Abstract

The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.

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Acknowledgements

We would like to thank S. Nimer and N. Rosen for their critical advice and helpful suggestions. We would also like to thank A. Viale and the MSKCC sequencing core for all their support. We would like to thank the Lowe laboratory (Memorial Sloan Kettering Cancer Center) for their generous gift of the RN2 cells26 and the CRISPR–Cas9 constructs. We would like to thank Y. Neelamraju for technical support. M.G.K. was supported by the US National Institutes of Health National Institute of Diabetes, Digestive and Kidney Diseases Career Development Award, NIDDK NIH R01-DK101989-01A1, NCI 1R01CA193842-01, Louis V. Gerstner Young Investigator Award, American Society of Hematology Junior Scholar Award, Kimmel Scholar Award, V-Scholar Award, Geoffrey Beene Award and Alex's Lemonade Stand A Award and the Starr Foundation (M.G.K. and L.C.). L.P.V. is supported by the Damon Runyon-Sohn Pediatric Cancer Fellowship Award, E.P. was supported by grants U24 CA114737 and U10 CA180827. C.J.L. was supported by an R01 grant from the National Cancer Institute (NIH) and a fellowship from the W.W. Smith Charitable Trust. The research was funded in part through NIH/NCI Cancer Support Core Grant P30 CA08748 to M.G.K. and R.G.

Author information

Affiliations

  1. Molecular Pharmacology Program, Center for Cell Engineering, Center for Stem Cell Biology, and Center for Experimental Therapeutics, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Ly P Vu
    • , Camila Prieto
    • , Elianna M Amin
    • , Timothy Chou
    • , Arthur Chow
    • , Gerard Minuesa
    • , Sun Mi Park
    • , Trevor S Barlowe
    • , James Taggart
    • , Patrick Tivnan
    •  & Michael G Kharas
  2. Physiology, Biophysics and Systems Biology Graduate Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, New York, USA.

    • Camila Prieto
    •  & Sagar Chhangawala
  3. Computational Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Sagar Chhangawala
    • , Yuheng Lu
    •  & Christina Leslie
  4. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Andrei Krivtsov
    •  & Scott A Armstrong
  5. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Andrei Krivtsov
    • , Scott A Armstrong
    •  & Leandro Cerchietti
  6. Department of Medicine, Division of Hematology/Oncology, Weill Cornell Medical College, New York, New York, USA.

    • M Nieves Calvo-Vidal
  7. Harvard Medical School, Boston, Massachusetts, USA.

    • Raquel P Deering
    •  & Nir Hacohen
  8. Division of Hematology, Brigham and Woman's Hospital, Boston, Massachusetts, USA.

    • Lisa P Chu
    •  & Benjamin L Ebert
  9. Whitehead Institute, Boston, Massachusetts, USA.

    • Jeong-Ah Kwon
  10. Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.

    • Cem Meydan
  11. Translational Bioinformatics Unit, Clinical Research Programme, Spanish National Cancer Research Centre, Madrid, Spain.

    • Javier Perales-Paton
    •  & Fatima Al-Shahrour
  12. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Arora Arshi
    •  & Mithat Gönen
  13. Human Oncology and Pathogenesis Program, Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Christopher Famulare
    • , Minal Patel
    •  & Ross Levine
  14. Department of Medicine, Montefiore Medical Center, Bronx, New York, USA.

    • Elisabeth Paietta
  15. Leukemia Service, Department of Medicine, Memorial Sloan Kettering Hospital, New York, New York, USA.

    • Martin S Tallman
    •  & Jacob Glass
  16. Department of Medicine and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA.

    • Francine E Garret-Bakelman
  17. Division of Hematology and Medical Oncology, Departments of Medicine and Pharmacology, Weill Cornell Medical College, Cornell University, New York, New York, USA.

    • Francine E Garret-Bakelman
    •  & Ari Melnick
  18. Department of Clinical Genetics, Lund University, Lund, Sweden.

    • Marcus Järås
  19. RNAi Core, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Alexia Hwang
    •  & Ralph Garippa
  20. Department of Animal Biology, Department of Cell and Developmental Biology, and Institute for Regenerative Medicine, Schools of Veterinary Medicine and Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Christopher J Lengner
  21. Discovery Sciences, Janssen Research and Development, Spring House, Pennsylvania, USA.

    • Glenn S Cowley
  22. Broad Institute, Boston, Massachusetts, USA.

    • David Root
    •  & John Doench

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Contributions

M.G.K. directed the project, performed experiments, analyzed data, and wrote the manuscript. L.P.V. led the project, performed experiments, analyzed data, and wrote the manuscript. C.P., E.M.A., M.N.C.-V., T.C., A.C., G.M., T.S.B., J.T., P.T., R.P.D., L.P.C., and J.-A.K. performed experiments. J.P.-P., F.A.-S. and M.J. performed shRNA screening data analysis. S.C. and Y.L. performed RNA sequencing data analysis. C.M., A.A., M.G., C.F., M.P., E.P., M.S.T., J.G., and F.E.G.-B. provided clinical data and analysis. A.H. and R.G. provided critical reagents. S.M.P., A.M., R.L., N.H., C.J.L., S.A.A. L.C., G.S.C., D.R., J.D., C.L. and B.L.E. provided suggestions and project support.

Competing interests

There is a patent pending (M.G.K. and L.P.V.).

Corresponding author

Correspondence to Michael G Kharas.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

Excel files

  1. 1.

    Supplementary Table 1

    Mass spectrometry list of MSI2 protein interactions.

  2. 2.

    Supplementary Table 2

    Top 1,000 targets bound by MSI1 in the intestine.

  3. 3.

    Supplementary Table 3

    List of gene sets used that are hematopoietic and MSI related.

  4. 4.

    Supplementary Table 4

    Prioritization matrix gene list for pool 1a.

  5. 5.

    Supplementary Table 5

    Prioritization matrix gene list for pool 1b.

  6. 6.

    Supplementary Table 6

    Prioritization matrix gene list for pool 2.

  7. 7.

    Supplementary Table 7

    Prioritization matrix for pool 3.

  8. 8.

    Supplementary Table 8

    List of genes included in the in vivo shRNA screen associated with the MSI2 interactome.

  9. 9.

    Supplementary Table 9

    Raw normalized read counts of all target sequences in the in vivo shRNA screen.

  10. 10.

    Supplementary Table 10

    List of hits from the in vivo MSI2 interactome shRNA screen.

  11. 11.

    Supplementary Table 11

    PCR primers for Syncrip CR-KO genotyping.

  12. 12.

    Supplementary Table 12

    List of differentially expressed genes in Syncrip-shRNA leukemia cells with log2 (fold change) >1.5.

  13. 13.

    Supplementary Table 13

    Ranked list of all genes expressed in Syncrip-shRNA leukemia cells with log2 (fold change).

  14. 14.

    Supplementary Table 14

    GSEA analysis of enrichment for pathways that are negatively regulated by SYNCRIP.

  15. 15.

    Supplementary Table 15

    GSEA analysis of enrichment for pathways that are positively regulated by SYNCRIP.

  16. 16.

    Supplementary Table 16

    List of additional GSEA analysis for stem cell–associated pathways.

  17. 17.

    Supplementary Table 17

    Information on primary AML samples from patients used in Figure 7f.

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DOI

https://doi.org/10.1038/ng.3854

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