Article

Small-molecule inhibitors targeting INK4 protein p18INK4C enhance ex vivo expansion of haematopoietic stem cells

  • Nature Communications volume 6, Article number: 6328 (2015)
  • doi:10.1038/ncomms7328
  • Download Citation
Received:
Accepted:
Published:

Abstract

Among cyclin-dependent kinase inhibitors that control the G1 phase in cell cycle, only p18 and p27 can negatively regulate haematopoietic stem cell (HSC) self-renewal. In this manuscript, we demonstrate that p18 protein is a more potent inhibitor of HSC self-renewal than p27 in mouse models and its deficiency promoted HSC expansion in long-term culture. Single-cell analysis indicated that deleting p18 gene favoured self-renewing division of HSC in vitro. Based on the structure of p18 protein and in-silico screening, we further identified novel small-molecule inhibitors that can specifically block the activity of p18 protein. Our selected lead compounds were able to expand functional HSCs in a short-term culture. Thus, these putative small-molecule inhibitors for p18 protein are valuable for further dissecting the signalling pathways of stem cell self-renewal and may help develop more effective chemical agents for therapeutic expansion of HSC.

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Cell cycle inhibitors in normal and tumor stem cells. Oncogene 23, 7256–7266 (2004).

  2. 2.

    & The CDK inhibitors: potential targets for therapeutic stem cell manipulations? Gene Ther. 15, 117–125 (2008).

  3. 3.

    et al. p57 is required for quiescence and maintenance of adult hematopoietic stem cells. Cell Stem Cell 9, 262–271 (2011).

  4. 4.

    et al. p57(Kip2) and p27(Kip1) cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70. Cell Stem Cell 9, 247–261 (2011).

  5. 5.

    & Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9, 1149–1163 (1995).

  6. 6.

    , , , & In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nat. Cell. Biol. 6, 436–442 (2004).

  7. 7.

    , , & Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners. Blood 107, 1200–1206 (2006).

  8. 8.

    et al. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 443, 421–426 (2006).

  9. 9.

    , , , & Cyclin dependent kinase inhibitors differentially modulate synergistic cytokine responsiveness of hematopoietic progenitor cells. Stem Cells Dev. 21, 1597–1603 (2012).

  10. 10.

    , , & Steel factor responsiveness regulates the high self-renewal phenotype of fetal hematopoietic stem cells. Blood 109, 5043–5048 (2007).

  11. 11.

    , , , & The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells. Nat. Cell. Biol. 12, 1213–1219 (2010).

  12. 12.

    , , , & Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells. Nat. Cell. Biol. 7, 172–178 (2005).

  13. 13.

    , , , & Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nature Med. 6, 1235–1240 (2000).

  14. 14.

    , , & Asymmetric division and lineage commitment at the level of hematopoietic stem cells: inference from differentiation in daughter cell and granddaughter cell pairs. J. Exp. Med. 199, 295–302 (2004).

  15. 15.

    , , & In vitro self-renewal division of hematopoietic stem cells. J. Exp. Med. 192, 1281–1288 (2000).

  16. 16.

    et al. M-CSF instructs myeloid lineage fate in single haematopoietic stem cells. Nature 497, 239–243 (2013).

  17. 17.

    , & GPCR structure-based virtual screening approach for CB2 antagonist search. J. Chem. Inf. Model. 47, 1626–1637 (2007).

  18. 18.

    , & 3D structural model of the G-protein-coupled cannabinoid CB2 receptor. Proteins 53, 307–319 (2003).

  19. 19.

    , & Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations. Nature Struct. Biol. 5, 74–81 (1998).

  20. 20.

    , & Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes Dev. 14, 3115–3125 (2000).

  21. 21.

    & Data-mining a small molecule drug screening representative subset from NIH PubChem database. J. Comput. Inf. Model. 48, 465–475 (2008).

  22. 22.

    , & In vitro proliferation of hemopoietic stem cells in long-term marrow cultures: principles in mouse applied to man. Exp. Hematol. 7(Suppl 5), 135–148 (1979).

  23. 23.

    et al. Stem cell traits in long-term co-culture revealed by time-lapse imaging. Leukemia 24, 153–161 (2010).

  24. 24.

    et al. IGF binding protein 2 supports the survival and cycling of hematopoietic stem cells. Blood 118, 3236–3243 (2011).

  25. 25.

    , , , & Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation. Blood 111, 3415–3423 (2008).

  26. 26.

    , , & Small molecules, big roles -- the chemical manipulation of stem cell fate and somatic cell reprogramming. J. Cell. Sci. 125, 5609–5620 (2012).

  27. 27.

    , , , & Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Med. 10, 55–63 (2004).

  28. 28.

    , , & Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation. Nature Med. 12, 89–98 (2006).

  29. 29.

    et al. Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion. Genes Dev. 25, 1928–1942 (2011).

  30. 30.

    , , , & Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways. Nature Med. 18, 1778–1785 (2012).

  31. 31.

    et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 329, 1345–1348 (2010).

  32. 32.

    et al. Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 345, 1509–1512 (2014).

  33. 33.

    et al. An opposite effect of the CDK inhibitor, p18(INK4c) on embryonic stem cells compared with tumor and adult stem cells. PLoS ONE 7, e45212 (2012).

  34. 34.

    et al. Hematopoietic stem cells are not the direct target of spontaneous leukemic transformation in p18(INK4C)-null reconstituted mice. Cancer Res. 66, 343–351 (2006).

  35. 35.

    , & Mouse hematopoietic stem cell transplantation. Methods Mol. Biol. 976, 25–35 (2013).

  36. 36.

    , & Flow cytometry-based cell cycle measurement of mouse hematopoietic stem and progenitor cells. Methods Mol. Biol. 430, 77–86 (2008).

  37. 37.

    , , , & Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity. J. Biol. Chem. 277, 48827–48833 (2002).

Download references

Acknowledgements

This work was funded by the National Basic Research Program of China (NO. 2011CB964800, 2012CB966600), the National Natural Science Foundation of China (NSFC 90913018, 81090410, 81421002, 30825017 and 81170465), the key technologies R&D Program of Tianjin (13RCGFSF19500) and NIH R21HL1096541.

Author information

Author notes

    • Yingdai Gao
    • , Peng Yang
    •  & Hongmei Shen

    These authors contributed equally to this work

Affiliations

  1. State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China

    • Yingdai Gao
    • , Liyan Zhang
    • , Sha Hao
    • , Fang Dong
    • , Shihui Ma
    • , Qing Ji
    • , Yahui Ding
    • , Yanxin Li
    • , Hui Cheng
    • , Weimin Miao
    • , Weiping Yuan
    • , Youzhong Yuan
    •  & Tao Cheng
  2. Department of Pharmaceutical Sciences, Computational Chemical Genomics Screening Center, School of Pharmacy, NIH National Center of Excellence for Drug Abuse Research, Drug Discovery Institute, Pittsburgh, Pennsylvania 15260, USA

    • Peng Yang
    • , Peng Zhang
    • , Haizi Cheng
    • , Zhaojun Xie
    • , Patrick Bartlow
    • , Lirong Wang
    • , Haibin Liu
    •  & Xiang-Qun Xie
  3. Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA

    • Peng Yang
    • , Peng Zhang
    • , Haizi Cheng
    • , Zhaojun Xie
    • , Patrick Bartlow
    • , Lirong Wang
    • , Haibin Liu
    •  & Xiang-Qun Xie
  4. Department of Radiation Oncology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA

    • Hongmei Shen
    •  & Hui Yu
  5. Department of Hematology, Changhai Hospital, Secondary Military Medical University, Shanghai 200433, China

    • Xianmin Song

Authors

  1. Search for Yingdai Gao in:

  2. Search for Peng Yang in:

  3. Search for Hongmei Shen in:

  4. Search for Hui Yu in:

  5. Search for Xianmin Song in:

  6. Search for Liyan Zhang in:

  7. Search for Peng Zhang in:

  8. Search for Haizi Cheng in:

  9. Search for Zhaojun Xie in:

  10. Search for Sha Hao in:

  11. Search for Fang Dong in:

  12. Search for Shihui Ma in:

  13. Search for Qing Ji in:

  14. Search for Patrick Bartlow in:

  15. Search for Yahui Ding in:

  16. Search for Lirong Wang in:

  17. Search for Haibin Liu in:

  18. Search for Yanxin Li in:

  19. Search for Hui Cheng in:

  20. Search for Weimin Miao in:

  21. Search for Weiping Yuan in:

  22. Search for Youzhong Yuan in:

  23. Search for Tao Cheng in:

  24. Search for Xiang-Qun Xie in:

Contributions

Y.G., P.Y. and H.S. designed the study, performed the experiments, analysed/interpreted the data and wrote the manuscript; H.Y., X.S., L.Z., P.Z., H.C., Z.X., S.H., F.D., S.M., Q.J., P.B., Y.D., L.W., H.L, Y.L., H.C., W.M., W.Y, and Y.Y. performed the study, analysed the data. T.C. and X.-Q.X. conceived and designed the research, analysed the data, and wrote and edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Tao Cheng or Xiang-Qun Xie.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1-5, Supplementary Table 1, Supplementary Note and Supplementary References

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.