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:

Sensitivity and Resistance to Therapy

Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

Leukemia volume 26, pages 757–768 (2012)Cite this article

Abstract

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the β5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant β5 subunit of the proteasome. These newly identified β5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the β5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant β5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

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. Navon A, Ciechanover A . The 26 S proteasome: from basic mechanisms to drug targeting. J Biol Chem 2009; 284: 33713–33718.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ortiz-Navarrete V, Seelig A, Gernold M, Frentzel S, Kloetzel PM, Hammerling GJ . Subunit of the ‘20S’ proteasome (multicatalytic proteinase) encoded by the major histocompatibility complex. Nature 1991; 353: 662–664.

    Article  CAS  PubMed  Google Scholar 

  3. Rivett AJ, Bose S, Brooks P, Broadfoot KI . Regulation of proteasome complexes by gamma-interferon and phosphorylation. Biochimie 2001; 83: 363–366.

    Article  CAS  PubMed  Google Scholar 

  4. Seifert U, Bialy LP, Ebstein F, Bech-Otschir D, Voigt A, Schroter F et al. Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell 2010; 142: 613–624.

    Article  CAS  PubMed  Google Scholar 

  5. Crawford LJ, Walker B, Ovaa H, Chauhan D, Anderson KC, Morris TC et al. Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132. Cancer Res 2006; 66: 6379–6386.

    Article  CAS  PubMed  Google Scholar 

  6. Ruschak AM, Slassi M, Kay LE, Schimmer AD . Novel proteasome inhibitors to overcome bortezomib resistance. J Natl Cancer Inst 2011; 103: 1007–1017.

    Article  CAS  PubMed  Google Scholar 

  7. Mitsiades N, Mitsiades CS, Richardson PG, Poulaki V, Tai YT, Chauhan D et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood 2003; 101: 2377–2380.

    Article  CAS  PubMed  Google Scholar 

  8. Hideshima T, Mitsiades C, Akiyama M, Hayashi T, Chauhan D, Richardson P et al. Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341. Blood 2003; 101: 1530–1534.

    Article  CAS  PubMed  Google Scholar 

  9. Obeng EA, Carlson LM, Gutman DM, Harrington Jr WJ, Lee KP, Boise LH . Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood 2006; 107: 4907–4916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Fanourakis G, Gu X et al. Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci USA 2002; 99: 14374–14379.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res 2001; 61: 3071–3076.

    CAS  PubMed  Google Scholar 

  12. Vink J, Cloos J, Kaspers GJ . Proteasome inhibition as novel treatment strategy in leukaemia. Br J Haematol 2006; 134: 253–262.

    Article  CAS  PubMed  Google Scholar 

  13. Shah JJ, Orlowski RZ . Proteasome inhibitors in the treatment of multiple myeloma. Leukemia 2009; 23: 1964–1979.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Orlowski RZ, Kuhn DJ . Proteasome inhibitors in cancer therapy: lessons from the first decade. Clin Cancer Res 2008; 14: 1649–1657.

    Article  CAS  PubMed  Google Scholar 

  15. McConkey DJ, Zhu K . Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist Updat 2008; 11: 164–179.

    Article  CAS  PubMed  Google Scholar 

  16. Messinger Y, Gaynon P, Raetz E, Hutchinson R, Dubois S, Glade-Bender J et al. Phase I study of bortezomib combined with chemotherapy in children with relapsed childhood acute lymphoblastic leukemia (ALL): a report from the therapeutic advances in childhood leukemia (TACL) consortium. Pediatr Blood Cancer 2010; 55: 254–259.

    Article  PubMed  Google Scholar 

  17. Kraus M, Ruckrich T, Reich M, Gogel J, Beck A, Kammer W et al. Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells. Leukemia 2007; 21: 84–92.

    Article  CAS  PubMed  Google Scholar 

  18. Rückrich T, Kraus M, Gogel J, Beck A, Ovaa H, Verdoes M et al. Characterization of the ubiquitin-proteasome system in bortezomib-adapted cells. Leukemia 2009; 23: 1098–1105.

    Article  PubMed  Google Scholar 

  19. Lu S, Yang J, Song X, Gong S, Zhou H, Guo L et al. Point mutation of the proteasome beta5 subunit gene is an important mechanism of bortezomib resistance in bortezomib-selected variants of Jurkat T cell lymphoblastic lymphoma/leukemia line. J Pharmacol Exp Ther 2008; 326: 423–431.

    Article  CAS  PubMed  Google Scholar 

  20. Oerlemans R, Franke NE, Assaraf YG, Cloos J, van ZI, Berkers CR et al. Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein. Blood 2008; 112: 2489–2499.

    Article  CAS  PubMed  Google Scholar 

  21. Ri M, Iida S, Nakashima T, Miyazaki H, Mori F, Ito A et al. Bortezomib-resistant myeloma cell lines: a role for mutated PSMB5 in preventing the accumulation of unfolded proteins and fatal ER stress. Leukemia 2010; 24: 1506–1512.

    Article  CAS  PubMed  Google Scholar 

  22. Groll M, Berkers CR, Ploegh HL, Ovaa H . Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure 2006; 14: 451–456.

    Article  CAS  PubMed  Google Scholar 

  23. Groll M, Kim KB, Huber R, Crews C . Crystal structure of epoxomicin:20s proteasome reveals a molecular basis for selectivity of alpha, beta-epoxyketone proteasome inhibitors. J Am Chem Soc 2000; 122: 1237–1238.

    Article  CAS  Google Scholar 

  24. Unno M, Mizushima T, Morimoto Y, Tomisugi Y, Tanaka K, Yasuoka N et al. The structure of the mammalian 20S proteasome at 2.75 A resolution. Structure 2002; 10: 609–618.

    Article  CAS  PubMed  Google Scholar 

  25. Wang J, Cieplak P, Kollman PA . How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comput Chem 2000; 21: 1049–1074.

    Article  CAS  Google Scholar 

  26. Chauhan D, Singh AV, Aujay M, Kirk CJ, Bandi M, Ciccarelli B et al. A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma. Blood 2010; 116: 4906–4915.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Demo SD, Kirk CJ, Aujay MA, Buchholz TJ, Dajee M, Ho MN et al. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res 2007; 67: 6383–6391.

    Article  CAS  PubMed  Google Scholar 

  28. Muchamuel T, Basler M, Aujay MA, Suzuki E, Kalim KW, Lauer C et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat Med 2009; 15: 781–787.

    Article  CAS  PubMed  Google Scholar 

  29. Li X, Wood TE, Sprangers R, Jansen G, Franke NE, Mao X et al. Effect of noncompetitive proteasome inhibition on bortezomib resistance. J Natl Cancer Inst 2010; 102: 1069–1082.

    Article  CAS  PubMed  Google Scholar 

  30. Elsasser S, Schmidt M, Finley D . Characterization of the proteasome using native gel electrophoresis. Methods Enzymol 2005; 398: 353–363.

    Article  CAS  PubMed  Google Scholar 

  31. Parlati F, Lee SJ, Aujay M, Suzuki E, Levitsky K, Lorens JB et al. Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome. Blood 2009; 114: 3439–3447.

    Article  CAS  PubMed  Google Scholar 

  32. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD et al. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 1997; 386: 463–471.

    Article  CAS  PubMed  Google Scholar 

  33. Borissenko L, Groll M . 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem Rev 2007; 107: 687–717.

    Article  CAS  PubMed  Google Scholar 

  34. Lu S, Yang J, Chen Z, Gong S, Zhou H, Xu X et al. Different mutants of PSMB5 confer varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the Jurkat cell line. Exp Hematol 2009; 37: 831–837.

    Article  PubMed  Google Scholar 

  35. Heinemeyer W, Gruhler A, Mohrle V, Mahe Y, Wolf DH . PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins. J Biol Chem 1993; 268: 5115–5120.

    CAS  PubMed  Google Scholar 

  36. Princiotta MF, Schubert U, Chen W, Bennink JR, Myung J, Crews CM et al. Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival. Proc Natl Acad Sci USA 2001; 98: 513–518.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. La RP, Deininger MW . Resistance to imatinib: mutations and beyond. Semin Hematol 2010; 47: 335–343.

    Article  Google Scholar 

  38. Engelman JA, Janne PA . Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Clin Cancer Res 2008; 14: 2895–2899.

    Article  PubMed  Google Scholar 

  39. Vassetzky YS, Alghisi GC, Gasser SM . DNA topoisomerase II mutations and resistance to anti-tumor drugs. Bioessays 1995; 17: 767–774.

    Article  CAS  PubMed  Google Scholar 

  40. Lu S, Chen Z, Yang J, Chen L, Gong S, Zhou H et al. Overexpression of the PSMB5 gene contributes to bortezomib resistance in T-lymphoblastic lymphoma/leukemia cells derived from Jurkat line. Exp Hematol 2008; 36: 1278–1284.

    Article  CAS  PubMed  Google Scholar 

  41. Ashworth A . Drug resistance caused by reversion mutation. Cancer Res 2008; 68: 10021–10023.

    Article  CAS  PubMed  Google Scholar 

  42. Paquet AC, Baxter J, Weidler J, Lie Y, Lawrence J, Kim R et al. Differences in reversion of resistance mutations to wild-type under structured treatment interruption and related increase in replication capacity. PLoS One 2011; 6: e14638.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 2005; 352: 2487–2498.

    Article  CAS  PubMed  Google Scholar 

  44. Mateos MV, Richardson PG, Schlag R, Khuageva NK, Dimopoulos MA, Shpilberg O et al. Bortezomib plus melphalan and prednisone compared with melphalan and prednisone in previously untreated multiple myeloma: updated follow-up and impact of subsequent therapy in the phase III VISTA trial. J Clin Oncol 2010; 28: 2259–2266.

    Article  CAS  PubMed  Google Scholar 

  45. Hrusovsky I, Emmerich B, von RA, Voegeli J, Taverna C, Olie RA et al. Bortezomib retreatment in relapsed multiple myeloma - results from a retrospective multicentre survey in Germany and Switzerland. Oncology 2010; 79: 247–254.

    Article  CAS  PubMed  Google Scholar 

  46. Wang L, Kumar S, Fridley BL, Kalari KR, Moon I, Pelleymounter LL et al. Proteasome beta subunit pharmacogenomics: gene resequencing and functional genomics. Clin Cancer Res 2008; 14: 3503–3513.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Shaughnessy Jr JD, Qu P, Usmani S, Heuck CJ, Zhang Q, Zhou Y et al. Pharmacogenomics of bortezomib test-dosing identifies hyperexpression of proteasome genes, especially PSMD4, as novel high-risk feature in myeloma treated with total therapy 3. Blood 2011; e-pub ahead of print 31 May 2011.

Download references

Acknowledgements

This study was supported by grants from the Stichting Translational Research, Cancer Center Amsterdam (to JC), NWO-AGIKO (to NEF). YGA is recipient of visiting professor fellowships from the Royal Netherlands Academy of Arts and Sciences (KNAW) and the Netherlands Organization for Scientific Research (NWO) and his scientific contributions are also supported by the Dutch Foundation Children Cancer-free (KIKA).

Author information

Authors and Affiliations

  1. Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands

    N E Franke, D Niewerth, J van Meerloo, K Vojtekova, C H van Zantwijk, G J L Kaspers & J Cloos

  2. Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa, Israel

    Y G Assaraf

  3. Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands

    J van Meerloo, K Vojtekova, C H van Zantwijk, S Zweegman & J Cloos

  4. Research Department, Onyx Pharmaceuticals, Inc, South San Francisco, CA, USA

    E T Chan & C J Kirk

  5. Division of Molecular and Computational Toxicology, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit, Amsterdam, The Netherlands

    D P Geerke

  6. Ontario Cancer Institute, Princess Margaret Hospital Toronto, Toronto, ON, Canada

    A D Schimmer

  7. Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands

    G Jansen

Authors
  1. N E Franke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D Niewerth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Y G Assaraf
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J van Meerloo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K Vojtekova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C H van Zantwijk
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S Zweegman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. E T Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. C J Kirk
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. D P Geerke
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. A D Schimmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. G J L Kaspers
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. G Jansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. J Cloos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J Cloos.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Franke, N., Niewerth, D., Assaraf, Y. et al. Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells. Leukemia 26, 757–768 (2012). https://doi.org/10.1038/leu.2011.256

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2011.256

Keywords

This article is cited by

Search

Advanced search

Quick links