Cancer Cell Article

A bis-Benzylidine Piperidone Targeting Ubiquitin Receptor RPN13/ADRM1 as a Therapy for Cancer

Ravi K. Anchoori,1 Balasubramanyam Karanam,2 Shiwen Peng,2 Joshua W. Wang,2 Rosie Jiang,2 Toshihiko Tanno,1 Robert Z. Orlowski,4 William Matsui,1 Ming Zhao,1 Michelle A. Rudek,1 Chien-fu Hung,2 Xiang Chen,5,6 Kylie J. Walters,5,6 and Richard B.S. Roden1,2,3,* 1Department of Oncology, The Johns Hopkins University, Baltimore, MD 21231, USA 2Department of Pathology, The Johns Hopkins University, Baltimore, MD 21231, USA 3Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD 21231, USA 4Division of Cancer Medicine, Department of Lymphoma/Myeloma, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA 5Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA 6Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccr.2013.11.001

SUMMARY

The bis-benzylidine piperidone RA190 covalently binds to cysteine 88 of ubiquitin receptor RPN13 in the 19S regulatory particle and inhibits proteasome function, triggering rapid accumulation of polyubiquitinated pro- teins. Multiple myeloma (MM) lines, even those resistant to bortezomib, were sensitive to RA190 via endo- plasmic reticulum stress-related apoptosis. RA190 stabilized targets of human papillomavirus (HPV) E6 oncoprotein, and preferentially killed HPV-transformed cells. After oral or intraperitoneal dosing of mice, RA190 distributed to plasma and major organs except the brain and inhibited proteasome function in skin and muscle. RA190 administration profoundly reduced growth of MM and ovarian cancer xenografts, and oral RA190 treatment retarded HPV16+ syngeneic mouse tumor growth, without affecting spontaneous HPV-specific CD8+ T cell responses, suggesting its therapeutic potential.

INTRODUCTION recognized by two proteasome subunits, RPN10 and RPN13, within the 19S regulatory particle (RP; Deveraux et al., 1994; Husn- degradation is exquisitely regulated within the cell to main- jak et al., 2008; Sakata et al., 2012; Schreiner et al., 2008). The RP tain protein homeostasis and eliminate misfolded or damaged recycles ubiquitin by its removal from the target protein, which is (Schwartz and Ciechanover, 2009). Targeted degrada- unfolded upon transit into the 20S core particle (CP) of the tion of regulatory proteins by the ubiquitin-proteasome system proteasome, where it is degraded. RPN13 docks into the RP via is central to many signaling cascades, including those that govern RPN2 with its ubiquitin-binding Pru domain and uses a nine helix cell proliferation, and is exploited by many infectious agents C-terminal domain (Chen et al., 2010b) to recruit UCH37 to the (Ciechanover, 1998; Ruschak et al., 2011). Typically, degradation proteasome, where it enhances its deubiquitinase activity (Hama- of a target protein is signaled by repeated covalent linkage of zaki et al., 2006; Qiu et al., 2006; Yao et al., 2006). ubiquitin, as mediated by an E3 ubiquitin ligase. Proteins marked The CP contains three catalytic subunits, b1, b2, and b5, with with ubiquitin chains, especially those linked via lysine 48, are caspase, trypsin, and chymotrypsin-like activities, respectively

Significance

A greater requirement by cancer cells for proteasome function provides a therapeutic window for bortezomib and carfilzo- mib by triggering endoplasmic reticulum stress, an unresolved unfolded protein response (UPR), and cell death. Bortezomib and carfilzomib inhibit 20S proteasome activity and are licensed for the treatment of multiple myeloma, but side effects of thrombocytopenia and neuropathy, intravenous administration, and disease resistance suggest a need for alternative inhib- itors. RPN13/ADRM1 encodes a proteasome ubiquitin receptor and is frequently amplified in ovarian and colon cancer. Bis-benzylidine piperidone, RA190, adducts cysteine 88 of RPN13, triggering rapid accumulation of high-molecular-weight polyubiquitinated proteins, UPR and apoptosis. RA190 is orally available, exhibits a promising toxicity profile, and is active against cervical and ovarian cancer and bortezomib-resistant MM cells.

Cancer Cell 24, 791–805, December 9, 2013 ª2013 Elsevier Inc. 791 Cancer Cell RPN13 Inhibitor RA190 for Cancer Therapy

(Schwartz and Ciechanover, 2009). Degradation occurs proces- studies suggested the value of two chlorine atoms on each sively via nucleophilic attack of the substrate amide bond by a phenyl moiety and thus we synthesized RA190 and RA190Ac, Thr within the b-subunit active site. Bortezomib and carfilzomib which differ in the phenylalanine and amide conjugation principally inhibit chymotrypsin-like proteolysis (Bedford et al., compared to the urea conjugation in our early generation mole- 2011). cule RA1 (Bazzaro et al., 2011). Bortezomib was approved for the treatment of relapsed multi- Using a variety of cancer cell lines, cell viability was deter- ple myeloma (MM) and mantle cell lymphoma (Bedford et al., mined with an 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5- 2011), and carfilzomib was recently approved for patients with [(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay MM progression while on or after treatment with bortezomib after 48-hr treatment with titrations of each compound (Table and an immunomodulatory agent. Their efficacy has been attrib- S1). Activity against several cell lines known to be sensitive to uted to the induction of endoplasmic reticulum stress and an proteasome inhibitors was observed, including those derived unfolded protein response (UPR) upon accumulation of protein from cervical cancer (HeLa, CaSki, and SiHa), MM (ANBL6, aggregates, inhibition of nuclear factor-kappa light chain MM.1S, NCI-H929, U266, and RPMI-8226), colon cancer enhancer of activated B cells and tumor necrosis factor alpha (HCT116), and ovarian cancer (ES2 and OVCAR3; Table S1; Fig- signaling, increases in reactive oxygen species, and stabilization ures 1A and 1B). Because RA190 consistently exhibited the of tumor suppressors such as p53 (Chen et al., 2010a; Scheffner most potent antiproliferative effects against MM lines (half- et al., 1993). In human papillomavirus (HPV)-related cancers, the maximal inhibitory concentration [IC50] % 0.1 mM) and HPV- E6 viral oncoprotein drives transformation by coopting the transformed cells (IC50 % 0.3 mM), it was the focus for further À cellular E3 ubiquitin ligase E6AP to polyubiquitinate targets, analysis. RA190 was less efficacious against HPV (IC50 > notably p53 and PDZ-family members, for rapid degradation 5 mM for HT3 and C33A, Table S1) than HPV+ (HeLa, CaSki, (Moody and Laimins, 2010), and HPV-transformed cells are pref- and SiHa) cervical cancer cell lines. Likewise, the HPV16- erentially sensitive to bortezomib (Lin et al., 2009; Schwartz and immortalized oral keratinocyte line HOK-16B was more sensi- Ciechanover, 2009). tive to RA190 than either HaCaT cells (HPVÀ, spontaneously Bortezomib induces thrombocytopenia and neuropathy asso- immortalized keratinocytes) or FaDu (HPVÀ head and neck can- ciated with off-target activity (Arastu-Kapur et al., 2011), and the cer cells). emergence of disease resistance remains a clinically significant MM cells may acquire bortezomib resistance by several problem (Ruschak et al., 2011). Carfilzomib has similar issues. mechanisms (Kuhn et al., 2012; Ri et al., 2010). We tested Additional orally delivered drugs targeting distinct activities of RA190 potency against two MM cell lines that developed resis- the proteasome are needed to increase dosing flexibility, over- tance after extended culture in bortezomib (Kuhn et al., 2012), come resistance, and reduce side effects (Chauhan et al., 2005). and it was equally efficacious against both the bortezomib-resis- tant derivative lines and the parental lines, consistent with a RESULTS mode of action distinct from bortezomib (Figure 1A). Further- more, the combination of RA190 and bortezomib provides a Activity of bis-Benzylidine Piperidone Derivatives synergistic effect on the loss of cervical cancer cell viability against Cancer Cells (Figure S1A). We recently described a series of 1,3-diphenylpropen-1-one (chalcone)-based derivatives bearing a variety of amino acid RA190 Triggers Accumulation of Polyubiquitinated substitutions on the amino group of the 4-piperidone, including Proteins RA1, which inhibits ubiquitin-mediated protein degradation and Because compounds related to RA190 are proteasome inhibi- preferentially kills cervical cancer cells (Bazzaro et al., 2011). tors (Anchoori et al., 2011), we examined its impact on the levels The a,b ketone represents the minimal molecular determinant of polyubiquitinated proteins in HeLa and CaSki cells by anti- for inhibition, which occurs without affecting CP activity (An- K48-linked ubiquitin immunoblot analysis. RA190 treatment of choori et al., 2011). To improve activity and solubility, and to HeLa cells (4 hr) dramatically increased the levels of K48-linked probe their pharmacophore, we generated a series of derivatives polyubiquitinated proteins similarly to bortezomib (Figure 1C) with varying substituents in the aromatic ring to modulate the and in a dose-dependent manner. However, accumulated K48 acceptor character of the enone system including o- and polyubiquitinated proteins observed following exposure to p-halogens, and different amino acids at the amine functionality RA190 exhibited a higher molecular weight than that seen in bor- of 4-piperidone (see Supplemental Experimental Procedures tezomib-treated cells (Figure 1C) and occurred more rapidly (Fig- available online). Because our previous work suggested its ure S1B). These results suggest that the toxicity exerted by importance for proteasome inhibitory activity (Bazzaro et al., RA190 for cervical cancer cells is associated with a prior accu- 2011), most RA compounds incorporated phenylalanine and/or mulation of high-molecular-weight polyubiquitinated proteins substituted phenylalanine. To overcome the poor solubility and and occurs by a mechanism distinct to bortezomib. Indeed, un- pharmacokinetics of our previous generation molecules, we em- like bortezomib, RA190 does not inhibit CP chymotryptic, tryptic, ployed an amide in lieu of a urea linkage between amino acids and PGPH activities (Figure S1C). Inhibition of RP deubiquitinase and 4-piperidone. We synthesized RA166 and RA201 with chlo- activity can produce a similar accumulation of high-molecular- rine and fluorine at the ortho position of the aromatic rings and weight polyubiquitinated protein as seen for RA190 (Koulich phenylalanine attached to the 4-piperidone, and additional com- et al., 2008). However, the degradation of Ub-AMC by either pu- pounds in which histidine or tyrosine (RA213) are substituted for rified recombinant UCH37 (with or without the addition of phenylalanine, as well as RA181 with no substituent. Earlier RPN13) or purified RP was minimally affected by RA190,

792 Cancer Cell 24, 791–805, December 9, 2013 ª2013 Elsevier Inc. Cancer Cell RPN13 Inhibitor RA190 for Cancer Therapy

Figure 1. RA190 Causes a Toxic Accumulation of Polyubiquitinated Proteins

(A) RPMI-8226, ANBL6, and their respective in vitro selected bortezomib-resistant cell lines RPMI-8226-V10R and ANBL6-V10R were treated with the indicated compounds for 48 hr, and percent cell viability was measured by XTT assay and presented as mean ± SD. (B) The indicated MM cell lines was treated with the indicated compounds for 48 hr, and cell viability was measured by XTT assay and presented as mean ± SD. (C) HeLa cells were treated with RA190 (190), RA190ME (190ME), or bortezomib (Bz) for 4 hr (left) or 12 hr (right) at the concentrations indicated and their lysates were probed with anti-K48-linked ubiquitin antibody by immunoblot. An immunoblot of b-tubulin was used to confirm equivalent protein loading. (D) HeLa cells were transiently transfected with either tetraubiquitin-fused firefly luciferase (4UbFL) or FL plasmids. After 48 hr, the transfected cells were treated with various concentrations of the indicated compounds for 4 hr, and luciferase activity was measured. Data are shown as a ratio of 4UbFL to FL and expressed as a fold change ± SD compared to untreated cells. See also Figure S1 and Table S1. suggesting that it does not inhibit the RP deubiquitinases (Fig- providing a validated approach to assess proteasome function ures S1D and S1E). in live cells (Luker et al., 2003). Two days after transfection with either 4UbFL or FL expression vectors, HeLa cells were treated RA190 Stabilizes Tetraubiquitin-Fused Luciferin for 4 hr with bortezomib and luciferase-driven bioluminescence A tetraubiquitin-firefly luciferase (4UbFL) reporter, in which four was dramatically increased in cells expressing 4UbFL but not copies of ubiquitin (G76V) are genetically fused to the N terminus FL (Figure 1D). Thus, the ratio of bioluminescence observed in of firefly luciferase (FL), is rapidly degraded by the proteasome cells transfected with 4UbFL versus FL was used to assess the whereas FL alone has a much longer half-life. Importantly, treat- proteasome inhibition by the active compounds in the series, ment of cells expressing 4UbFL with proteasome inhibitors re- revealing RA190 as more potent than others, including RA166 sults in its stabilization and an increase in luciferase activity, and RA201 (Figure 1D).

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carboxyl moiety to oxime (RA190O) dramatically reduced activity in cell killing and functional assays (Figure S1F; see Figure S1G for chemical structures). Furthermore, washout studies are also consistent with RA190 acting as an irreversible inhibitor (Figure S1H). To identify its cellular target, biotin was covalently linked to RA190 via its free amine functionality (RA190B). Biotinylation of RA190 did not affect its potency in cell killing and functional as- says (Figures S2A–S2C). HeLa cell lysate was treated with RA190B, subjected to SDS-PAGE, and probed with streptavi- din-peroxidase following protein transfer to a polyvinylidene di- fluoride (PVDF) membrane. The streptavidin-peroxidase bound to biotinylated cellular proteins, but a striking new band at 42 kDa appeared in RA190B-treated samples (Figure 2A). Impor- tantly, RA190 was competitive for this interaction, suggesting specificity. RA190B bound to the 42 kDa protein in a purified RP preparation, and the interaction was similarly competed by RA190 (Figure 2B). Within the RP there are four proteins (intrinsic ubiquitin recep- tors RPN10 and RPN13, deubiquitinase UCH37, and shuttling ubiquitin receptor HHR23B) with a molecular weight similar (37–45 kDa) to the cellular target of RA190. These proteins were overexpressed separately in 293TT cells, and the lysates labeled with RA190B and probed with streptavidin-peroxidase. Enhanced labeling of a specific band with RA190B was observed in cell lysates overexpressing RPN13, but not the others (Figure 2C). To eliminate the possibility that another RP component was required for RA190B interaction, RPN13 or an irrelevant protein (L2) was overexpressed in bacteria. Cell lysate harvested from bacteria either with or without isopropylthio-b-galactoside in- duction of ectopic protein expression was labeled with RA190B and probed by blotting with streptavidin-peroxidase. Figure 2. RA190 Binds Covalently to RPN13 RA190B reacted strongly with a 42 kDa protein only in lysates (A) HeLa cell lysate was labeled with RA190B alone or in the presence of of bacteria expressing RPN13 (Figure 2D). Furthermore, this competitor RA190 at the indicated concentrations for 1 hr at 4C. After interaction was competed with by unlabeled RA190 and the labeling, equal aliquots were boiled in SDS sample buffer, separated by SDS- presence of RPN13 was confirmed by western blot with PAGE, transferred to a PVDF membrane, and probed with streptavidin- RPN13-specific monoclonal antibody (Figures 2E and 2F). These peroxidase for the recognition of biotinylated proteins. (B) As for (A) except that the labeling was performed with 500 ng purified 19S findings suggest that RA190 covalently binds directly to RPN13. proteasome, 10 mM RA190B (190B), and 100 mM RA190 (190). However, glycerol gradient separation studies indicate that (C) 293TT cells were transfected with plasmid expressing RPN13, RPN10, RA190 does not displace RPN13 from proteasome (Figure S2D). UCH37 or HHR23B, or luciferase as a control. At 48 hr posttransfection, cell lysates were labeled with RA190B (20 mM), separated by SDS-PAGE, trans- RA190 Ligates to RPN13 Pru Domain ferred to a PVDF membrane, and probed with streptavidin-peroxidase. RPN13 contains an N-terminal Pru (pleckstrin-like receptor for (D) Lysates of IPTG induced and uninduced bacteria transduced with ubiquitin) domain that binds to ubiquitin (Husnjak et al., 2008; expression vector for RPN13 or L2 were labeled with RA190B (20 mM), separated by SDS-PAGE, transferred to a PVDF membrane, and probed with Schreiner et al., 2008) and the RP (Gandhi et al., 2006; Hamazaki streptavidin-peroxidase. et al., 2006; Ito et al., 2001; Schreiner et al., 2008), and a C-ter- (E) Competition for labeling of RPN13 expressed in bacterial cell lysate with minal domain that recruits UCH37 to the proteasome (Hamazaki 200 mM RA190 (190), 20 mM RA190B (190B), or both. et al., 2006; Qiu et al., 2006; Yao et al., 2006). We used nuclear (F) The membrane from (D) was stripped and reprobed with RPN13 antibody. magnetic resonance (NMR) to determine whether RA190 targets See also Figure S2. a specific RPN13 functional domain. Unlabeled RA190 was incubated overnight at 10-fold molar excess and 4C with RA190 Covalently Binds to the RP Subunit RPN13 15N-labeled RPN13; 15N-labeled RPN13 (1–150), which includes Removal of the olefin bond from RA190 by treatment with b-mer- its Pru domain; or 15N-labeled RPN13 (253–407), which includes captoethanol (forming RA190ME) significantly reduced its its UCH37-binding domain. Unreacted RA190 was removed by potency in both cell killing and polyubiquitin accumulation as- dialysis and heteronuclear single quantum coherence (HSQC) says (Figure 1C; Figure S1F), suggesting it is a Michael acceptor spectra were acquired to evaluate the effect of RA190 on the with olefin bonds that are susceptible to nucleophilic attack. three RPN13 constructs. The spectrum acquired on full-length Elimination of the enone moiety (RA190R) or conversion of the RPN13 after incubation with RA190 exhibited significant signal

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loss for specific amino acids in its Pru domain, but not its domain abuts the Pru domain (Chen et al., 2010b), and the re- UCH37-binding domain (Figures 3A and S3A). Moreover, gion targeted by RA190 is within the interdomain contact sur- RA190 significantly affected NMR spectra recorded on the face (Figure S4D). An NMR spectrum from a mixture of RPN13 Pru domain (Figures 3B and S3B), but not RPN13 (253– RA190-modified and unmodified RPN13 provides evidence 407; Figure S3C). These data indicate that RA190 interacts that the RPN13 interdomain interactions are abrogated by with the RPN13 Pru domain. RA190 (Figure S4E). b-mercaptoethanol prevented the effect of RA190 on the 15N-labeled RPN13 Pru domain (Figure S3D). To test whether RA190 Causes Endoplasmic Reticulum Stress RA190 interacts covalently with the RPN13 Pru domain, we The accumulation of unfolded proteins in the endoplasmic retic- compared liquid chromatography high-resolution mass spectra ulum triggers the UPR, which attempts to restore homeostasis acquired on our 15N-labeled RPN13 samples with and without by translation attenuation and upregulation of chaperones. How- RA190 incubation (Figure 3C). Unmodified RPN13 was present ever when the UPR fails to restore homeostasis, it promotes in each of the samples exposed to RA190 along with an addi- apoptosis. Proteasome inhibition creates endoplasmic reticulum tional species at a molecular weight shifted by 561.4 Da for the stress by blocking the removal of misfolded proteins, enhancing full-length protein (Figure 3C, upper right panel) and 559.8 Da IRE1a-mediated splicing of the mRNA coding for active tran- for the RPN13 Pru domain (Figure 3C, lower left panel); the ex- scription factor XBP1, one of the main UPR branches, and pected molecular weight shift caused by RA190 attachment is elevating expression of activating transcription factor-4 (ATF-4) 561.31 Da. Thus, one RA190 molecule adducted to the RPN13 and C/EBP-homologous protein (CHOP)-10, both transcription Pru domain. factors driving apoptosis. Treatment of MM.1S and HeLa cells with RA190 caused upregulation of ATF-4 protein levels and RA190 Adducts to RPN13 C88 CHOP-10 and XBP1s mRNA levels prior to apoptosis (Figures We used site-directed mutagenesis and NMR to determine the 5A–5E). Bax is a critical element in the induction of apoptosis site of RA190 ligation. RA190 no longer interacts with RPN13 by UPR, and RA190 treatment of MM.1S cells significantly Pru domain when its four native cysteines are replaced with elevated Bax protein levels (Figure 5F). Conversely, UPR- alanine (RPN13 Pru C/A). 15N-labeled RPN13 Pru C/A incubated induced cell death is typically p53-independent. Isogenic with RA190 exhibited no changes in HSQC experiments HCT116 cells in which both alleles of wild-type TP53 had been compared to RPN13 Pru C/A alone (Figure S3E). eliminated by homologous recombination, or HCT-116 cells Inspection of RPN13 NMR spectra acquired with and without into which mutant p53 was introduced, exhibited similar sensi- RA190 revealed that C88 was significantly affected (Figures 3A tivity to bortezomib and RA190 as the parental line (Figure S5A), and 3B), and we tested whether this cysteine is required for consistent with p53-independent cell death in response to an un- the interaction. RA190 did not cause changes to NMR spectra resolved UPR. recorded on 15N-labeled RPN13 Pru C88A (Figure S3F), and only one species of the correct molecular weight for RPN13 RA190 Elevates p53 and p53-Regulated Pru C88A was observed by mass spectrometry (MS; Figure S3G). in Cervical Cancer Cells By contrast, RPN13 Pru C60,80,121A, in which only C88 was pre- E6-mediated degradation of p53 and other targets via the pro- served, exhibited significant spectral changes upon incubation teasome is a hallmark of high-risk HPV types and critical to trans- with RA190 (Figure 3D) and a molecular weight shift of formation (Chen et al., 2010a; Scheffner et al., 1993), suggesting 559.9 Da by MS (Figure 3C, lower right). These data indicate that stabilization of E6 targets and consequent pro-apoptotic that RA190 ligates to RPN13 C88. signaling may account for the greater sensitivity of HPV-trans- formed cells to RA190. However, combination of RA190 and bor- Model of RPN13 Pru Adducted with RA190 tezomib was synergistic (CI = 0.4) for killing of HeLa cells in vitro, We quantified the RA190 effect on RPN13 Pru by integrating the suggesting distinct targets (Figure S1A). We therefore investi- NMR signal of spectra acquired on free and RA190-adducted gated whether RA190, like bortezomib, could restore the levels RPN13 Pru (Figures 3B and S3B). The ratio of these values of wild-type p53 in HPV-transformed cervical cancer cells (Lin was plotted for each backbone (Figure 4A) and side chain (Fig- et al., 2009). Treatment of HeLa, CaSki, and SiHa cells for ure 4B) amide group. This analysis highlighted RPN13 Pru amino 24 hr with RA190 elevated p53 levels as with bortezomib (Fig- groups that are significantly affected by RA190 and was used to ure 5G, top panel). Rapid and time-dependent recovery of p53 generate model structures of RPN13 Pru adducted with RA190 levels was observed within 2 hr of RA190 treatment, reaching in HADDOCK (Dominguez et al., 2003). The amino acids most maximal levels by 6 hr (Figure 5G, bottom panel). p53-targets affected by RA190 map to a region opposite RPN13 ubiquitin- p21 and Puma (Vousden and Lu, 2002), including their ubiquiti- binding loops that includes C88. Despite its small size and cova- nated forms, were also increased in a time-dependent manner lent attachment, RA190 addition to RPN13 led to signal loss (Figures 5H and S5B). Treatment of HeLa cells with RA190 or (Figures 3A and 3B), which suggests that it may adopt multiple bortezomib increased levels of pro-apoptotic factors targeted configurations when adducted to RPN13. Our structure calcula- by E6 for degradation (Moody and Laimins, 2010), notably Bax tions yielded four major RA190 conformations when adducted to and Bak (Figure 5H), and the tumor suppressor hDLG-1 in RPN13 C88 Sg (Supplemental Experimental Procedures; Fig- HeLa (Figure 5H) and CaSki cells (not shown). Thus RA190 ures 4C and S4A–S4C). For depiction, we selected the lowest stabilizes multiple E6 targets (Moody and Laimins, 2010), energy structure (Figure 4C), which was also most consistent including pro-apoptotic and tumor suppressor proteins, through with our NMR data (Figures 4A and 4B). Rpn13 UCH37-binding proteasome inhibition.

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(legend on next page)

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RA190 Induces Apoptosis and Display of HSP90 on the and RA190-treated animals. Similar studies performed in mice Surface of Dying Tumor Cells bearing TC-1 tumors and treated with either RA190 or bortezomib The rapid upregulation of pro-apoptotic factors and loss of also suggest that RA190 has a promising safety profile (Table S3). viability upon RA190 treatment may reflect apoptosis. Annexin- V flow cytometric measurements made 12 hr after treating MM Proteasome Inhibition In Vivo by RA190 and HeLa cells indicate that RA190 and bortezomib trigger Naked 4UbFL reporter plasmid DNA was delivered either by extensive apoptosis (Figures 6A and 6B). Activation of ICE family gun into the skin or in vivo electroporation in muscle (Fig- members such as caspase-3 and -7 results in cleavage of poly ure 7), and the level of luciferase in the mouse was assessed ADP ribose polymerase (PARP) to 85 kDa and 25 kDa fragments before and after drug treatment by bioluminescence imaging. and drives apoptosis. The ability of RA190 to trigger caspase-3 To examine whether RA190 was able to inhibit proteasome func- activity (Figures 6C and 6D) and PARP cleavage (Figures 6E tion in vivo, mice (ten per group) were injected i.p. with RA190 and 6F) in MM lines and cervical cancer is consistent with induc- (40 mg/kg), bortezomib (1.5 mg/kg), or vehicle 1 day after intra tion of apoptotic cell death. muscular (i.m.) electroporation with 4UbFL reporter plasmid (Fig- Bortezomib treatment of MM cells elevates expression and ure 7A). At 4 hr after treatment, mice treated with RA190 ex- surface exposure of HSP90 in association with ‘‘immunogenic’’ hibited 4-fold higher levels of bioluminescence compared to cell death (Spisek and Dhodapkar, 2007). RA190 treatment of vehicle alone, whereas bortezomib elevated it only 2-fold. One HeLa cells for 24 hr produced cell-surface HSP90 on 54.2% of day posttreatment, both RA190 and bortezomib-treated animals cells, whereas 12.8% of bortezomib-treated cells and only 3% exhibited a similar 4-fold increase in bioluminescence, suggest- of control cells displayed HSP90, demonstrating that this phe- ing continued inhibition of proteasome function (Figure 7A). The nomenon is not restricted to MM cells. Notably, cisplatin did level of bioluminescence observed in the vehicle-treated mice not induce surface display of HSP90, suggesting that not all decreased steadily over 48 hr, suggesting a slow loss of trans- types of killing affect cells in this way (Figure 6G). A time course fected cells. At 48 hr after RA190 treatment, the mice still show experiment in HeLa cells demonstrated initiation of surface a significant increase in bioluminescence despite the short HSP90 by 6 hr and strong upregulation by 12 hr following half-life of RA190 in blood (Figure 7A), possibly reflecting slow RA190 treatment (Figure 6G), indicating a similar time course regeneration of new or the continued presence of to Annexin V-staining (not shown), and more rapid onset than RA190 or active metabolites in tissues. Because papillomavirus that for bortezomib treatment. infections are typically restricted to skin and mucosa, the 4UbFL reporter plasmid was delivered into the skin by gene gun (Best Pharmacokinetics and Safety of RA190 et al., 2009; Trimble et al., 2003). Similar stabilization of the Upon formulation in 20% (w/v) b-hydroxyisopropyl-cyclodextrin 4UbFL reporter was observed, albeit of shorter duration, sug- in water, mice were treated with various single oral (p.o.) and gesting that both RA190 and bortezomib were also active in intraperitoneal (i.p.) doses of RA190. With i.p. administration of skin (Figure 7B).

10 mg/kg RA190, peak plasma levels (Cmax) were observed in Oral administration of RA190 increased the bioluminescence 2 hr, and then declined multi-exponentially with distribution produced by mice transduced with 4UbFL plasmid either i.m.

(T1/2, a) and terminal (T1/2, b) half-lives of 4.2 and 25.5 hr, respec- by electroporation (Figure 7C) or in skin via gene gun (Figure 7D). tively (Figure S6). After p.o. administration of 20 mg/kg, RA190 Again, the elevation of bioluminescence in skin was of shorter plasma concentrations rose rapidly during the first hour and duration than that for the i.m. studies, possibly reflecting a then declined exponentially with a T1/2, b of 2.6 hr. Based on more rapid turnover of skin cells, as compared to transduced the RA190 AUC values, the bioavailability of RA190 delivered muscle cells of electroporated animals. Topical administration p.o. relative to i.p. was 7.2%. RA190 was detected in kidney of RA190 at 4% in Cremophor-EL also stabilized the 4UbFL re- (0.61 mg/g), liver (0.57 mg/g), lung (0.66 mg/g), and spleen porter in skin (Figure 7E). (0.59 mg/g), but not brain 48 hr after the mice were given a single i.p. dose of 10 mg/kg RA190. RA190 Treatment Inhibits Tumor Growth in Mice To examine its safety/toxicity profile, mice were given three NOG (NOD/Shi-scid/IL-2Rgnull) mice carrying an NCI-H929 MM doses of 40 mg/kg RA190 p.o. or vehicle alone every third day line that expresses luciferase received RA190 or vehicle 20 mg/ and euthanized on day 12. Blood was harvested and blood chem- kg/day i.p. daily for 7 days. Prior to and at the end of the treat- istry and hematologic analyses performed (Table S2). No signifi- ment, mice were imaged for bioluminescence. RA190 showed cant difference between the panels of tests was observed potent antitumor activity, even with a 3-day break in the middle between the vehicle and RA190-treated groups, except for a of treatment (Figures 8A and 8B). We have previously described small reduction in triglycerides. The histopathology of the lungs, the susceptibility of human ovarian cancer lines to proteasome kidney, spleen, and liver was unremarkable in both the vehicle inhibition. Nude mice carrying ES2-luciferase tumor i.p. were

Figure 3. RA190 Interacts with the RPN13 Pru Domain (A and B) Enlarged regions of HSQC spectra for 15N-labeled human RPN13 (A, black) or RPN13 Pru domain (B, black) and after RA190 incubation (A and B, orange). Labels for signals affected by RA190 are italicized and bold. (C) LC-MS experiments for RPN13 Pru domain (top, left) and RA190-exposed RPN13 (top, right), RPN13 Pru domain (bottom, left), and RPN13 Pru C60,80,121A (bottom, right). The samples used for these experiments are identical to the ones used for the corresponding NMR experiments (A, B, and D). (D) HSQC spectra of 15N-labeled RPN13 Pru C60,80,121A (black) and after RA190 incubation (orange). See also Figure S3.

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treated with RA190 or vehicle for 14 days and bioluminescence was imaged weekly. Treatment with RA190 significantly inhibited the ES2 tumor growth (Figures 8C and 8D). C57BL6 mice carrying HPV16 E6 and E7-transformed and syngeneic tumor TC-1, which spontaneously induces E7-spe- cific CD8+ T cell responses, were treated with either RA190 (i.p., 20 mg/kg) or vehicle daily for 7 days and the tumors were harvested. Tumor lysates probed by western blot for polyubiqui- tinated proteins were dramatically elevated in the RA190 treat- ment group, suggesting RA190 can access solid tumor to block proteasome function (Figure S7A). Oral administration to tumor- bearing mice of RA190 (40 mg/kg every third day) significantly inhibited (p < 0.001) TC-1 tumor growth as compared to treat- ment with vehicle alone (Figure 8E). The final tumor weights after the 14-day treatment period were 1,873 ± 180 mg in vehicle- treated mice and 727 ± 101 mg in the RA190-treated mice (p < 0.001). Weight gain and the spontaneous E7-specific CD8+ T cell response did not differ significantly between the vehicle- and RA190-treated mice (Figure 8F; Figure S7B).

DISCUSSION

Bortezomib improves clinical outcomes for patients with MM or mantle cell lymphoma, but a significant fraction of tumors are resistant or become resistant. This has driven efforts to develop second-generation pharmaceuticals with a distinct mechanism of proteasome inhibition (Ruschak et al., 2011). The empty p-orbital on boron of bortezomib accepts electrons from the hydroxyl group of a key active site threonine to form a pseudoco- valent bond. Several epoxide or peptide aldehyde proteasome inhibitors bind to the active site covalently, but the reactivity of the aldehyde and epoxide groups toward nonspecific targets and consequent short half-life in vivo are important pharmaco- logic limitations (Ruschak et al., 2011). Covalent inhibitors typi- cally contain Michael acceptors that can be more readily modi- fied structurally to react selectively to target nucleophiles than highly reactive alkylating agents. The bis-benzylidine piperidone RA190 is a Michael acceptor that we have engineered over several iterations and it exhibits specificity against proteasome ubiquitin receptor RPN13. We show that RA190 adducts to RPN13 cysteine 88 in the ubiquitin- and proteasome-binding Pru domain, rather than the UCH37-interaction domain. Furthermore, we have implicated residues surrounding C88 as interacting with RA190, and found evidence of conformational freedom in the docking site. Although C88 and the surrounding interaction surface of RPN13 have been implicated in proteasome binding (Chen et al., 2010b; Schreiner et al., 2008), we were unable to detect RPN13 displacement from proteasome by RA190. RPN13 binds sub-stoichiometrically to proteasome (Sakata et al., 2012) and exists free of proteasome in cells (Hamazaki et al., 2006). In this free form, its Pru and UCH37-binding domains interact, Figure 4. A Surface Opposite RPN13 Ubiquitin-Binding Loops Is which restricts accessibility to the ubiquitin-binding loops and Implicated in RA190 Binding reduces affinity of RPN13 for ubiquitin 26-fold (Chen et al., (A and B) Normalized peak intensity attenuation (D) of RPN13 Pru domain backbone (A) and side chain (B) amide groups upon binding RA190. The dashed line indicates one SD above average. Unassigned, overlapping, or amino acids most affected by RA190 are highlighted in darkest red (see scale proline groups are excluded from this analysis and indicated (*). bar for D from A). RA190 carbon, nitrogen, oxygen, and chlorine atoms are (C) The lowest energy modeled structure for human RPN13 PruRA190 with colored light blue, indigo, red, and green, respectively. RPN13 Pru domain colored according to the data in (A) and (B), such that See also Figure S4.

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Figure 5. RA190 Upregulates UPR and Targets of HPV E6 (A) Immunoblot analysis for ATF-4 and actin in MM.1S cells either untreated (C), or treated with RA190 (190) or bortezomib (Bz) for the indicated times. (B) Immunoblot analysis for ATF-4 in HeLa cells either untreated (C), or treated with 1 mM of RA190 (190) or 1 mM bortezomib (Bz) for 6 hr. (C–E) CHOP-10 (C and D) and XBP1s (E) mRNA levels were assessed by quantitative RT-PCR and normalized to GAPDH expression after treating HeLa cells with 1 mM RA190 (190) or 1 mM bortezomib (Bz). Results are expressed as fold change over control-treated cells and represent the average and SD of three inde- pendent experiments. (F) Immunoblot analysis for Bax protein levels in MM.1S cells treated as for (A). (G) Immunoblot analysis for p53 and b-tubulin in the indicated cell line either untreated (C) or treated with 1 mM RA190 (190) or Bortezomib (Bz) for 24 hr (top panel) or for the indicated times (bottom panel). (H) After treatment with 1 mM RA190 (190) or 1 mM bortezomib (Bz), HeLa cell lysates were analyzed by immunoblot for p21, Puma, Bax, Bak, and hDLG-1 at the time points indicated. See also Figure S5.

2010b). Docking into proteasome appears to abrogate RPN13 therefore promote more promiscuous interactions between interdomain interaction and thus promote ubiquitin binding ubiquitinated proteins and extra-proteasomal RPN13 as well (Chen et al., 2010b). Our results provide evidence that RPN13 in- as interfere with substrate exchange to factors downstream of terdomain interactions are abrogated by RA190, which could RPN13. Future experiments are needed to test this model.

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Figure 6. RA190 Triggers Apoptosis and Cell Surface Presentation of HSP90 (A and B) HeLa cells treated with 1 mM RA190 (190) or botezomib (Bz) for 12 hr (A) or MM.1S cells treated with 0.5 mM RA190 or bortezomib for 6 hr (B) were analyzed by flow cytometry after staining with fluorescein-labeled Annexin-V. (C) HeLa cells treated as in (A) were analyzed by flow cytometry after staining for active caspase-3 and the percent of positive cells ± SD was determined. (D) A representative flow cytometry analysis of MM.1S cells treated as in (B) and stained for active caspase-3. (E) Lysates of HeLa cells either untreated (C) or treated with 1 mM RA190 (190) or bortezomib (Bz) after the periods of treatment indicated were immunobloted with PARP-specific antibody. (F) MM.1S cells were untreated (C) or treated with 0.5 mM RA190 or botezomib for 6 hr, then lysed for immunoblot analysis with PARP-specific antibody. (G) HeLa cells were treated with 1 mM RA190, botezomib, or cisplatin and stained at the indicated time points for cell surface HSP90 and analyzed by flow cytometry.

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Figure 7. Oral, Intraperitoneal, and Topical Administration of RA190 Inhibits Proteaso- mal Activity in Mice (A–E) Mice received 4UbFL DNA delivered either i.m. by electroporation (A and C), or intradermally via gene gun (B, D, and E). The transduced mice were treated with vehicle, RA190, or bortezomib (ten mice per group) respectively. After the indi- cated time points of treatment, bioluminescence was measured by injection of luciferin and imaging with an IVIS 200 (fold change ± SD, *p < 0.05, **p < 0.01). RA190 was given i.p. (A and B) or orally (C and D) at 40 mg/kg; bortezomib was given i.p. at 1.5 mg/kg dose (A and B). (E) RA190 was given 4% in Cremophor-EL topically. See also Figure S6.

tivity to RA190 compared to HPVÀ lines suggests recovery of E6 targets is a fac- tor. Indeed, stabilization of p53 is associ- ated with the expression of downstream mediators such as p21 and Puma, which arrest cell cycle progression and promote apoptosis. However, an siRNA knock- down study did not support a role for the recovery of wild-type p53 levels alone in bortezomib-mediated death of HeLa cells (Chen et al., 2010a). Thus other E6 targets recovered by RA190, such as Bak, Bax, and hDLG-1, may act in con- cert with p53 and UPR to trigger rapid apoptosis upon RA190 treatment. Most cell lines of HPVÀ solid tumors were less sensitive to proteasome inhibi- tors. However, some of the most rapidly growing with high rates of protein synthe- To understand the pharmacophore of RA190, we evaluated sis, including those derived from ovarian and colon cancer, were analogs while keeping the basic benzylidine piperidone skeleton similarly sensitive as HPV+ lines. This likely reflects a rapid intact. Substitution of L- for D-phenylalanine or conversion of its buildup of toxic polyubiquitinated protein aggregates seen in carboxyl moiety to oxime reduced the potency of RA190. Elimi- MM, termed aggresomes (Hideshima et al., 2005), stressing the nation of Michael acceptor properties by the addition of thiol or endoplasmic reticulum and triggering of the UPR (Bazzaro complete removal of the enone moiety nullified drug activity in et al., 2006, 2008). Interestingly, frequent amplification of cytotoxic and functional assays, demonstrating the importance RPN13 has been described in both ovarian (Fejzo et al., 2013) of the enone moiety. and colon cancer (Chen et al., 2009), suggesting a dependence

Although the IC50 of RA190 is higher than that of the licensed upon the proteasome that may confer sensitivity to RA190 and proteasome inhibitors, oral administration was effective. When other proteasome inhibitors. formulated in 20% (w/v) b-hydroxyisopropyl-cyclodextrin in wa- The side effects of bortezomib and carfilzomib, including neu- ter, the oral availability was only approximately 7% that of the i.p. ropathy and thrombocytopenia, remain important clinical con- delivery of RA190. Athough this suggests additional formulation cerns (Ruschak et al., 2011). Alternative proteasome inhibitors studies are required for RA190, it proved sufficient for significant with distinct mechanisms of action are potential solutions. antitumor effects and proteasomal inhibition in vivo. RA190 was RPN13 is one of two major ubiquitin receptors in the RP (Dever- also effective via topical administration at 4% in Cremophor, aux et al., 1994; Husnjak et al., 2008; Sakata et al., 2012; suggesting potential for treatment of HPV+ intraepithelial Schreiner et al., 2008), and Rpn13 KO mice are viable, suggest- neoplasia for cancer prevention. ing that RA190 may have a favorable toxicity profile (Al-Shami The mechanism of MM killing by licensed proteasome inhibi- et al., 2010). Indeed, oral RA190 treatment was well tolerated, tors via the induction of endoplasmic reticulum stress, UPR, producing no significant difference in hematologic or clinical and p53-independent apoptosis is well documented, and is chemistry parameters as compared with vehicle, and it did not also seen in response to RA190. This mechanism is potentially affect weight gain or compromise spontaneous antitumor immu- also active in HPV+ cervical cancer cells, but their relative sensi- nity, further suggesting a promising safety profile. Tumor specific

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Figure 8. RA190 Inhibits Tumor Growth In Vivo (A) NOG mice carrying NCI-H929-GFP-luc human tumor cells were treated once per day with 20 mg/kg RA190 (i.p.) or vehicle alone (n = 5) on the days indicated. The mice were imaged before and at the end of the treatment for bioluminescence levels. (B) Representative bioluminescence images of mice in (A) before (top panel) and after (bottom panel) treatment. (C) Percentage change of bioluminescence ± SD in nude mice bearing ES2-luciferase tumor cells (tumor cells injected i.p.) receiving 10 mg/kg RA190 (i.p.) or vehicle alone (n = 8) every day. Prior to and 7 and 14 days after initiation of treatment, the mice were imaged for luciferase activity. (D) Representative bioluminescence images of mice in (C) before (upper panel) and after (lower panel) treatment. (E) Mice bearing palpable TC-1 tumors received oral treatment with 40 mg/kg RA190 or vehicle alone (n = 8 per group) every third day. The mean tumor volumes are plotted ± SD and significant differences indicated (*p < 0.05, **p < 0.01). (F) After 2 weeks of treatment, the spleens were harvested. The mean number ± SD of IFNg+ CD8+ T cells per 3 3 105 splenocytes elicited with or without E7 peptide stimulation (E7pep) was determined by flow cytometry and plotted. See also Figure S7 and Tables S2 and S3.

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cellular immunity is associated with better clinical outcomes, but Western Blots most chemotherapies are immunosuppressive. That the sponta- Antibodies were obtained from commercial sources: anti-ubiquitin, p53 neous E7-specific CD8+ T cell response to TC-1 tumor was not (FL393), p21(WAF1), hDLG-1(2D11), Puma, Bak, Bax, and UCH37 (Cell Signaling Technology); anti-I-kB-a (C-15), and ATF-4 (SC-200; Santa Cruz compromised by RA190 therapy is important given the potential Biotechnology); anti-PARP (BD PharMingen); anti-HSP90 (Stressgen); antitu- + for such responses to help control HPV disease. Indeed, it may bulin (Sigma); anti-Actin, RPN13, and RPN2 (Sigma-Aldrich); anti-RPN10 therefore be possible to combine treatment of cervical cancer and RPN1 (Thermo Scientific); streptavidin Dyna beads and HRP-streptavi- with RA190 and therapeutic vaccines targeting HPV E6 and/or din (Invitrogen); or peroxidase-linked anti-mouse or rabbit IgG (GE Health- E7 (Welters et al., 2008). care UK), and utilized at the concentration recommended by the Clinical resistance to bortezomib often develops, and this manufacturer. has driven further efforts to generate new proteasome inhibi- tors with distinct chemistry and proteasome targets (Ruschak Enzymatic Assays Reaction mixtures contained 500 nM of the purified 19S RP (R&D Systems) in et al., 2011). While carfilzomib has a distinct chemistry, it 19S enzyme assay buffer and 1 mM Ub-AMC. Release of free AMC by the also targets PSMB5, and resistance has been associated enzyme reaction was determined by measuring its fluorescence at 380 nm with PSMB5 mutation and/or overexpression. RA190 was syn- (excitation) and 440 nm (emission). The 20S proteolytic activity assays were ergistic with bortezomib for killing of HeLa cells in vitro, sug- performed as described elsewhere (Bazzaro et al., 2011). gesting different targets. With a distinct chemistry and target, RA190 can overcome at least some forms of resistance to bor- Biotin Labeling Assay 6 tezomib because its potency remained in two bortezomib- Human cells (5 3 10 HeLa or 293TT cells) were lysed using 500 ml mammalian resistant MM lines. Importantly, while RA190 induces a similar protein extraction reagent (M-PER; Pierce). Escherichia coli-expressing RPN13 or L2 (500 ml culture) were harvested by centrifgation and resuspended buildup of polyubiquitinated proteins, the molecular weight of in 500 ml BPER (Pierce). Lysates were centrifuged (10,000 3 g, 2 min, 4C) to the accumulated polyubiquitinated proteins was higher than remove cell debris. Lysate supernatant (200 ml) was pre-cleared with strepta- that for bortezomib. Accumulation of polyubiquitinated pro- vidin-Dynabeads (20 ml) for 1 hr at 4C, and incubated with compounds at 4C teins, cell surface display of HSP90, and apoptosis occurred for 1 hr. Alternatively, pure 19S RP (R&D Systems, E-366; 500 ng) in 20 mM more rapidly upon treatment with RA190 than for bortezomib, HEPES, 20 mM NaCl, 1 mM dithiothreitol, and 15% glycerol was incubated consistent with blockade of the proteasomal recognition of pol- with compounds for 1 hr at room temperature. Samples were boiled in Laemmli buffer, separated using 4%–15% SDS-PAGE, and transferred to a yubiquitinated proteins and/or trimming of the ubiquitin chains  PVDF membrane overnight at 4 C (24 V). The membrane was blocked with by the RP. 5% BSA in phosphate-buffered saline with Tween for 1 hr, washed and probed In conclusion, the ubiquitin receptor RPN13 is a promising with HRP-streptavidin (1:10,000 in PBST) for 1 hr at room temperature. After target for cancer treatment. Intriguingly, we note that RPN13 washing, the blot was developed using HyGLO chemiluminescent detection is amplified, overexpressed, and required in both ovarian (Fejzo reagent (Denville). et al., 2013) and colon cancer (Chen et al., 2009), suggesting its importance in cancer biology. Given the issues of drug resis- Recombinant RPN13 Preparation and NMR tance, route of delivery, and significant side effects of the Human RPN13 full-length, RPN13 (1–150), RPN13 (253–407), and His-tagged UCH37 were prepared as described elsewhere (Chen et al., 2010b; Schreiner licensed proteasome inhibitors, RA190 may be complementary   et al., 2008). NMR spectra were acquired at 25 Cor10C and at 850 or to, or advantageous over, targeting the 20S proteasome 900 MHz. RPN13 PruRA190 complexes were generated by HADDOCK 2.1 subunit. (Dominguez et al., 2003) and CNS (Bru¨ nger et al., 1998). A homology model of human RPN13 Pru domain was generated by Schro¨ dinger based on murine EXPERIMENTAL PROCEDURES RPN13 Pru ( entry 2R2Y) (Schreiner et al., 2008). Ambiguous interaction restraints were imposed to restrict RPN13 Pru active residues to be Expanded experimental procedures are provided in the Supplemental Exper- within 2.0 A˚ of any RA190 atom (Dominguez et al., 2003). imental Procedures. Animal Studies Cell Culture and Reagents All animal experiments were performed in accordance with protocols Cell lines were from ATCC and cultured as described elsewhere (Anchoori approved by the Animal Care and Use Committee of Johns Hopkins Univer- et al., 2011). Cell viability was assayed using CellTiter 96 AQueous One Solution sity. Nude, C57BL/6, Balb/c female mice were purchased from the National Reagent (Promega). 293TT cells were transfected with RPN13, RPN10 Cancer Institute (NCI) and NOG mice were bred in-house. DNA delivery (Origene), UCH37, and HHR23B (Addgene) expression vectors using was via a helium-driven gun (Bio-Rad) as described previously (Trimble TransIT-2020 transfection reagent per Mirus Bio’s instructions. Compounds et al., 2003) or electroporation (ElectroSquarePorator 833, BTX-2 Needle were generated using approaches described elsewhere (Anchoori et al., array 5 mm gap, Harvard apparatus) delivered as eight pulses at 106 V for 2011; Bazzaro et al., 2011). 20 ms with 200 ms intervals. Bioluminescence images were acquired for 10 min with a Xenogen IVIS 200 (Caliper; Luker et al., 2003). C57BL/6 mice 4 Quantitative PCR to Measure mRNA Levels were challenged subcutaneously with 5 3 10 TC-1 cells/mice and E7-spe- + Quantitative PCR was performed per the manufacturer’s instructions (Applied cific CD8 T cell response and tumor size were measured as described 6 Biosystems), according to the Livak method and normalized to reference gene previously (Trimble et al., 2003). Nude mice were inoculated with 1 3 10 GAPDH. ES2-luciferase cells i.p. in 100 ml PBS. At day 3, mice were imaged for basal level luciferase activity. NOG mice (five per group) were inoculated with 1 3 6 Flow Cytometry 10 NCI-H929-GFP-luc cells i.v., and imaged for basal luciferase activity after An annexin V-PE apoptosis detection kit I (BD PharMingen) was used accord- 4 weeks. ing to the manufacturer’s protocol. Cell surface HSP90 was stained with anti- HSP90 mAb (Stressgen), followed by PE-conjugated anti-mouse IgG1. The Statistical Analysis data were acquired with a FACSCalibur and analyzed using CellQuest Results are reported as mean ± SD. Statistical significance was calculated us- software. ing Prism (V.5 Graphpad) as p % 0.05.

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SUPPLEMENTAL INFORMATION (1998). Crystallography & NMR system: A new software suite for macromolec- ular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. Supplemental Information includes Supplemental Experimental Procedures, Chauhan, D., Catley, L., Li, G., Podar, K., Hideshima, T., Velankar, M., seven figures, and three tables and can be found with this article online at Mitsiades, C., Mitsiades, N., Yasui, H., Letai, A., et al. (2005). A novel orally http://dx.doi.org/10.1016/j.ccr.2013.11.001. active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib. Cancer Cell 8, 407–419. ACKNOWLEDGMENTS Chen, W., Hu, X.T., Shi, Q.L., Zhang, F.B., and He, C. (2009). Knockdown of the novel proteasome subunit Adrm1 located on the 20q13 amplicon inhibits We thank B. Vogelstein (Johns Hopkins University) for the HCT116 lines and colorectal cancer cell migration, survival and tumorigenicity. Oncol. Rep. 21, D. Piwnica-Worms (Washington University, St. Louis, MO) for the 4UbFL and 531–537. FL plasmids. Grant support was provided by National Institutes of Health ATIP Program grants P50 CA098252 and CA136472 and the M.D. Anderson Chen, S., Blank, J.L., Peters, T., Liu, X.J., Rappoli, D.M., Pickard, M.D., Menon, Cancer Center Specialized Program of Research Excellence in Multiple S., Yu, J., Driscoll, D.L., Lingaraj, T., et al. (2010a). Genome-wide siRNA screen Myeloma (P50 CA142509). The project was supported in part by the Sidney for modulators of cell death induced by proteasome inhibitor bortezomib. Kimmel Comprehensive Cancer Center Analytical Pharmacology Core at Cancer Res. 70, 4318–4326. Johns Hopkins (P30 CA006973 and UL1 RR 025005). We also thank the Chen, X., Lee, B.H., Finley, D., and Walters, K.J. (2010b). Structure of protea- HERA Foundation and the Ephraim and Wilma Shaw Roseman Foundation some ubiquitin receptor hRpn13 and its activation by the scaffolding protein for support. NMR data were acquired in the NMR facility at the University of hRpn2. Mol. Cell 38, 404–415. Minnesota, and data processing and depiction occurred in the Minnesota Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on protein death Supercomputing Institute Basic Sciences Computing Lab. We also thank P. and cell life. EMBO J. 17, 7151–7160. Villalta (University of Minnesota) for his help with the LC-MS and E. Arriaga Deveraux, Q., Ustrell, V., Pickart, C., and Rechsteiner, M. (1994). A 26 S pro- (University of Minnesota) for allowing us to use his spectrofluorometer. tease subunit that binds ubiquitin conjugates. J. Biol. Chem. 269, 7059–7061.

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