, Volume 15, Issue 7, pp 795–804 | Cite as

Inhibition of MEK/ERK signaling induces apoptosis of acute myelogenous leukemia cells via inhibition of eukaryotic initiation factor 4E-binding protein 1 and down-regulation of Mcl-1

  • Chie Nishioka
  • Takayuki Ikezoe
  • Jing Yang
  • Akihito Yokoyama
Original Paper


We previously showed that the MEK inhibitor AZD6244 induced apoptosis in acute myelogenous leukemia (AML) HL60 cells. However, the mechanisms of AZD6244 to induce apoptosis remain to be fully elucidated. This study found that exposure of HL60 cells to AZD6244 down-regulated the levels of phosphor (p)-4E-binding protein 1 (4E-BP1), a substrate of mammalian target of rapamycin complex 1 (mTORC1), and anti-apoptotic protein Mcl-1. On the other hand, exposure of EOL-1 and MOLM13 cells to AZD6244 failed to induce apoptosis and levels of p-4E-BP1 and Mcl-1 were not down-regulated in these cells. These observations prompted us to hypothesize that down-regulation od 4E-BP1 and Mcl-1 might play an important role in AZD6244-mediated apoptosis. As expected, down-regulation of 4E-BP1 by an siRNA sensitized EOL-1 cells to AZD6244-mediated apoptosis in parallel with down-regulation of Mcl-1. Moreover, we found that blockade of mTORC1 by RAD001 synergistically enhanced the action of AZD6244 in leukemia cells.


ERK 4E-BP1 Mcl-1 AZD6244 RAD001 



Acute myelogenous leukemia


Mitogen-activated protein kinase


Extracellular signal-regulated kinase


Phosphoinositide-3 kinase


Mammalian target of rapamycin


Peripheral blood mononuclear cells


Eukaryotic initiation factor 4E-binding protein 1


Flow cyctometry



This work was supported in part by Grant-in-Aid from the Ministry of Education, Culture Sports, Science, and Technology of Japan (to T.I), The Kochi University President’s Discretionary Grant (to T.I), Takeda Science Foundation (to T.I), AstraZeneca Research Grant 2008 (to T.I), and Sagawa Foundation for Promotion of Cancer Research (to T.I). C.N. is grateful for a JSPS Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.


  1. 1.
    Bacher U, Haferlach T, Schoch C, Kern W, Schnittger S (2006) Implications of NRAS mutations in AML: a study of 2502 patients. Blood 107:3847–3853CrossRefPubMedGoogle Scholar
  2. 2.
    Zhao S, Konopleva M, Cabreira-Hansen M, Xie Z, Hu W, Milella M, Estrov Z, Mills GB, Andreeff M (2004) Inhibition of phosphatidylinositol 3-kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias. Leukemia 18:267–275CrossRefPubMedGoogle Scholar
  3. 3.
    Gregorj C, Ricciardi MR, Petrucci MT, Scerpa MC, De Cave F, Fazi P, Vignetti M, Vitale A, Mancini M, Cimino G, Palmieri S, Di Raimondo F, Specchia G, Fabbiano F, Cantore N, Mosna F, Camera A, Luppi M, Annino L, Miraglia E, Fioritoni G, Ronco F, Meloni G, Mandelli F, Andreeff M, Milella M, Foà R, Tafuri A, GIMEMA Acute Leukemia Working Party (2007) ERK1/2 phosphorylation is an independent predictor of complete remission in newly diagnosed adult acute lymphoblastic leukemia. Blood 109:5473–5476CrossRefPubMedGoogle Scholar
  4. 4.
    Milella M, Konopleva M, Precupanu CM, Tabe Y, Ricciardi MR, Gregorj C, Collins SJ, Carter BZ, D’Angelo C, Petrucci MT, Foà R, Cognetti F, Tafuri A, Andreeff M (2007) MEK blockade converts AML differentiating response to retinoids into extensive apoptosis. Blood 109:2121–2129CrossRefPubMedGoogle Scholar
  5. 5.
    Milella M, Kornblau SM, Estrov Z, Carter BZ, Lapillonne H, Harris D, Konopleva M, Zhao S, Estey E, Andreeff M (2001) Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia. J Clin Invest 108:851–859PubMedGoogle Scholar
  6. 6.
    Staber PB, Linkesch W, Zauner D, Beham-Schmid C, Guelly C, Schauer S, Sill H, Hoefler G (2004) Common alterations in gene expression and increased proliferation in recurrent acute myeloid leukemia. Oncogene 23:894–904CrossRefPubMedGoogle Scholar
  7. 7.
    Kornblau SM, Womble M, Qiu YH, Jackson CE, Chen W, Konopleva M, Estey EH, Andreeff M (2006) Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia. Blood 108:2358–2365CrossRefPubMedGoogle Scholar
  8. 8.
    Steelman LS, Abrams SL, Whelan J, Bertrand FE, Ludwig DE, Bäsecke J, Libra M, Stivala F, Milella M, Tafuri A, Lunghi P, Bonati A, Martelli AM, McCubrey JA (2008) Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia. Leukemia 22:686–707CrossRefPubMedGoogle Scholar
  9. 9.
    Yeh TC, Marsh V, Bernat BA, Ballard J, Colwell H, Evans RJ, Parry J, Smith D, Brandhuber BJ, Gross S, Marlow A, Hurley B, Lyssikatos J, Lee PA, Winkler JD, Koch K, Wallace E (2007) Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res 13:1576–1583CrossRefPubMedGoogle Scholar
  10. 10.
    Tai YT, Fulciniti M, Hideshima T, Song W, Leiba M, Li XF, Rumizen M, Burger P, Morrison A, Podar K, Chauhan D, Tassone P, Richardson P, Munshi NC, Ghobrial IM, Anderson KC (2007) Targeting MEK induces myeloma-cell cytotoxicity and inhibits osteoclastogenesis. Blood 110:1656–1663CrossRefPubMedGoogle Scholar
  11. 11.
    Friday BB, Yu C, Dy GK, Smith PD, Wang L, Thibodeau SN, Adjei AA (2008) BRAF V600E disrupts AZD6244-induced abrogation of negative feedback pathways between extracellular signal-regulated kinase and Raf proteins. Cancer Res 68:6145–6153CrossRefPubMedGoogle Scholar
  12. 12.
    Nishioka C, Ikezoe T, Yang J, Koeffler HP, Yokoyama A (2008) Inhibition of MEK/ERK signaling synergistically potentiates histone deacetylase inhibitor-induced growth arrest, apoptosis and acetylation of histone H3 on p21waf1 promoter in acute myelogenous leukemia cell. Leukemia 22:1449–1452CrossRefPubMedGoogle Scholar
  13. 13.
    Guertin DA, Sabatini DM (2007) Defining the role of mTOR in cancer. Cancer Cell 12:9–22CrossRefPubMedGoogle Scholar
  14. 14.
    Schmelzle T, Hall MN (2000) mTOR, a central controller of cell growth. Cell 103:253–262CrossRefPubMedGoogle Scholar
  15. 15.
    Brown EJ, Schreiber SL (1996) A signaling pathway to translational control. Cell 86:517–520CrossRefPubMedGoogle Scholar
  16. 16.
    Wang X, Beugnet A, Murakami M, Yamanaka S, Proud CG (2005) Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins. Mol Cell Biol 25:2558–2572CrossRefPubMedGoogle Scholar
  17. 17.
    Castellvi J, Garcia A, Rojo F, Ruiz-Marcellan C, Gil A, Baselga J, Ramon y Cajal S (2006) Phosphorylated 4E binding protein 1: a hallmark of cell signaling that correlates with survival in ovarian cancer. Cancer 107:1801–1811CrossRefPubMedGoogle Scholar
  18. 18.
    Chen CS, Weng SC, Tseng PH, Lin HP, Chen CS (2005) Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes. J Biol Chem 280:38879–38887CrossRefPubMedGoogle Scholar
  19. 19.
    Oudard S, Medioni J, Aylllon J, Barrascourt E, Elaidi RT, Balcaceres J, Scotte F (2009) Everolimus (RAD001): an mTOR inhibitor for the treatment of metastatic renal cell carcinoma. Expert Rev Anticancer Ther 9:705–717CrossRefPubMedGoogle Scholar
  20. 20.
    Ikezoe T, Nishioka C, Tasaka T, Yang Y, Komatsu N, Togitani K, Koeffler HP, Taguchi H (2006) The antitumor effects of sunitinib (formerly SU11248) against a variety of human hematologic malignancies: enhancement of growth inhibition via inhibition of mammalian target of rapamycin signaling. Mol Cancer Ther 5:2522–2530CrossRefPubMedGoogle Scholar
  21. 21.
    Nishioka C, Ikezoe T, Yang J, Koeffler HP, Taguchi H (2007) Fludarabine induces apoptosis of human T-cell leukemia virus type 1-infected T cells via inhibition of the nuclear factor-kappaB signal pathway. Leukemia 21:1044–1049CrossRefPubMedGoogle Scholar
  22. 22.
    Ikezoe T, Bandobashi K, Yang Y, Takeuchi S, Sekiguchi N, Sakai S, Koeffler HP, Taguchi H (2006) HIV-1 protease inhibitor ritonavir potentiates the effect of 1, 25-dihydroxyvitamin D3 to induce growth arrest and differentiation of human myeloid leukemia cells via down-regulation of CYP24. Leuk Res 30:1005–1011CrossRefPubMedGoogle Scholar
  23. 23.
    Nishioka C, Ikezoe T, Takeshita A, Yang J, Tasaka T, Yang Y, Kuwayama Y, Komatsu N, Togitani K, Koeffler HP, Taguchi H (2007) ZD6474 induces growth arrest and apoptosis of human leukemia cells, which is enhanced by concomitant use of a novel MEK inhibitor, AZD6244. Leukemia 21:1308–1310CrossRefPubMedGoogle Scholar
  24. 24.
    Chou TC, Talalay P (1984) Quantitative analysis of dose–effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55CrossRefPubMedGoogle Scholar
  25. 25.
    Wergeland L, Sjøholt G, Haaland I, Hovland R, Bruserud Ø, Gjertsen BT (2007) Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells. Mol Cancer 6:33CrossRefPubMedGoogle Scholar
  26. 26.
    Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC (1998) Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 91:991–1000PubMedGoogle Scholar
  27. 27.
    Bos JL, Verlaan-de Vries M, Jansen AM, Veeneman GH, van Boom JH, van der Eb AJ (1984) Three different mutations in codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization. Nucleic Acids Res 12:9155–9163CrossRefPubMedGoogle Scholar
  28. 28.
    Kinkade CW, Castillo-Martin M, Puzio-Kuter A, Yan J, Foster TH, Gao H, Sun Y, Ouyang X, Gerald WL, Cordon-Cardo C, Abate-Shen C (2008) Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model. J Clin Invest 118:3051–3064PubMedGoogle Scholar
  29. 29.
    Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R, Maira M, McNamara K, Perera SA, Song Y, Chirieac LR, Kaur R, Lightbown A, Simendinger J, Li T, Padera RF, García-Echeverría C, Weissleder R, Mahmood U, Cantley LC, Wong KK (2008) Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med 14:1351–1356CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Chie Nishioka
    • 1
    • 2
  • Takayuki Ikezoe
    • 1
  • Jing Yang
    • 1
  • Akihito Yokoyama
    • 1
  1. 1.Department of Hematology and Respiratory Medicine, Kochi Medical SchoolKochi UniversityOko-cho, NankokuJapan
  2. 2.Japanese Society for the Promotion of Science (JSPS)Chiyodaku, TokyoJapan

Personalised recommendations