mosse et al 2008; Soda et al 2007). The most frequent aberration in ALK+ ALCL is the NPM-ALK fusion (Morris et al 1994; Swerdlow et al 2008). There are currently four ongoing clinical trials of crizotinib (NCT00939770, NCT01606878, NCT01524926, NCT00585195) and one of a dual ALK/EGFR inhibitor AP26113 (NCT01449461) in ALCL patients

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