Letter | Published:

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

Nature volume 516, pages 423427 (18 December 2014) | Download Citation

  • A Corrigendum to this article was published on 24 June 2015

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Chromosomal translocations in cancer and their relevance for therapy. Curr. Opin. Oncol. 18, 62–68 (2006)

  2. 2.

    et al. Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature 448, 561–566 (2007)

  3. 3.

    et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N. Engl. J. Med. 363, 1693–1703 (2010)

  4. 4.

    & Technologically advanced cancer modeling in mice. Curr. Opin. Genet. Dev. 12, 105–110 (2002)

  5. 5.

    & The mighty mouse: genetically engineered mouse models in cancer drug development. Nature Rev. Drug Discov. 5, 741–754 (2006)

  6. 6.

    et al. Acquired resistance of EGFR-mutant lung adenocarcinomas to afatinib plus cetuximab is associated with activation of mTORC1. Cell Rep. 7, 999–1008 (2014)

  7. 7.

    & Modes of resistance to anti-angiogenic therapy. Nature Rev. Cancer 8, 592–603 (2008)

  8. 8.

    et al. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer. Proc. Natl Acad. Sci. USA 104, 12117–12122 (2007)

  9. 9.

    et al. Acute leukaemia in bcr/abl transgenic mice. Nature 344, 251–253 (1990)

  10. 10.

    et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev. 23, 877–889 (2009)

  11. 11.

    et al. Overexpression of NPM–ALK induces different types of malignant lymphomas in IL-9 transgenic mice. Oncogene 22, 517–527 (2003)

  12. 12.

    et al. NPM–ALK transgenic mice spontaneously develop T-cell lymphomas and plasma cell tumors. Blood 101, 1919–1927 (2003)

  13. 13.

    et al. A mouse model for EML4–ALK-positive lung cancer. Proc. Natl Acad. Sci. USA 105, 19893–19897 (2008)

  14. 14.

    et al. An Mll–AF9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes. Cell 85, 853–861 (1996)

  15. 15.

    et al. A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nature Genet. 9, 376–385 (1995)

  16. 16.

    , , , & Inter-chromosomal recombination of Mll and Af9 genes mediated by cre-loxP in mouse development. EMBO Rep. 1, 127–132 (2000)

  17. 17.

    et al. Cancer translocations in human cells induced by zinc finger and TALE nucleases. Genome Res. 23, 1182–1193 (2013)

  18. 18.

    et al. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. Nature Commun. 5, 3964 (2014)

  19. 19.

    et al. Chromosomal translocations induced at specified loci in human stem cells. Proc. Natl Acad. Sci. USA 106, 10620–10625 (2009)

  20. 20.

    & Targeted genomic rearrangements using CRISPR/Cas technology. Nature Commun. 5, 3728 (2014)

  21. 21.

    et al. Identification of novel isoforms of the EML4–ALK transforming gene in non-small cell lung cancer. Cancer Res. 68, 4971–4976 (2008)

  22. 22.

    & CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010)

  23. 23.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

  24. 24.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  25. 25.

    et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science 263, 1281–1284 (1994)

  26. 26.

    , & Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nature Protocols 4, 1064–1072 (2009)

  27. 27.

    et al. Histologic and cytomorphologic features of ALK-rearranged lung adenocarcinomas. Modern Pathol. 25, 1462–1472 (2012)

  28. 28.

    et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001)

  29. 29.

    , , , & The anaplastic lymphoma kinase in the pathogenesis of cancer. Nature Rev. Cancer 8, 11–23 (2008)

  30. 30.

    et al. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J. Biol. Chem. 289, 21312–21324 (2014)

Download references

Acknowledgements

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.

Author information

Affiliations

  1. Memorial Sloan Kettering Cancer Center, Cancer Biology and Genetics Program, 1275 York Avenue, New York, New York 10065, USA

    • Danilo Maddalo
    • , Eusebio Manchado
    • , Carla P. Concepcion
    • , Ciro Bonetti
    • , Joana A. Vidigal
    • , Yoon-Chi Han
    • , Paul Ogrodowski
    • , Scott W. Lowe
    •  & Andrea Ventura
  2. Weill Cornell Graduate School of Medical Sciences of Cornell University, 1300 York Avenue, New York, New York 10065, USA

    • Carla P. Concepcion
  3. Milano-Bicocca University, Department of Medical Oncology, San Gerardo Hospital, 20052, Via G B Pergolesi 33, Monza, Italy

    • Alessandra Crippa
  4. Memorial Sloan Kettering Cancer Center, Thoracic Pathology and Cytopathology, 1275 York Avenue, New York, New York 10065, USA

    • Natasha Rekhtman
  5. Memorial Sloan Kettering Cancer Center, Molecular Pharmacology Program, 1275 York Avenue, New York, New York 10065, USA

    • Elisa de Stanchina
  6. Howard Hughes Medical Institute, 1275 York Avenue, New York, New York 10065, USA

    • Scott W. Lowe

Authors

  1. Search for Danilo Maddalo in:

  2. Search for Eusebio Manchado in:

  3. Search for Carla P. Concepcion in:

  4. Search for Ciro Bonetti in:

  5. Search for Joana A. Vidigal in:

  6. Search for Yoon-Chi Han in:

  7. Search for Paul Ogrodowski in:

  8. Search for Alessandra Crippa in:

  9. Search for Natasha Rekhtman in:

  10. Search for Elisa de Stanchina in:

  11. Search for Scott W. Lowe in:

  12. Search for Andrea Ventura in:

Contributions

D.M. and A.V. conceived the project, designed and analysed the experiments, and wrote the manuscript. S.W.L. contributed to the interpretation of the results and the writing of the manuscript. D.M. generated and tested the constructs, performed the cell-based experiments, and characterized the Eml4Alk tumours. E.M., D.M., C.B., Y.-C.H. and P.O. performed the in vivo experiments. E.d.S. supervised the crizotinib treatment experiments and analysed the results. J.A.V., D.M., C.P.C. and A.V. microdissected and analysed lung tumours to detect the Eml4Alk inversion. C.B., D.M. and A.C. performed the immunostainings. N.R. reviewed the histopathology.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andrea Ventura.

Extended data

Supplementary information

Videos

  1. 1.

    Vehicle-treatment day 0 (OP1259)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1259 at day 0 of treatment with vehicle (water).

  2. 2.

    Vehicle-treatment week 2 (OP1259)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1259 after 2 weeks of treatment with vehicle (water).

  3. 3.

    Crizotinib-treatment day 0 (OP1300)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1300 at day 0 of treatment with crizotinib.

  4. 4.

    Crizotinib-treatment week 2 (OP1300)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1300 after 2 weeks of treatment with crizotinib.

  5. 5.

    Vehicle-treatment day 0 (OP1280)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1280 at day 0 of treatment with vehicle (water).

  6. 6.

    Vehicle-treatment week 2 (OP1280)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1280 after 2 weeks of treatment with vehicle (water).

  7. 7.

    Crizotinib-treatment day 0 (OP1290)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1290 at day 0 of treatment with crizotinib.

  8. 8.

    Crizotinib-treatment week 2 (OP1290)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1290 after 2 weeks of treatment with crizotinib.

  9. 9.

    Crizotinib-treatment day 0 (OP1293)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1293 at day 0 of treatment with crizotinib.

  10. 10.

    Crizotinib-treatment week 2 (OP1293)

    Video generated from axial µCT scans of Ad-EA-infected mouse OP1293 after 2 weeks of treatment with crizotinib.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13902

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.