Skip to main content
SpringerLink
Log in

A first-in-class inhibitor, MLN4924 (pevonedistat), induces cell-cycle arrest, senescence, and apoptosis in human renal cell carcinoma by suppressing UBE2M-dependent neddylation modification

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

MLN4924 is a second-generation inhibitor that targets ubiquitin–proteasome system by inhibiting neddylation activation enzyme (NAE), and subsequently blocking the neddylation-dependent activation of Cullin-RING E3 ligases (CRLs), which leads to the accumulation of CRLs substrates and hence, suppressing diverse tumor development. In this study, we investigated the potential application of this first-in-class inhibitor MLN4924 in the treatment of human renal cell carcinoma both in vitro and in vivo.

Methods

The impact of MLN4924 on renal cancer cells was determined by measuring viability (MTS), proliferation cell count test and clonogenic assays, cell cycle progression (flow cytometry with propidium iodide staining), apoptosis (flow cytometry with annexin V-FITC labeling) and DNA damage (immunofluorescent staining). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration was analyzed by wound healing assays. A well-established SCID xenograft mouse model was used to evaluate the effects of MLN4924 on tumor growth in vivo.

Results

The data showed that MLN4924 induced a dose-dependent cytotoxicity, anti-proliferation, anti-migration, and apoptosis in human renal cancer cells; and caused cell cycle arrested at the G2 phase. In addition, the E2 conjugating enzymes of Neddylation UBE2M played a major role in the proliferation control of renal cancer cells. Finally, we confirmed MLN4924 inhibited tumor growth in a RCC xenograft mouse model with minimal general toxicity.

Conclusion

We concluded that MLN4924 induces apoptosis and cell cycle arrest. These findings implied that MLN4924 provides a novel strategy for the treatment of RCC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, Negrier S, Chevreau C, Solska E, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Freeman S, Schwartz B, Shan M, Simantov R, Bukowski RM (2007) Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 356:125–134

    Article  PubMed  CAS  Google Scholar 

  2. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Staehler M, Negrier S, Chevreau C, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Gore M, Anderson S, Hofilena G, Shan M, Pena C, Lathia C, Bukowski RM (2009) Sorafenib for treatment of renal cell carcinoma: final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. J Clin Oncol 27:3312–3318

    Article  PubMed  CAS  Google Scholar 

  3. Motzer RJ, Hutson TE, Cella D, Reeves J, Hawkins R, Guo J, Nathan P, Staehler M, de Souza P, Merchan JR, Boleti E, Fife K, Jin J, Jones R, Uemura H, De Giorgi U, Harmenberg U, Wang J, Sternberg CN, Deen K, McCann L, Hackshaw MD, Crescenzo R, Pandite LN, Choueiri TK (2013) Pazopanib versus sunitinib in metastatic renal-cell carcinoma. N Engl J Med 369:722–731

    Article  PubMed  CAS  Google Scholar 

  4. Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Oudard S, Negrier S, Szczylik C, Pili R, Bjarnason GA, Garcia-del-Muro X, Sosman JA, Solska E, Wilding G, Thompson JA, Kim ST, Chen I, Huang X, Figlin RA (2009) Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 27:3584–3590

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Rini BI, Wilding G, Hudes G, Stadler WM, Kim S, Tarazi J, Rosbrook B, Trask PC, Wood L, Dutcher JP (2009) Phase II study of axitinib in sorafenib-refractory metastatic renal cell carcinoma. J Clin Oncol 27:4462–4468

    Article  PubMed  CAS  Google Scholar 

  6. Adams J (2004) The development of proteasome inhibitors as anticancer drugs. Cancer cell 5:417–421

    Article  PubMed  CAS  Google Scholar 

  7. Schwartz AL, Ciechanover A (2009) Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 49:73–96

    Article  PubMed  CAS  Google Scholar 

  8. Nakayama KI, Nakayama K (2006) Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6:369–381

    Article  PubMed  CAS  Google Scholar 

  9. Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6:9–20

    Article  PubMed  CAS  Google Scholar 

  10. Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M, Conaway RC, Conaway JW, Harper JW, Pavletich NP (2002) Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416:703–709

    Article  PubMed  CAS  Google Scholar 

  11. Yang D, Li L, Liu H, Wu L, Luo Z, Li H, Zheng S, Gao H, Chu Y, Sun Y, Liu J, Jia L (2013) Induction of autophagy and senescence by knockdown of ROC1 E3 ubiquitin ligase to suppress the growth of liver cancer cells. Cell Death Differ 20:235–247

    Article  PubMed  CAS  Google Scholar 

  12. Jia L, Bickel JS, Wu J, Morgan MA, Li H, Yang J, Yu X, Chan RC, Sun Y (2011) RBX1 (RING box protein 1) E3 ubiquitin ligase is required for genomic integrity by modulating DNA replication licensing proteins. J Biol Chem 286:3379–3386

    Article  PubMed  CAS  Google Scholar 

  13. Watson IR, Irwin MS, Ohh M (2011) NEDD8 pathways in cancer. Sine Quibus Non Cancer cell 19:168–176

    Article  PubMed  CAS  Google Scholar 

  14. Milhollen MA, Traore T, Adams-Duffy J, Thomas MP, Berger AJ, Dang L, Dick LR, Garnsey JJ, Koenig E, Langston SP, Manfredi M, Narayanan U, Rolfe M, Staudt LM, Soucy TA, Yu J, Zhang J, Bolen JB, Smith PG (2010) MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-{kappa}B-dependent lymphoma. Blood 116:1515–1523

    Article  PubMed  CAS  Google Scholar 

  15. Nawrocki ST, Kelly KR, Smith PG, Espitia CM, Possemato A, Beausoleil SA, Milhollen M, Blakemore S, Thomas M, Berger A, Carew JS (2013) Disrupting protein NEDDylation with MLN4924 is a novel strategy to target cisplatin resistance in ovarian cancer. Clin Cancer Res 19:3577–3590

    Article  PubMed  CAS  Google Scholar 

  16. Xie P, Zhang M, He S, Lu K, Chen Y, Xing G, Lu Y, Liu P, Li Y, Wang S, Chai N, Wu J, Deng H, Wang HR, Cao Y, Zhao F (2014) Cui Y3, Wang J3, He F3, Zhang L8. The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis. Nat Commun 5:3733

    Article  PubMed  CAS  Google Scholar 

  17. Zhao Y, Morgan MA, Sun Y (2014) Targeting Neddylation pathways to inactivate cullin-RING ligases for anticancer therapy. Antioxid Redox Signal 21:2383–2400

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Bhatia S, Pavlick AC, Boasberg P, Thompson JA, Mulligan G, Pickard MD, Faessel H, Dezube BJ, Hamid O (2016) A phase I study of the investigational NEDD8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924) in patients with metastatic melanoma. Invest New Drugs 34:439–449

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Shah JJ, Jakubowiak AJ, O’Connor OA, Orlowski RZ, Harvey RD, Smith MR, Lebovic D, Diefenbach C, Kelly K, Hua Z, Berger AJ, Mulligan G, Faessel HM, Tirrell S, Dezube BJ, Lonial S (2016) Phase I study of the novel investigational NEDD8-activating enzyme inhibitor pevonedistat (MLN4924) in patients with relapsed/refractory multiple myeloma or lymphoma. Clin Cancer Res 22:34–43

    Article  PubMed  CAS  Google Scholar 

  20. Sarantopoulos J, Shapiro GI, Cohen RB, Clark JW, Kauh JS, Weiss GJ, Cleary JM, Mahalingam D, Pickard MD, Faessel HM, Berger AJ, Burke K, Mulligan G, Dezube BJ, Harvey RD (2016) Phase I study of the investigational NEDD8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924) in patients with advanced solid tumors. Clin Cancer Res 22:847–857

    Article  PubMed  CAS  Google Scholar 

  21. Swords RT, Erba HP, DeAngelo DJ, Bixby DL, Altman JK, Maris M, Hua Z, Blakemore SJ, Faessel H, Sedarati F, Dezube BJ, Giles FJ, Medeiros BC (2015) Pevonedistat (MLN4924), a first-in-class NEDD8-activating enzyme inhibitor, in patients with acute myeloid leukaemia and myelodysplastic syndromes: a phase 1 study. Br J Haematol 169:534–543

    Article  PubMed  CAS  Google Scholar 

  22. Wei W, Guo H, Chang J, Yu Y, Liu G, Zhang N, Willard SH, Zheng S, Yu XF (2016) ICAM-5/telencephalin is a functional entry receptor for enterovirus D68. Cell Host Microbe 20:631–641

    Article  PubMed  CAS  Google Scholar 

  23. Chow WH, Dong LM, Devesa SSD (2010) Epidemiology and risk factors for kidney cancer. Nat Rev Urol 7:245–257

    Article  PubMed  PubMed Central  Google Scholar 

  24. Siegel RL, Miller KD, Jemal A. Cancer Statistics (2017) CA Cancer J Clin 2017 67:7–30

    Article  PubMed  Google Scholar 

  25. Linehan WM (2012) Genetic basis of kidney cancer: role of genomics for the development of disease-based therapeutics. Genome Res 22:2089–2100

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Chen Z, Zhang J, Zhang Z, Feng Z, Wei J, Lu J, Fang Y, Liang Y, Cen J, Pan Y, Huang Y, Zhou F, Chen W, Luo J (2017) The putative tumor suppressor microRNA-30a-5p modulates clear cell renal cell carcinoma aggressiveness through repression of ZEB2. Cell Death Dis 8:e2859

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Chen W, Hill H, Christie A, Kim MS, Holloman E, Pavia-Jimenez A, Homayoun F, Ma Y, Patel N, Yell P, Hao G, Yousuf Q, Joyce A, Pedrosa I, Geiger H, Zhang H, Chang J, Gardner KH, Bruick RK, Reeves C, Hwang TH, Courtney K, Frenkel E, Sun X, Zojwalla N, Wong T, Rizzi JP, Wallace EM, Josey JA, Xie Y, Xie XJ, Kapur P, McKay RM, Brugarolas J (2016) Targeting renal cell carcinoma with a HIF-2 antagonist. Nature 539:112–117

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Cancer Genome Atlas Research N (2013) Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 499:43–49

    Article  CAS  Google Scholar 

  29. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, Brownell JE, Burke KE, Cardin DP, Critchley S, Cullis CA, Doucette A, Garnsey JJ, Gaulin JL, Gershman RE, Lublinsky AR, McDonald A, Mizutani H, Narayanan U, Olhava EJ, Peluso S, Rezaei M, Sintchak MD, Talreja T, Thomas MP, Traore T, Vyskocil S, Weatherhead GS, Yu J, Zhang J, Dick LR, Claiborne CF, Rolfe M, Bolen JB, Langston SP (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458:732–736

    Article  PubMed  CAS  Google Scholar 

  30. Blank JL, Liu XJ, Cosmopoulos K, Bouck DC, Garcia K, Bernard H, Tayber O, Hather G, Liu R, Narayanan U, Milhollen MA, Lightcap ES (2013) Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924. Cancer Res 73:225–234

    Article  PubMed  CAS  Google Scholar 

  31. Milhollen MA, Narayanan U, Soucy TA, Veiby PO, Smith PG, Amidon B (2011) Inhibition of NEDD8-activating enzyme induces rereplication and apoptosis in human tumor cells consistent with deregulating CDT1 turnover. Cancer Res 71:3042–3051

    Article  PubMed  CAS  Google Scholar 

  32. Lin JJ, Milhollen MA, Smith PG, Narayanan U, Dutta A (2010) NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells. Cancer Res 70:10310–10320

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Emanuele MJ, Elia AE, Xu Q, Thoma CR, Izhar L, Leng Y, Guo A, Chen YN, Rush J, Hsu PW, Yen HC, Elledge SJ (2011) Global identification of modular cullin-RING ligase substrates. Cell 147:459–474

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Yao WT, Wu JF, Yu GY, Wang R, Wang K, Li LH, Chen P, Jiang YN, Cheng H, Lee HW, Yu J, Qi H, Yu XJ, Wang P, Chu YW, Yang M, Hua ZC, Ying HQ, Hoffman RM, Jeong LS, Jia LJ (2014) Suppression of tumor angiogenesis by targeting the protein neddylation pathway. Cell Death Dis 5:e1059

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Chen Y, Yang Z, Meng M, Zhao Y, Dong N, Yan H, Liu L, Ding M, Peng HB, Shao F (2009) Cullin mediates degradation of RhoA through evolutionarily conserved BTB adaptors to control actin cytoskeleton structure and cell movement. Mol Cell 35:841–855

    Article  PubMed  CAS  Google Scholar 

  36. Yang D, Zhao Y, Liu J, Sun Y, Jia L (2012) Protective autophagy induced by RBX1/ROC1 knockdown or CRL inactivation via modulating the DEPTOR-MTOR axis. Autophagy 8:1856–1858

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Cukras S, Morffy N, Ohn T, Kee Y (2014) Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases. PLoS One 9:e101844

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Huang DT, Ayrault O, Hunt HW, Taherbhoy AM, Duda DM, Scott DC, Borg LA, Neale G, Murray PJ, Roussel MF, Schulman BA (2009) E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification. Mol Cell 33:483–495

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Haagenson KK, Tait L, Wang J, Shekhar MP, Polin L, Chen W, Wu GS (2012) Cullin-3 protein expression levels correlate with breast cancer progression. Cancer Biol Ther 13:1042–1046

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Hung MS, Chen IC, You L, Jablons DM, Li YC, Mao JH, Xu Z, Lung JH, Yang CT, Liu ST (2016) Knockdown of cullin 4A inhibits growth and increases chemosensitivity in lung cancer cells. J Cell Mol Med 20:1295–1306

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  42. Durie BG, Hoering A, Abidi MH, Rajkumar SV, Epstein J, Kahanic SP, Thakuri M, Reu F, Reynolds CM, Sexton R, Orlowski RZ, Barlogie B, Dispenzieri A (2017) Bortezomib with lenalidomide and dexamethasone versus lenalidomide and dexamethasone alone in patients with newly diagnosed myeloma without intent for immediate autologous stem-cell transplant (SWOG S0777): a randomised, open-label, phase 3 trial. Lancet 389:519–527

    Article  PubMed  CAS  Google Scholar 

  43. Arastu-Kapur S, Anderl JL, Kraus M, Parlati F, Shenk KD, Lee SJ, Muchamuel T, Bennett MK, Driessen C, Ball AJ, Kirk CJ (2011) Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events. Clin Cancer Res 17:2734–2743

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

The study was funded by the National Natural Science Foundation of China (81772183 and 31600132), the Chinese Ministry of Science and Technology (2018ZX10731101-003-004), Department of Science and Technology of Jilin Province (No. 20180101127JC) and the Fundamental Research Funds for the Central Universities and Program for JLU Science and Technology Innovative Research Team.

Author information

Authors and Affiliations

  1. Department of Urology, The First Hospital of Jilin University, Changchun, 130021, Jilin, China

    Bo Xu, Yuyou Deng, Ran Bi & Chunxi Wang

  2. Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130021, Jilin, China

    Bo Xu, Yuyou Deng, Ran Bi, Haoran Guo, Junliang Chang, Guanchen Liu & Wei Wei

  3. Department of Obstetrics and Gynecology, The First Hospital of Jilin University, Changchun, 130021, Jilin, China

    Chang Shu & Neelam Kumari Shah

  4. Department of Nephrology, First Hospital of Jilin University, Changchun, 130021, Jilin, China

    Yujun Du

Authors
  1. Bo Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuyou Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ran Bi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Haoran Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chang Shu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Neelam Kumari Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Junliang Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guanchen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yujun Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Chunxi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yujun Du, Wei Wei or Chunxi Wang.

Ethics declarations

Conflict of interest

All authors declare that they have no conflicts of interest.

Ethical approval

All procedures performed in studies involving animal experiments were in accordance with the ethical standards of China research committee and the protocol approved by the Ethics Committee of the First Hospital of Jilin University.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, B., Deng, Y., Bi, R. et al. A first-in-class inhibitor, MLN4924 (pevonedistat), induces cell-cycle arrest, senescence, and apoptosis in human renal cell carcinoma by suppressing UBE2M-dependent neddylation modification. Cancer Chemother Pharmacol 81, 1083–1093 (2018). https://doi.org/10.1007/s00280-018-3582-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-018-3582-z

Keywords

Search

Navigation