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.

  • Original Article
  • Published:

Chronic Myeloproliferative Neoplasias

Grb10 is involved in BCR-ABL-positive leukemia in mice

Leukemia volume 29, pages 858–868 (2015)Cite this article

Subjects

Abstract

The SH2-containing adaptor protein Grb10 was first identified in a yeast screen as a new binding partner for BCR-ABL and associates with BCR-ABL in a tyrosine-dependent manner. However, its function in BCR-ABL-mediated leukemogenesis in vivo is still unknown. Here we describe an important role of Grb10 in BCR-ABL-induced leukemia by using a versatile system for efficient oncogene expression and simultaneous Grb10 knockdown from a single vector. Primary bone marrow (BM) cells coexpressing Grb10-miR/BCR-ABL showed a significant decrease in colony formation and cell cycle progression. Transplantation of Grb10miR/BCR-ABL- or control-miR/BCR-ABL- transduced BM leads to a CML/B-ALL-like phenotype with significantly delayed disease onset and progression resulting in prolonged overall survival in Grb10-miR-transplanted mice. Methylcellulose experiments exhibit additive effects of imatinib treatment and Grb10 knockdown. Cell cycle analysis suggests an anti-proliferative effect of Grb10 knockdown in BCR-ABL+ primary BM cells. However, Grb10 abrogation was not capable of completely abolishing the BCR-ABL-induced disease. Our findings were confirmed in the human BCR-ABL+ cell line K562, where we demonstrate reduced viability, cell cycle progression and induction of apoptosis by stable Grb10 microRNA expression. Taken together, our results suggest that Grb10 knockdown in vivo leads to impaired proliferation, longer survival and reduced colony formation, suggesting an important role of Grb10 in BCR-ABL-mediated leukemogenesis.

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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001; 344: 1031–1037.

    Article  CAS  Google Scholar 

  2. Bhatia R, Holtz M, Niu N, Gray R, Snyder DS, Sawyers CL et al. Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment. Blood 2003; 101: 4701–4707.

    Article  CAS  Google Scholar 

  3. Mahon FX, Rea D, Guilhot J, Guilhot F, Huguet F, Nicolini F et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 2010; 11: 1029–1035.

    Article  CAS  Google Scholar 

  4. de Lavallade H, Apperley JF, Khorashad JS, Milojkovic D, Reid AG, Bua M et al. Imatinib for newly diagnosed patients with chronic myeloid leukemia: incidence of sustained responses in an intention-to-treat analysis. J Clin Oncol 2008; 26: 3358–3363.

    Article  Google Scholar 

  5. Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med. 2006; 355: 2408–2417.

    Article  CAS  Google Scholar 

  6. Kantarjian H, Giles F, Wunderle L, Bhalla K, O'Brien S, Wassmann B et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N Engl J Med. 2006; 354: 2542–2551.

    Article  Google Scholar 

  7. Quintas-Cardama A, Kantarjian H, Jones D, Nicaise C, O'Brien S, Giles F et al. Dasatinib (BMS-354825) is active in Philadelphia chromosome-positive chronic myelogenous leukemia after imatinib and nilotinib (AMN107) therapy failure. Blood 2007; 109: 497–499.

    Article  CAS  Google Scholar 

  8. Kantarjian H, Shah NP, Hochhaus A, Cortes J, Shah S, Ayala M et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2010; 362: 2260–2270.

    Article  CAS  Google Scholar 

  9. Saglio G, Kim DW, Issaragrisil S, le Coutre P, Etienne G, Lobo C et al. Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia. N Engl J Med. 2010; 362: 2251–2259.

    Article  CAS  Google Scholar 

  10. Kantarjian HM, Giles F, Gattermann N, Bhalla K, Alimena G, Palandri F et al. Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance. Blood 2007; 110: 3540–3546.

    Article  CAS  Google Scholar 

  11. Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, Paquette R et al. Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med. 2006; 354: 2531–2541.

    Article  CAS  Google Scholar 

  12. Kantarjian HM, Hochhaus A, Saglio G, De Souza C, Flinn IW, Stenke L et al. Nilotinib versus imatinib for the treatment of patients with newly diagnosed chronic phase, Philadelphia chromosome-positive, chronic myeloid leukaemia: 24-month minimum follow-up of the phase 3 randomised ENESTnd trial. Lancet Oncol 2011; 12: 841–851.

    Article  CAS  Google Scholar 

  13. Gontarewicz A, Balabanov S, Keller G, Colombo R, Graziano A, Pesenti E et al. Simultaneous targeting of Aurora kinases and Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is effective against imatinib-resistant BCR-ABL mutations including T315I. Blood 2008; 111: 4355–4364.

    Article  CAS  Google Scholar 

  14. Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ, Wen SC, Zon G et al. Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells. Blood 1995; 86: 726–736.

    CAS  PubMed  Google Scholar 

  15. Mohi MG, Boulton C, Gu TL, Sternberg DW, Neuberg D, Griffin JD et al. Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs. Proc Natl Acad Sci USA 2004; 101: 3130–3135.

    Article  CAS  Google Scholar 

  16. Klejman A, Rushen L, Morrione A, Slupianek A, Skorski T . Phosphatidylinositol-3 kinase inhibitors enhance the anti-leukemia effect of STI571. Oncogene 2002; 21: 5868–5876.

    Article  CAS  Google Scholar 

  17. Yu C, Krystal G, Dent P, Grant S . Flavopiridol potentiates STI571-induced mitochondrial damage and apoptosis in BCR-ABL-positive human leukemia cells. Clin Cancer Res 2002; 8: 2976–2984.

    CAS  PubMed  Google Scholar 

  18. Peters DG, Hoover RR, Gerlach MJ, Koh EY, Zhang H, Choe K et al. Activity of the farnesyl protein transferase inhibitor SCH66336 against BCR/ABL-induced murine leukemia and primary cells from patients with chronic myeloid leukemia. Blood 2001; 97: 1404–1412.

    Article  CAS  Google Scholar 

  19. Borthakur G, Kantarjian H, Daley G, Talpaz M, O'Brien S, Garcia-Manero G et al. Pilot study of lonafarnib, a farnesyl transferase inhibitor, in patients with chronic myeloid leukemia in the chronic or accelerated phase that is resistant or refractory to imatinib therapy. Cancer 2006; 106: 346–352.

    Article  CAS  Google Scholar 

  20. Jahn T, Seipel P, Urschel S, Peschel C, Duyster J . Role for the adaptor protein Grb10 in the activation of Akt. Mol Cell Biol 2002; 22: 979–991.

    Article  CAS  Google Scholar 

  21. Nantel A, Mohammad-Ali K, Sherk J, Posner BI, Thomas DY . Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases. J Biol Chem 1998; 273: 10475–10484.

    Article  CAS  Google Scholar 

  22. Deng Y, Bhattacharya S, Swamy OR, Tandon R, Wang Y, Janda R et al. Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action. J Biol Chem 2003; 278: 39311–39322.

    Article  CAS  Google Scholar 

  23. Langlais P, Dong LQ, Hu D, Liu F . Identification of Grb10 as a direct substrate for members of the Src tyrosine kinase family. Oncogene 2000; 19: 2895–2903.

    Article  CAS  Google Scholar 

  24. Murdaca J, Treins C, Monthouel-Kartmann MN, Pontier-Bres R, Kumar S, Van Obberghen E et al. Grb10 prevents Nedd4-mediated vascular endothelial growth factor receptor-2 degradation. J Biol Chem 2004; 279: 26754–26761.

    Article  CAS  Google Scholar 

  25. Charalambous M, Smith FM, Bennett WR, Crew TE, Mackenzie F, Ward A . Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism. Proc Natl Acad Sci USA 2003; 100: 8292–8297.

    Article  CAS  Google Scholar 

  26. Holt LJ, Turner N, Mokbel N, Trefely S, Kanzleiter T, Kaplan W et al. Grb10 regulates the development of fiber number in skeletal muscle. FASEB J 2012; 26: 3658–3669.

    Article  CAS  Google Scholar 

  27. Smith FM, Holt LJ, Garfield AS, Charalambous M, Koumanov F, Perry M et al. Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life. Mol Cell Biol 2007; 27: 5871–5886.

    Article  CAS  Google Scholar 

  28. Yu Y, Yoon S-O, Poulogiannis G, Yang Q, Ma XM, Villén J et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science 2011; 332: 1322–1326.

    Article  CAS  Google Scholar 

  29. Okino K, Konishi H, Doi D, Yoneyama K, Ota Y, Jin E et al. Up-regulation of growth factor receptor-bound protein 10 in cervical squamous cell carcinoma. Oncol Rep 2005; 13: 1069–1074.

    CAS  PubMed  Google Scholar 

  30. Casas S, Ollila J, Aventin A, Vihinen M, Sierra J, Knuutila S . Changes in apoptosis-related pathways in acute myelocytic leukemia. Cancer Genet Cytogenet 2003; 146: 89–101.

    Article  CAS  Google Scholar 

  31. Kazi JU, Ronnstrand L . FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. Mol Oncol 2013; 7: 402–418.

    Article  CAS  Google Scholar 

  32. Gutierrez NC, Lopez-Perez R, Hernandez JM, Isidro I, Gonzalez B, Delgado M et al. Gene expression profile reveals deregulation of genes with relevant functions in the different subclasses of acute myeloid leukemia. Leukemia 2005; 19: 402–409.

    Article  CAS  Google Scholar 

  33. Diaz-Blanco E, Bruns I, Neumann F, Fischer JC, Graef T, Rosskopf M et al. Molecular signature of CD34(+) hematopoietic stem and progenitor cells of patients with CML in chronic phase. Leukemia 2007; 21: 494–504.

    Article  CAS  Google Scholar 

  34. Bai RY, Jahn T, Schrem S, Munzert G, Weidner KM, Wang JY et al. The SH2-containing adapter protein GRB10 interacts with BCR-ABL. Oncogene 1998; 17: 941–948.

    Article  CAS  Google Scholar 

  35. von Bubnoff N, Veach DR, van der Kuip H, Aulitzky WE, Sanger J, Seipel P et al. A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor. Blood 2005; 105: 1652–1659.

    Article  CAS  Google Scholar 

  36. Albers C, Illert AL, Miething C, Leischner H, Thiede M, Peschel C et al. An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL-driven leukemogenesis. Blood 2011; 118: 2200–2210.

    Article  CAS  Google Scholar 

  37. Miething C, Feihl S, Mugler C, Grundler R, von Bubnoff N, Lordick F et al. The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib. Leukemia 2006; 20: 650–657.

    Article  CAS  Google Scholar 

  38. Haferlach T, Kohlmann A, Wieczorek L, Basso G, Kronnie GT, Bene MC et al. Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group. J Clin Oncol 2010; 28: 2529–2537.

    Article  CAS  Google Scholar 

  39. Coustan-Smith E, Song G, Clark C, Key L, Liu P, Mehrpooya M et al. New markers for minimal residual disease detection in acute lymphoblastic leukemia. Blood 2011; 117: 6267–6276.

    Article  CAS  Google Scholar 

  40. Maia S, Haining WN, Ansen S, Xia Z, Armstrong SA, Seth NP et al. Gene expression profiling identifies BAX-delta as a novel tumor antigen in acute lymphoblastic leukemia. Cancer Res 2005; 65: 10050–10058.

    Article  CAS  Google Scholar 

  41. Zeng Y, Wagner EJ, Cullen BR . Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 2002; 9: 1327–1333.

    Article  CAS  Google Scholar 

  42. Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 2005; 37: 1281–1288.

    Article  CAS  Google Scholar 

  43. Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ . A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 2005; 102: 13212–13217.

    Article  CAS  Google Scholar 

  44. Zeng Y, Cai X, Cullen BR . Use of RNA polymerase II to transcribe artificial microRNAs. Methods Enzymol 2005; 392: 371–380.

    Article  CAS  Google Scholar 

  45. Li S, Ilaria RL Jr, Million RP, Daley GQ, Van Etten RA . The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity. J Exp Med 1999; 189: 1399–1412.

    Article  CAS  Google Scholar 

  46. Van Etten RA . Retroviral transduction models of Ph+ leukemia: advantages and limitations for modeling human hematological malignancies in mice. Blood Cells Mol Dis 2001; 27: 201–205.

    Article  CAS  Google Scholar 

  47. Ren R . Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer 2005; 5: 172–183.

    Article  CAS  Google Scholar 

  48. Jiang X, Stuible M, Chalandon Y, Li A, Chan WY, Eisterer W et al. Evidence for a positive role of SHIP in the BCR-ABL-mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia. Blood 2003; 102: 2976–2984.

    Article  CAS  Google Scholar 

  49. Sattler M, Mohi MG, Pride YB, Quinnan LR, Malouf NA, Podar K et al. Critical role for Gab2 in transformation by BCR/ABL. Cancer Cell 2002; 1: 479–492.

    Article  CAS  Google Scholar 

  50. Di Cristofano A, Niki M, Zhao M, Karnell FG, Clarkson B, Pear WS et al. p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl). J Exp Med 2001; 194: 275–284.

    Article  CAS  Google Scholar 

  51. Kebache S, Ash J, Annis MG, Hagan J, Huber M, Hassard J et al. Grb10 and active Raf-1 kinase promote Bad-dependent cell survival. J Biol Chem 2007; 282: 21873–21883.

    Article  CAS  Google Scholar 

  52. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME . Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995; 270: 1326–1331.

    Article  CAS  Google Scholar 

  53. Chang L, Karin M . Mammalian MAP kinase signalling cascades. Nature 2001; 410: 37–40.

    Article  CAS  Google Scholar 

  54. Aceves-Luquero CI, Agarwal A, Callejas-Valera JL, Arias-Gonzalez L, Esparis-Ogando A, del Peso Ovalle L et al. ERK2, but not ERK1, mediates acquired and "de novo" resistance to imatinib mesylate: implication for CML therapy. PLoS One 2009; 4: e6124.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant to JD from the Deutsche Forschungsgemeinschaft (SFB684); ALI was supported by the FoKo Freiburg. We thank Melanie Thiede and Sabina Schaberg for technical assistance.

Author information

Author notes

  1. A L Illert and C Albers: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany

    A L Illert, C Albers, S Kreutmair, C Miething & J Duyster

  2. Department of Internal Medicine III, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany

    C Albers, H Leischner & C Peschel

  3. Department of Hematology, Oncology, and Tumor Immunology, HELIOS Klinikum Berlin-Buch, Berlin, Germany

    H Leischner

Authors
  1. A L Illert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C Albers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Kreutmair
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H Leischner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C Peschel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C Miething
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J Duyster
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A L Illert.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Illert, A., Albers, C., Kreutmair, S. et al. Grb10 is involved in BCR-ABL-positive leukemia in mice. Leukemia 29, 858–868 (2015). https://doi.org/10.1038/leu.2014.283

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2014.283

This article is cited by

Search

Advanced search

Quick links