Identification and characterization of ALK kinase splicing isoforms in non-small-cell lung cancer

Abstract

Introduction: Anaplastic lymphoma kinase (ALK) rearrangements are present in an important subset of non-small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC.

Methods: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations and in addition generated constructs with full-length echinoderm microtubule-associated protein-like 4 (EML4)-ALK (E13;A20) and a splicing isoform.

Results: Splicing isoforms of the kinase domain of ALK-including complete skipping of exon 23 (ALKdel23, ALK p.I1171fs*42) and exon 27 (ALKdel27, ALK p.T1312fs*0)-were identified in 11.1% (30 of 270 cases) of NSCLC, and these changes coexisted with ALK rearrangements, KRAS mutations, and EGFR mutations. ALK splicing isoforms were observed with full-length EML4-ALK in crizotinib-naive and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phosphotyrosine sites. Coexpression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, whereas coexpression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib.

Conclusions: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be nonfunctional variants of ALK.

FIGURE 1

Screening for ALK kinase domain splicing variants in non-small-cell lung cancer (NSCLC). A. Reverse transcriptase PCR using primers that flank exons 20 to 29 of ALK (encompass the kinase domain). Cases with or without ALK kinase domain expression, and those expressing ALK slicing variants; B. Sequence chromatogram of a tumor specimen with complete skipping of exon 27 of ALK (ALKdel27). Representative sequences from cDNA isolated from case no. 72 highlighting the ALK exon 26-exon 27 and ALK exon 26-exon 28 co-existing sequences; C. Sequence chromatogram of a tumor specimen with complete skipping of exon 23 of ALK (ALKdel23). Representative sequences from cDNA isolated from case no. 20 highlighting the ALK exon 22-exon 23 and ALK exon 22-exon 24 co-existing sequences. Reference ALK gene sequence (NM_004304.3; homo sapiens ALK mRNA); D. Proposed amino acid sequence of wild type (WT) ALK, ALKdel27 and ALKdel23. The latter truncated proteins generate early stop codons. *denotes a STOP codon sequence.

FIGURE 2

Evaluation of EML4-ALK T1312fs*0 in preclinical models. A. Sequence chromatogram of an EML4-ALK positive tumor specimen. Representative sequences from cDNA isolated from the aforomentioned lymph node. Nucleotide areas corresponding to anaplastic lymphoma kinase (ALK) exon 26, 27 and 28 are highlighted. Reference ALK gene sequence (NM_004304.3; homo sapiens ALK mRNA); the expected nucleotide and amino acid sequences for the wild-type ALK exon 26-exon 27 junction, and for the splicing variant with ALK exon 26-exon 28 junction are shown. The latter generates the early stop codon sequence p. T1312fs*0 (n.TGG->TGA). B. Western blot analysis of protein extracts from COS-7 cells transfected with EML4-ALK constructs and empty vector. EML-ALK E13;A20 constructs with a 3' Myc-His-Tag were cloned into pcDNA3.1. COS-7 cells were transfected with 1 μg of DNA and protein extracts were collected 24 hours later. The radiograph shows the expression of phosphorylated ALK (p-ALK Tyr1096) and EML4-ALK detected by using a Myc-Tag antibody. EML4-ALK T1312fs*0 results in an 80 KDa protein, different from the expected size - 117 kDa - of EML4-ALK WT and EML4-ALK containing the L1196M crizotinib-resistant mutation. C. Western blot analysis of protein extracts from COS-7 cells transfected with EML4-ALK-Myc-His-Tag constructs or empty vector and immunoprecipitated with Myc-Tag antibody. The upper figure shows the expression of phospho-tyrosine (p-TYR) and the lower figure shows the expression of EML4-ALK detected by using a Myc-Tag antibody. EML4-ALK T1312fs*0 results in an 80 KDa protein that is not phosphorylated. D. Western blot analysis of protein extracts from COS-7 cells co-transfected with different DNA amounts of EML4-ALK constructs and empty vector and treated with crizotinib or vehicle (DMSO). The left panel with E13;A20 EML4-ALK (0.4 μg) + empty vector (0.6 μg); the middle with EML4-ALK (0.4 μg) + empty vector (0.2 μg) + EML4-ALK T1312fs*0 (0.4 μg) in which EML4-ALK T1312fs*0 does not result in resistance to crizotinib as verified by ALK phosphorylation inhibition using Tyr1096 phospho-ALK antibody; the right with EML4-ALK (0.4 μg) + empty vector (0.2 μg) + EML4-ALK L1196M (0.4 μg) in which the co-transfection of EML4-ALK L1196M is shown in the right panel and discloses resistance to inhibition of phospho-ALK by crizotinib. Blots were cropped to highlight the referenced molecular weight.