antitumor activity in multiple myelomaSmall-molecule inhibition of proteasome and aggresome function induces synergisticL. Schreiber, and Kenneth C. Anderson Teru Hideshima, James E. Bradner, Jason Wong, Dharminder Chauhan, Paul Richardson, Stuartdoi:10.1073/pnas.0503221102 2005;102;8567-8572; originally published online Jun 3, 2005; PNAS This information is current as of November 2006. & ServicesOnline Information www.pnas.org/cgi/content/full/102/24/8567etc., can be found at: High-resolution figures, a citation map, links to PubMed and Google Scholar, Related Articles www.pnas.org/cgi/content/full/102/24/8393A related article has been published: References www.pnas.org/cgi/content/full/102/24/8567#BIBLThis article cites 29 articles, 15 of which you can access for free at: www.pnas.org/cgi/content/full/102/24/8567#otherarticlesThis article has been cited by other articles: E-mail Alerts. click hereat the top right corner of the article orReceive free email alerts when new articles cite this article - sign up in the box Rights & Permissions www.pnas.org/misc/rightperm.shtmlTo reproduce this article in part (figures, tables) or in entirety, see: Reprints www.pnas.org/misc/reprints.shtmlTo order reprints, see: Notes:Small-molecule inhibition of proteasome andaggresome function induces synergisticantitumor activity in multiple myelomaTeru Hideshima*, James E. Bradner*†, Jason Wong†, Dharminder Chauhan*, Paul Richardson*, Stuart L. Schreiber†‡§,and Kenneth C. Anderson*§*Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana–Farber Cancer Institute, Harvard Medical School, 44 Binney Street,Boston, MA 02115;†Broad Institute of Harvard University and Massachusetts Institute of Technology, 320 Charles Street, Cambridge, MA 02141; and‡Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138Contributed by Stuart L. Schreiber, April 20, 2005We have shown that the proteasome inhibitor bortezomib (for-merly known as PS-341) triggers significant antitumor activity inmultiple myeloma (MM) in both preclinical models and patientswith relapsed refractory disease. Recent studies have shown thatunfolded and misfolded ubiquitinated proteins are degraded notonly by proteasomes, but also by aggresomes, dependent onhistone deacetylase 6 (HDAC6) activity. We therefore hypothesizedthat inhibition of both mechanisms of protein catabolism couldinduce accumulation of ubiquitinated proteins followed by signif-icant cell stress and cytotoxicity in MM cells. To prove this hypoth-esis, we used bortezomib and tubacin to inhibit the proteasomeand HDAC6, respectively. Tubacin specifically triggers acetylationof␣-tubulin as a result of HDAC6 inhibition in a dose- andtime-dependent fashion. It induces cytotoxicity in MM cells at 72 hwith an IC50of 5–20␮M, which is mediated by caspase-dependentapoptosis; no toxicity is observed in normal peripheral bloodmononuclear cells. Tubacin inhibits the interaction of HDAC6 withdynein and induces marked accumulation of ubiquitinated pro-teins. It synergistically augments bortezomib-induced cytotoxicityby c-Jun NH2-terminal kinase兾caspase activation. Importantly, thiscombination also induces significant cytotoxicity in plasma cellsisolated from MM patient bone marrow. Finally, adherence of MMcells to bone marrow stromal cells confers growth and resistanceto conventional treatments; in contrast, the combination of tubacinand bortezomib triggers toxicity even in adherent MM cells. Ourstudies therefore demonstrate that tubacin combined with bort-ezomib mediates significant anti-MM activity, providing the frame-work for clinical evaluation of combined therapy to improvepatient outcome in MM.histone deacetylaseMultiple myeloma (MM) is a plasma cell malignancy thatremains incurable despite conventional treatment (1) orhigh-dose therapy and stem cell transplant ation (2). Novelagents have recently been developed that t arget not only MMcells, but also the bone marrow (BM) microenvironment, andcan overcome conventional drug resistance (3). For example, theproteasome inhibitor bortezomib (formally PS-341) inducessign ificant antitumor activity in human MM cell lines and freshlyisolated patient MM cells (3–11) associated with c-Jun NH2-ter minal kinase (JNK) (also known as stress-activated proteink inase) and caspase activation, followed by apoptosis (4, 5, 9).Bortezomib also inhibits adherence of MM cells to BM stromalcells (BMSCs) by down-regulating adhesion molecules (inter-cellular adhesion molecule-1 and vascular cell adhesion molecule1) (12) and induces cleavage of DNA-protein kinase cataly ticsubun it, suggesting that bortezomib also inhibits DNA repair.Neither IL-6 nor adherence of MM cells to BMSCs protectsagainst bortezomib-induced apoptosis. These data further sup-port our observation that bortezomib enhances sensitivit y andcan overcome resistance in MM cells to conventional chemo-therapeutic agents, especially to DNA-damaging agents (7).Import antly, a phase II trial of bortezomib treatment of 202patients with refractory relapsed MM demonstrated 35% re-sponses, including 10% c omplete and near-complete responses(13); however, 65% of patients did not respond. However,resistance to bortezomib, to date, is not fully understood. Wehave recently demonstrated that heat shock protein (hsp)-27mediates bortezomib resist ance; conversely, inhibiting hsp-27ex pression by using hsp-27 antisense, p38 mitogen-activatedprotein k inase (MAPK) small interfering RNA (siRNA), or p38M APK inhibitor to down-regulate hsp-27 can restore MM cellsusceptibilit y to bortezomib (8, 11).Recent studies have revealed an alternative system to theproteasome for degradation of polyubiquitinated misfolded兾unfolded proteins, termed the aggresome (14, 15). Agg resomefor mation ultimately induces autophagic clearance, which ter-minates in lysosomal degradation. The aggresome pathwaytherefore likely provides a novel system for delivery of aggre-gated proteins from cy toplasm to lysosomes for degradation(16). In aggresomal protein degradation, histone deacetylase 6(HDAC6) has an essential role because it can bind both polyu-biquitinated proteins and dynein motors, thereby acting torecr uit protein cargo to dynein motors for transport to aggre-somes (17). In the present study, we hypothesized that inhibitionof both proteasomal and aggresomal protein degradation sys-tems could induce