Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions1. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4–ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC)2 and is clinically relevant because it confers sensitivity to ALK inhibitors3. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4–Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4–Alk inversion, express the Eml4–Alk fusion gene, display histopathological and molecular features typical of ALK+ human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.
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We would like to thank M. Fazio, M. Ladanyi, G. Riely, S. Armstrong, and the members of the Ventura, Lowe and Jacks laboratories for discussion and comments. We also thank J. Hollenstein for editing the manuscript, T. Jacks for providing tumour samples from K-RasG12D mice, and the Cytogenetic Core Facility of MSKCC for tissue processing and histology. This work was supported by grants from the Geoffrey Beene Cancer Research Foundation (A.V.), NCI (Cancer Center Support Grant P30 CA008748, E.d.S.), HHMI (S.W.L.), NCI Project Grant (S.W.L.); and by fellowships from the American Italian Cancer Foundation (D.M.), the Foundation Blanceflor Boncompagni Ludovisi, née Bildt (D.M.), and the Jane Coffin Childs Foundation (E.M.). C.P.C. was supported by an NCI training grant.
The authors declare no competing financial interests.
Extended data figures and tables
a, Alignment of human EML4 exon 13 and mouse Eml4 exon 14. b, Alignment of the junction between the human EML4–ALK (variant 1) and the predicted murine Eml4–Alk proteins.
a, Schematic of the Npm1–Alk translocation. Red arrows indicate the sites recognized by the sgRNAs. b, Sequences recognized by the sgRNAs and location of primers used to detect the Npm1–Alk and Alk–Npm1 rearrangement (top panel). PCR on genomic DNA extracted from NIH/3T3 co-transfected with pX330 constructs expressing the indicated sgRNAs (middle panel). Sequences of four independent subclones obtained from the PCR products and representative chromatogram (bottom panel). c, Detection of the Npm1–Alk fusion transcript by RT–PCR on total RNAs extracted from NIH/3T3 cells co-transfected with the indicated pX330 constructs (left panel). The PCR band was extracted and sequenced to confirm the presence of the correct Npm1–Alk junction (bottom-right panel). Representative results from two independent experiments.
a, Schematic of pX330 (A) and its derivatives (B–E) used in these experiments. NIH/3T3 were transfected with these constructs and lysed to extract total RNA and genomic DNA. b, RNAs were analysed by northern blotting with probes against the Alk (left) or Eml4 (right) sgRNAs. c, d, The DNA samples were subjected to surveyor assays (c), or amplified by PCR to detect the Eml4–Alk inversion (d).
Extended Data Figure 4 Induction of the Eml4–Alk inversion in primary MEFs using an adenoviral vector expressing Flag–Cas and tandem sgRNAs.
a, Schematic of the adenoviral vectors. b, Immunoblot using an anti-Flag antibody on lysates from MEFs infected with the indicated adenoviruses. c, Small-RNA northerns using probes against sgEml4 and sgAlk on total RNAs from cells infected with Ad-Cas9 or Ad-EA. d, PCR-mediated detection of the Eml4–Alk inversion in MEFs infected with Ad-Cas9 or Ad-EA for the indicated number of days. e, Standard curve generated performing quantitative PCR analysis on genomic DNA containing a known fraction of Eml4–Alk alleles. Average of two independent experiments. f, Quantification of the fraction of MEFs harbouring the Eml4–Alk inversion at the indicated time points after infection with Ad-EA or Ad-Cas9. Values are mean of three independent infections ± s.d.
µCT images from crizotinib- or vehicle-treated mice at day 0 and after 2 weeks of treatment.
Video generated from axial µCT scans of Ad-EA-infected mouse OP1259 at day 0 of treatment with vehicle (water). (MOV 9878 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1259 after 2 weeks of treatment with vehicle (water). (MOV 9574 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1300 at day 0 of treatment with crizotinib. (MOV 11508 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1300 after 2 weeks of treatment with crizotinib. (MOV 10605 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1280 at day 0 of treatment with vehicle (water). (MOV 8885 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1280 after 2 weeks of treatment with vehicle (water). (MOV 9429 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1290 at day 0 of treatment with crizotinib. (MOV 5581 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1290 after 2 weeks of treatment with crizotinib. (MOV 5166 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1293 at day 0 of treatment with crizotinib. (MOV 5537 kb)
Video generated from axial µCT scans of Ad-EA-infected mouse OP1293 after 2 weeks of treatment with crizotinib. (MOV 5128 kb)
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Maddalo, D., Manchado, E., Concepcion, C. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014). https://doi.org/10.1038/nature13902