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Establishment of a Conditional Transgenic Mouse Model Recapitulating EML4-ALK– Positive Human Non–Small Cell Lung Cancer  Kyoung Ho Pyo, PhD, Sun Min Lim,

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Presentation on theme: "Establishment of a Conditional Transgenic Mouse Model Recapitulating EML4-ALK– Positive Human Non–Small Cell Lung Cancer  Kyoung Ho Pyo, PhD, Sun Min Lim,"— Presentation transcript:

1 Establishment of a Conditional Transgenic Mouse Model Recapitulating EML4-ALK– Positive Human Non–Small Cell Lung Cancer  Kyoung Ho Pyo, PhD, Sun Min Lim, MD, Hye Ryun Kim, MD, Young Hoon Sung, PhD, Mi Ran Yun, PhD, Sung-Moo Kim, PhD, Hwan Kim, PhD, Han Na Kang, PhD, Ji Min Lee, BSc, Sang Gyun Kim, BSc, Chae Won Park, PhD, Hyun Chang, MD, Hyo Sup Shim, MD, Han-Woong Lee, PhD, Byoung Chul Cho, MD, PhD  Journal of Thoracic Oncology  Volume 12, Issue 3, Pages (March 2017) DOI: /j.jtho Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

2 Figure 1 Genotyping of echinoderm microtubule associated protein like 4 gene (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) transgenic mice. (A) In a wild-type (WT) mouse, echinoderm microtubule associated protein like 4 (EML4)–intron F primer binds on mouse intron 1, and EML4-R common primer binds on both human and mouse high homologous EML4 exon 2 region (the product size is 632 base pairs [bp]). In transgenic mouse, pCB-F primer binds (pCB vector region), and EML4-R-common primer can produce 279-bp polymerase chain reaction products. (B) Genotypes of EML4-ALK transgenic mice were confirmed by polymerase chain reaction. WT allele shows a 632-bp band, whereas the transgenic allele shows a 279-bp band. The copy number of EML4-ALK transgene plasmid was serially diluted with WT genomic DNA (0–1000 copies). Two positive transgenic mice (mice 3 and 4) and two WT mice (mice 1 and 2) are seen. CMV, cytomegalovirus; PGK, phosphoglycerate kinase. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

3 Figure 2 Mechanism of tumor generation in surfactant protein C (SPC)–Cre–estrogen receptor T2 (Cre-ERT2)/echinoderm microtubule associated protein like 4 (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) transgenic mouse. Spontaneous expression of Cre-ERT2 is derived by lung-specific SPC promoter. Cre-ERT2 gene is located between equine rhinitis A virus gene (E2A) and Thosea asigna virus 2A gene (T2A). Both genes have self-cleaving effects. The expression of echinoderm microtubule associated protein like 4 (EML4)–anaplastic lymphoma kinase (ALK) is not initiated because of tissue-type plasminogen activator (tpA) stop signaling. Cre-ERT2 is activated by tamoxifen or 4-OHT. The translocated Cre-ERT2 binds to two LoxP sites to remove phosphoglycerate kinase (PGK)-NEOR-tpA sequence. After removal of the PGK-NEOR-tpA sequence through Cre/LoxP complex, cytomegalovirus (CMV) immediate early enhancer (CMVie) and chicken β-actin promoter promote the expression of EML4-ALK. EML4-ALK is tagged with FLAGS on the N-terminus. rtTA, reverse tetracycline-controlled transactivator; FRT, recombination site for integration of Neomycin (Neo) resistance gene into SPC targeting gene cassette. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

4 Figure 3 Expression of echinoderm microtubule associated protein like 4 gene (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) in transgenic mice. (A) The expression of echinoderm microtubule associated protein like 4 (EML4)–anaplastic lymphoma kinase (ALK) was not initiated by tissue-type plasminogen activator (tpA) stop signaling. Cre–estrogen receptor T2 is activated by tamoxifen or 4-OHT. The translocated Cre–estrogen receptor T2 binds to two LoxP sites to remove PGK-NEOR-tpA sequence. (B) On Western blot, EML4-ALK variant type 1 was detected at 120 KDa. Protein lysates from multiple organs were collected and loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When anti-ALK antibody was applied, only lung showed its expression. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

5 Figure 4 Immunohistochemistry of echinoderm microtubule associated protein like 4 gene (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) transgenic mouse tissues. (A) Gross view of tumor morphologic structure was observed after 1 week of 4-OHT treatment in EML4-ALK mice and was compared with that in transgenic mice without 4-OHT treatment. Numerous tumor nodules were observed in both lungs of EML4-ALK mice. (B) On hematoxylin and eosin (H&E) stain, the normal alveolar epithelia were shown, and after tamoxifen treatment, development of acinar or papillary type of lung adenocarcinoma was seen (C). WT, wild type. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

6 Figure 5 Initial response and development of acquired resistance in transgenic mice. (A) Treatment plan of tamoxifen in surfactant protein C (SPC)–Cre–estrogen receptor T2 (Cre-ERT2)/echinoderm microtubule associated protein like 4 (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) transgenic mouse is shown. During the first week, tamoxifen injection was performed on days 3 and 6. After 2 weeks, magnetic resonance imaging (MRI) was performed to examine lung tumorigenesis. Then, crizotinib (200 mg/kg) was administered daily, and its efficacy was monitored by MRI every 2 weeks. MRI was followed up until progressive disease (PD) was observed. (B) MRI scan of SPC–Cre-ERT2/EML4-ALK transgenic mice exhibited multiple tumor nodules after treatment with tamoxifen. After 2 weeks of treatment with crizotinib (150 mg/kg), lung tumor nodules showed complete response (CR) in two mice and partial response (PR) in one mouse. After 4 weeks of treatment, PD was observed in the form of numerous nodules throughout both lungs in all three mice. (C) On hematoxylin and eosin stain, tumor nodules were selected with laser microdissection and sequenced for ALK kinase domain mutation from M876 and M869. (D) Development of secondary mutation in the ALK kinase domain was checked with Sanger sequencing. (E) A gatekeeper mutation G1202R was identified from resistant tumor nodules in M876. ALK, anaplastic lymphoma kinase; TKI, tyrosine kinase inhibitor. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

7 Supplementary Figure 1 Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

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