A mouse model for EML4-ALK-positive lung cancer

Abstract

EML4-ALK is a fusion-type protein tyrosine kinase that is generated in human non-small-cell lung cancer (NSCLC) as a result of a recurrent chromosome inversion, inv (2)(p21p23). Although mouse 3T3 fibroblasts expressing human EML4-ALK form transformed foci in culture and s.c. tumors in nude mice, it has remained unclear whether this fusion protein plays an essential role in the carcinogenesis of NSCLC. To address this issue, we have now established transgenic mouse lines that express EML4-ALK specifically in lung alveolar epithelial cells. All of the transgenic mice examined developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth, confirming the potent oncogenic activity of the fusion kinase. Although such tumors underwent progressive enlargement in control animals, oral administration of a small-molecule inhibitor of the kinase activity of ALK resulted in their rapid disappearance. Similarly, whereas i.v. injection of 3T3 cells expressing EML4-ALK induced lethal respiratory failure in recipient nude mice, administration of the ALK inhibitor effectively cleared the tumor burden and improved the survival of such animals. These data together reinforce the pivotal role of EML4-ALK in the pathogenesis of NSCLC in humans, and they provide experimental support for the treatment of this intractable cancer with ALK inhibitors.

Fig. 1.

Generation of transgenic mouse lines for EML4-ALK. (A) A cDNA for FLAG-tagged EML4-ALK was inserted between the SPC promoter and both splicing and polyadenylation [poly(A)] signal sequences. (B) Genomic DNA was isolated from the tail of founder mice generated from pronuclear-stage C57BL/6J embryos and was subjected to Southern blot analysis with full-length EML4-ALK cDNA as a probe. Control samples on the right comprised mouse genomic DNA with 0, 1, 10, or 100 copies of the transgene per diploid genome. The ID numbers of founder mice positive for the transgene are shown at the top. (C) Oligo(dT)-primed cDNA was synthesized from total RNA isolated from lung (Lu), liver (Lv), esophagus (Es), stomach (St), colon (Co), brain (Br), kidney (Kd), and uterus (Ut) of an F₁ mouse of the 502-4 line, with the reaction being performed in the presence (+) or absence (−) of reverse transcriptase. The cDNA preparations were then subjected to PCR with primer sets for EML4-ALK or for GAPDH, and the PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. The positions of the PCR products are indicated on the left. RT-PCR was also performed for a no-template control (NTC) and for a human NSCLC specimen harboring EML4-ALK variant 1 as a positive control (PC).

Fig. 2.

Development of lung adenocarcinoma in EML4-ALK transgenic mice. (A) Hundreds of adenocarcinoma nodules (arrows) were apparent in the lungs of a founder mouse (503-6) that died 3 weeks after birth. H, heart. (B) Microscopic examination of the nodules shown in A after staining with H&E. Images at low (Left) and high (Right) magnification are shown with scale bars of 200 and 40 μm, respectively. Some tumors exhibited obvious scar formation, suggesting that they were invasive carcinomas. (C) Immunohistochemical analysis with antibodies to ALK of one of the nodules shown in A revealed a pattern of cytoplasmic staining with granular accentuations. (Scale bar, 50 μm.) (D) Immunoprecipitates prepared with antibodies to FLAG from the indicated tissues of an F₁ mouse of the 502-4 line were subjected to immunoblot analysis with the same antibodies. The position of EML4-ALK is shown at the left.

Fig. 3.

Treatment of EML4-ALK transgenic mice with a specific ALK inhibitor. (A and B) Transgenic mice were subjected to daily peroral administration of vehicle (A) or ALK inhibitor (B) beginning at 4 weeks of age and were examined by CT scanning of the chest on days 0, 11, and 25. The ID numbers of the mice are shown at the top. H, heart. Tumors (indicated by broken lines) in both lungs underwent progressive enlargement in all control mice but became progressively smaller in all treated animals. (C) Macroscopic examination of the lungs from mice of the control and treatment groups at 2 months after the onset of treatment. The tissue was stained with H&E. The ID numbers of the mice are shown at the bottom. Cancer nodules are indicated by arrows. Thy, thymus.

Fig. 4.

Treatment with the ALK inhibitor of mice injected with EML4-ALK/3T3 cells. (A) Nude mice were injected i.v. with 2 × 10⁵ 3T3 cells expressing EML4-ALK variant 1 and were then immediately subjected to daily peroral administration of vehicle (control, n = 10) or ALK inhibitor (treatment, n = 10). Survival of the 2 cohorts is shown as a Kaplan–Meier plot and was compared by the log-rank test, with the calculated P value indicated. (B) Macroscopic examination of lungs isolated from mice of the control group at death or of the treatment group after treatment for 31 days. The tissue was stained with H&E. Most of the lungs in both control animals were occupied with transformed EML4-ALK/3T3 cells, whereas such cells were rarely observed in the treated animal. (C) Microscopic examination of lung tissue from a mouse of the control group after H&E staining. Images of low (Left) and high (Right) magnification are shown with scale bars of 500 and 50 μm, respectively. (D) Immunohistochemical analysis with antibodies to ALK of the nodules of EML4-ALK/3T3 cells that formed in the lungs of a mouse in the control group. Images of low (Left) and high (Right) magnification are shown with scale bars of 200 and 50 μm, respectively.