Original Article | Published:

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance

Oncogene volume 36, pages 56315638 (05 October 2017) | Download Citation

Abstract

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription–PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

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.

    , , , , . Trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes. Mol Cell Proteomics 2011; 10: 003871.

  2. 2.

    , , , , , . Complete subunit architecture of the proteasome regulatory particle. Nature 2012; 482: 186–191.

  3. 3.

    , , , , , et al. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc Natl Acad Sci USA 2012; 109: 1380–1387.

  4. 4.

    . Protein degradation and protection against misfolded or damaged proteins. Nature 2003; 426: 895–899.

  5. 5.

    , . Ubiquitin: structures, functions, mechanisms. Biochim Biophys Acta 2004; 1695: 55–72.

  6. 6.

    . The proteasome: a suitable antineoplastic target. Nat Rev Cancer 2004; 4: 349–360.

  7. 7.

    , , . Proteasome inhibition in multiple myeloma: therapeutic implication. Annu Rev Pharmacol Toxicol 2005; 45: 465–476.

  8. 8.

    . The ubiquitin system for protein degradation and some of its roles in the control of the cell-division cycle (Nobel lecture). Angew Chem Int Ed Engl 2005; 44: 5932–5943.

  9. 9.

    . Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 2009; 78: 477–513.

  10. 10.

    , , . DUBs and cancer: the role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors. Cell Cycle 2009; 8: 1688–1697.

  11. 11.

    , , , , , et al. A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell 2012; 22: 345–358.

  12. 12.

    , . Deubiquitinating enzymes as therapeutic targets in cancer. Curr Pharm Des 2013; 19: 4039–4052.

  13. 13.

    , . Functions of the 19S complex in proteasomal degradation. Trends Biochem Sci 2013; 38: 103–110.

  14. 14.

    , , , . Regulated protein turnover: snapshots of the proteasome in action. Nat Rev Mol Cell Biol 2014; 15: 122–133.

  15. 15.

    , , , , , et al. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med 2011; 17: 1636–1640.

  16. 16.

    , , , , , . A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J 2001; 20: 5187–5196.

  17. 17.

    , , , , , et al. A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance. Blood 2014; 123: 706–716.

  18. 18.

    , . A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 2002; 419: 403–407.

  19. 19.

    , , , , , et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 2002; 298: 611–615.

  20. 20.

    , , , . MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. BMC Biochem 2002; 3: 28.

  21. 21.

    , , . Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol Biol Cell 2008; 19: 1072–1082.

  22. 22.

    , , , , , et al. The JAMM motif of human deubiquitinase Poh1 is essential for cell viability. Mol Cancer Ther 2007; 6: 262–268.

  23. 23.

    , , , . Endoplasmic reticulum stress compromises the ubiquitin-proteasome system. Hum Mol Genet 2005; 14: 2787–2799.

  24. 24.

    , , , , , et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood 1996; 87: 1104–1112.

  25. 25.

    , , , , , et al. Targeting PD1-PDL1 immune checkpoint in plasmacytoid dendritic cell interactions with T cells, natural killer cells and multiple myeloma cells. Leukemia 2015; 29: 1441–1444.

  26. 26.

    , , , , , . Characterization of a novel human cell-cycle-regulated homologue of Drosophila dlg1. Genomics 2001; 77: 5–7.

  27. 27.

    , , , , , . Changes in expression of the human homologue of the Drosophila discs large tumour suppressor protein in high-grade premalignant cervical neoplasias. Carcinogenesis 2002; 23: 1791–1796.

  28. 28.

    , , , , , et al. Functional interaction of plasmacytoid dendritic cells with multiple myeloma cells: a therapeutic target. Cancer Cell 2009; 16: 309–323.

  29. 29.

    , , , , , et al. Targeting proteasome ubiquitin receptor Rpn13 in multiple myeloma. Leukemia 2016; 30: 1877–1886.

  30. 30.

    , , , , , et al. POH1 deubiquitylates and stabilizes E2F1 to promote tumour formation. Nat Commun 2015; 6: 8704.

  31. 31.

    , . Molecular pathogenesis and a consequent classification of multiple myeloma. J Clin Oncol 2005; 23: 6333–6338.

  32. 32.

    , , , , , . Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma. Proc Natl Acad Sci USA 1996; 93: 13931–13936.

  33. 33.

    , , , , , et al. Characterization of the MM.1 human multiple myeloma (MM) cell lines: a model system to elucidate the characteristics, behavior, and signaling of steroid-sensitive and -resistant MM cells. Exp Hematol 2003; 31: 271–282.

  34. 34.

    , , , , , et al. Genetic abnormalities and survival in multiple myeloma: the experience of the Intergroupe Francophone du Myelome. Blood 2007; 109: 3489–3495.

  35. 35.

    , , , , , et al. Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma. Hematol J 2001; 2: 42–53.

  36. 36.

    , , , , , et al. Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26S proteasome subunit. J Biol Chem 1997; 272: 30470–30475.

  37. 37.

    , , . The essential 26S proteasome subunit Rpn11 confers multidrug resistance to mammalian cells. Anticancer Res 2002; 22: 3905–3909.

  38. 38.

    , , , , . A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology. Mol Biol Cell 1998; 9: 2917–2931.

  39. 39.

    , , , . Mitochondrial effects of the pleiotropic proteasomal mutation mpr1/rpn11: uncoupling from cell cycle defects in extragenic revertants. Gene 2002; 286: 43–51.

  40. 40.

    , , , , , et al. Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain. Biochem J 2004; 381: 275–285.

  41. 41.

    , , , , , et al. A phase 2 trial of lenalidomide, bortezomib, and dexamethasone in patients with relapsed and relapsed/refractory myeloma. Blood 2014; 123: 1461–1469.

  42. 42.

    , , , , , et al. Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma. Blood 2010; 116: 679–686.

  43. 43.

    , , , , , et al. Multicenter, phase I, dose-escalation trial of lenalidomide plus bortezomib for relapsed and relapsed/refractory multiple myeloma. J Clin Oncol 2009; 27: 5713–5719.

  44. 44.

    , , , , , et al. Pomalidomide alone or in combination with low-dose dexamethasone in relapsed and refractory multiple myeloma: a randomized phase 2 study. Blood 2014; 123: 1826–1832.

  45. 45.

    , , , , , et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood 2007; 110: 3281–3290.

  46. 46.

    , , , , , et al. Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma. Blood 2008; 111: 1654–1664.

  47. 47.

    , , , , , et al. In vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells. Clin Cancer Res 2013; 19: 3019–3031.

Download references

Acknowledgements

This investigation was supported by National Institutes of Health Specialized Programs of Research Excellence (SPORE) Grants P50100707, R01CA207237 and RO1CA050947. KCA is an American Cancer Society Clinical Research Professor.

Author contributions

DC conceived the project, designed research, analyzed data and wrote the manuscript; YS designed and performed the experiments and interpreted data; SL helped in RT–PCR and animal studies; AR and DSD helped in cytotoxicity assays; JQ helped in western studies; MKS performed the GEP data set analyses; NM and Y-TT provided patient samples; RDC helped in immunohistochemistry analysis; and KCA reviewed the manuscript.

Author information

Author notes

    • D Chauhan
    •  & K C Anderson

    Joint senior authors.

Affiliations

  1. LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    • Y Song
    • , A Ray
    • , D S Das
    • , M K Samur
    • , Y-T Tai
    • , N Munshi
    • , R D Carrasco
    • , D Chauhan
    •  & K C Anderson
  2. Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    • S Li
  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    • J Qi

Authors

  1. Search for Y Song in:

  2. Search for S Li in:

  3. Search for A Ray in:

  4. Search for D S Das in:

  5. Search for J Qi in:

  6. Search for M K Samur in:

  7. Search for Y-T Tai in:

  8. Search for N Munshi in:

  9. Search for R D Carrasco in:

  10. Search for D Chauhan in:

  11. Search for K C Anderson in:

Competing interests

KCA is on advisory board of Celgene, Millenium, Gilead and Bristol Myers Squibb, and is a Scientific Founder of Acetylon, Oncopep and C4 Therapeutics. The other authors declare no conflict of interest.

Corresponding authors

Correspondence to D Chauhan or K C Anderson.

Supplementary information

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/onc.2017.172

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)