Article | Published:

Stem cell transplantation

Rational identification of a Cdc42 inhibitor presents a new regimen for long-term hematopoietic stem cell mobilization

Leukemia (2018) | Download Citation

Abstract

Mobilization of hematopoietic stem cells (HSCs) from bone marrow (BM) to peripheral blood (PB) by cytokine granulocyte colony-stimulating factor (G-CSF) or the chemical antagonist of CXCR4, AMD3100, is important in the treatment of blood diseases. Due to clinical conditions of each application, there is a need for continued improvement of HSC mobilization regimens. Previous studies have shown that genetic ablation of the Rho GTPase Cdc42 in HSCs results in their mobilization without affecting survival. Here we rationally identified a Cdc42 activity-specific inhibitor (CASIN) that can bind to Cdc42 with submicromolar affinity and competitively interfere with guanine nucleotide exchange activity. CASIN inhibits intracellular Cdc42 activity specifically and transiently to induce murine hematopoietic stem/progenitor cell egress from the BM by suppressing actin polymerization, adhesion, and directional migration of stem/progenitor cells, conferring Cdc42 knockout phenotypes. We further show that, although, CASIN administration to mice mobilizes similar number of phenotypic HSCs as AMD3100, it produces HSCs with better long-term reconstitution potential than that by AMD3100. Our work validates a specific small molecule inhibitor for Cdc42, and demonstrates that signaling molecules downstream of cytokines and chemokines, such as Cdc42, constitute a useful target for long-term stem cell mobilization.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Morrison SJ, Spradling AC. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell. 2008;132:598–611.

  2. 2.

    Boulais PE, Frenette PS. Making sense of hematopoietic stem cell niches. Blood. 2015;125:2621–9.

  3. 3.

    Li L, Xie T. Stem cell niche: structure and function. Annu Rev Cell Dev Biol. 2005;21:605–31.

  4. 4.

    Bernitz JM, Kim HS, MacArthur B, Sieburg H, Moore K. Hematopoietic stem cells count and remember self-renewal divisions. Cell. 2016;167:1296–1309.e10.

  5. 5.

    Crane GM, Jeffery E, Morrison SJ. Adult haematopoietic stem cell niches. Nat Rev Immunol. 2017;17:573–90.

  6. 6.

    Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci USA. 1999;96:3120–5.

  7. 7.

    van den Brink MR, Burakoff SJ. Cytolytic pathways in haematopoietic stem-cell transplantation. Nat Rev Immunol. 2002;2:273–81.

  8. 8.

    Wagner JE, Eapen M, MacMillan ML, Harris RE, Pasquini R, Boulad F, et al. Unrelated donor bone marrow transplantation for the treatment of Fanconi anemia. Blood. 2007;109:2256–62.

  9. 9.

    Gluckman E, Wagner JE. Hematopoietic stem cell transplantation in childhood inherited bone marrow failure syndrome. Bone Marrow Transplant. 2010;41:127–32.

  10. 10.

    Nikiforow S, Ritz J. (2016) Dramatic expansion of HSCs: new possibilities for HSC transplants? Cell Stem Cell. 2008;18:10–12.

  11. 11.

    Dufour C. How I manage patients with Fanconi anaemia. Br J Haematol. 2017;178:32–47.

  12. 12.

    de Haan G, Lazare SS. Aging of hematopoietic stem cells. Blood. 2018;131:479–87.

  13. 13.

    Papayannopoulou T. Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization. Blood. 2004;103:1580–5.

  14. 14.

    Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ, Winkler I. et al. G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow. Blood. 2005;106:3020–7.

  15. 15.

    Cancelas JA, Williams DA. Stem cell mobilization by beta2-agonists. Nat Med. 2006;12:278–9.

  16. 16.

    Hopman RK, DiPersio JF. Advances in stem cell mobilization. Blood Rev. 2014;8:31–40.

  17. 17.

    Cao LQ, Liu L, Xu LP, Zhang XH, Wang Y, Fan QZ, et al. Correlation between pediatric donor characteristics and cell compositions in mixture allografts of combined G-CSF-mobilized PBSCs and bone marrow allografts. Bone Marrow Transplant. 2018;53:108–10.

  18. 18.

    Flomenberg N, Devine SM, Dipersio JF, Liesveld JL, McCarty JM, Rowley SD. et al. The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood. 2005;106:1867–74.

  19. 19.

    Leotta S, Poidomani M, Mauro E, Spadaro A, Marturano E, Milone G. AMD3100 for urgent PBSC mobilization and allogeneic transplantation from a normal donor after failed marrow harvest. Bone Marrow Transplant. 2011;46:314–6.

  20. 20.

    Dar A, Schajnovitz A, Lapid K, Kalinkovich A, Itkin T, Ludin A, et al. Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells. Leukemia. 2011;25:1286–96.

  21. 21.

    Liu L, Yu Q, Fu S, Wang B, Hu K, Wang L, et al. The CXCR4 antagonist AMD3100 promotes mesenchymal stem cell mobilization in rats preconditioned with the hypoxia-mimicking agent cobalt chloride. Stem Cells Dev. 2018;27:466–478.

  22. 22.

    Hoggatt J, Singh P, Tate TA, Chou BK, Datari SR, Fukuda S, et al. Rapid mobilization reveals a highly engraftable hematopoietic stem cell. Cell. 2018;172:191–204.

  23. 23.

    Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;420:629–35.

  24. 24.

    Mulloy JC, Cancelas JA, Filippi MD, Kalfa TA, Guo F, Zheng Y. Rho GTPases in hematopoiesis and hemopathies. Blood. 2010;115:936–47.

  25. 25.

    Yang L, Wang L, Geiger H, Cancelas JA, Mo J, Zheng Y. Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow. Proc Natl Acad Sci USA. 2007;104:5091–6.

  26. 26.

    Yang L, Zheng Y. Cdc42 - a signal coordinator in hematopoietic stem cell maintenance. Cell Cycle. 2007;6:1445–50.

  27. 27.

    Florian MC, Dörr K, Niebel A, Daria D, Schrezenmeier H, Rojewski M, et al. Cdc42 activity regulates hematopoietic stem cell aging and rejuvenation. Cell Stem Cell. 2012;10:520–30.

  28. 28.

    Gu Y, Filippi MD, Cancelas JA, Siefring JE, Williams EP, Jasti AC, et al. Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases. Science. 2003;302:445–9.

  29. 29.

    Cancelas JA, Lee AW, Prabhakar R, Stringer KF, Zheng Y, Williams DA. Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization. Nat Med. 2005;11:886–91.

  30. 30.

    Wang L, Yang L, Filippi MD, Williams DA, Zheng Y. Genetic deletion of Cdc42GAP reveals a role of Cdc42 in erythropoiesis and hematopoietic stem/progenitor cell survival, adhesion, and engraftment. Blood. 2006;107:98–105.

  31. 31.

    Peterson JR, Lebensohn AM, Pelish HE, Kirschner MW. Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components. Chem Biol. 2006;13:443–52.

  32. 32.

    Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun. 2010;1:100.

  33. 33.

    Adams GB, Scadden DT. The hematopoietic stem cell in its place. Nat Immunol. 2006;7:333–7.

  34. 34.

    Pitchford SC, Furze RC, Jones CP, Wengner AM, Rankin SM. Differential mobilization of subsets of progenitor cells from the bone marrow. Cell Stem Cell. 2009;4:62–72.

  35. 35.

    Christopherson KW 2nd, Hangoc G, Mantel CR, Broxmeyer HE. Modulation of hematopoietic stem cell homing and engraftment by CD26. Science. 2004;305:1000–3.

  36. 36.

    Shiozawa Y, Pedersen EA, Havens AM, Jung Y, Mishra A, Joseph J, et al. Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow. J Clin Invest. 2011;121:1298–312.

  37. 37.

    Surviladze Z, Waller A, Strouse JJ, Bologa C, Ursu O, Salas V, et al. A potent and selective inhibitor of Cdc42 GTPase. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-2010 Feb 27.

  38. 38.

    Hong L, Kenney SR, Phillips GK, Simpson D, Schroeder CE, Nöth J, et al. Characterization of a Cdc42 protein inhibitor and its use as a molecular probe. J Biol Chem. 2013;288:8531–43.

  39. 39.

    Yamao M, Naoki H, Kunida K, Aoki K, Matsuda M, Ishii S. Distinct predictive performance of Rac1 and Cdc42 in cell migration. Sci Rep. 2015;5:17527.

  40. 40.

    Zins K, Lucas T, Reichl P, Abraham D, Aharinejad S. A Rac1/Cdc42 GTPase-specific small molecule inhibitor suppresses growth of primary human prostate cancer xenografts and prolongs survival in mice. PLoS ONE. 2013;8:e74924.

  41. 41.

    Friesland A, Zhao Y, Chen YH, Wang L, Zhou H, Lu Q. Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility. Proc Natl Acad Sci USA. 2013;110:1261–6.

  42. 42.

    Nur-E-Kamal MS, Kamal JM, Qureshi MM, Maruta H. The CDC42-specific inhibitor derived from ACK-1 blocks v-Ha-Ras-induced transformation. Oncogene. 1999;18:7787–93.

  43. 43.

    Yang W, Lo CG, Dispenza T, Cerione RA. The Cdc42 target ACK2 directly interacts with clathrin and influences clathrin assembly. J Biol Chem. 2001;276:17468–73.

  44. 44.

    Chen C, Song X, Ma S, Wang X, Xu J, Zhang H, et al. Cdc42 inhibitor ML141 enhances G-CSF-induced hematopoietic stem and progenitor cell mobilization. Int J Hematol. 2015;101:5–12.

  45. 45.

    Du W, Liu W, Mizukawa B, Shang X, Sipple J, Wunderlich M, et al. A non-myeloablative conditioning approach for long-term engraftment of human and mouse hematopoietic stem cells. Leukemia. 2018. https://doi.org/10.1038/s41375-018-0200-3. [Epub ahead of print] PMID:29959415.

Download references

Acknowledgements

We thank James F. Johnson, Victoria Summey, and Jeff Bailey for assistance in xenograft experiments. This work was supported in part by the NIH grants R01 CA193350, DK104814, CA204895, and HL085362.

Author contributions

W.L. and W.D. designed and performed the research, analyzed the data, and wrote the paper. X.S., L.W., C.E., M.C.F., M.A.R., A.R., X.Z., K.S., F.G., performed some of the experiments. N.N., J.M., H.G., and Q.P. designed the research. Y.Z. designed the research, analyzed the data, and wrote the paper.

Author information

Author notes

  1. These authors contributed equally: Wei Liu, Wei Du

Affiliations

  1. Cancer and Blood Diseases Institute, University of Cincinnati, Cincinnati, OH, 45229, USA

    • Wei Liu
    • , Xun Shang
    • , Lei Wang
    • , Chris Evelyn
    • , Marnie A. Ryan
    • , Ahmad Rayes
    • , Fukun Guo
    • , Nicolas Nassar
    • , Hartmut Geiger
    • , Qishen Pang
    •  & Yi Zheng
  2. Department of Pharmaceutical Sciences, West Virginia University School of Pharmacy, Morgantown, WV, 26506, USA

    • Wei Du
  3. Institute of Molecular Medicine, University of Ulm, Ulm, Germany

    • Maria Carolina Florian
    •  & Hartmut Geiger
  4. Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

    • Xueheng Zhao
    •  & Kenneth Setchell
  5. Division of Biomedical Informatics, Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH, 45229, USA

    • Jarek Meller

Authors

  1. Search for Wei Liu in:

  2. Search for Wei Du in:

  3. Search for Xun Shang in:

  4. Search for Lei Wang in:

  5. Search for Chris Evelyn in:

  6. Search for Maria Carolina Florian in:

  7. Search for Marnie A. Ryan in:

  8. Search for Ahmad Rayes in:

  9. Search for Xueheng Zhao in:

  10. Search for Kenneth Setchell in:

  11. Search for Jarek Meller in:

  12. Search for Fukun Guo in:

  13. Search for Nicolas Nassar in:

  14. Search for Hartmut Geiger in:

  15. Search for Qishen Pang in:

  16. Search for Yi Zheng in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Yi Zheng.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41375-018-0251-5