A Small-Molecule Inhibitor Targeting TRIP13 Suppresses Multiple

Published OnlineFirst November 15, 2019; DOI: 10.1158/0008-5472.CAN-18-3987

CANCER RESEARCH | TRANSLATIONAL SCIENCE

A Small-Molecule Inhibitor Targeting TRIP13 Suppresses Multiple Myeloma Progression Yingcong Wang1, Jing Huang2,BoLi3, Han Xue2, Guido Tricot4, Liangning Hu1, Zhijian Xu3, Xiaoxiang Sun5, Shuaikang Chang1, Lu Gao1, Yi Tao1, Hongwei Xu4, Yongsheng Xie1, Wenqin Xiao1, Dandan Yu1, Yuanyuan Kong1, Gege Chen1, Xi Sun1, Fulin Lian3, Naixia Zhang3, Xiaosong Wu1, Zhiyong Mao5, Fenghuang Zhan4, Weiliang Zhu3, and Jumei Shi1,6

ABSTRACT ◥ The AAA-ATPase TRIP13 drives multiple myeloma progres- The inhibitor impaired nonhomologous end joining repair and sion. Here, we present the crystal structure of wild-type human inhibited NF-kB activity. Moreover, combining DCZ0415 with TRIP13 at a resolution of 2.6 Å. A small-molecule inhibitor the multiple myeloma chemotherapeutic melphalan or the targeting TRIP13 was identified on the basis of the crystal HDAC inhibitor panobinostat induced synergistic antimyeloma structure. The inhibitor, designated DCZ0415, was confirmed activity. Therefore, targeting TRIP13 may be an effective ther- to bind TRIP13 using pull-down, nuclear magnetic resonance apeutic strategy for multiple myeloma, particularly refractory or spectroscopy, and surface plasmon resonance–binding assays. relapsed multiple myeloma. DCZ0415 induced antimyeloma activity in vitro, in vivo,andin Significance: These findings identify TRIP13 as a potentially primary cells derived from drug-resistant patients with myeloma. new therapeutic target in multiple myeloma.

Introduction plexity and clonal heterogeneity are the main reasons for cancer treatment failure in patients with multiple myeloma (4). Thus, the Multiple myeloma is characterized by clonal proliferation of malig- identification of a key driver gene for multiple myeloma may enable the nant monoclonal plasma cells in the bone marrow (1). Genomic specific targeting of these malignant cells. instability, defined by a higher rate of acquisition of genomic changes Accumulating evidence has shown that dysregulated thyroid per cell division compared with normal cells, is a prominent feature of hormone receptor–interacting protein 13 (TRIP13) protein levels multiple myeloma cells. Approximately 86,000 new patients with are operational in several tumors, including breast, liver, gastric, multiple myeloma are diagnosed worldwide each year (2). Although lung, prostate cancer, human chronic lymphocytic leukemia, and the prognosis of patients with multiple myeloma has improved with Wilmstumor(5,6).TRIP13isthemouse ortholog of pachytene the increased use of autologous stem cell transplantation and combi- checkpoint 2 (7). During mitosis, TRIP13 regulates the spindle nations of approved antimyeloma agents such as proteasome inhibi- assembly checkpoint via remodeling of its effector MAD2 from a tors (bortezomib and carfilzomib), immunomodulatory drugs (lena- “closed” (active) into an “open” (inactive) form (8). During meiosis, lidomide and pomalidomide), and mAbs (daratumumab and elotu- TRIP13 was found to regulate meiotic recombination in Saccharo- zumab), 5-year overall survival rate is only 45% (3). Genetic com- myces cerevisiae, Caenorhabditis elegans,andDrosophila (9). A recent study indicated that TRIP13 enhanced nonhomologous end joining (NHEJ) repair and induced treatment resistance via binding 1 Department of Hematology, Shanghai Tenth People's Hospital, Tongji Univer- to NHEJ proteins KU70/KU80/DNA-PKcs in head and neck sity School of Medicine, Shanghai, China. 2Shanghai Institute of Precision cancer (10). Medicine, The Ninth People's Hospital, Shanghai Jiao Tong University School fi of Medicine, Shanghai, China. 3CAS Key Laboratory of Receptor Research, Drug In our previous study, TRIP13 was identi ed as a chromosome Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese instability gene that was correlated with multiple myeloma drug Academy of Sciences, and University of Chinese Academy of Sciences, Shanghai, resistance, disease relapse, and poor outcomes in patients with mul- China. 4Department of Internal Medicine, University of Iowa Carver College of tiple myeloma (11). TRIP13 was first identified by yeast two-hybrid 5 Medicine, Iowa City, Iowa. Clinical and Translational Research Center of Shang- screening as a protein fragment that was associated with thyroid hai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and hormone receptor in a hormone-independent fashion (12). Over- Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China. 6Tongji University Cancer Center, Tongji University, Shanghai, expressing TRIP13 in cancer cells prompted cell growth and drug China. resistance, while targeting TRIP13 by TRIP13 shRNA inhibited mul- Note: Supplementary data for this article are available at Cancer Research tiple myeloma cell growth, induced cell apoptosis, and reduced the Online (http://cancerres.aacrjournals.org/). tumor burden in xenograft multiple myeloma mice (11). Our previous results suggested that TRIP13 might serve as a biomarker for multiple Y. Wang, J. Huang, B. Li, and H. Xue are the co-first authors of this article. myeloma disease development and prognosis, making it a potential Corresponding Authors: Jumei Shi, Shanghai Tenth People's Hospital, Tongji target for future therapies. University School of Medicine, 301 Yanchang Road, Shanghai 200072, China. To identify a TRIP13 inhibitor, detailed structural information of Phone: 8618-9176-83490; Fax: 8621-6630-6641; E-mail: [email protected]; and Weiliang Zhu, [email protected] TRIP13 is essential. Although the reported crystal structure of the TRIP13 mutant (E253Q or E253A) provided insight into the mech- Cancer Res 2020;80:536–48 anism of substrate recognition (8), further structural information doi: 10.1158/0008-5472.CAN-18-3987 of the wild-type TRIP13 protein is needed for specific inhibitor 2019 American Association for Cancer Research. development. In this study, we determined the crystal structure of

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the wild-type human TRIP13 at a resolution of 2.6 Å. We then were performed at 25C with Biacore T2000 (GE Healthcare). In this identified small-molecule inhibitors of TRIP13 based on its crystal step, compounds were diluted at different concentrations in PBS buffer structure via molecular docking and bioassay. A small-molecule (10 mmol/L HEPES pH ¼ 7.4, 150 mmol/L NaCl, 3 mmol/L EDTA), inhibitor, designated DCZ0415, was confirmed to bind to TRIP13 by and were flowed over the chip at rate of 30 mL/minute. The combining pull-down, nuclear magnetic resonance (NMR) spectroscopy, surface time and dissociation time was set at 120 and 150 seconds, respectively. plasmon resonance (SPR) assays. DCZ0415 exhibited significant Data analysis was finished via the state model of T2000 evaluation antimyeloma activity in vitro, in vivo, and in patient multiple myeloma software (GE Healthcare). cells. Importantly, DCZ0415 also synergized with melphalan and the histone deacetylase (HDAC) inhibitor panobinostat in multiple mye- Cell viability assay loma cells. Cell viability assay was performed as described previously (13). Briefly, cells were seeded in triplicate in 96-well plates and then treated with DCZ0415. Cell viability was measured using the Cell Counting Kit Materials and Methods (CCK)-8 assays. Cell lines and patient samples U266, HEK293T, MOPC-315, and HS-5 cells were commercially Apoptosis assay obtained from the ATCC. ARP-1, OCI-MY5, RPMI-8226, and H929 Apoptosis assay was performed as described previously (14). Briefly, cells were provided by Dr. Fenghuang Zhan (University of Iowa, Iowa cells were treated with or without DCZ0415. Then, cells were collected City, IA). Cell lines were certificated by short tandem repeat analysis and stained with Annexin-V for 15 minutes and then PI for 5 minutes (Shanghai Biotechnology Co., Ltd.). Mycoplasma testing was per- at room temperature. Stained cells were detected via using flow formed using MycoAlert Mycoplasma Detection Kit according to the cytometry. manufacturer's recommended protocols. Multiple myeloma cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% Crystallization, data collection, and structural determination FBS (Gibco) and 1% penicillin–streptomycin (Gibco). Human HS-5, Wild-type TRIP13 protein was mixed with AMP-PNP at a molar HEK293T and mouse MOPC-315 cells were maintained in DMEM ratio of 1:2 and incubated on ice for 1 hour to allow complex (Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin. formation. Crystallization was achieved by sitting-drop vapor dif- fi All cells were maintained in a humidi ed atmosphere of 5% CO2 at fusion at 4 C with the well solution containing 0.1 mol/L bicine, 37C, subcultured every 3 days and passaged routinely for use until pH 9.0, and 10% (v/v) ()-2-Methyl-2,4-pentanediol. Crystals passage 20. Bone marrow samples were obtained from patients with were gradually transferred to a harvesting solution containing multiple myeloma after obtaining written informed consent at the the precipitant solution and 25% glycerol, prior to flash-freezing Department of Hematology Shanghai Tenth People's Hospital (Shang- them in liquid nitrogen for storage. Native and Se-Met-SAD hai, China). The protocol for collection and usage of clinical samples datasets were collected under cryogenic conditions (100 K) at the was approved by the Shanghai Tenth People's Hospital Ethics Com- beamlines BL18U1 and BL19U1 of the Shanghai Synchrotron mittee. Informed consent was obtained in accordance with the Dec- Radiation Facility, and were processed using the program laration of Helsinki. HKL3000 (15). The single-wavelength anomalous diffraction (SAD) data phases were calculated using the CCP4i (16) suite and four Reagents and antibody selenium atoms were located and refined. The initial SAD map was DCZ0415 was synthesized by Shanghai Institute of Materia Medica, significantly improved by solvent flattening. A model was built into Chinese Academy of Sciences, Shanghai, China. Antibodies for cas- the experimental electron density using the programs CCP4i and pase-3, caspase-8, caspase-9, mouse CD4, CD3, and CD8 were pur- Coot (16) and further refined in the program Phenix (17). The chased from Cell Signaling Technology; TRIP13, BAX, BCL2, CDK4, native structure was determined by molecular replacement using CDK6, cyclin D, p-p65, a-tubulin, TUNEL, Ki-67 and b-actin were the crystal structure of Se-Met TRIP13 as the initial model, and from Abcam. Annexin V-FITC and propidium iodide (PI) detection further refined in Coot and Phenix (17). Figures of the crystal kit was purchased from BD Pharmingen. Penicillin–streptomycin was structures were generated with the program PyMOL (Schrodinger purchased from Invitrogen. Puromycin and biotin were purchased L. The PyMOL Molecular Graphics System, Version 1.8. 2015). from Sigma. Plasmids for TRIP13 WT and TRIP13 MT expression Pull-down assay The oligonucleotide sequence specific for TRIP13 silencing Cells were harvested, aspirated, and washed with cold PBS; they (sgRNA) was designed. The packaging plasmids VSVG and psPAX2 were then lysed and centrifuged at 8,000 g at 4C. The lysate was then were used to produce recombinant lentivirus by transfecting incubated with 10 mL of DCZ0415-biotin (50 mmol/L) or biotin (50 HEK293T cells. Lentiviral transduction of myeloma cell lines was mmol/L) for 2 hours in the presence of neutrAvidin agarose resins performed using polybrene. Stable cell lines were selected with puro- (Thermo Fisher Scientific) with rotation at 4C. The solution was then mycin (2.5 mg/mL). The efficiency of viral transduction was >95%. centrifuged at 1,500 g and the supernatant was discarded; it was then Then, PCDH constructs were used to generate human TRIP13 WT washed twice with PBS, centrifuging after each wash, resuspended in and TRIP13 MT overexpression plasmids for transfection into SDS, and analyzed by immunoblotting. sgTRIP13 cells.

Surface plasmon resonance DNA double-strand break repair assay TRIP13 protein was prepared in 10 mmol/L sodium acetate (pH ¼ DNA double-strand break (DSB) repair assay was performed as 5.5) and then covalently immobilized on CM5 sensor chip xia amine- described previously (18). Brieﬂy, the efﬁciency of NHEJ and homol- coupling procedure. The rest binding sites of the sensor chip were ogous recombination (HR) was measured using a GFP-based reporter blocked by ethanolamine. The kinetic measurements of compounds system.

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Tumor xenograft models for crystallization, we further purified wt_hTRIP13 monomer using Nude mice (6 weeks old) were purchased from Shanghai SLAC mono-Q ion exchange chromatography and gel filtration chromatog- Laboratory Animal Co., Ltd. (Shanghai, China). Human H929 cells raphy. We used AMP-PNP (a nonhydrolyzable analogue of ATP) (1 106) in 100 mL of serum-free culture medium were subcutane- to cocrystallize with wt_hTRIP13. AMP-PNP has a binding affinity of ously injected into the upper flank region of the nude mice. After the 49 mmol/L to wt_hTRIP13 (Supplementary Fig. S1B). We determined tumor growth of mice, mice were randomly assigned to two groups: the the crystal structure of wt_hTRIP13 by single-wavelength anomalous control group (DMSO, Tween-80 and saline) and 50 mg/kg DCZ0415- dispersion at a resolution of 2.6 Å (Supplementary Table S1). There is treated group (dissolved in DMSO, Tween-80 and saline solution). one copy of wt_hTRIP13 per asymmetric unit and the crystal packing BALB/c mice (6 weeks old) were injected subcutaneously in the right belongs to the space group P65. Similar to recently reported mutant flank with or without 5 106 MOPC-315 cells in a volume of 0.1 mL. structure of human TRIP13 (TRIP13E253Q; ref. 8), wt_hTRIP13 After tumor growth of mice, mice were randomly assigned to two assembled into a helical filament instead of the classic hexamer ring þ groups: the control group (DMSO, Tween-80, and saline), 25 mg/kg of the AAA ATPase family, probably due to the crystal packing DCZ0415-treated group (dissolved in DMSO, Tween-80, and saline (Supplementary Fig. S1C). solution). Mice were then administered with or without 25 mg/kg The wt_hTRIP13 monomer comprises three domains: An N- DCZ0415 via intraperitoneal injection every day for 15 days. All mice terminal domain that is involved in substrate recognition, and the were euthanized at the end of the experiment and tumors were large and small AAA domains that form the catalytic site for ATP photographed. Tumor volumes were measured using a Vernier caliper hydrolysis (Fig. 1A and B). Positive electron densities could be and calculated using the formula: Tumor volume (mm3) ¼ 1/2 observed inside the ATP-binding cleft of TRIP13 in the Fo-Fc map (relatively shorter diameter)2 (relatively longer diameter). All of the wt_hTRIP13 structure, but we failed to fit the AMP-PNP animal studies were approved by the Institutional Review Board of molecule into this density. Superposition of the wt_hTRIP13 structure Shanghai Tenth People's Hospital (ID: SYXK 2011-0111). with the structure of TRIP13E253Q mutant in complex with ATP (PDB:5VQA) revealed that the base and phosphate groups of ATP Statistical analysis happens to occupy the positive electron densities (Fig. 1C). The key Statistical analyses were performed using Prism software (Graph- residues that participate in coordinating and hydrolyzing ATP, such as Pad). Data are expressed as means SD. Data were considered K185/T186 of the Walker A motif, N300 of the Sensor 1 motif, and statistically significant at P < 0.05. The Student t test was used to R386 of the Sensor 2 motif, adopt almost identical conformations compare two groups. The log-rank test was used for survival curves. between the two structures (Fig. 1C). However, as for the residue E253 The combination index (CI) values were calculated by median dose- of the Walker B motif, its side chain sticks outward from the ATP- effect analysis using commercially available software (CalcuSyn; Bio- binding cleft and seems not to participate in ATP-binding as the soft). All tests of statistical significance were two sided. residue Q253 in the TRIP13E253Q mutant (Fig. 1C). Residue E253 has a higher b factor compared with the other parts of the structure, indicating that its conformation might be dynamic and prone to Results alternate. Determination of the crystal structure of wild-type human TRIP13 TRIP13 inhibitor DCZ0415 binds to TRIP13 We set out to study the crystal structure of wild-type human TRIP13 On the basis of our wt_hTRIP13 structure, structure-based molec- (wt_hTRIP13) to aid the discovery and rational design of TRIP13 ular docking was applied to virtually screen our in-house compound inhibitors for multiple myeloma therapeutics. Wild-type human library with 8,000 compounds using Smina, a fork of AutoDock Vina, TRIP13 protein exists as a mixture of monomer and oligomers in with default parameters (19, 20). Of the compounds identified, 76 were solution (Supplementary Fig. S1A). To obtain homogeneous sample selected for biological testing (Supplementary Table S2). Viability test

Figure 1. Crystal structure of wt_hTRIP13. A, Domain organization of the wt_ hTRIP13 monomer. The N-terminal domain (NTD) is shown in yellow, the large AAA domain in blue, the small AAA domain in green and other regions in gray. B, Crystal structure of wt_hTRIP13 monomer at 2.6 Å. The N- terminal domain (NTD) is shown in yellow, the large AAA domain in blue, and the small AAA domain in green. The ATP-binding cleft is shown in the box. C, Enlarged view of the ATP- binding cleft as boxed in B. The structure (colored as in B) is superposed onto the structure of TRIP13E253Q-ATP (PDB: 5VQA). Positive electron density of the Fo- Fc map inside the ATP-binding cleft of wt_hTRIP13 is contoured at 3.0 s and colored in gray. See also Sup- plementary Fig. S1.

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Figure 2. Binding of DCZ0415 to TRIP13. A, The structure of DCZ0415 (top) and its binding mode to TRIP13 as determined by molecular docking (bottom). Three residues forming hydrogen bonds are shown as gray sticks, DCZ0415 as yellow sticks, and hydrogen bonds as yellow dashes. B, A pull-down assay was used to detect the binding of DCZ0415-biotin to TRIP13. Right, immunoblotting analyses of the input proteins. C, CPMG spectra was acquired using 200 mmol/L of DCZ0415 alone (red) and 200 mmol/L of DCZ0415 with the addition of 5, 8, or 10 mmol/L of TRIP13 (cyan, green, and blue, respectively). D, The STD spectrum was acquired using 200 mmol/L of DCZ0415 with the addition of 5 mmol/L of TRIP13. E and F, SPR bio- sensor was used to detect the binding of DCZ0415-biotin to TRIP13. Apparent

Kd value is calculated by SPR the data. The ﬁtted Kd is 2.42 1.26 mmol/L.

of multiple myeloma cells revealed some active compounds, of which, dose-dependent decrease in viability (Fig. 3A). Using CCK-8 assay, – DCZ0415 was the most promising inhibitor based on biology screen- the IC50 value of DCZ0415 was 1.0 10 mmol/L calculated by CalcuSyn ing (Fig. 2A). in multiple myeloma cell lines. To further investigate the antimyeloma To further validate whether DCZ0415 targeted TRIP13, a series activity of DCZ0415, two representative cell lines were treated with of assays were performed. We employed an affinity pull-down target DCZ0415 (0–40 mmol/L) for 24, 48, and 72 hours. We observed that verification system, in which DCZ0415 was conjugated with a DCZ0415 decreased cell viability in a time- and dose-dependent biotin. Compared with the unconjugated biotin, the addition of manner (Supplementary Fig. S2A). To examine the effect of DCZ0415 DCZ0415-biotin to the cell lysate brought down endogenous on the colony formation of multiple myeloma cells, soft-agar clono- TRIP13 (Fig. 2B). And a 2% input of total cell lysate was tested genic assays were performed. Multiple myeloma cells treated with by immunoblotting analyses (right; Fig. 2B). In this study, we DCZ0415 showed a significant decrease in colony formation, indicat- measured the interaction between DCZ0415 and TRIP13 using ing that this compound inhibits cell proliferation (Fig. 3B; Supple- NMR. We observed that the CPMG spectra of 200 mmol/L mentary Fig. S2B). EdU assays were employed to examine whether DCZ0415 with the addition of 5, 8, 10 mmol/L TRIP13 (Fig. 2C) DCZ0415 affects DNA synthesis. Compared with that in the control and the STD spectrum of 200 mmol/L DCZ0415 with the addition of group, the percentage of EdU-positive cells was significantly decreased 5 mmol/L TRIP13 both showed the interaction (Fig. 2D). In our with DCZ0415 treatment, indicating that DCZ0415 exerts cytotoxic study, we measured the interaction between TRIP13 and DCZ0415 effects by inhibiting DNA synthesis in multiple myeloma cells (Sup- fi using SPR assay. The binding af nity (Kd) was calculated from SPR plementary Fig. S2C). fi data. The measurements displayed a strong af nity with a Kd value Bone marrow stromal cells (BMSC) mediated the paracrine growth of 2.42 1.26 mmol/L (Fig. 2E and F). of multiple myeloma cells and protect against the cytotoxicity of antimyeloma agents via cytokine secretion (21). To determine whether DCZ0415 inhibits multiple myeloma cell growth and induces DCZ0415 could overcome the protective effects of the BM microen- apoptosis vironment, multiple myeloma cells were cultured with or without HS-5 To evaluate the inhibitory effect of DCZ0415, multiple myeloma BMSCs in the presence or absence of DCZ0415 (Fig. 3C; Supplemen- cells were treated with DCZ0415 for 72 hours and cell viability tary Fig. S2D). As a positive control, cells were treated with melphalan was assessed. Data showed that DCZ0415 induced a significant with or without BMSCs for the same period of time. Results revealed

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Figure 3. DCZ0415 inhibits the proliferation of multiple myeloma cell lines and induces apoptosis. A, Cell viability of multiple myeloma cells with DCZ0415 treatment for

72 hours at the indicated concentrations. IC50 values were the means from three independent experiments. B, Soft agar colony formation of ARP-1 cells with DMSO or DCZ0415 treatment at the indicated concentrations. Left, representative images of colonies. (Continued on the following page.)

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Figure 4. The anti-MM activity of DCZ0415 depends on TRIP13. A, The viability of multiple myeloma cells transfected with empty vector or TRIP13-sgRNA with DCZ0415 treatment (0, 5, 10, 20, and 40 mmol/L, 48 hours) was analyzed by a CCK-8 assay. SgCon- trol represents nontarget scramble– transfected cells. TRIP13 sgRNA represents TRIP13-silenced cells. The result is expressed as means SD of three independent experiments. B, A pull-down assay was used to test the binding of DCZ0415-biotin with TRIP13 WT cells or TRIP13 MT cells. TRIP13 WT represents TRIP13 wild- type overexpression in TRIP13- silenced cells, whereas TRIP13 MT represents TRIP13 V140/S187/ R386A overexpression in TRIP13- silenced cells. C, Cell viability in TRIP13 WT and TRIP13 MT groups treated with DCZ0415, as analyzed by a CCK-8 assay. The result is expressed as means SD of three independent experiments.

that DCZ0415 treatment significantly inhibited multiple myeloma cell inhibition of IL6- or IGF1-induced multiple myeloma cell growth viability in the presence and absence of BMSCs, but melphalan- was observed with DCZ0415 treatment (Fig. 3D; Supplementary induced cytotoxicity could be abrogated by BMSCs (Supplementary Fig. S2F), suggesting that this compound not only directly targets Fig. S2E). multiple myeloma cells, but can also overcome the cytoprotective Cytokines IL6 and insulin growth factor-1 (IGF1), which are effects of the host BM microenvironment. secreted by multiple myeloma cells and BMSCs, have been reported We next examined whether DCZ0415 functioned by inducing to promote multiple myeloma cell proliferation, migration, and drug apoptotic cell death (23). Multiple myeloma cells were exposed to resistance (22). We therefore examined the effect of DCZ0415 on IL6- various concentrations of DCZ0415, leading to significant increases in þ or IGF1–induced multiple myeloma cell growth. Multiple myeloma the proportion of early- (Annexin-V /PI ) and late-stage (Annexin- þ þ cells were cultured either alone or with IL6 or IGF1. Significant V /PI ) apoptotic cells compared with that in control cells exposed to

(Continued.) Right, quantiﬁcation of colony numbers. The y-axis represents the percentage of colonies relative to the number of DMSO-treated cells. Statistical evaluation was performed using the Student t tests. C, Activity of DCZ0415 against ARP-1 cell lines cultured in the presence or absence of the HS-5 stromal cell line for 48 hours. The result is expressed as means SD of three independent experiments. D, Activity of DCZ0415 against ARP-1 cell lines cultured in the presence or absence of IL6 and IGF1 for 48 hours. Error bars, SD. The result is expressed as means SD of three independent experiments. E, Flow cytometry evaluation of Annexin-V– positive apoptotic cells in DCZ0415-treated ARP-1 cells. F, Flow cytometry evaluation of apoptosis in patient multiple myeloma cells after DCZ0415 treatment for 48 hours. Normal PBMCs from healthy donors (PBMCs#1–PBMCs#3) were treated with the indicated concentrations of DCZ0415 for 48 hours and then apoptosis was analyzed. Protein levels of TRIP13 were evaluated in PBMCs#1, PBMCs#2, Pt#2, Pt#3, Pt#9, and Pt#10 cells. G, ARP-1 cells were incubated with or without pan-caspase inhibitor Z-VAD-FMK for 1 hour and then treated with DCZ0415 (0 or 10 mmol/L) for 48 hours, followed by assessment of cell apoptosis using Annexin V/PI staining. Right, columns represent the average percentage of Annexin V–positive cells from three independent experiments, which are shown as the mean SD. H, Cell-cycle analysis of DCZ0415 (0, 10, and 20 mmol/L, 24 hours)-treated ARP-1 cells. P values were calculated using the Student t tests. , P < 0.05; , P < 0.01; , P < 0.001. See also Supplementary Figs. S2 and S3.

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DMSO (Fig. 3E; Supplementary Fig. S3A). To determine whether manner but increased the expression of BAX (Supplementary DCZ0415 could induce apoptosis in patient multiple myeloma cells, Fig. S3B). Also reduction of TRIP13 induced similar increase in BAX þ purified CD138 cells were examined from six newly diagnosed and decrease in BCL2 (Supplementary Fig. S3C). To determine the patients and four refractory/relapsed patients with multiple myeloma dependence of DCZ0415-induced apoptosis on the caspase pathway, who were refractory to bortezomib. Patient cells showed a dose- we assessed the ability of the pan-caspase inhibitor Z-VAD-FMK to dependent relationship between DCZ0415 treatment and apoptotic protect against cell apoptosis. As shown in Fig. 3G, Z-VAD-FMK cell death. This confirmed that DCZ0415 can trigger cytotoxicity in partially blocked DCZ0415-induced cell apoptosis as determined by bortezomib-resistant primary myeloma cells. In contrast, DCZ0415 Annexin V–PI staining. These data demonstrate that DCZ0415 trig- does not significantly induce normal peripheral blood mononuclear gers caspase-dependent apoptosis in multiple myeloma cells. In addi- cells’ (PBMC) apoptosis (Fig. 3F). We tested the sensitivity of cells of tion to apoptosis, effects on cell-cycle progression might be important PBMCs and patients to TRIP13 expression and the results showed that for the action of anticancer drugs. We therefore evaluated the effects of cells with high expression of TRIP13 were more sensitive to DCZ0415 DCZ0415 on cell-cycle progression by flow cytometry analysis. Treat- fi – than those with low expression of TRIP13 (Fig. 3F). Because BCL2 ment induced a signi cant accumulation in G0 G1 multiple myeloma family proteins affect apoptosis via the regulation of cytochrome C cells (Fig. 3H; Supplementary Fig. S3D). As essential components of release, which then mediates caspase activation (24), the effects of the cell-cycle machinery, cyclins function to bind and activate specific DCZ0415 on the expression of caspase enzymes, antiapoptotic BCL2, cyclin-dependent kinase (CDK) partners. Thus, protein levels of and proapoptotic protein BAX were evaluated. Caspase-8 and -9 CDK4, CDK6, and cyclin D were evaluated. In agreement with flow activities increased in a dose-dependent manner in multiple myeloma cytometry data, we observed a marked dose-dependent decrease in cells treated with DCZ0415 for 48 hours. Furthermore, DCZ0415 cyclin D, CDK4, and CDK6 after DCZ0415 administration (Supple- treatment decreased the expression of BCL2 in a dose-dependent mentary Fig. S3E).

Figure 5. DCZ0415 impaired DNA repair and inhibited NF-kB activity in multiple myeloma cells. A, Immunofluores- cence staining of cellular gH2AX in ARP-1 and H929 cells with or without DCZ0415 treatment for 24 hours. B, NHEJ was quantified by GFP and DsRed expression as analyzed by flow cytometry. Error bars, SD. The result is expressed as means SD of three independent experiments. C, TRIP13 WT and TRIP13 MT cells were separately treated with DCZ0415 (0 and 10 mmol/L) for 24 hours. TRIP13 WT represents TRIP13 wild-type overexpression in TRIP13-silenced cells, whereas TRIP13 MT represents TRIP13 V140/S187/R386A overexpression in TRIP13-silenced cells. Expression of gH2AX was tested by immunoblotting analyses. D, TRIP13 WT and TRIP13 MT cells were separately treated with or without DCZ0415 for 48 hours. NHEJ was quantified by GFP and DsRed expression as analyzed by flow cytometry. Error bars, SD. The result is expressed as means SD of three independent experiments. E, Protein level of p-ikBa,ikBa, p-p65, and p65 was evaluated in whole-cell lysates from ARP-1 and OCI-MY5 cells after treatment with or without 10 mmol/L DCZ0415 for 48 hours. F, NF-kB luciferase reporter was transfected in ARP- 1 cells. The cells were treated with or without 10 mmol/L DCZ0415 for 24 hours and then the relative luciferase activity was analyzed. The result is expressed as means SD of three independent experiments. G, ARP-1 cells were treated with or without 20 mmol/L DCZ0415 for 24 hours, with or without 20 ng/mL TNFa for 1 hour, and then analyzed by flow cytometry. , P < 0.05: , P < 0.01.

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A TRIP13 Inhibitor Suppresses Multiple Myeloma Progression

– The antimyeloma activity of DCZ0415 depends on TRIP13 sgControl cells were blocked at G0 G1 stage under DCZ0415 treat- To determine whether the antimyeloma activity of DCZ0415 is ment. However, compared with in sgControl cells, sgTRIP13 cells dependent on TRIP13, TRIP13-sgRNA and point mutation plasmids showed no significant changes in the cell cycle under DCZ0415 were designed. Treatment of sgTRIP13 cells with DCZ0415 led to the treatment (Supplementary Fig. S4C). These results suggested that the loss of sensitivity compared with that of sgControl-transfected wild- antimyeloma activity of DCZ0415 depends on TRIP13. On the basis of type cells (Fig. 4A). sgTRIP13 and sgControl cells were separately the structure of TRIP13 and DCZ0415, we speculated that valine treated with melphalan or left untreated. The results showed that (V140), serine (S187), and arginine (R386) of TRIP13 are essential for sgTRIP13 and sgControl cells were both sensitive to melphalan DCZ0415 binding. We thus mutated TRIP13 V140/S187/R386 (Supplementary Fig. S4A and S4B). This finding suggested that (TRIP13 WT) to TRIP13 alanine (A) 140/187/386 (TRIP13 MT); we cells with deleted TRIP13 were specifically resistant to DCZ0415. then downregulated endogenous TRIP13 expression using the We evaluated the effects of DCZ0415 on cell-cycle progression in sgTRIP13 target intron sequence and overexpressed TRIP13 WT or sgControl and sgTRIP13 cells by using flow cytometry analysis. The TRIP13 MT. Pull-down assay shown that DCZ0145 bound to TRIP13

Figure 6. DCZ0415 in combination with melphalan or panobinostat functions synergistically to exert cytotoxicity. ARP-1 (A)andOCI-MY5(B) cells were treated with DCZ0415 (10–80 mmol/L) plus melphalan (2.5–20 mmol/L) for 48 hours, which was followed by a CCK-8 assay to determine cell viability. The synergistic cytotoxic effects of DCZ0415 and melphalan are shown. CI < 1 indicated synergistic activity, as determined using CalcuSyn software. The Fa fraction represented the cells affected. ARP-1 (C)andOCI-MY5(D) cells were treated with DCZ0415 (10–80 mmol/L) plus panobinostat (2.5–20 nmol/L) for 48 hours, which was followed by a CCK-8 assay to determine cell viability. Synergistic antimyeloma activity was analyzed. Error bars, SD. All results are expressed as means SD of three independent experiments.

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A TRIP13 Inhibitor Suppresses Multiple Myeloma Progression

WT cells, but not TRIP13 MT cells (Fig. 4B). Cell viability of was treatment, greater levels of NHEJ repair were detected in TRIP13 MT compared among the groups (TRIP13 WT and TRIP13 MT) with cells compared with TRIP13 WT cells (Fig. 5D). This suggested that DCZ0415 treatment. We found that DCZ0415 decreased cell viability TRIP13 MT cells were resistant to DCZ0415, associated with dimin- in the TRIP13 WT group, whereas TRIP13 MT cells were resistant to ished DNA damage and greater NHEJ repair compared with TRIP13 DCZ0415, as compared with TRIP13 WT cells. The protein expression WT cells. Besides, to address the impact of DCZ0415 on mitotic of TRIP13 was also examined among these groups (Fig. 4C). This spindles, a-tubulin/DAPI staining was performed. As shown in suggests that DCZ0415–TRIP13 binding is essential for the antimye- Supplementary Fig. S5D, DCZ0415 induced spindle multipolarity, loma activity of DCZ0415. Our findings suggested that TRIP13 is suggesting that DCZ0415 acted as a spindle poison, which supported essential for the antimyeloma activity of DCZ0415. the involvement of TRIP13 in regulation of the mitotic spindle. NF-kB is aberrantly activated in MM and promotes cell survival and DCZ0415 impaired DNA repair and inhibited NF-kB activity in malignancy by upregulating antiapoptotic genes (29). To investigate multiple myeloma cells the possible contribution of the NF-kB signaling pathway to patho- Some anticancer agents sensitize cancer cells by inducing DNA genesis, we investigated whether ikBa and NF-kB p65 phosphoryla- DSBs and DNA damage responses (25). In the response of mammalian tion were decreased by DCZ0415 treatment. Compared with the cells to DNA DSBs, phosphorylation of histone H2AX (gH2AX) at untreated control cells, the protein levels of phosphorylated (p)-ikBa sites proximal to the DNA breaks has been reported (26). In this study, and phosphorylated (p)-NF-kB p65 were decreased in multiple mye- gH2AX levels were evaluated in multiple myeloma cells following loma cells treated with DCZ0415 (Fig. 5E), and DCZ0415 showed a DCZ0415 treatment by immunofluorescence analysis. The results strong inhibitory effect on NF-kB–promoter luciferase activity revealed that gH2AX levels were higher in multiple myeloma cells (Fig. 5F). As shown in Fig. 5G, DCZ0415-induced cell apoptosis treated with DCZ0415 than baseline gH2AX levels (untreated control), could be partially rescued by TNFa. These results suggested that which reflected ongoing DNA damage (Fig. 5A). Ataxia telangiectasia cell death induced by DCZ0415 may be mediated via the inhibition mutated (ATM) protein kinase is a key mediator of this DNA damage of the NF-kB signaling pathway. Importantly, TRIP13 increased NF- response, which induces cell-cycle arrest and facilitates DNA repair by kB–promoter luciferase activity (Supplementary Fig. S5E). activating downstream targets such as the cell-cycle checkpoint kinase (CHK2; refs. 27, 28). The protein levels of phosphorylated (p)-ATM Combined treatment with DCZ0415 and melphalan or HDAC and phosphorylated (p)-CHK2 were found to be increased in a dose- inhibitor panobinostat induces synergistic antimyeloma activity dependent manner in DCZ0415-treated multiple myeloma cells com- To further evaluate its preclinical efficacy, we investigated the effects pared with the untreated control cells (Supplementary Fig. S5A). A of DCZ0415 on multiple myeloma cell growth when used in combi- DNA damage response accompanied by efficient and appropriate nation with other antimyeloma agents. The cytotoxicity of combined repair of DSBs is essential for the preservation of genomic integrity. DCZ0415 and melphalan (30) was examined in multiple myeloma However, in cancer, the repair of anticancer agent-induced DSBs by cells. Melphalan-induced growth inhibition was enhanced with the NHEJ or HR repair pathways promotes treatment resistance and increasing concentrations of DCZ0415. Calculation of the CI values subsequent relapse in patients (26). TRIP13 also enhanced NHEJ using CalcuSyn software revealed synergistic effects of DCZ0415 on repair and induced treatment resistance via binding to NHEJ proteins melphalan against multiple myeloma cells (Fig. 6A and B). Then we KU70/KU80/DNA-PKcs in head and neck cancer (10). Thus, we tested DCZ0415 in combination with HDAC inhibitor panobino- evaluated whether DCZ0415 impaired DNA repair via the NHEJ stat (31) in multiple myeloma cells. Isobologram analysis revealed repair pathways using GFP-based reporter assay, which was an synergy between DCZ0415 and panobinostat with a CI less than 1 excellent tool to measure the efficiency of NHEJ repair. The results (Fig. 6C and D). This provided evidence for the beneficial effects of indicated that DCZ0415 suppressed the NHEJ repair pathway combination therapy, which could effectively reduce the required (Fig. 5B), which was consistent with TRIP13 promoting DSB- concentration of other antimyeloma agents, thereby reducing the induced NHEJ repair and we confirmed that TRIP13 interacted with potential side effects. the NHEJ key regulator KU70/KU80 (Supplementary Fig. S5B). Besides, our results have shown that 10 mmol/L DCZ0415 had a little Multiple myeloma xenografts are sensitive to DCZ0415 effect on HR repair pathway, but 20 mmol/L DCZ0415 could signif- To investigate the therapeutic potential of DCZ0415 in vivo,an icantly inhibit the HR repair (Supplementary Fig. S5C). With multiple myeloma mouse xenograft model was employed. The DCZ0415 treatment, the protein level of gH2AX increased in TRIP13 administration of DCZ0415 significantly reduced the growth of WT cells. However, the protein level of gH2AX in TRIP13 MT cells, multiple myeloma cells-induced tumors in immunodeficient mice which did not bind DCZ0415, showed no significant change under compared with control mice (Fig. 7A). There were no significant DCZ0415 treatment (Fig. 5C). We studied NHEJ repair in TRIP13 MT differences in the body weight of nude mice in each group, and WT cells using a GFP-based reporter assay. Following DCZ0415 suggesting that DCZ0415 was well tolerated (Fig. 7B). The toxicity

Figure 7. Antitumorigenic effects of DCZ0415 in a xenograft model of multiple myeloma. A, Average and SD of the tumor volumes (cm3) versus time. H929 cells were injected subcutaneously into mice (n ¼ 7/group) and then mice were treated with or without 50 mg/kg DCZ0415 every day for 14 days. Tumor sizes were measured every 2 days. B, Averages and SDs of nude mouse weights versus the time (mean weight SD; 7/group). C, Graphs of the percentage of survival over time (until the tumor volume reached 2,000 mm3) for the duration of the experiment. “Control” and “DCZ0415” represent mice bearing tumors that were treated with the vehicle or DCZ0415, respectively. Kaplan–Meier plots of mice treated with the vehicle or DCZ0415. Survival was signiﬁcantly increased in DCZ0415-treated mice compared with the control group (n ¼ 9/group). P < 0.001 versus the control group. D, Representative images of Ki-67, caspase-3, TUNEL, p-p65, and gH2AX IHC staining of tumor tissues after 14 days of treatment with vehicle or DCZ0415. E, Average and SD of the tumor volumes (cm3) versus time. MOPC-315 cells were injected subcutaneously into BALB/c mice (n ¼ 5/group) and then mice were treated with or without 25 mg/kg DCZ0415 every day for 15 days. Tumor sizes were measured every 2 days. F, Averages and SDs of BALB/c mice weights versus the time (mean weight SD; 5/group). G, Representative images of CD3, CD4, and CD8 IHC staining of tumor tissues after 15 days of treatment with vehicle or DCZ0415.

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of DCZ0415 was also examined by hematoxylin and eosin (H&E) TRIP13 inhibitor DCZ0415. DCZ0415 displayed highly potent staining of major organs and no significant histologic changes were antimyeloma activity against a large panel of multiple myeloma observed in the liver and kidney of the mice (Supplementary cell lines. Furthermore, DCZ0415 results in a significant reduction Fig. S6A), indicating that the side effects of DCZ0415 were minimal. of the proliferation rate in multiple myeloma cells as evidenced by Significantly, treatment with DCZ0415 resulted in a significant colony formation and EdU expression assays. The DCZ0415- prolongation in overall survival compared with vehicle-treated induced reduction in proliferation was associated with inducing animals (Fig. 7C). apoptosis, arresting the cell cycle. Furthermore, we performed a pharmacodynamic study whereby the We previously reported that TRIP13 was an oncogene in multiple harvested tumors were analyzed for antiproliferation and apoptosis myeloma; however, the detailed mechanism by which TRIP13 pro- markers. DCZ0415-treated mice exhibited decreased tumor Ki-67 and motes cell growth was not fully explained. In this study, we found that p-p65 levels compared with control mice. However, we observed an DCZ0415 (a TRIP13 inhibitor) induced cell death via inhibition of the increase in cleaved caspase-3, TUNEL (apoptosis markers), and NF-kB signaling pathway. Our results suggest that TRIP13 acts as an gH2AX following DCZ0415 treatment of multiple myeloma cells oncogene by activating the NF-kB signaling pathway in multiple compared with control cells (Fig. 7D). myeloma. Using a cellular assay to mimic multiple myeloma in its To examine DCZ0415 effect on immunocompetent mice, we microenvironment, we observed that DCZ0415 inhibited growth injected MOPC-315 cells subcutaneously into BALB/c mouse, and inhibition of multiple myeloma cells even in the presence of BMSCs treated with 25 mg/kg DCZ0415 or not. With DCZ0415 treatment, and the cytokines IL6 and IGF1. This suggested that besides its the growth of multiple myeloma cells induced tumors significantly cytotoxicity on multiple myeloma cells, DCZ0415 also targets the BM was reduced compared with control mice (Fig. 7E). There were no microenvironment and overcomes the proliferative effects of BMSCs significant changes in the body weight of BALB/c mice in each (Fig. 3C). In BM microenvironment, multiple myeloma cell adhesion group (Fig. 7F).IHCstudiesforCD4,CD3,andCD8expression to BMSCs triggers the NF-kB–dependent transcription and secretion from mice postsacrifice show that treatment with DCZ0415 signif- of cytokines such as IL6 in BMSCs, which further stimulate multiple icantly increased immune effect cells infiltration, as evidenced by myeloma cell growth, survival, and drug resistance (22, 33, 34). More- much greater CD4, CD3, and CD8 staining surrounding MOPC- over, activation of NF-kB by cell adhesion and cytokines augments the 315 cells remains (Fig. 7G). There was no significant histologic binding of multiple myeloma cells to BMSCs, which in turn induces change in the liver and kidneys of mice, as detected by H&E IL6 transcription and secretion in BMSCs. Conversely, the inhibition staining, indicating that the side effects of DCZ0415 were minimal of NF-kB activity abrogates this response (22, 33, 34). Our data (Supplementary Fig. S6B). demonstrate that DCZ0415 inhibits NF-kB activity (Fig. 5E and F) These findings suggest that DCZ0415 yields potent antimyeloma and TRIP13 activated NF-kB pathway (Supplementary Fig. S5E). responses in mice, prolonging their survival times. These data indicate Thus, DCZ0415 might prevent multiple myeloma progression by that TRIP13 inhibitors developed using this approach may provide a targeting TRIP13 and inhibiting NF-kB–dependent transcription and roadmap for candidate therapeutic agents in multiple myeloma. secretion of cytokines in BMSCs and the expression of many cell adhesion molecules on both multiple myeloma cells and BMSCs, thereby disrupting the tumor-BM microenvironment interactions that Discussion contribute to multiple myeloma progression (33). Our results sug- TRIP13 is thought to function as an oncogene based on meta- gested that treatment of cells with DCZ0415 was correlated with analysis of gene expression datasets from various cell lines, and is inhibition of NF-kB activity, cell-cycle blockade, and apoptosis, significantly amplified in patients with high-risk multiple myelo- although further in-depth study is required. In the future, we will ma (32). We previously showed that knockdown of TRIP13 inhibited identify the mechanism underlying DCZ0415 treatment by using the growth of multiple myeloma both in vitro and in vivo (11). We genetic tools. found that TRIP13 was overexpressed in human multiple myeloma cell Some anticancer agents induce apoptosis by enhancing DNA lines, further supporting a role for TRIP13 as an oncogene in multiple damage; however, this is amended by DNA repair, which increases myeloma. Therefore, we suggested that TRIP13 might represent an cell survival and induces drug resistance (35, 36). Thus, DNA repair antimyeloma target and inhibition of TRIP13 could be a promising inhibitors have received increased attention in recent years. For strategy in multiple myeloma therapy. example, it was reported that SCR7 was a putative inhibitor of NHEJ, Protein crystal structures can offer invaluable insight into the blocking end-joining by interfering with ligase IV binding to DNA, molecular mechanisms of action of specific proteins. The mechanisms thereby leading to accumulation of DSBs within the cells and culmi- of substrate recognition and remodeling of TRIP13 were revealed by nating in cytotoxicity (36). DCZ0415 not only enhanced DNA damage, the crystal structure of human TRIP13E253Q (8). However, it remained but also impeded DNA repair. As an inhibitor of TRIP13, the necessary to analyze the full-length, wild-type, human TRIP13. In this impediment of DNA repair by DCZ0415 is understandable, which study, the crystal structure of the wild-type TRIP13 protein was is consistent with previous data that TRIP13 is essential for DSB resolved, which not only contributed toward our understanding of repair (10). For example, it was revealed that instead of having a the molecular mechanisms of TRIP13 activity, but also helped to checkpoint role, TRIP13 was required for one of the two major classes identify new inhibitors against TRIP13 activity. of recombination in meiosis that was required for repairing DNA DCZ0415 was identified via structure-based molecular docking breaks. TRIP13 was also shown to be involved in DNA repair induced and cellular screening from in-house compound library. Our proof- by programmed DSBs in meiotic recombination (37). Our study of-concept experiments, which include pull-down, NMR, and SPR indicated that DCZ0415 impaired NHEJ, as shown in Fig. 5B. NHEJ assays, provide evidence that DCZ0415 binds to TRIP13. Our is the major DSB repair pathway in mammalian cells and is activated studies employed multiple myeloma cell lines, patient multiple by DSB ends being recognized by the KU (KU70 and KU80) hetero- myeloma cells and xenograft models, along with biochemical and dimer (38). It was recently reported that TRIP13 promoted NHEJ genetic models, to demonstrate the antimyeloma activity of the repair in head and neck cancer via binding of KU70 and KU80 (10).

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A TRIP13 Inhibitor Suppresses Multiple Myeloma Progression

Consistent with this, we observed endogenous binding between Acquisition of data (provided animals, acquired and managed patients, provided TRIP13 and KU70/KU80 in multiple myeloma cells, which also facilities, etc.): Y. Wang, B. Li, H. Xue, L. Hu, X. Sun, S. Chang, L. Gao, Y. Xie, W. Xiao, indicated that TRIP13 played a key role in NHEJ repair. D. Yu, G. Chen, N. Zhang, Z. Mao Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Resistance mechanisms present the largest hurdle to the cure computational analysis): Y. Wang, B. Li, H. Xue, Z. Xu, S. Chang, L. Gao, of multiple myeloma and it can arise initially or emerge during the Y. Tao, H. Xu course of treatment (39). Thus, to examine the synergistic effects of Writing, review, and/or revision of the manuscript: Y. Wang, J. Huang, B. Li, DCZ0415 and other antimyeloma agents, we tested the effects of H. Xue, G. Tricot, Z. Xu, F. Zhan, W. Zhu, J. Shi DCZ0415 in combination with melphalan and panobinostat. Prom- Administrative, technical, or material support (i.e., reporting or organizing data, isingly, DCZ0415 was able to enhance the cytotoxic effects of both constructing databases): Y. Kong, X. Sun, F. Lian, X. Wu Study supervision: J. Huang, W. Zhu, J. Shi melphalan and panobinostat in multiple myeloma cells. This suggests that DCZ0415 may have promising antimyeloma activity both alone and combined with other antimyeloma agents. In summary, our Acknowledgments studies presented crystal structure of the wild-type human TRIP13 This work was supported by grants from the National Natural Science Foundation fi of China (grant no. 81870158, 81570190, 31570766, U1632130, 81602515, 81529001, and identi ed an inhibitor of TRIP13. The inhibitor, named as and 81670194), National Key R&D Program of China (grant no. 2016YFA0501803, DCZ0415, effectively induced antimyeloma activity in multiple mye- 2017YFA0504504, and 2016YFA0502301), and Chinese Pharmaceutical Association loma, both in vitro and in vivo by impairing DSB repair and inhibiting - Yiling Biopharmaceutical Innovation Project (CPAYLJ201908). We thank D Yao, L NF-kB pathway. Our present data suggest that TRIP13 could be an Wu, W Qin, and R Zhang from the beamlines BL18U1 and BL19U1 at National important target for refractory/relapsed multiple myeloma. Center for Protein Science Shanghai (NCPSS) and Shanghai Synchrotron Radiation Facility (SSRF) for help with crystal data collection. fl Disclosure of Potential Con icts of Interest The costs of publication of this article were defrayed in part by the payment of page fl No potential con icts of interest were disclosed. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors’ Contributions Conception and design: J. Huang, W. Zhu, J. Shi Received December 26, 2018; revised July 30, 2019; accepted November 11, 2019; Development of methodology: Y. Wang, H. Xue, L. Hu, Z. Xu published first November 15, 2019.

References 1. Weaver AJ, Flannelly KJ, Koenig HG, Smith FD Jr. A review of research on 14. Xiao W, Li B, Sun X, Yu D, Xie Y, Wu H, et al. DCZ3301, a novel aryl-guanidino chaplains and community-based clergy in the Journal of the American Medical inhibitor, induces cell apoptosis and cell cycle arrest via suppressing the PI3K/ Association, Lancet, and the New England Journal of Medicine: 1998–2000. AKT pathway in T-cell leukemia/lymphoma. Acta Biochim Biophys Sin 2018;50: J Pastoral Care Counsel 2004;58:343–50. 643–50. 2. Becker N. Epidemiology of multiple myeloma. Recent Results Cancer Res 2011; 15. Minor W, Cymborowski M, Otwinowski Z, Chruszcz M. HKL-3000: the 183:25–35. integration of data reduction and structure solution–from diffraction images 3. Joseph NS, Gentili S, Kaufman JL, Lonial S, Nooka AK. High-risk multiple to an initial model in minutes. Acta Crystallogr D Biol Crystallogr 2006;62:859– myeloma: deﬁnition and management. Clin Lymphoma Myeloma Leuk 2017; 66. 17S:S80–S7. 16. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. 4. Manier S, Salem KZ, Park J, Landau DA, Getz G, Ghobrial IM. Genomic Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol complexity of multiple myeloma and its clinical implications. Nat Rev Clin Crystallogr 2011;67:235–42. Oncol 2017;14:100–13. 17. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. 5. Dazhi W, Mengxi Z, Fufeng C, Meixing Y. Elevated expression of thyroid PHENIX: a comprehensive Python-based system for macromolecular structure hormone receptor-interacting protein 13 drives tumorigenesis and affects solution. Acta Crystallogr D Biol Crystallogr 2010;66:213–21. clinical outcome. Biomark Med 2017;11:19–31. 18. Mao Z, Bozzella M, Seluanov A, Gorbunova V. Comparison of nonhomologous 6. Yost S, de Wolf B, Hanks S, Zachariou A, Marcozzi C, Clarke M, et al. Biallelic end joining and homologous recombination in human cells. DNA Repair 2008;7: TRIP13 mutations predispose to Wilms tumor and chromosome missegrega- 1765–71. tion. Nat Genet 2017;49:1148–51. 19. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking 7. Bhalla N, Dernburg AF. A conserved checkpoint monitors meiotic chromosome with a new scoring function, efﬁcient optimization, and multithreading. synapsis in Caenorhabditis elegans. Science 2005;310:1683–6. J Comput Chem 2010;31:455–61. þ 8. Ye Q, Kim DH, Dereli I, Rosenberg SC, Hagemann G, Herzog F, et al. The AAA 20. Koes DR, Baumgartner MP, Camacho CJ. Lessons learned in empirical scoring ATPase TRIP13 remodels HORMA domains through N-terminal engagement with smina from the CSAR 2011 benchmarking exercise. J Chem Inf Model 2013; and unfolding. EMBO J 2017;36:2419–34. 53:1893–904. 9. San-Segundo PA, Roeder GS. Pch2 links chromatin silencing to meiotic check- 21. Anderson KC. Targeted therapy of multiple myeloma based upon tumor- point control. Cell 1999;97:313–24. microenvironmental interactions. Exp Hematol 2007;35:155–62. 10. Banerjee R, Russo N, Liu M, Basrur V, Bellile E, Palanisamy N, et al. TRIP13 22. Chauhan D, Uchiyama H, Akbarali Y, Urashima M, Yamamoto K, Libermann promotes error-prone nonhomologous end joining and induces chemoresis- TA, et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in tance in head and neck cancer. Nat Commun 2014;5:4527. bone marrow stromal cells involves activation of NF-kappa B. Blood 1996;87: 11. Tao Y, Yang G, Yang H, Song D, Hu L, Xie B, et al. TRIP13 impairs mitotic 1104–12. checkpoint surveillance and is associated with poor prognosis in multiple 23. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, et al. Control of myeloma. Oncotarget 2017;8:26718–31. apoptosis and mitotic spindle checkpoint by survivin. Nature 1998;396:580–4. 12. Li XC, Schimenti JC. Mouse pachytene checkpoint 2 (trip13) is required 24. Renault TT, Dejean LM, Manon S. A brewing understanding of the regulation of for completing meiotic recombination but not synapsis. PLos Genet 2007; Bax function by Bcl-xL and Bcl-2. Mech Ageing Dev 2017;161:201–10. 3:e130. 25. Qie S, Diehl JA. Cyclin D1, cancer progression, and opportunities in cancer 13. Gao M, Li B, Sun X, Zhou Y, Wang Y, Tompkins VS, et al. Preclinical activity of treatment. J Mol Med 2016;94:1313–26. DCZ3301, a novel aryl-guanidino compound in the therapy of multiple mye- 26. Lindahl T, Barnes DE. Repair of endogenous DNA damage. Cold Spring Harb loma. Theranostics 2017;7:3690–9. Symp Quant Biol 2000;65:127–33.

AACRJournals.org Cancer Res; 80(3) February 1, 2020 547

Wang et al.

27. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM phosphorylates 33. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. Under- histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001;276: standing multiple myeloma pathogenesis in the bone marrow to identify new 42462–7. therapeutic targets. Nat Rev Cancer 2007;7:585–98. 28. Chehab NH, Malikzay A, Appel M, Halazonetis TD. Chk2/hCds1 functions as a 34. Hideshima T, Chauhan D, Schlossman R, Richardson P, Anderson KC. The role DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev 2000;14:278–88. of tumor necrosis factor alpha in the pathophysiology of human multiple 29. Staudt LM. Oncogenic activation of NF-kappaB. Cold Spring Harb Perspect Biol myeloma: therapeutic applications. Oncogene 2001;20:4519–27. 2010;2:a000109. 35. Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the 30. Esma F, Salvini M, Troia R, Boccadoro M, Larocca A, Pautasso C. Melphalan cancer connection. Nat Genet 2001;27:247–54. hydrochloride for the treatment of multiple myeloma. Expert Opin Pharmac- 36. Srivastava M, Nambiar M, Sharma S, Karki SS, Goldsmith G, Hegde M, et al. An other 2017;18:1127–36. inhibitor of nonhomologous end-joining abrogates double-strand break repair 31. San-Miguel JF, Hungria VT, Yoon SS, Beksac M, Dimopoulos MA, Elghandour and impedes cancer progression. Cell 2012;151:1474–87. A, et al. Panobinostat plus bortezomib and dexamethasone versus placebo plus 37. Roig I, Dowdle JA, Toth A, de Rooij DG, Jasin M, Keeney S. Mouse TRIP13/ bortezomib and dexamethasone in patients with relapsed or relapsed and PCH2 is required for recombination and normal higher-order chromosome refractory multiple myeloma: a multicentre, randomised, double-blind phase structure during meiosis. PLos Genet 2010;6. doi: 10.1371/journal.pgen.1001062. 3 trial. Lancet Oncol 2014;15:1195–206. 38. Lieber MR. The mechanism of double-strand DNA break repair by the non- 32. Shaughnessy JD Jr, Zhan F, Burington BE, Huang Y, Colla S, Hanamura I, et al. A homologous DNA end-joining pathway. Annu Rev Biochem 2010;79:181–211. validated gene expression model of high-risk multiple myeloma is deﬁned by 39. Hideshima T, Qi J, Paranal RM, Tang W, Greenberg E, West N, et al. Discovery of deregulated expression of genes mapping to chromosome 1. Blood 2007;109: selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor 2276–84. resistance in multiple myeloma. Proc Natl Acad Sci U S A 2016;113:13162–7.

548 Cancer Res; 80(3) February 1, 2020 CANCER RESEARCH

A Small-Molecule Inhibitor Targeting TRIP13 Suppresses Multiple Myeloma Progression

Yingcong Wang, Jing Huang, Bo Li, et al.

Cancer Res 2020;80:536-548. Published OnlineFirst November 15, 2019.

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