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Identification and Characterization of ALK Kinase Splicing Isoforms in Non–Small-Cell Lung Cancer  Lorena Lobo de Figueiredo-Pontes, MD, PhD, Daisy Wing-Sze.

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Presentation on theme: "Identification and Characterization of ALK Kinase Splicing Isoforms in Non–Small-Cell Lung Cancer  Lorena Lobo de Figueiredo-Pontes, MD, PhD, Daisy Wing-Sze."— Presentation transcript:

1 Identification and Characterization of ALK Kinase Splicing Isoforms in Non–Small-Cell Lung Cancer 
Lorena Lobo de Figueiredo-Pontes, MD, PhD, Daisy Wing-Sze Wong, MPhil, Vicky Pui-Chi Tin, MMedSci, Lap-Ping Chung, DPhil, Hiroyuki Yasuda, MD, Norihiro Yamaguchi, MD, MPH, Sohei Nakayama, MD, Pasi Antero Jänne, MD, PhD, Maria Pik Wong, MD, Susumu Soeda Kobayashi, MD, PhD, Daniel Botelho Costa, MD, PhD  Journal of Thoracic Oncology  Volume 9, Issue 2, Pages (February 2014) DOI: /JTO Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

2 FIGURE 1 Screening for ALK kinase domain splicing variants in 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 exons 26 to 27 and ALK exons 26 to 28 coexisting 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 exons 22 to 23 and ALK exons 22 to 24 coexisting sequences. Reference ALK gene sequence (NM_ ; homo sapiens ALK mRNA). D, Proposed amino acid sequence of WT ALK, ALKdel27 and ALKdel23. The latter truncated proteins generate early stop codons. *, A STOP codon sequence; ALK, anaplastic lymphoma kinase; NSCLC, non–small-cell lung cancer; PCR, polymerase chain reaction; WT, wild type; EML4, echinoderm microtubule-associated protein-like 4. Journal of Thoracic Oncology 2014 9, DOI: ( /JTO ) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

3 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 aforementioned lymph node. Nucleotide areas corresponding to ALK exons 26, 27, and 28 are highlighted. Reference ALK gene sequence (NM_ ; homo sapiens ALK mRNA); the expected nucleotide and amino acid sequences for the wild-type ALK exons 26 to 27 junction and for the splicing variant with ALK exons 26 to 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. EML4-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 a 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 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 cotransfected 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 cotransfection 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. EML4, echinoderm microtubule-associated protein-like 4; ALK, anaplastic lymphoma kinase; p-TYR, phosphotyrosine. Journal of Thoracic Oncology 2014 9, DOI: ( /JTO ) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

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