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PMID: 23568838 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Cancer translocations in human cells induced by zinc finger and TALE nucleases.

Genome research ·Vol. 23 ·No. 7 ·2013-07-00 ·Pages 1182-93

Piganeau M, Ghezraoui H, De Cian A, Guittat L, Tomishima M, Perrouault L, René O, Katibah GE, Zhang L, Holmes MC, Doyon Y, Concordet JP, Giovannangeli C, Jasin M, Brunet E

Abstract

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.

MeSH Terms
Cell Line Chromosome Breakpoints Endonucleases/metabolism Humans Neoplasms/enzymology,genetics Nucleophosmin Protein-Tyrosine Kinases/genetics Sarcoma, Ewing/genetics,metabolism Translocation, Genetic Zinc Fingers
Chemicals
NPM1 protein, human Nucleophosmin p80(NPM-ALK) protein Protein-Tyrosine Kinases Endonucleases
Authors & Affiliations
15 authors, click to expand affiliations / ORCID
Piganeau Marion
Museum National d'Histoire Naturelle, CNRS UMR7196, Inserm U565, 75005 Paris, France.
Ghezraoui Hind
De Cian Anne
Guittat Lionel
Tomishima Mark
Perrouault Loic
René Oliver
Katibah George E
Zhang Lei
Holmes Michael C
Doyon Yannick
Concordet Jean-Paul
Giovannangeli Carine
Jasin Maria
Brunet Erika
References (43)
43 references, click to expand
  1. A rapid and general assay for monitoring endogenous gene modification.
    Methods Mol Biol. 2010;649:247-56 PMID: 20680839
  2. Mesenchymal stem cell features of Ewing tumors.
    Cancer Cell. 2007 May;11(5):421-9 PMID: 17482132
  3. NPM mutations in acute myelogenous leukemia.
    N Engl J Med. 2005 Jan 20;352(3):291-2 PMID: 15659732
  4. Chromosome translocation based on illegitimate recombination in human tumors.
    Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11786-91 PMID: 9751743
  5. Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases.
    Genome Res. 2012 Mar;22(3):539-48 PMID: 22183967
  6. Targeted chromosomal deletions in human cells using zinc finger nucleases.
    Genome Res. 2010 Jan;20(1):81-9 PMID: 19952142
  7. Genomic structure of the EWS gene and its relationship to EWSR1, a site of tumor-associated chromosome translocation.
    Genomics. 1993 Dec;18(3):609-15 PMID: 8307570
  8. TALE nucleases: tailored genome engineering made easy.
    Curr Opin Biotechnol. 2012 Oct;23(5):644-50 PMID: 22342754
  9. Derivation of multipotent mesenchymal precursors from human embryonic stem cells.
    PLoS Med. 2005 Jun;2(6):e161 PMID: 15971941
  10. Transcripts of the npm-alk fusion gene in anaplastic large cell lymphoma, Hodgkin's disease, and reactive lymphoid lesions.
    Blood. 1995 Nov 1;86(9):3517-21 PMID: 7579458
  11. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.
    Science. 1994 Mar 4;263(5151):1281-4 PMID: 8122112
  12. EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3.
    Mol Cell Biol. 2004 Aug;24(16):7275-83 PMID: 15282325
  13. Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line.
    J Exp Med. 1980 Dec 1;152(6):1709-19 PMID: 6778951
  14. Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis.
    Oncogene. 2001 Nov 1;20(50):7386-97 PMID: 11704868
  15. Mechanisms of nonhomologous recombination in mammalian cells.
    Mol Cell Biol. 1985 Oct;5(10):2599-607 PMID: 3016509
  16. Genome engineering with zinc-finger nucleases.
    Genetics. 2011 Aug;188(4):773-82 PMID: 21828278
  17. Triggers for genomic rearrangements: insights into genomic, cellular and environmental influences.
    Nat Rev Genet. 2010 Dec;11(12):819-29 PMID: 21045868
  18. Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) oncoprotein induces the T regulatory cell phenotype by activating STAT3.
    Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9964-9 PMID: 16766651
  19. Frequent chromosomal translocations induced by DNA double-strand breaks.
    Nature. 2000 Jun 8;405(6787):697-700 PMID: 10864328
  20. Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation.
    Nat Struct Mol Biol. 2010 Apr;17(4):410-6 PMID: 20208544
  21. Genome editing with engineered zinc finger nucleases.
    Nat Rev Genet. 2010 Sep;11(9):636-46 PMID: 20717154
  22. A TALE nuclease architecture for efficient genome editing.
    Nat Biotechnol. 2011 Feb;29(2):143-8 PMID: 21179091
  23. FLASH assembly of TALENs for high-throughput genome editing.
    Nat Biotechnol. 2012 May;30(5):460-5 PMID: 22484455
  24. Molecular pathogenesis of Ewing sarcoma: new therapeutic and transcriptional targets.
    Annu Rev Pathol. 2012;7:145-59 PMID: 21942527
  25. Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation.
    Cancer Cell. 2004 May;5(5):465-75 PMID: 15144954
  26. Formation of NHEJ-derived reciprocal chromosomal translocations does not require Ku70.
    Nat Cell Biol. 2007 Aug;9(8):978-81 PMID: 17643113
  27. Temporal and spatial control of nucleophosmin by the Ran-Crm1 complex in centrosome duplication.
    Nat Cell Biol. 2005 Aug;7(8):823-30 PMID: 16041368
  28. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.
    Nat Methods. 2011 Jan;8(1):74-9 PMID: 21131970
  29. Retrovirus-mediated gene transfer of NPM-ALK causes lymphoid malignancy in mice.
    Blood. 1997 Oct 15;90(8):2901-10 PMID: 9376569
  30. EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells.
    Cancer Res. 2008 Apr 1;68(7):2176-85 PMID: 18381423
  31. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.
    Nucleic Acids Res. 2000 Sep 1;28(17):3361-9 PMID: 10954606
  32. IgH class switching and translocations use a robust non-classical end-joining pathway.
    Nature. 2007 Sep 27;449(7161):478-82 PMID: 17713479
  33. Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant.
    Cancer Res. 2006 Mar 15;66(6):3044-50 PMID: 16540653
  34. Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma.
    Cancer Cell. 2006 May;9(5):405-16 PMID: 16697960
  35. Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma.
    Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5831-6 PMID: 19321746
  36. EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.
    Genes Dev. 2010 May;24(9):916-32 PMID: 20382729
  37. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.
    PLoS Genet. 2011 Jun;7(6):e1002080 PMID: 21655080
  38. Nucleophosmin interacts directly with c-Myc and controls c-Myc-induced hyperproliferation and transformation.
    Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18794-9 PMID: 19033198
  39. The impact of translocations and gene fusions on cancer causation.
    Nat Rev Cancer. 2007 Apr;7(4):233-45 PMID: 17361217
  40. Targeting of EWS/FLI-1 by RNA interference attenuates the tumor phenotype of Ewing's sarcoma cells in vitro.
    J Orthop Res. 2004 Jul;22(4):910-7 PMID: 15183454
  41. Genetic engineering of human pluripotent cells using TALE nucleases.
    Nat Biotechnol. 2011 Jul 07;29(8):731-4 PMID: 21738127
  42. Chromosomal translocations induced at specified loci in human stem cells.
    Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10620-5 PMID: 19549848
  43. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.
    Nucleic Acids Res. 2011 Nov;39(21):9283-93 PMID: 21813459
Article Info
Journal
Genome research
Abbr.
Genome Res
ISSN
1549-5469
DOI
10.1101/gr.147314.112
Published
2013-07-00
Epub
2013-00-08
Pages
1182-93
Language
English
Region
United States
NLM ID
9518021
PMCID
PMC3698511
Subset
IM
Grants
NIGMS NIH HHS · R01 GM054668 · United States
NIGMS NIH HHS · GM54668 · United States
External Links
PubMed source DOI: 10.1101/gr.147314.112 PMC Full Text View journal details
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