Abstract

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells1. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks2,3,4, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma5,6, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL–AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4−/− MLL–AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL–AF9 blasts, which requires cyclin-dependent kinase inhibitor p21Cip1 (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

Data sets have been deposited in the Gene Expression Omnibus under accession number GSE57147.

References

  1. 1.

    , & Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21, 283–296 (2012)

  2. 2.

    et al. DNA repair is limiting for haematopoietic stem cells during ageing. Nature 447, 686–690 (2007)

  3. 3.

    et al. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447, 725–729 (2007)

  4. 4.

    et al. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 128, 325–339 (2007)

  5. 5.

    et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476, 298–303 (2011)

  6. 6.

    et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nature Genet. 43, 830–837 (2011)

  7. 7.

    & MLL translocations, histone modifications and leukaemia stem-cell development. Nature Rev. Cancer 7, 823–833 (2007)

  8. 8.

    et al. Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors. Cell Stem Cell 1, 324–337 (2007)

  9. 9.

    & Grist for the MLL: how do MLL oncogenic fusion proteins generate leukemia stem cells? Int. J. Hematol. 91, 735–741 (2010)

  10. 10.

    et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495–501 (2014)

  11. 11.

    et al. H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation. eLife 2, e01503 (2013)

  12. 12.

    et al. A PML-PPAR-δ pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance. Nature Med. 18, 1350–1358 (2012)

  13. 13.

    et al. Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells. Cell 154, 1112–1126 (2013)

  14. 14.

    et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321 (2002)

  15. 15.

    et al. AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell 146, 697–708 (2011)

  16. 16.

    et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL–AF9. Nature 442, 818–822 (2006)

  17. 17.

    et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell 20, 66–78 (2011)

  18. 18.

    & Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation. Nature 461, 537–541 (2009)

  19. 19.

    et al. A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage. Cell 148, 1001–1014 (2012)

  20. 20.

    et al. BRCA1 induces antioxidant gene expression and resistance to oxidative stress. Cancer Res. 64, 7893–7909 (2004)

  21. 21.

    et al. BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival. J. Exp. Med. 210, 1529–1544 (2013)

  22. 22.

    et al. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nature Med. 12, 446–451 (2006)

  23. 23.

    , , & I-PpoI and I–CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment. J. Mol. Biol. 280, 345–353 (1998)

  24. 24.

    et al. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010)

  25. 25.

    et al. Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation. Cell 137, 1088–1099 (2009)

  26. 26.

    , & An oncogene-induced DNA damage model for cancer development. Science 319, 1352–1355 (2008)

  27. 27.

    et al. Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR. J. Clin. Invest. 122, 241–252 (2012)

  28. 28.

    , , , & Positive feedback between PU.1 and the cell cycle controls myeloid differentiation. Science 341, 670–673 (2013)

  29. 29.

    et al. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nature Cell Biol. 5, 675–679 (2003)

  30. 30.

    et al. ATM prevents the persistence and propagation of chromosome breaks in lymphocytes. Cell 130, 63–75 (2007)

  31. 31.

    et al. Leukemia-associated NOTCH1 alleles are weak tumor initiators but accelerate K-ras-initiated leukemia. J. Clin. Invest. 118, 3181–3194 (2008)

  32. 32.

    et al. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. Nature Struct. Mol. Biol. 18, 721–727 (2011)

  33. 33.

    et al. RNA-seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516–535 (2010)

  34. 34.

    , & TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009)

  35. 35.

    et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004)

  36. 36.

    et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature Genet. 25, 25–29 (2000)

  37. 37.

    et al. Reactome: a knowledge base of biologic pathways and processes. Genome Biol. 8, R39 (2007)

  38. 38.

    et al. Pathway Commons, a web resource for biological pathway data. Nucleic Acids Res. 39, D685–D690 (2011)

  39. 39.

    & Controlling the False discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995)

  40. 40.

    Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Applic. Genet. Molec. Biol. 3, 3 (2004)

Download references

Acknowledgements

We thank all members of the A. Nussenzweig laboratory, J. Daniel and P. Love for discussions; L. Granger for flow cytometry; M. J. Kruhlak for microscopy; K. Zhao for RNA sequencing; R. Anderson and K. Smith for animal care; K. Naka for the FOXO3 retrovirus; G. Legube for the pBABE-AsiSI-ER plasmid; J. Zuber for pLEPG and pRT3GEPIR plasmids; O. Fernandez-Capetillo for ATRi; and S. John for suggestions. S.A.A. was supported by the Leukemia and Lymphoma Society and National Cancer Institute grants CA66996 and CA140575. This work was supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute and the Center for Cancer Research, and an Ellison Medical Foundation Senior Scholar in Aging Award to A.N.

Author information

Author notes

    • Robert B. Faryabi
    • , Aysegul V. Ergen
    • , Amanda M. Day
    •  & Amy Malhowski

    These authors contributed equally to this work.

Affiliations

  1. Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Margarida A. Santos
    • , Robert B. Faryabi
    • , Aysegul V. Ergen
    • , Amanda M. Day
    • , Amy Malhowski
    • , Andres Canela
    • , Elsa Callen
    • , Hua-Tang Chen
    • , Nancy Wong
    • , Nadia Finkel
    •  & André Nussenzweig
  2. The Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Masahiro Onozawa
    •  & Peter D. Aplan
  3. Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Ji-Eun Lee
    •  & Kai Ge
  4. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, Massachusetts 02115, USA

    • Paula Gutierrez-Martinez
    •  & Derrick J. Rossi
  5. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Paula Gutierrez-Martinez
    •  & Derrick J. Rossi
  6. Human Oncology and Pathogenesis Program and Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Aniruddha Deshpande
    •  & Scott A. Armstrong
  7. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Susan Sharrow
  8. Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Departments of Cell Biology and Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    • Keisuke Ito

Authors

  1. Search for Margarida A. Santos in:

  2. Search for Robert B. Faryabi in:

  3. Search for Aysegul V. Ergen in:

  4. Search for Amanda M. Day in:

  5. Search for Amy Malhowski in:

  6. Search for Andres Canela in:

  7. Search for Masahiro Onozawa in:

  8. Search for Ji-Eun Lee in:

  9. Search for Elsa Callen in:

  10. Search for Paula Gutierrez-Martinez in:

  11. Search for Hua-Tang Chen in:

  12. Search for Nancy Wong in:

  13. Search for Nadia Finkel in:

  14. Search for Aniruddha Deshpande in:

  15. Search for Susan Sharrow in:

  16. Search for Derrick J. Rossi in:

  17. Search for Keisuke Ito in:

  18. Search for Kai Ge in:

  19. Search for Peter D. Aplan in:

  20. Search for Scott A. Armstrong in:

  21. Search for André Nussenzweig in:

Contributions

M.A.S., R.B.F., P.D.A., S.A.A. and A.N. participated in the study design. M.A.S., A.V.E., A.M.D., A.M., N.F., H.C. and N.W. performed mouse breeding, HSC analysis, transplantation and leukaemia experiments, and analysed data. R.B.F. performed computational experiments. E.C. led the genome stability experiments and analysed data. A.C. generated and performed experiments with AsiSI-ER-Tet-on and MLL4 shRNA and qPCR. P.G.-M. and D.J.R. supervised HSC experiments and performed serial colony assays; S.S. supervised flow cytometry. J.-E.L. and K.G. generated targeting vector and MLL4-deficient mice. K.I. performed the in vitro immunophenotypic division assay. M.O. quantified colony morphology in cytospins. A.D. generated and tested MLL–AF9 vectors. M.A.S. and A.N. wrote the manuscript and all authors reviewed it. A.N. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to André Nussenzweig.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13483

Further reading 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.