Abstract
Abstract Introduction: Anaplastic lymphoma kinase (ALK) translocations are present in an important subset of non-small-cell lung cancer (NSCLC) and predict for response to the multitargeted tyrosine kinase inhibitor (TKI) crizotinib. Splicing isoforms of ALK - including complete skipping of exon 27 - have been observed in association with full length EML4-ALK in crizotinib-naïve and treated NSCLCs; however their preclinical and clinical significance have not been previously determined. Methods: We generated pCDNA3.1-Myc-His-Tag and other constructs with full length EML4-ALK (E13;A20 - variant 1), EML4-ALK with the crizotinib-resistant ALK p.L1196M mutation and EML4-ALK with a stop codon following exon 26 (equivalent to the consequence of EML4-ALKdel27 [exon 26-28 sequence], ALK p.T1312fs*0). Protein extracts obtained from cells transfected with the aforementioned constructs were analyzed for their molecular weight, ability to auto-phosphorylate ALK and other tyrosine residues, and impact on the biochemical inhibition of crizotinib. Results: EML4-ALK T1312fs resulted in an ∼80kDa protein, while EML4-ALK and EML4-ALK L1196M were proteins of the predicted 117kDa molecular weight. The immunoprecipitation of the aforementioned EML4-ALK proteins disclosed that, unlike EML4-ALK and EML4-ALK L1196M, the truncated EML4-ALK T1312fs did not auto-phosphorylate ALK or other phospho-tyrosine sites. In addition, in contrast to EML4-ALK and EML4-ALK L1196M, EML4-ALK T1312fs was unable to induce transformation when constitutively expressed in a cell line. Co-expression of equal DNA amounts of EML4-ALK T1312fs and EML4-ALK did not result in resistance to crizotinib, as verified by ALK phosphorylation. In comparison, co-expression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. Conclusions: Our group performed the first comprehensive characterization of an ALK splicing variant in the context of the EML4-ALK translocation, a driver oncogene in NSCLC. EML4-ALKdel27 generated the protein EML4-ALK T1312fs, which lacked the full tyrosine kinase domain of ALK and had a molecular weight of ∼80kDa. This truncated EML4-ALK variant was unable to auto-phosphorylate itself, other tyrosine residues and had no transforming ability. In addition, EML4-ALKdel27 (i.e., EML4-ALK T1312fs) did not affect the exquisite sensitivity of full length EML4-ALK to the ALK TKI crizotinib. Therefore, we conclude that EML4-ALKdel27 is a non-functional splicing variant. Our observation is noteworthy - since routine next generation tumor DNA/RNA sequencing has progressively expanded into the care of NSCLC - and it suggests that if EML4-ALKdel27 is identified in tumor samples it should not alter therapeutic decisions or the selection of ALK inhibitors. Citation Format: Lorena F. Pontes, Hiroyuki Yasuda, Pasi A. Jänne, Daniel B. Costa, Susumu Kobayashi. Characterization of ALK splicing isoforms in EML4-ALK-translocated lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2013-1196
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