Maddalo D et al. (2014), In vivo engineering of oncogenic chromosomal re...

ECB-ART-50041

Nature 2014 Dec 18;5167531:423-7. doi: 10.1038/nature13902.

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In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.

Maddalo D , Manchado E , Concepcion CP , Bonetti C , Vidigal JA , Han YC , Ogrodowski P , Crippa A , Rekhtman N , de Stanchina E , Lowe SW , Ventura A .

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Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. 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) and is clinically relevant because it confers sensitivity to ALK inhibitors. 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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	Extended Data Figure 2. Induction of the Npm1-Alk translocation in NIH/3T3 cells(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 cotransfected 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.
	Extended Data Figure 3. Comparison of dual and single sgRNA-expressing plasmids(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 analyzed by Northern blotting with probes against the Alk (left) or Eml4 (right) sgRNAs. (c) The DNA samples were subjected to surveyor assays, or (d) amplified by PCR to detect the Eml4-Alk inversion.
	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 a 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 known fraction of Eml4-Alk alleles. Average of two independent experiments. (f) Quantification of the fraction of MEFs harboring 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.
	Extended Data Figure 5. Radiologic response of Ad-EA-induced tumors to crizotinib treatmentμCT images from crizotinib- or vehicle-treated mice at day 0 and after 2 weeks of treatement.
	Figure 1. Induction of Eml4-Alk rearrangement in murine cells using the CRISPR-Cas9 system(a) Schematic of the In(17) involving the Eml4 and Alk loci. Red arrows indicate the sites recognized by the sgRNAs. (b) A schematic of the loci before and after the inversion with the location of the primers used (top panel). PCRs were performed on genomic DNA extracted from NIH/3T3 cells transfected with the indicated pX330 constructs (middle panels). The PCR bands were sub-cloned and the sequences of four independent clones and a representative chromatogram are shown in the lower panels. (c) Schematic of the Eml4-Alk fusion transcript (top panel). Detection of the Eml4-Alk fusion transcript by RT-PCR on total RNAs extracted from NIH/3T3 cells transfected with the indicated pX330 constructs (bottom left panel). Sequence of the PCR product showing the correct Eml4-Alk junction (bottom-right panel). (d) Schematic of the break-apart interphase FISH strategy. In cells with the Eml4-Alk inversion, the red and green probes become separated, and the green and orange probes become juxtaposed. (e) Break-apart interphase FISH assay on a NIH/3T3 clone selected from cells cotransfected with pX330-Eml4 and pX330-Alk. Both wild type (white arrows) and the In(17) Eml4-Alk allele (red arrow) are detected.
	Figure 2. Intratracheal delivery of Ad-EA leads to lung cancer formation in mice(a) Hematoxylin-eosin staining of lungs from mice at the indicated times after intratracheal instillation of Ad-EA. (b) Representative μCT scans (top) and macroscopic appearance (bottom) of lungs from mice at 8 weeks post-infection with Ad-Cre or Ad-EA. Numerous neoplastic lesions are evident in the Ad-EA-infected lung. (c) Representativeimmunostainings of Ad-EA-induced lung tumors with the indicated antibodies. (d-j’) Tumor architecture and cytology of Ad-EA induced tumors. Representative micrographs showing: papillary (d) or acinar (e) tumors, lesions originating in proximity of intrabronchial hyperplasia (f), atypical adenomatous hyperplasia (g), mild to moderate nuclear atypia (h, h’), cells with large cytoplasmic vacuole and eccentric nuclei (i, i’), and PAS-positive tumors (j, j’).
	Figure 3. Lung tumors induced by Ad-EA infection harbor the Eml4-Alk inversion(a) Break-apart interphase FISH showing the presence of the Eml4-Alk inversion in a tumor from an Ad-EA-infected mouse (8 weeks post-infection) and (b) wild type configuration of the Eml4 and Alk loci in a control tumor from a conditional K-RasG12D mouse. (i) Bright Field. (i’, ii, iii increasing magnifications of merged fluorescent channels) (c) Detection of the wild type Eml4 locus and Eml4-Alk inversion in micro-dissected tumors from Ad-EA-infected mice using a three-primer PCR strategy. (d) RNAs extracted from the same tumors shown in (c) were reverse-transcribed and amplified using a three-primer strategy to detect the Eml4 and Eml4-Alk transcripts. (e) RT-PCR detection (left) of the full length Eml4-Alk cDNA (~3.2 Kb) in the tumors shown in (c). The full-length PCR products were sequenced on both strands. A chromatogram of the Eml4-Alk junction is shown (right). (f) Representative immunohistochemistry of Ad-EA-induced lung tumors decorated with antibodies against the indicated phospho-proteins. A bar-plot of staining intensity for the indicated phospho-proteins is also shown. Tumors from two mice for each group were scored.
	Figure 4. Ad-EA-induced lung tumors respond to crizotinib treatment(a) Schematic of the experiment. (b) Representative μCT of the lungs of mice treated with crizotinib or vehicle at day 0 and after 2 weeks of treatement. Lung tumors are indicated by arrows. Red asterisks mark the hearts. (c) Macroscopic appearance of the lungs after 2 weeks of treatment. (d) Low magnification of lung sections from two crizotinib- and 2 vehicle-treated mice (hematoxylin eosin). (e) Higher magnification of representative hematoxylin-eosin stained lung sections from crizotinib-treated mice showing residual atrofic foci of tumor cells (left) or necrotic-inflammatory debris (right).

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