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:

Multiple Myeloma, Gammopathies

Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo

Leukemia volume 30, pages 390–398 (2016)Cite this article

Subjects

Abstract

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2’s capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

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
Figure 7

Similar content being viewed by others

References

  1. Michigami T, Shimizu N, Williams PJ, Niewolna M, Dallas SL, Mundy GR et al. Cell-cell contact between marrow stromal cells and myeloma cells via VCAM-1 and alpha(4)beta(1)-integrin enhances production of osteoclast-stimulating activity. Blood 2000; 96: 1953–1960.

    CAS  PubMed  Google Scholar 

  2. Sanz-Rodriguez F, Teixido J . VLA-4-dependent myeloma cell adhesion. Leuk Lymphoma 2001; 41: 239–245.

    Article  CAS  Google Scholar 

  3. Okada T, Hawley RG, Kodaka M, Okuno H . Significance of VLA-4-VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model. Clin Exp Metastasis 1999; 17: 623–629.

    Article  CAS  Google Scholar 

  4. Kim MS, Day CJ, Selinger CI, Magno CL, Stephens SR, Morrison NA . MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption. J Biol Chem 2006; 281: 1274–1285.

    Article  CAS  Google Scholar 

  5. Giuliani N, Rizzoli V . Myeloma cells and bone marrow osteoblast interactions: role in the development of osteolytic lesions in multiple myeloma. Leuk Lymphoma 2007; 48: 2323–2329.

    Article  Google Scholar 

  6. Hiruma Y, Honjo T, Jelinek D, Windle JJ, Shin J, Roodman GD et al. Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation. Blood 2009; 113: 4894–4902.

    Article  CAS  Google Scholar 

  7. Teramachi J, Windle JJ, Roodman D, Kurihara N . The ZZ domain of sequestosome-1/p62 plays an important role in stromal cell support of myeloma cell growth and osteoclast formation. ASH Annual Meeting Abstracts 2010; 116, 128.

  8. Yang P, Teramachi J, Feng R, Wang L, Beumer JH, Eiseman JL et al. A Novel Chemical Inhibitor Targeting Sequestosme-1/p62 Suppresses Multiple Myeloma Cell Growth and Osteoclast Formation Poster Presentation at the 2014 AAPS Annual Meeting and Exposition; November 2014; San Diego, CA Poster T3045, 2014.

  9. Teramachi J, Myint KZY, Feng R, Xie X, Windle JJ, Roodman D et al. Blocking the ZZ domain of sequestosome 1/p62 suppress the enhancement of myeloma cell growth and osteoclast formation by marrow stromal cells. ASH Annual Meeting Abstracts 2011; 118, 888.

  10. Kurihara N, Chenu C, Miller M, Civin C, Roodman GD . Identification of committed mononuclear precursors for osteoclast-like cells formed in long term human marrow cultures. Endocrinology 1990; 126: 2733–2741.

    Article  CAS  Google Scholar 

  11. Kurihara N, Civin C, Roodman GD . Osteotropic factor responsiveness of highly purified populations of early and late precursors for human multinucleated cells expressing the osteoclast phenotype. J Bone Miner Res 1991; 6: 257–261.

    Article  CAS  Google Scholar 

  12. D'Souza S, del Prete D, Jin S, Sun Q, Huston AJ, Kostov FE et al. Gfi1 expressed in bone marrow stromal cells is a novel osteoblast suppressor in patients with multiple myeloma bone disease. Blood 2011; 118: 6871–6880.

    Article  CAS  Google Scholar 

  13. Cumova J, Kovarova L, Potacova A, Buresova I, Kryukov F, Penka M et al. Optimization of immunomagnetic selection of myeloma cells from bone marrow using magnetic activated cell sorting. Int J Hematol 2010; 92: 314–319.

    Article  CAS  Google Scholar 

  14. Cackowski FC, Anderson JL, Patrene KD, Choksi RJ, Shapiro SD, Windle JJ et al. Osteoclasts are important for bone angiogenesis. Blood 2010; 115: 140–149.

    Article  CAS  Google Scholar 

  15. D'Argenio D, Schumitzky A, Wang X . ADAPT 5 User's Guide: Pharmacokinetic/Pharmacodynamic Systems Analysis Software. Los Angeles, CA: Biomedical Simulations Resource, 2014.

    Google Scholar 

  16. Kurihara N, Hiruma Y, Yamana K, Michou L, Rousseau C, Morissette J et al. Contributions of the measles virus nucleocapsid gene and the SQSTM1/p62(P392L) mutation to Paget's disease. Cell Metab 2011; 13: 23–34.

    Article  CAS  Google Scholar 

  17. National Research Council (US), Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (US), National Academies Press (US). Guide for the Care and Use of Laboratory Animals. National Academies Press: Washington, DC, 2011; Available from: http://www.ncbi.nlm.nih.gov/books/NBK54050, ebrary http://site.ebrary.com/id/10443276, National Academies Press http://www.nap.edu/catalog.php?record_id=12910, National Academies Press http://www.nap.edu/catalog.php?record_id=12910-toc, http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=nap12910, http://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf.

  18. Mohammad KS, Javelaud D, Fournier PG, Niewolna M, McKenna CR, Peng XH et al. TGF-beta-RI kinase inhibitor SD-208 reduces the development and progression of melanoma bone metastases. Cancer Res 2011; 71: 175–184.

    Article  CAS  Google Scholar 

  19. Mitsiades CS, Mitsiades NS, Munshi NC, Richardson PG, Anderson KC . The role of the bone microenvironment in the pathophysiology and therapeutic management of multiple myeloma: interplay of growth factors, their receptors and stromal interactions. Eur J Cancer 2006; 42: 1564–1573.

    Article  CAS  Google Scholar 

  20. Ye H, Arron JR, Lamothe B, Cirilli M, Kobayashi T, Shevde NK et al. Distinct molecular mechanism for initiating TRAF6 signalling. Nature 2002; 418: 443–447.

    Article  CAS  Google Scholar 

  21. Sanz L, Sanchez P, Lallena MJ, Diaz-Meco MT, Moscat J . The interaction of p62 with RIP links the atypical PKCs to NF-kappaB activation. EMBO J 1999; 18: 3044–3053.

    Article  CAS  Google Scholar 

  22. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G . Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997; 89: 747–754.

    Article  CAS  Google Scholar 

  23. Xu G, Liu K, Anderson J, Patrene K, Lentzsch S, Roodman GD et al. Expression of XBP1s in bone marrow stromal cells is critical for myeloma cell growth and osteoclast formation. Blood 2012; 119: 4205–4214.

    Article  CAS  Google Scholar 

  24. Tai YT, Podar K, Mitsiades N, Lin B, Mitsiades C, Gupta D et al. CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling. Blood 2003; 101: 2762–2769.

    Article  CAS  Google Scholar 

  25. Hideshima T, Chauhan D, Schlossman R, Richardson P, Anderson KC . The role of tumor necrosis factor alpha in the pathophysiology of human multiple myeloma: therapeutic applications. Oncogene 2001; 20: 4519–4527.

    Article  CAS  Google Scholar 

  26. Puls A, Schmidt S, Grawe F, Stabel S . Interaction of protein kinase C zeta with ZIP, a novel protein kinase C-binding protein. Proc Natl Acad Sci USA 1997; 94: 6191–6196.

    Article  CAS  Google Scholar 

  27. Gilbert L, He X, Farmer P, Boden S, Kozlowski M, Rubin J et al. Inhibition of osteoblast differentiation by tumor necrosis factor-alpha. Endocrinology 2000; 141: 3956–3964.

    Article  CAS  Google Scholar 

  28. Duran A, Serrano M, Leitges M, Flores JM, Picard S, Brown JP et al. The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis. Dev Cell 2004; 6: 303–309.

    Article  CAS  Google Scholar 

  29. Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR et al. Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling. Cell 2009; 137: 721–735.

    Article  CAS  Google Scholar 

  30. Rodriguez A, Duran A, Selloum M, Champy MF, Diez-Guerra FJ, Flores JM et al. Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62. Cell Metab 2006; 3: 211–222.

    Article  CAS  Google Scholar 

  31. Painuly U, Kumar S . Efficacy of bortezomib as first-line treatment for patients with multiple myeloma. Clin Med Insights Oncol 2013; 7: 53–73.

    Article  CAS  Google Scholar 

  32. Kapoor P, Ramakrishnan V, Rajkumar SV . Bortezomib combination therapy in multiple myeloma. Semin Hematol 2012; 49: 228–242.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Drs Rosen, Oyajobi, Giuliani, Stancato and Jelinek for MM cell lines. This project used the University of Pittsburgh Cancer Institute (UPCI) Clinical Pharmacology Analytical Facility (CPAF) and UPCI Small Animal Facility, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA047904. Services and products in support of the research project were also generated by the VCU Massey Cancer Center Transgenic/Knock-out Mouse Facility, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059. Funding was also received from the Multiple Myeloma Research Foundation (NK and GDR), the NIH R21CA141426, 5R01 AR059679-05, 5R21CA179017-02 (GDR) and R01DA025612, R21HL109654 (XQX), and VA Merit Review Grant 5101CX000623-04 (GDR).

Author contributions

JT and NK designed and performed in vitro research and analyzed and interpreted data; PY, RF, KM and XQX designed, synthesized and characterized XRK3F2; RS and WZ performed μCT analysis to characterize new bone formation; KSM performed histology and immunohistochemistry; NM interpreted radiographs; JG, JHB and JLE designed, performed and analyzed in vivo pharmacokinetic and pharmacodynamic experiments; JJW provided C57BL/KaLwRij mice; and RS and GDR designed the research, analyzed and interpreted data, and wrote the manuscript.

Author information

Author notes

  1. J Teramachi, R Silbermann and P Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Hematology Oncology, Indiana University, Indianapolis,, IN, USA

    J Teramachi, R Silbermann, W Zhao, J L Anderson, D Zhou, G D Roodman & N Kurihara

  2. Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh,, PA, USA

    P Yang, R Feng, K-Z Myint, J H Beumer & X-Q Xie

  3. Computational Chemical Genomics Screening Center, University of Pittsburgh,, Pittsburgh,, PA, USA

    P Yang & X-Q Xie

  4. Division of Endocrinology, Department of Medicine, Indiana University, Indianapolis,, IN, USA

    K S Mohammad

  5. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh,, PA, USA

    J Guo & J L Eiseman

  6. Department of Radiology, Indiana University, Indianapolis,, IN, USA

    N Maertz

  7. Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh,, Pittsburgh,, PA, USA

    J H Beumer & J L Eiseman

  8. Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond,, VA, USA

    J J Windle

  9. Department of Medicine, Richard L. Roudebush VA Medical Center, Indianapolis,, IN, USA

    G D Roodman

Authors
  1. J Teramachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R Silbermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. W Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K S Mohammad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J L Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. D Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. K-Z Myint
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. N Maertz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. J H Beumer
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. J L Eiseman
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. J J Windle
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. X-Q Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. G D Roodman
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. N Kurihara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to R Silbermann, X-Q Xie or G D Roodman.

Ethics declarations

Competing interests

GDR is a consultant for Amgen. XQX is a consultant for Oxis Biotech. The remaining authors declare no competing financial interests.

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

Teramachi, J., Silbermann, R., Yang, P. et al. Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo. Leukemia 30, 390–398 (2016). https://doi.org/10.1038/leu.2015.229

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2015.229

Search

Advanced search

Quick links