Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

Nature Genetics volume 46, pages 116–125 (2014)Cite this article

Subjects

Abstract

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1–positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Acquired mutations in ETV6-RUNX1 ALL.
Figure 2: Evaluation of V(D)J recombination motifs.
Figure 3: Chromatin segmentation of all somatic structural variations in ETV6-RUNX1 ALL.
Figure 4: Clonal heterogeneity in ETV6-RUNX1 ALL.
Figure 5: Characterization of structural variation in ETV6-RUNX1 ALL.
Figure 6: Acquired somatic events in ETV6-RUNX1 ALL.
Figure 7: Mutational signatures in ETV6-RUNX1 ALL.

References

  1. Bhojwani, D. et al. ETV6-RUNX1–positive childhood acute lymphoblastic leukemia: improved outcome with contemporary therapy. Leukemia 26, 265–270 (2012).

    CAS  PubMed  Google Scholar 

  2. Greaves, M.F. & Wiemels, J. Origins of chromosome translocations in childhood leukaemia. Nat. Rev. Cancer 3, 639–649 (2003).

    CAS  PubMed  Google Scholar 

  3. Mori, H. et al. Chromosome translocations and covert leukemic clones are generated during normal fetal development. Proc. Natl. Acad. Sci. USA 99, 8242–8247 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Bateman, C.M. et al. Acquisition of genome-wide copy number alterations in monozygotic twins with acute lymphoblastic leukemia. Blood 115, 3553–3558 (2010).

    CAS  PubMed  Google Scholar 

  5. Mullighan, C.G. et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 446, 758–764 (2007).

    CAS  PubMed  Google Scholar 

  6. Zhang, M. & Swanson, P.C. V(D)J recombinase binding and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies. J. Biol. Chem. 283, 6717–6727 (2008).

    CAS  PubMed  Google Scholar 

  7. Mullighan, C.G. et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 322, 1377–1380 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Raschke, S., Balz, V., Efferth, T., Schulz, W.A. & Florl, A.R. Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines. Genes Chromosom. Cancer 42, 58–67 (2005).

    CAS  PubMed  Google Scholar 

  9. Waanders, E. et al. The origin and nature of tightly clustered BTG1 deletions in precursor B-cell acute lymphoblastic leukemia support a model of multiclonal evolution. PLoS Genet. 8, e1002533 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Holmfeldt, L. et al. The genomic landscape of hypodiploid acute lymphoblastic leukemia. Nat. Genet. 45, 242–252 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hübner, S. et al. High incidence and unique features of antigen receptor gene rearrangements in TEL-AML1–positive leukemias. Leukemia 18, 84–91 (2004).

    PubMed  Google Scholar 

  12. Schatz, D.G. & Swanson, P.C. V(D)J recombination: mechanisms of initiation. Annu. Rev. Genet. 45, 167–202 (2011).

    CAS  PubMed  Google Scholar 

  13. Fugmann, S.D., Lee, A.I., Shockett, P.E., Villey, I.J. & Schatz, D.G. The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu. Rev. Immunol. 18, 495–527 (2000).

    CAS  PubMed  Google Scholar 

  14. Komori, T., Okada, A., Stewart, V. & Alt, F.W. Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science 261, 1171–1175 (1993).

    CAS  PubMed  Google Scholar 

  15. Raghavan, S.C., Swanson, P.C., Ma, Y. & Lieber, M.R. Double-strand break formation by the RAG complex at the Bcl-2 major breakpoint region and at other non-B DNA structures in vitro. Mol. Cell. Biol. 25, 5904–5919 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Tsai, A.G. et al. Human chromosomal translocations at CpG sites and a theoretical basis for their lineage and stage specificity. Cell 135, 1130–1142 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Hesse, J.E., Lieber, M.R., Mizuuchi, K. & Gellert, M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 3, 1053–1061 (1989).

    CAS  PubMed  Google Scholar 

  18. Mullighan, C.G. et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 453, 110–114 (2008).

    CAS  PubMed  Google Scholar 

  19. Zhang, J. et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 481, 157–163 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Nik-Zainal, S. et al. Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Campbell, P.J. et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 467, 1109–1113 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Baca, S.C. et al. Punctuated evolution of prostate cancer genomes. Cell 153, 666–677 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Bailey, T.L., Williams, N., Misleh, C. & Li, W.W. MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res. 34, W369–W373 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Stephens, P.J. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462, 1005–1010 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Berger, M.F. et al. The genomic complexity of primary human prostate cancer. Nature 470, 214–220 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  27. Stephens, P.J. et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 144, 27–40 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Shimazaki, N., Tsai, A.G. & Lieber, M.R. H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations. Mol. Cell 34, 535–544 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Anderson, K. et al. Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature 469, 356–361 (2011).

    CAS  PubMed  Google Scholar 

  30. Potter, N.E. et al. Single cell mutational profiling and clonal phylogeny in cancer. Genome Res. 23, 2115–2125 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Familiades, J. et al. PAX5 mutations occur frequently in adult B-cell progenitor acute lymphoblastic leukemia and PAX5 haploinsufficiency is associated with BCR-ABL1 and TCF3-PBX1 fusion genes: a GRAALL study. Leukemia 23, 1989–1998 (2009).

    CAS  PubMed  Google Scholar 

  32. Kempski, H. et al. An investigation of the t(12;21) rearrangement in children with B-precursor acute lymphoblastic leukaemia using cytogenetic and molecular methods. Br. J. Haematol. 105, 684–689 (1999).

    CAS  PubMed  Google Scholar 

  33. van Delft, F.W. et al. Clonal origins of relapse in ETV6-RUNX1 acute lymphoblastic leukemia. Blood 117, 6247–6254 (2011).

    CAS  PubMed  Google Scholar 

  34. Hurlin, P.J., Steingrimsson, E., Copeland, N.G., Jenkins, N.A. & Eisenman, R.N. Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif. EMBO J. 18, 7019–7028 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N. Engl. J. Med. 368, 2059–2074 (2013).

  36. Chen, S. et al. Novel non-TCR chromosome translocations t(3;11)(q25;p13) and t(X;11)(q25;p13) activating LMO2 by juxtaposition with MBNL1 and STAG2. Leukemia 25, 1632–1635 (2011).

    CAS  PubMed  Google Scholar 

  37. Jaffe, J.D. et al. Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia. Nat. Genet. 45, 1386–1391 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Harris, R.S., Petersen-Mahrt, S.K. & Neuberger, M.S. RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Mol. Cell 10, 1247–1253 (2002).

    CAS  PubMed  Google Scholar 

  39. Neuberger, M.S. & Rada, C. Somatic hypermutation: activation-induced deaminase for C/G followed by polymerase eta for A/T. J. Exp. Med. 204, 7–10 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Alexandrov, L.B. et al. Signatures of mutational processes in human cancer. Nature 500, 415–421 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Roberts, S.A. et al. Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions. Mol. Cell 46, 424–435 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Roberts, K.G. et al. Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia. Cancer Cell 22, 153–166 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Tsai, A.G. & Lieber, M.R. RAGs found “not guilty”: cleared by DNA evidence. Blood 111, 1750 (2008).

    CAS  PubMed  Google Scholar 

  44. Boubakour-Azzouz, I., Bertrand, P., Claes, A., Lopez, B.S. & Rougeon, F. Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells. Nucleic Acids Res. 40, 8381–8391 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Hong, D. et al. Initiating and cancer-propagating cells in TEL-AML1–associated childhood leukemia. Science 319, 336–339 (2008).

    CAS  PubMed  Google Scholar 

  46. Tsuzuki, S., Seto, M., Greaves, M. & Enver, T. Modeling first-hit functions of the t(12;21) TEL-AML1 translocation in mice. Proc. Natl. Acad. Sci. USA 101, 8443–8448 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Ross, M.E. et al. Classification of pediatric acute lymphoblastic leukemia by gene expression profiling. Blood 102, 2951–2959 (2003).

    CAS  PubMed  Google Scholar 

  48. Kozarewa, I. et al. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nat. Methods 6, 291–295 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    PubMed  PubMed Central  Google Scholar 

  50. Varela, I. et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539–542 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Ye, K., Schulz, M.H., Long, Q., Apweiler, R. & Ning, Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25, 2865–2871 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Van Loo, P. et al. Allele-specific copy number analysis of tumors. Proc. Natl. Acad. Sci. USA 107, 16910–16915 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Alexandrov, L.B., Nik-Zainal, S., Wedge, D.C., Campbell, P.J. & Stratton, M.R. Deciphering signatures of mutational processes operative in human cancer. Cell Rep. 3, 246–259 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Greenman, C., Wooster, R., Futreal, P.A., Stratton, M.R. & Easton, D.F. Statistical analysis of pathogenicity of somatic mutations in cancer. Genetics 173, 2187–2198 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Klipper-Aurbach, Y. et al. Mathematical formulae for the prediction of the residual β cell function during the first two years of disease in children and adolescents with insulin-dependent diabetes mellitus. Med. Hypotheses 45, 486–490 (1995).

    CAS  PubMed  Google Scholar 

  56. van Dongen, J.J. et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17, 2257–2317 (2003).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Kay Kendall Leukaemia Fund (KKLF; grant reference KKL407), Leukemia and Lymphoma Research (grant reference 11021) and the Wellcome Trust (grant reference 077012/Z/05/Z). P.J.C. is personally funded through a Wellcome Trust Senior Clinical Research Fellowship (grant reference WT088340MA). P.V.L. is supported by a postdoctoral research fellowship from Research Foundation–Flanders (FWO). S.N.-Z. is a Wellcome Trust Intermediate Clinical Fellow (grant reference WT100183MA). F.W.v.D. is funded by KKLF (grant reference KKL417). J.Z. is supported by University Hospital Motol (grant MH-CR DRO 00064203).

Author information

Author notes

  1. Mel Greaves and Peter J Campbell: These authors jointly directed this work.

Authors and Affiliations

  1. Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK

    Elli Papaemmanuil, Yilong Li, David C Wedge, Jose Tubio, Ludmil B Alexandrov, Peter Van Loo, Susanna L Cooke, John Marshall, Inigo Martincorena, Jonathan Hinton, Gunes Gundem, Serena Nik-Zainal, David R Jones, Manasa Ramakrishna, Lucy Stebbings, Catherine Leroy, Andrew Menzies, John Gamble, Ben Robinson, Laura Mudie, Keiran Raine, Sarah O'Meara, Jon W Teague, Adam P Butler, Michael R Stratton & Peter J Campbell

  2. Hospital Universitario 12 de Octubre, Madrid, Spain

    Inmaculada Rapado

  3. Institute for Cancer Research, Sutton, London, UK

    Nicola E Potter, Frederik W van Delft, Ian Titley, Anthony M Ford & Mel Greaves

  4. Department of Human Genetics, VIB and University of Leuven, Leuven, Belgium

    Peter Van Loo

  5. Northern Institute for Cancer Research, University of Newcastle, Newcastle-upon-Tyne, UK

    Frederik W van Delft

  6. Centro Ricerca Tettamanti, Hospital San Gerardo, Monza, Italy

    Giovanni Cazzaniga & Andrea Biondi

  7. Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic

    Jan Zuna

  8. Paediatric Malignancy Unit, Molecular Haematology & Cancer Biology Unit, Camelia Botnar Laboratories, Great Ormond Street Hospital for Children and University College London (UCL) Institute of Child Health, London, UK

    Helena Kempski

  9. Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA

    Markus Muschen

  10. Addenbrooke's National Health Service (NHS) Foundation Trust, Cambridge, UK

    Peter J Campbell

  11. Department of Haematology, University of Cambridge, Cambridge, UK

    Peter J Campbell

Authors
  1. Elli Papaemmanuil
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Inmaculada Rapado
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yilong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicola E Potter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David C Wedge
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jose Tubio
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ludmil B Alexandrov
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter Van Loo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Susanna L Cooke
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. John Marshall
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Inigo Martincorena
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jonathan Hinton
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Gunes Gundem
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Frederik W van Delft
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Serena Nik-Zainal
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. David R Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Manasa Ramakrishna
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Ian Titley
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Lucy Stebbings
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Catherine Leroy
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Andrew Menzies
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. John Gamble
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Ben Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Laura Mudie
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Keiran Raine
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Sarah O'Meara
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Jon W Teague
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Adam P Butler
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Giovanni Cazzaniga
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Andrea Biondi
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Jan Zuna
    View author publications

    You can also search for this author in PubMed Google Scholar

  32. Helena Kempski
    View author publications

    You can also search for this author in PubMed Google Scholar

  33. Markus Muschen
    View author publications

    You can also search for this author in PubMed Google Scholar

  34. Anthony M Ford
    View author publications

    You can also search for this author in PubMed Google Scholar

  35. Michael R Stratton
    View author publications

    You can also search for this author in PubMed Google Scholar

  36. Mel Greaves
    View author publications

    You can also search for this author in PubMed Google Scholar

  37. Peter J Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.P., M.G. and P.J.C. designed the study and wrote the manuscript, with assistance from M.R.S. E.P. designed experiments, performed experiments, analyzed sequencing data and performed and reviewed bioinformatics and statistical analyses. I.R. performed sample preparation, validation experiments and evaluation of sequencing data. Y.L. performed bioinformatics and statistical analyses and wrote the manuscript. D.C.W., L.B.A., I.M. and P.V.L. performed statistical analysis. N.E.P., I.T., F.W.v.D. and A.M.F. performed experiments. G.G., J.T., C.L., S.L.C., J.M., J.H., A.M., K.R., S.N.-Z., M.R., L.S., D.R.J., A.P.B., J.G. and J.W.T. support variant calling algorithms and sequencing analysis platforms. L.M., B.R. and S.O. performed sample preparation and experiments. J.Z., H.K., G.C., M.M. and A.B. provided and prepared samples and experimental materials. All authors reviewed the manuscript during its preparation.

Corresponding authors

Correspondence to Mel Greaves or Peter J Campbell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 (PDF 13180 kb)

Supplementary Tables 1–19

Supplementary Tables 1–19 (XLSX 2768 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Papaemmanuil, E., Rapado, I., Li, Y. et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nat Genet 46, 116–125 (2014). https://doi.org/10.1038/ng.2874

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.2874

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing