Cancer Chemotherapy and Pharmacology

, Volume 61, Issue 6, pp 923–932 | Cite as

IPI-504, a novel and soluble HSP-90 inhibitor, blocks the unfolded protein response in multiple myeloma cells

  • Jon Patterson
  • Vito J. Palombella
  • Christian Fritz
  • Emmanuel NormantEmail author
Original Article



Inhibitors of heat shock protein (Hsp) 90 induce apoptosis in multiple myeloma (MM) cells, but the molecular mechanisms underlying this cytotoxic outcome are not clear. Here, we investigate the effect of IPI-504, a novel and highly soluble inhibitor of the Hsp90 ATPase activity, on the unfolded protein response (UPR) in MM cells. The UPR is a stress response pathway triggered by sensors located at the endoplasmic reticulum (ER) membrane whose function is to reduce an excessive accumulation of misfolded protein in the ER. During normal development of B-lymphocytes to antibody-producing plasma cells, a partial UPR has been described, where IREα and ATF-6 are stimulated, whereas the third sensor, PERK, is not induced.


Levels of the activated forms of the three main UPR sensors ATF-6, XBP-1 and PERK/eIF-2 were monitored in two different MM cells lines and one non-MM cell lines under various experimental conditions including incubation with increasing concentration of IPI-504. Also, MM cells were incubated with IPI-504 and several apoptosis markers were monitored.


We show here that a partial UPR is constitutively activated in plasma cell-derived MM cells and that IPI-504 can potently inhibit this pathway. IPI-504 achieves this by inactivating the transcription factors XBP1 and ATF6. In addition, IPI-504 also blocks the tunicamycin-induced phosphorylation of eIF2 by PERK. Dose-response and time course experiments reveal that IPI-504’s inhibitory effect on the UPR parallels its cytotoxic and pro-apoptotic effects on MM cells.


The results presented here suggest that the IPI-504-induced apoptosis might be, in part, mediated by the inhibition of the partial UPR. Other malignancies that rely on intact and efficient UPR to survive could be considered as new indications for Hsp90 inhibitors.


Unfolded protein response Hsp90 Multiple myeloma 


  1. 1.
    Sirohi B, Powles R (2004) Multiple myeloma. Lancet 363(9412):875–887PubMedCrossRefGoogle Scholar
  2. 2.
    Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569(1–2):29–63PubMedGoogle Scholar
  3. 3.
    Ma Y, Hendershot LM (2004) The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 4(12):966–977PubMedCrossRefGoogle Scholar
  4. 4.
    Brewer JW, Hendershot LM (2005) Building an antibody factory: a job for the unfolded protein response. Nat Immunol 6(1):23–29PubMedCrossRefGoogle Scholar
  5. 5.
    Kaufman RJ (2002) Orchestrating the unfolded protein response in health and disease. J Clin Invest 110(10):1389–1398PubMedGoogle Scholar
  6. 6.
    Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R et al (2000) ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 6(6):1355–1364PubMedCrossRefGoogle Scholar
  7. 7.
    Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M et al (2000) ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol Cell Biol 20(18):6755–6767PubMedCrossRefGoogle Scholar
  8. 8.
    Haze K, Yoshida H, Yanagi H, Yura T, Mori K (1999) Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 10(11):3787–3799PubMedGoogle Scholar
  9. 9.
    Yoshida H, Haze K, Yanagi H, Yura T, Mori K (1998) Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J Biol Chem 273(50):33741–33749PubMedCrossRefGoogle Scholar
  10. 10.
    Wang Y, Shen J, Arenzana N, Tirasophon W, Kaufman RJ, Prywes R (2000) Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J Biol Chem 275(35):27013–27020PubMedGoogle Scholar
  11. 11.
    Roy B, Lee AS (1999) The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex. Nucleic Acids Res 27(6):1437–1443PubMedCrossRefGoogle Scholar
  12. 12.
    Yamamoto K, Yoshida H, Kokame K, Kaufman RJ, Mori K (2004) Differential contributions of ATF6 and XBP1 to the activation of endoplasmic reticulum stress-responsive cis-acting elements ERSE, UPRE and ERSE-II. J Biochem (Tokyo) 136(3):343–350Google Scholar
  13. 13.
    Lawson B, Brewer JW, Hendershot LM (1998) Geldanamycin, an hsp90/GRP94-binding drug, induces increased transcription of endoplasmic reticulum (ER) chaperones via the ER stress pathway. J Cell Physiol 174(2):170–178PubMedCrossRefGoogle Scholar
  14. 14.
    Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107(7):881–891PubMedCrossRefGoogle Scholar
  15. 15.
    Liou HC, Boothby MR, Finn PW, Davidon R, Nabavi N, Zeleznik-Le NJ et al (1990) A new member of the leucine zipper class of proteins that binds to the HLA DR alpha promoter. Science 247(4950):1581–1584PubMedCrossRefGoogle Scholar
  16. 16.
    Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP et al (2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415(6867):92–96PubMedCrossRefGoogle Scholar
  17. 17.
    Cox JS, Shamu CE, Walter P (1993) Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73(6):1197–1206PubMedCrossRefGoogle Scholar
  18. 18.
    Shen X, Ellis RE, Lee K, Liu CY, Yang K, Solomon A et al (2001) Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development. Cell 107(7):893–903PubMedCrossRefGoogle Scholar
  19. 19.
    Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397(6716):271–274PubMedCrossRefGoogle Scholar
  20. 20.
    Shaffer AL, Shapiro-Shelef M, Iwakoshi NN, Lee AH, Qian SB, Zhao H et al (2004) XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity 21(1):81–93PubMedCrossRefGoogle Scholar
  21. 21.
    Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D (2000) Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 5(5):897–904PubMedCrossRefGoogle Scholar
  22. 22.
    Gass JN, Gifford NM, Brewer JW (2002) Activation of an unfolded protein response during differentiation of antibody-secreting B cells. J Biol Chem 277(50):49047–49054PubMedCrossRefGoogle Scholar
  23. 23.
    Iwakoshi NN, Lee AH, Vallabhajosyula P, Otipoby KL, Rajewsky K, Glimcher LH (2003) Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat Immunol 4(4):321–329PubMedCrossRefGoogle Scholar
  24. 24.
    Ubeda M, Habener JF (2000) CHOP gene expression in response to endoplasmic-reticular stress requires NFY interaction with different domains of a conserved DNA-binding element. Nucleic Acids Res 28(24):4987–4997PubMedCrossRefGoogle Scholar
  25. 25.
    Neckers L (2002) Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med 8(4 Suppl):S55–S61PubMedCrossRefGoogle Scholar
  26. 26.
    Maloney A, Workman P (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther 2(1):3–24PubMedCrossRefGoogle Scholar
  27. 27.
    Goetz MP, Toft DO, Ames MM, Erlichman C (2003) The Hsp90 chaperone complex as a novel target for cancer therapy. Ann Oncol 14(8):1169–1176PubMedCrossRefGoogle Scholar
  28. 28.
    Fumo G, Akin C, Metcalfe DD, Neckers L (2004) 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is effective in down-regulating mutated, constitutively activated KIT protein in human mast cells. Blood 103(3):1078–1084PubMedCrossRefGoogle Scholar
  29. 29.
    Minami Y, Kiyoi H, Yamamoto Y, Yamamoto K, Ueda R, Saito H et al (2002) Selective apoptosis of tandemly duplicated FLT3-transformed leukemia cells by Hsp90 inhibitors. Leukemia 16(8):1535–1540PubMedCrossRefGoogle Scholar
  30. 30.
    Gorre ME, Ellwood-Yen K, Chiosis G, Rosen N, Sawyers CL (2002) BCR-ABL point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein 90. Blood 100(8):3041–3044PubMedCrossRefGoogle Scholar
  31. 31.
    Nimmanapalli R, O’Bryan E, Bhalla K (2001) Geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers Bcr-Abl levels and induces apoptosis and differentiation of Bcr-Abl-positive human leukemic blasts. Cancer Res 61(5):1799–1804PubMedGoogle Scholar
  32. 32.
    Shimamura T, Lowell AM, Engelman JA, Shapiro GI (2005) Epidermal growth factor receptors harboring kinase domain mutations associate with the heat shock protein 90 chaperone and are destabilized following exposure to geldanamycins. Cancer Res 65(14):6401–6408PubMedCrossRefGoogle Scholar
  33. 33.
    Sydor JR, Normant E, Pien CS, Porter JR, Ge J, Grenier L et al (2006) Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90. Proc Natl Acad Sci U S A 103(46):17408–17413PubMedCrossRefGoogle Scholar
  34. 34.
    Ge J, Normant E, Porter JR, Ali J, Dembski MS, Gao Y et al (2006) Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent water-soluble inhibitors of Hsp90. J Med Chem 49:4606–4615PubMedCrossRefGoogle Scholar
  35. 35.
    Guo W, Reigan P, Siegel D, Zirrolli J, Gustafson D, Ross D (2005) Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition. Cancer Res 65(21):10006–10015PubMedCrossRefGoogle Scholar
  36. 36.
    Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Kung AL, Davies FE, et al (2006) Anti-myeloma activity of heat shock protein-90 inhibition. BloodPubMedCrossRefGoogle Scholar
  37. 37.
    Pittet JF, Lee H, Pespeni M, O’Mahony A, Roux J, Welch WJ (2005) Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90. J Immunol 174(1):384–394PubMedGoogle Scholar
  38. 38.
    Broemer M, Krappmann D, Scheidereit C (2004) Requirement of Hsp90 activity for IkappaB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-kappaB activation. Oncogene 23(31):5378–5386PubMedCrossRefGoogle Scholar
  39. 39.
    Marcu MG, Doyle M, Bertolotti A, Ron D, Hendershot L, Neckers L (2002) Heat shock protein 90 modulates the unfolded protein response by stabilizing IRE1alpha. Mol Cell Biol 22(24):8506–8513PubMedCrossRefGoogle Scholar
  40. 40.
    Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143(7):1883–1898PubMedCrossRefGoogle Scholar
  41. 41.
    Plemper RK, Wolf DH (1999) Retrograde protein translocation: eradication of secretory proteins in health and disease. Trends Biochem Sci 24(7):266–270PubMedCrossRefGoogle Scholar
  42. 42.
    Mimnaugh EG, Xu W, Vos M, Yuan X, Isaacs JS, Bisht KS et al (2004) Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity. Mol Cancer Ther 3(5):551–566PubMedGoogle Scholar
  43. 43.
    Lee AH, Iwakoshi NN, Anderson KC, Glimcher LH (2003) Proteasome inhibitors disrupt the unfolded protein response in myeloma cells. Proc Natl Acad Sci U S A 100(17):9946–9951PubMedCrossRefGoogle Scholar
  44. 44.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning : a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  45. 45.
    Lee AH, Iwakoshi NN, Glimcher LH (2003) XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23(21):7448–7459PubMedCrossRefGoogle Scholar
  46. 46.
    Jenner RG, Maillard K, Cattini N, Weiss RA, Boshoff C, Wooster R et al (2003) Kaposi’s sarcoma-associated herpesvirus-infected primary effusion lymphoma has a plasma cell gene expression profile. Proc Natl Acad Sci U S A 100(18):10399–10404PubMedCrossRefGoogle Scholar
  47. 47.
    Munshi NC, Hideshima T, Carrasco D, Shammas M, Auclair D, Davies F et al (2004) Identification of genes modulated in multiple myeloma using genetically identical twin samples. Blood 103(5):1799–1806PubMedCrossRefGoogle Scholar
  48. 48.
    Nakamura M, Gotoh T, Okuno Y, Tatetsu H, Sonoki T, Uneda S et al (2006) Activation of the endoplasmic reticulum stress pathway is associated with survival of myeloma cells. Leuk Lymphoma 47(3):531–539PubMedCrossRefGoogle Scholar
  49. 49.
    Yoneda T, Imaizumi K, Oono K, Yui D, Gomi F, Katayama T et al (2001) Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress. J Biol Chem 276(17):13935–13940PubMedGoogle Scholar
  50. 50.
    Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R et al (2004) CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev 18(24):3066–3077PubMedCrossRefGoogle Scholar
  51. 51.
    McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ (2001) Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol 21(4):1249–1259PubMedCrossRefGoogle Scholar
  52. 52.
    Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA et al (2000) Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403(6765):98–103PubMedCrossRefGoogle Scholar
  53. 53.
    Obeng EA, Boise LH (2005) Caspase-12 and caspase-4 are not required for caspase-dependent endoplasmic reticulum stress-induced apoptosis. J Biol Chem 280(33):29578–29587PubMedCrossRefGoogle Scholar
  54. 54.
    Di Sano F, Ferraro E, Tufi R, Achsel T, Piacentini M, Cecconi F (2006) Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism. J Biol Chem 281(5):2693–2700PubMedCrossRefGoogle Scholar
  55. 55.
    Li J, Lee B, Lee AS (2006) Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem 281(11):7260–7270PubMedCrossRefGoogle Scholar
  56. 56.
    Nawrocki ST, Carew JS, Pino MS, Highshaw RA, Dunner K Jr, Huang P et al (2005) Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis. Cancer Res 65(24):11658–11666PubMedCrossRefGoogle Scholar
  57. 57.
    Nawrocki ST, Carew JS, Dunner K Jr, Boise LH, Chiao PJ, Huang P et al (2005) Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells. Cancer Res 65(24):11510–11519PubMedCrossRefGoogle Scholar
  58. 58.
    Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Fanourakis G, Gu X et al (2002) Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci U S A 99(22):14374–14379PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Jon Patterson
    • 1
  • Vito J. Palombella
    • 1
  • Christian Fritz
    • 1
  • Emmanuel Normant
    • 1
    Email author
  1. 1.Infinity Pharmaceuticals, Inc.CambridgeUSA

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