Abstract 5595: ALK mutant-driven cell lines develop crizotinib resistance by secondary mutations and ALK-independent means

Abstract

Abstract The clinical success of crizotinib against mutant ALK-expressing cancers has generated great interest; however, the emergence of drug resistance is limiting its durability in some patients. In NSCLC, crizotinib resistance is linked to secondary mutations within EML4-ALK at amino acids L1196M, C1156Y, L1152R. A mutation at amino acid F1174L reduced the sensitivity to criozotinib in a patient with an inflammatory myofibroblastic tumor harboring a RANBP2-ALK translocation. The goal of our studies was to evaluate mechanisms of crizotinib resistance in a range of ALK-mutant expressing cell lines. Cells from ALCL (Karpas299), NSCLC (NCI-H3122 and NCI-H2228) or neuroblastoma (SKNSH and KELLY) developed resistance to crizotinib after being exposed to incremental increases in the compound over several weeks. NPM-ALK expressing Karpas299 cells independently acquired point mutations in ALK at amino acids N1178H and I1171N. As the concentration of drug was increased, cells that harbored the NPM-ALK N1178H mutation also expressed a mutation at G1269A. Each mutation enhanced the temperature dependent auto-phosphorylation activity of the kinase and diminished the inhibitory effect of crizotinib on downstream ALK signaling. Mutations in Karpas299 cells were also accompanied by up regulation of a drug efflux transporter, BCRP, whose heightened expression level was dependent on the presence of the compound. In contrast no additional mutations were discovered in ALK in crizotinib resistant NCI-H3122 and NCI-H2228 (EML4-ALK) or SKNSH and KELLY (ALK F1174L) cell lines. Instead both NCI-H3122 and NCI-H2228 resistant cell lines displayed increased levels of phospho-HER2 and HER3 and activation of AKT and ERK that may be due to autocrine secretion of a growth factor. NSCLC resistant lines also displayed an apparent change in phenotype with a mesenchymal-to-epithelial transition. SKNSH and KELLY are inherently less sensitive to crizotinib. We found that these cells may survive exposure to high levels of the compound by altering RET expression. Our observations represent potential mechanisms of resistance demonstrated in a small number of cell lines. It will be important to align these results with emerging data from drug-resistant patients to understand the multiple mechanisms by which resistance might develop. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5595. doi:1538-7445.AM2012-5595