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.

  • Article
  • Published:

Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia

Nature Cell Biology volume 12, pages 372–379 (2010)Cite this article

Subjects

Abstract

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17–92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17–92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17–92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: miR-19 enhances cytokine independent survival in vitro.
Figure 2: Pooled miRNA screen for tumorigenic activities within the 17–92 cluster and its paralogues.
Figure 3: miR-19 is a novel T-ALL oncogene.
Figure 4: Gene expression analysis of parental and miR-19-transduced FL5-12 cells.
Figure 5: Genetic screen for shRNAs that phenocopy miR-19 in lymphocyte survival.
Figure 6: Summary of the screen result.
Figure 7: The identified genes are actual targets of miR-19.
Figure 8: miR-19 acts through multiple negative regulators of PI(3)K-related survival signals.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    Article  CAS  Google Scholar 

  2. Friedman, R.C., Farh, K.K., Burge, C.B. & Bartel, D.P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105 (2009).

    Article  CAS  Google Scholar 

  3. Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007).

    Article  CAS  Google Scholar 

  4. Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, C.B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).

    Article  CAS  Google Scholar 

  5. Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).

    Article  CAS  Google Scholar 

  6. Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).

    Article  CAS  Google Scholar 

  7. Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature 460, 479–486 (2009).

    Article  CAS  Google Scholar 

  8. Xiao, C. & Rajewsky, K. MicroRNA control in the immune system: basic principles. Cell 136, 26–36 (2009).

    Article  CAS  Google Scholar 

  9. Xiao, C. et al. Lymphoproliferative disease and autoimmunity in mice with increased miR-17–92 expression in lymphocytes. Nature Immunol. 9, 405–414 (2008).

    Article  CAS  Google Scholar 

  10. He, L. et al. A microRNA polycistron as a potential human oncogene. Nature 435, 828–833 (2005).

    Article  CAS  Google Scholar 

  11. Mendell, J.T. miRiad roles for the miR-17–92 cluster in development and disease. Cell 133, 217–222 (2008).

    Article  CAS  Google Scholar 

  12. Ventura, A. et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 132, 875–886 (2008).

    Article  CAS  Google Scholar 

  13. Mu, P. et al. Genetic dissection of the miR-17∼92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 23, 2806–2811 (2009).

    Article  CAS  Google Scholar 

  14. Olive, V. et al. miR-19 is a key oncogenic component of mir-17–92. Genes Dev. 23, 2839–2849 (2009).

    Article  CAS  Google Scholar 

  15. O'Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V. & Mendell, J.T. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843 (2005).

    Article  CAS  Google Scholar 

  16. Petrocca, F. et al. E2F1-regulated microRNAs impair TGFβ-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 13, 272–286 (2008).

    Article  CAS  Google Scholar 

  17. Plas, D.R., Talapatra, S., Edinger, A.L., Rathmell, J.C. & Thompson, C.B. Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology. J. Biol. Chem. 276, 12041–12048 (2001).

    Article  CAS  Google Scholar 

  18. Malumbres, R. et al. Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas. Blood 113, 3754–3764 (2009).

    Article  CAS  Google Scholar 

  19. Calin, G.A. & Croce, C.M. Investigation of microRNA alterations in leukemias and lymphomas. Methods Enzymol. 427, 193–213 (2007).

    CAS  PubMed  Google Scholar 

  20. Palomero, T. et al. Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias. Leukemia 20, 1963–1966 (2006).

    Article  CAS  Google Scholar 

  21. Ellisen, L.W. et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66, 649–661 (1991).

    Article  CAS  Google Scholar 

  22. Weng, A.P. et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306, 269–271 (2004).

    Article  CAS  Google Scholar 

  23. Pear, W.S. et al. Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J. Exp. Med. 183, 2283–2291 (1996).

    Article  CAS  Google Scholar 

  24. Edgar, R., Domrachev, M. & Lash, A.E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).

    Article  CAS  Google Scholar 

  25. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    Article  CAS  Google Scholar 

  26. Silva, J.M. et al. Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319, 617–620 (2008).

    Article  CAS  Google Scholar 

  27. Chang, K., Elledge, S.J. & Hannon, G.J. Lessons from Nature: microRNA-based shRNA libraries. Nature Methods 3, 707–714 (2006).

    Article  CAS  Google Scholar 

  28. Bouillet, P. et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286, 1735–1738 (1999).

    Article  CAS  Google Scholar 

  29. Paddison, P.J., Caudy, A.A., Sachidanandam, R. & Hannon, G.J. Short hairpin activated gene silencing in mammalian cells. Methods Mol. Biol. 265, 85–100 (2004).

    CAS  PubMed  Google Scholar 

  30. John, B. et al. Human microRNA targets. PLoS Biol. 2, e363 (2004).

    Article  Google Scholar 

  31. Mavrakis, K.J. et al. Tumorigenic activity and therapeutic inhibition of Rheb GTPase. Genes Dev. 22, 2178–2188 (2008).

    Article  CAS  Google Scholar 

  32. Wendel, H.G. et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 428, 332–337 (2004).

    Article  CAS  Google Scholar 

  33. Yang, Y.H., Paquet, A. & Dudoit, S. R package version 1.12.0 (2006).

  34. Tusher, V.G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001).

    Article  CAS  Google Scholar 

  35. Efron, B. & Tibshirani, R. On testing the significance of sets of genes. Ann. Appl. Statist. 1, 107–129 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

We thank Lin He, S.W. Lowe and G. Hannon for access to shRNA and miRNA screening technologies; W. Pear, J. Huse and E.C. Holland for plasmids; H. Zhu for technical assistance; the Memorial Sloan-Kettering (MSK) animal facility and Research Animal Resource Center (RARC), A. Viale of the MSK Genomics Core, H. Zhao of the cBIO program, and J. Schatz and J. Massagué for editorial advice; and V. Murty for cytogenetic and fluorescence in situ hybridization analyses. This work was supported by grants from the American Cancer Society, the Geoffrey Beene Cancer Center, the Leukemia Research Foundation, the Louis V. Gerstner Foundation, the May and Samuel Rudin Foundation, the NY Community Trust and the William and Blanche Foundation (to H.-G.W.), from the Andrew Seligson Memorial Clinical Fellowship (to J.Z.), and NYStar (to P.J.P.). A.F. is supported by R01CA120196, the WOLF Foundation, the Rally across America Foundation and the Leukemia and Lymphoma Society (grants 1287-08 and 6237-08), and A.F. is a Leukemia & Lymphoma Society Scholar.

Author information

Author notes

  1. Taneisha James

    Present address: Present address: Department of Gene and Cell Medicine & Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York 10029, USA.,

  2. Konstantinos J. Mavrakis and Andrew L. Wolfe: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Biology & Genetics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10021, New York, USA

    Konstantinos J. Mavrakis, Andrew L. Wolfe, Elisa Oricchio & Hans-Guido Wendel

  2. Weill Cornell Graduate School of Medical Science, New York, 10065, New York, USA

    Andrew L. Wolfe & Aly A. Khan

  3. Department of Pathology, Columbia University, New York, 10032, New York, USA

    Teresa Palomero & Adolfo Ferrando

  4. Institute for Cancer Genetics, Columbia University, New York, 10032, New York, USA

    Teresa Palomero, Kim de Keersmaecker & Adolfo Ferrando

  5. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 11724, USA

    Katherine McJunkin, Johannes Zuber & Taneisha James

  6. Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 11724, USA

    Katherine McJunkin

  7. Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Aly A. Khan & Christina S. Leslie

  8. Expression Analysis Inc., Durham, 27502, North Carolina, USA

    Joel S. Parker

  9. Fred Hutchinson Cancer Research Center, Seattle, 98109, Washington, USA

    Patrick J. Paddison

  10. Department of Pathology and Laboratory Medicine, Joan and Sanford I. Weill Medical College of Cornell University, New York, 10025, New York, USA

    Wayne Tam

Authors
  1. Konstantinos J. Mavrakis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew L. Wolfe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elisa Oricchio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Teresa Palomero
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kim de Keersmaecker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katherine McJunkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Johannes Zuber
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Taneisha James
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Aly A. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christina S. Leslie
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Joel S. Parker
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Patrick J. Paddison
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Wayne Tam
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Adolfo Ferrando
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Hans-Guido Wendel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.J.M, A.L.W. and E.O. performed experimental design and analysis. K.d.K., T.P. and A.F. conducted T-ALL translocation analysis. K.M., J.Z., T.J. and K.C. performed the screen and analysis. A.A.K., C.S.L. and J.S.P. did data analysis. P.J.P. generated the shRNA library. W.T. was responsible for clinical specimens. H.-G.W. designed the study and wrote the paper.

Corresponding author

Correspondence to Hans-Guido Wendel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 924 kb)

Supplementary Information

Supplementary Table 1 (XLS 18 kb)

Supplementary Information

Supplementary Table 2 (XLS 31 kb)

Supplementary Information

Supplementary Table 3 (XLS 126 kb)

Supplementary Information

Supplementary Table 4 (XLS 109 kb)

Supplementary Information

Supplementary Table 5 (XLS 82 kb)

Supplementary Information

Supplementary Table 6 (XLS 1834 kb)

Supplementary Information

Supplementary Table 7 (XLS 280 kb)

Supplementary Information

Supplementary Table 8 (XLS 31 kb)

Supplementary Information

Supplementary Table 9 (XLS 30 kb)

Supplementary Information

Supplementary Table 10 (XLS 27 kb)

Supplementary Information

Supplementary Table 11 (XLS 16 kb)

Supplementary Information

Supplementary Information (PDF 105 kb)

Supplementary Information

Supplementary Information (PDF 142 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mavrakis, K., Wolfe, A., Oricchio, E. et al. Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia. Nat Cell Biol 12, 372–379 (2010). https://doi.org/10.1038/ncb2037

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2037

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