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Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease

Nature Medicine volume 16, pages 198–204 (2010)Cite this article

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Abstract

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.

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Figure 1: Hematopoietic reconstitution, gene marking and superoxide production in subjects 1 and 2 after gene therapy.
Figure 2: Proviral DNA methylation, clonal distribution of gene-marked cells and MDS1–EVI1 activation in blood samples of subjects 1 and 2.
Figure 3: Monosomy 7 in dominant clones of subjects 1 and 2.
Figure 4: Genomic instability in MDS1−EVI1-expressing cells.
Figure 5: EVI1 induces centrosomal aberrations in human cells.

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References

  1. Cavazzana-Calvo, M. et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669–672 (2000).

    Article  CAS  Google Scholar 

  2. Aiuti, A. et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410–2413 (2002).

    Article  CAS  Google Scholar 

  3. Gaspar, H.B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–2187 (2004).

    Article  CAS  Google Scholar 

  4. Gaspar, H.B. et al. Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessation of PEG-ADA and use of mild preconditioning. Mol. Ther. 14, 505–513 (2006).

    Article  CAS  Google Scholar 

  5. Aiuti, A. et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360, 447–458 (2009).

    Article  CAS  Google Scholar 

  6. Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

    Article  CAS  Google Scholar 

  7. Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003).

    Article  Google Scholar 

  8. Howe, S.J. et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118, 3143–3150 (2008).

    Article  CAS  Google Scholar 

  9. Hacein-Bey-Abina, S. et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest. 118, 3132–3142 (2008).

    Article  CAS  Google Scholar 

  10. Dinauer, M.C. The respiratory burst oxidase and the molecular genetics of chronic granulomatous disease. Crit. Rev. Clin. Lab. Sci. 30, 329–369 (1993).

    Article  CAS  Google Scholar 

  11. Roos, D. The genetic basis of chronic granulomatous disease. Immunol. Rev. 138, 121–157 (1994).

    Article  CAS  Google Scholar 

  12. Segal, A.W. The NADPH oxidase and chronic granulomatous disease. Mol. Med. Today 2, 129–135 (1996).

    Article  CAS  Google Scholar 

  13. Malech, H.L. & Hickstein, D.D. Genetics, biology and clinical management of myeloid cell primary immune deficiencies: chronic granulomatous disease and leukocyte adhesion deficiency. Curr. Opin. Hematol. 14, 29–36 (2007).

    Article  Google Scholar 

  14. Ott, M.G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1–EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).

    Article  CAS  Google Scholar 

  15. Fears, S. et al. Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family. Proc. Natl. Acad. Sci. USA 93, 1642–1647 (1996).

    Article  CAS  Google Scholar 

  16. Hamaguchi, H. et al. Establishment of a novel human myeloid leukaemia cell line (HNT-34) with t(3;3)(q21;q26), t(9;22)(q34;q11) and the expression of EVI1 gene, P210 and P190 BCR/ABL chimaeric transcripts from a patient with AML after MDS with 3q21q26 syndrome. Br. J. Haematol. 98, 399–407 (1997).

    Article  CAS  Google Scholar 

  17. Claus, R. & Lubbert, M. Epigenetic _targets in hematopoietic malignancies. Oncogene 22, 6489–6496 (2003).

    Article  CAS  Google Scholar 

  18. Nolte, F. & Hofmann, W.K. Myelodysplastic syndromes: molecular pathogenesis and genomic changes. Ann. Hematol. 87, 777–795 (2008).

    Article  Google Scholar 

  19. Christiansen, D.H., Andersen, M.K. & Pedersen-Bjergaard, J. Methylation of p15INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts a poor prognosis in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia 17, 1813–1819 (2003).

    Article  CAS  Google Scholar 

  20. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).

    Article  CAS  Google Scholar 

  21. Khanna, K.K. & Jackson, S.P. DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 27, 247–254 (2001).

    Article  CAS  Google Scholar 

  22. Fernandez-Capetillo, O., Celeste, A. & Nussenzweig, A. Focusing on foci: H2AX and the recruitment of DNA-damage response factors. Cell Cycle 2, 426–427 (2003).

    Article  CAS  Google Scholar 

  23. Kearns, W.G., Yamaguchi, H., Young, N.S. & Liu, J.M. Centrosome amplification and aneuploidy in bone marrow failure patients. Genes Chromosomes Cancer 40, 329–333 (2004).

    Article  CAS  Google Scholar 

  24. Neben, K., Giesecke, C., Schweizer, S., Ho, A.D. & Kramer, A. Centrosome aberrations in acute myeloid leukemia are correlated with cytogenetic risk profile. Blood 101, 289–291 (2003).

    Article  CAS  Google Scholar 

  25. Ganem, N.J., Godinho, S.A. & Pellman, D. A mechanism linking extra centrosomes to chromosomal instability. Nature 460, 278–282 (2009).

    Article  CAS  Google Scholar 

  26. Dinauer, M.C., Curnutte, J.T., Rosen, H. & Orkin, S.H. A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J. Clin. Invest. 84, 2012–2016 (1989).

    Article  CAS  Google Scholar 

  27. Wang, L., Robbins, P.B., Carbonaro, D.A. & Kohn, D.B. High-resolution analysis of cytosine methylation in the 5′ long terminal repeat of retroviral vectors. Hum. Gene Ther. 9, 2321–2330 (1998).

    Article  CAS  Google Scholar 

  28. Frank, O. et al. Tumor cells escape suicide gene therapy by genetic and epigenetic instability. Blood 104, 3543–3549 (2004).

    Article  CAS  Google Scholar 

  29. Burns, W.R., Zheng, Z., Rosenberg, S.A. & Morgan, R.A. Lack of specific gamma-retroviral vector long terminal repeat promoter silencing in patients receiving genetically engineered lymphocytes and activation upon lymphocyte restimulation. Blood 114, 2888–2899 (2009).

    Article  CAS  Google Scholar 

  30. Metais, J.Y. & Dunbar, C.E. The MDS1–EVI1 gene complex as a retrovirus integration site: impact on behavior of hematopoietic cells and implications for gene therapy. Mol. Ther. 16, 439–449 (2008).

    Article  CAS  Google Scholar 

  31. Du, Y., Jenkins, N.A. & Copeland, N.G. Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells. Blood 106, 3932–3939 (2005).

    Article  CAS  Google Scholar 

  32. Calmels, B. et al. Recurrent retroviral vector integration at the MDS1–EVI1 locus in non-human primate hematopoietic cells. Blood 106, 2530–2533 (2005).

    Article  CAS  Google Scholar 

  33. Li, Z. et al. Murine leukemia induced by retroviral gene marking. Science 296, 497 (2002).

    Article  CAS  Google Scholar 

  34. Modlich, U. et al. Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16. Leukemia 22, 1519–1528 (2008).

    Article  CAS  Google Scholar 

  35. Buonamici, S. et al. EVI1 induces myelodysplastic syndrome in mice. J. Clin. Invest. 114, 713–719 (2004).

    Article  CAS  Google Scholar 

  36. Du, Y., Spence, S.E., Jenkins, N.A. & Copeland, N.G. Cooperating cancer-gene identification through oncogenic-retrovirus-induced insertional mutagenesis. Blood 106, 2498–2505 (2005).

    Article  CAS  Google Scholar 

  37. Nucifora, G. The EVI1 gene in myeloid leukemia. Leukemia 11, 2022–2031 (1997).

    Article  CAS  Google Scholar 

  38. Dreyfus, F. et al. Expression of the Evi-1 gene in myelodysplastic syndromes. Leukemia 9, 203–205 (1995).

    CAS  PubMed  Google Scholar 

  39. Barjesteh van Waalwijk van Doorn-Khosrovani, S et al. High EVI1 expression predicts poor survival in acute myeloid leukemia: a study of 319 de novo AML patients. Blood 101, 837–845 (2003).

    Article  Google Scholar 

  40. Charrin, C. et al. Structural rearrangements of chromosome 3 in 57 patients with acute myeloid leukemia: clinical, hematological and cytogenetic features. Hematol. J. 3, 21–31 (2002).

    Article  Google Scholar 

  41. Lin, P., Medeiros, L.J., Yin, C.C. & Abruzzo, L.V. Translocation (3;8)(q26;q24): a recurrent chromosomal abnormality in myelodysplastic syndrome and acute myeloid leukemia. Cancer Genet. Cytogenet. 166, 82–85 (2006).

    Article  CAS  Google Scholar 

  42. Stevens-Kroef, M. et al. Translocation t(2;3)(p15–23;q26–27) in myeloid malignancies: report of 21 new cases, clinical, cytogenetic and molecular genetic features. Leukemia 18, 1108–1114 (2004).

    Article  CAS  Google Scholar 

  43. Trubia, M. et al. Characterization of a recurrent translocation t(2;3)(p15–22;q26) occurring in acute myeloid leukaemia. Leukemia 20, 48–54 (2006).

    Article  CAS  Google Scholar 

  44. Laricchia-Robbio, L. & Nucifora, G. Significant increase of self-renewal in hematopoietic cells after forced expression of EVI1. Blood Cells Mol. Dis. 40, 141–147 (2007).

    Article  Google Scholar 

  45. Perkins, A.S., Mercer, J.A., Jenkins, N.A. & Copeland, N.G. Patterns of Evi-1 expression in embryonic and adult tissues suggest that Evi-1 plays an important regulatory role in mouse development. Development 111, 479–487 (1991).

    CAS  PubMed  Google Scholar 

  46. Kustikova, O.S. et al. Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways. Blood 109, 1897–1907 (2007).

    Article  CAS  Google Scholar 

  47. Senyuk, V. et al. Repression of RUNX1 activity by EVI1: a new role of EVI1 in leukemogenesis. Cancer Res. 67, 5658–5666 (2007).

    Article  CAS  Google Scholar 

  48. Chi, Y., Senyuk, V., Chakraborty, S. & Nucifora, G. EVI1 promotes cell proliferation by interacting with BRG1 and blocking the repression of BRG1 on E2F1 activity. J. Biol. Chem. 278, 49806–49811 (2003).

    Article  CAS  Google Scholar 

  49. Cattaneo, F. & Nucifora, G. EVI1 recruits the histone methyltransferase SUV39H1 for transcription repression. J. Cell. Biochem. 105, 344–352 (2008).

    Article  CAS  Google Scholar 

  50. Spensberger, D. & Delwel, R. A novel interaction between the proto-oncogene Evi1 and histone methyltransferases, SUV39H1 and G9a. FEBS Lett. 582, 2761–2767 (2008).

    Article  CAS  Google Scholar 

  51. Spensberger, D. et al. Myeloid transforming protein Evi1 interacts with methyl-CpG binding domain protein 3 and inhibits in vitro histone deacetylation by Mbd3/Mi-2/NuRD. Biochemistry 47, 6418–6426 (2008).

    Article  CAS  Google Scholar 

  52. Kondo, Y. et al. Downregulation of histone H3 lysine 9 methyltransferase G9a induces centrosome disruption and chromosome instability in cancer cells. PLoS One 3, e2037 (2008).

    Article  Google Scholar 

  53. Krämer, A., Neben, K. & Ho, A.D. Centrosome replication, genomic instability and cancer. Leukemia 16, 767–775 (2002).

    Article  Google Scholar 

  54. Zhang, Y. et al. MLL5 contributes to hematopoietic stem cell fitness and homeostasis. Blood 113, 1455–1463 (2009).

    Article  CAS  Google Scholar 

  55. Heuser, M. et al. Loss of MLL5 results in pleiotropic hematopoietic defects, reduced neutrophil immune function, and extreme sensitivity to DNA demethylation. Blood 113, 1432–1443 (2009).

    Article  CAS  Google Scholar 

  56. Madan, V. et al. Impaired function of primitive hematopoietic cells in mice lacking the mixed-lineage-leukemia homolog MLL5. Blood 113, 1444–1454 (2009).

    Article  CAS  Google Scholar 

  57. Sebastian, S. et al. MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation. Proc. Natl. Acad. Sci. USA 106, 4719–4724 (2009).

    Article  CAS  Google Scholar 

  58. Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009).

    Article  CAS  Google Scholar 

  59. Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat. Methods 4, 1051–1057 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We are indebted to K. Bleuel and S. Wehner for contributions and advice during the study; to H. Holtgreve-Grez and B. Schoell for I-FISH and M-FISH analysis, respectively; to H. Konrad Müller-Hermelink for reference pathology; to B. Staehle for quantitative PCR; to M.C. Dinauer (Indiana University School of Medicine) for plasmid gp91phox(P415H) and to H. Kunkel, E. Rudolf, A. Dillmann, R. Kramer, B. Moreno, I. Vogler and L. Chen-Wichmann for assistance during this work. This work was supported by the Bundesministerium für Bildung und Forschung (grant 01GU0507, TP6b), the Chronic Granulomatous Disorder Research Trust, London (grant J4G/04B/GT), the Georg-Speyer-Haus (T300131), the EU (VIth Framework Program, CONSERT), the Research Priority Program 1230 from the Deutsche Forschungsgemeinschaft and, in part, by a grant of the Tumorzentrum Heidelberg/Mannheim to A.J. A.J.T. is also supported by the Wellcome Trust and the Department of Health (HTH/011/025/004). A. Krämer is supported by the Deutsche Krebshilfe (grant 108560). The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und Kunst.

Author information

Author notes

  1. Stefan Stein, Marion G Ott, Dieter Hoelzer, Reinhard Seger, Christof von Kalle and Manuel Grez: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany

    Stefan Stein, Stephan Schultze-Strasser, Andrea Kinner, Carolin Preiss & Manuel Grez

  2. Department of Hematology/Oncology, University Medical School, Frankfurt, Germany

    Marion G Ott, Joachim Schwäble, Hans Martin & Dieter Hoelzer

  3. Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany

    Anna Jauch

  4. Molecular Epidemiology Group, German Cancer Research Center, Heidelberg, Germany

    Barbara Burwinkel, Sandrine Tchatchou & Rongxi Yang

  5. Division Molecular Biology of Breast Cancer, University Women's Clinic, Heidelberg, Germany

    Barbara Burwinkel, Sandrine Tchatchou & Rongxi Yang

  6. Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany

    Manfred Schmidt, Hanno Glimm, Claudia Ball, Kerstin Schwarzwaelder & Christof von Kalle

  7. German Cancer Research Center and Department of Internal Medicine V, Clinical Cooperation Unit Molecular Hematology/Oncology, University of Heidelberg, Heidelberg, Germany

    Alwin Krämer & Kadin Karakaya

  8. Pediatric Hematology, Oncology and Hemostaseology, University Medical School, Frankfurt, Germany

    Ulrike Koehl

  9. Department of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany

    Gudrun Göhring & Brigitte Schlegelberger

  10. Department of Hematology, Oncology and Transfusion Medicine, Charité, Campus Benjamin Franklin, Berlin, Germany

    Wolf-Karsten Hofmann

  11. Institute of Pathology, Heinrich-Heine University, Düsseldorf, Germany

    Petra Reinecke

  12. EUFETS AG, Idar-Oberstein, Germany

    Klaus Kühlcke

  13. Centre for Immunodeficiency, UCL Institute of Child Health, and Great Ormond Street Hospital for Children NHS Trust London, UK

    Adrian J Thrasher

  14. Division of Immunology/Hematology, University Children's Hospital Zurich, Zurich, Switzerland

    Reinhard Seger

Authors
  1. Stefan Stein
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  2. Marion G Ott
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Contributions

S.S., M.G.O. and M.G. conceived, organized and supervised the study; S.S., A. Kinner, S.S.-S., C.P. and K. Kühlcke performed experiments; B.B., S.T. and R.Y. performed the SNP and methylation arrays and analyzed data; A. Kinner, A. Krämer, C.B., K. Karakaya. and H.G. generated and analyzed data on genomic instability; A.J. performed the I-FISH and M-FISH analysis; S.S.-S. performed the CpG methylation studies; M.G.O., J.S., H.M., G.G., W.-K.H., P.R., B.S., U.K., R.S. and D.H. were responsible for patient care, analyzed materials from the subjects and provided data on these samples; M.S., K.S. and C.v.K. provided data on retroviral integrations sites; A.J.T., R.S., U.K., M.S., C.v.K. and M.G. prepared and wrote the manuscript.

Corresponding author

Correspondence to Manuel Grez.

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Supplementary Notes, Supplementary Methods, Supplementary Figures 1–9, Supplementary Tables 1–3 (PDF 11311 kb)

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Stein, S., Ott, M., Schultze-Strasser, S. et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med 16, 198–204 (2010). https://doi.org/10.1038/nm.2088

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