A Small Molecule Inhibitor Targeting TRIP13 Suppresses Multiple Myeloma Progression

Author Manuscript Published OnlineFirst on November 15, 2019; DOI: 10.1158/0008-5472.CAN-18-3987 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 A Small Molecule Inhibitor Targeting TRIP13 suppresses

2 multiple myeloma progression

3 Yingcong Wang,1,* Jing Huang, 2,* Bo Li,3,* Han Xue,2,* Guido Tricot,4 , Liangning Hu,1

4 Zhijian Xu,3 Xiaoxiang Sun,5 Shuaikang Chang,1 Lu Gao,1 Yi Tao,1 Hongwei Xu,4

5 Yongsheng Xie,1 Wenqin Xiao,1 Dandan Yu,1 Yuanyuan Kong,1 Gege Chen,1 Xi Sun,1

6 Fulin Lian,3 Naixia Zhang,3 Xiaosong Wu,1 Zhiyong Mao,5 Fenghuang Zhan,4 Weiliang

7 Zhu,3, † and Jumei Shi1,6, †

8 1Department of Hematology, Shanghai Tenth People’s Hospital, Tongji University

9 School of Medicine, Shanghai 200072, China

10 2Shanghai Institute of Precision Medicine, The Ninth People’s Hospital, Shanghai Jiao

11 Tong University School of Medicine, Shanghai 200011, China

12 3CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center,

13 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and University

14 of Chinese Academy of Sciences, Shanghai 201203, China

15 4Department of Internal Medicine, University of Iowa Carver College of Medicine,

16 Iowa City, IA, USA

17 5Clinical and Translational Research Center of Shanghai First Maternity & Infant

18 Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of

19 Life Sciences and Technology, Tongji University, Shanghai 200092, China

20 6Tongji University Cancer Center, Tongji University, Shanghai 200092, China

21 *Co-first author.

22 †Correspondence Authors: Jumei Shi, MD & PhD. Department of Hematology

23 Shanghai Tenth People’s Hospital, Tongji University School of Medicine, 301

24 Yanchang Road, Shanghai 200072, China, Phone: +86-021-66306764,

25 [email protected], Weiliang Zhu, PhD. Drug Discovery and Design Center,

26 Shanghai Institute of Materia Medica, 555 Zuchongzhi Road, Shanghai 201203, China,

27 Phone: +86-021-50806600,[email protected]

28 1

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 15, 2019; DOI: 10.1158/0008-5472.CAN-18-3987 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

29 Running Title: A TRIP13 Inhibitor Suppresses Multiple myeloma Progression

30 Key words: TRIP13, Inhibitor, Multiple myeloma

31 Conflict of Interest: The authors declare that there is no conflict of interests.

32 Word count: 5422

33 Number of figures and tables: 7

49 Abstract

50 The AAA-ATPase TRIP13 drives multiple myeloma (MM) progression. Here we

51 present the crystal structure of wild-type human TRIP13 at a resolution of 2.6 Å. A

52 small molecule inhibitor targeting TRIP13 was identified based on the crystal structure.

53 The inhibitor, designated DCZ0415, was confirmed to bind TRIP13 using pull-down,

54 nuclear magnetic resonance spectroscopy, and surface plasmon resonance binding

55 assays. DCZ0415 induced anti-myeloma activity in vitro, in vivo, and in primary cells

56 derived from drug-resistant myeloma patients. The inhibitor impaired nonhomologous

57 end joining repair and inhibited NF-κB activity. Moreover, combining DCZ0415 with

58 the MM chemotherapeutic melphalan or the HDAC inhibitor panobinostat induced

59 synergistic anti-myeloma activity. Therefore, targeting TRIP13 may be an effective

60 therapeutic strategy for MM, particularly refractory or relapsed MM.

61 Significance

62 Findings identify TRIP13 as a potentially new therapeutic target in multiple myeloma.

63 Introduction

64 MM is characterized by clonal proliferation of malignant monoclonal plasma cells in

65 the bone marrow (1). Genomic instability, defined by a higher rate of acquisition of

66 genomic changes per cell division compared with normal cells, is a prominent feature

67 of MM cells. Approximately 86,000 new MM patients are diagnosed worldwide each

68 year (2). Although the prognosis of MM patients has improved with the increased use

69 of autologous stem cell transplantation and combinations of approved anti-myeloma

70 agents such as proteasome inhibitors (bortezomib, carfilzomib), immunomodulatory

71 drugs (lenalidomide, pomalidomide) and monoclonal antibodies (daratumumab,

72 elotuzumab), 5-year overall survival rate is only 45% (3). Genetic complexity and

73 clonal heterogeneity are the main reasons for cancer treatment failure in MM patients

74 (4). Thus, the identification of a key driver gene for MM may enable the specific

75 targeting of these malignant cells.

76 Accumulating evidence has shown that dysregulated thyroid hormone

77 receptor-interacting protein 13 (TRIP13) protein levels are operational in several

78 tumors, including breast, liver, gastric, lung, prostate cancer, human chronic

79 lymphocytic leukemia, and Wilms’ tumor (5,6). TRIP13 is the mouse ortholog of

80 pachytene checkpoint 2 (Pch2) (7). During mitosis, TRIP13 regulates the spindle

81 assembly checkpoint via remodeling of its effector MAD2 from a ‘closed’ (active) into

82 an ‘open’ (inactive) form (8). During meiosis, TRIP13 was found to regulate meiotic

83 recombination in Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila

84 (9). A recent study indicated that TRIP13 enhanced NHEJ repair and induced treatment

85 resistance via binding to NHEJ proteins KU70/KU80/DNA-PKcs in head and neck

86 cancer (10).

87 In our previous study, TRIP13 was identified as a chromosome instability gene

88 that was correlated with MM drug resistance, disease relapse and poor outcomes in

89 MM patients (11). TRIP13 was first identified by yeast two-hybrid screening as a

90 protein fragment that was associated with thyroid hormone receptor in a

91 hormone-independent fashion (12). Overexpressing TRIP13 in cancer cells prompted

92 cell growth and drug resistance, while targeting TRIP13 by TRIP13 shRNA inhibited

93 MM cell growth, induced cell apoptosis and reduced the tumor burden in xenograft

94 MM mice (11). Our previous results suggested that TRIP13 might serve as a biomarker

95 for MM disease development and prognosis, making it a potential target for future

96 therapies.

97 To identify a TRIP13 inhibitor, detailed structural information of TRIP13 is

98 essential. Although the reported crystal structure of the TRIP13 mutant (E253Q or

99 E253A) provided insight into the mechanism of substrate recognition (8), further

100 structural information of the wild-type TRIP13 protein is needed for specific inhibitor

101 development. In this study, we determined the crystal structure of the wild-type human

102 TRIP13 at a resolution of 2.6 Å. We then identified small molecular inhibitors of

103 TRIP13 based on its crystal structure via molecular docking and bioassay. A small

104 molecular inhibitor, designated DCZ0415, was confirmed to bind to TRIP13 by

105 pull-down, NMR spectroscopy, SPR assays. DCZ0415 exhibited significant 4

106 anti-myeloma activity in vitro, in vivo and in patient MM cells. Importantly, DCZ0415

107 also synergized with melphalan and the histone deacetylase (HDAC) inhibitor

108 panobinostat in MM cells.

109 Materials and Methods

110 Cell lines and patient samples

111 U266, HEK293T, MOPC-315 and HS-5 cells were commercially obtained from the

112 American Type Culture Collection (ATCC) (Mananssas, VA, USA). ARP-1,

113 OCI-MY5, RPMI-8226, and H929 cells were provided by Dr. Fenghuang Zhan

114 (University of Iowa, Iowa City, IA, USA). Cell lines were certificated by STR analysis

115 (Shanghai Biotechnology Co., Ltd., Shanghai, China). Mycoplasma testing was

116 performed using MycoAlert Mycoplasma Detection Kit (Basel, Switzerland) according

117 to the manufacturer’s recommended protocols. MM cells were maintained in RPMI

118 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum

119 (FBS; Gibco, BRL, USA) and 1% penicillin-streptomycin (PS; Gibco, Carlsbad, CA,

120 USA). Human HS-5, HEK293T and mouse MOPC-315 cells were maintained in

121 Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA)

122 supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were

123 maintained in a humidified atmosphere of 5% CO2 at 37 °C, subcultured every 3 days

124 and passaged routinely for use until passage 20. Bone marrow samples were obtained

125 from MM patients after obtaining written informed consent at the Department of

126 Hematology Shanghai Tenth People’s Hospital (Shanghai, China). The protocol for

127 collection and usage of clinical samples was approved by the Shanghai Tenth People’s

128 Hospital Ethics Committee. Informed consent was obtained in accordance with the

129 Declaration of Helsinki.

130 Reagents and antibody

131 DCZ0415 was synthesized by Shanghai Institute of Materia Medica, Chinese Academy

132 of Sciences, Shanghai, China. Antibodies for Caspase-3, Caspase-8, Caspase-9, mouse

133 CD4, CD3 and CD8 were purchased from Cell Signaling Technology (Danvers, MA, 5

134 USA); TRIP13, BAX, BCL2, CDK4, CDK6, Cyclin D, p-p65, α-tublin, TUNEL,

135 Ki-67 and β-actin were from Abcam (Cambridge, MA, USA). Annexin V-FITC and

136 propidium iodide (PI) detection kit was purchased from BD Pharmingen (San Diego,

137 CA, USA). Penicillin-streptomycin was purchased from Invitrogen (Carlsbad, CA,

138 USA). Puromycin and Biotin was purchased from Sigma (St.Louis, MO, USA).

139 Pull-down assay

140 Cells were harvested, aspirated, and washed with cold PBS; they were then lysed and

141 centrifuged at 8000g at 4°C. The lysate was then incubated with 10 µl of

142 DCZ0415-biotin (50 μM) or biotin (50 μM) for 2 h in the presence of neutrAvidin

143 agarose resins (Thermo Scientific) with rotation at 4°C. The solution was then

144 centrifuged at 1500 g and the supernatant was discarded; it was then washed twice with

145 PBS, centrifuging after each wash, resuspended in SDS, and analyzed by

146 immunoblotting.

147 Surface plasmon resonance (SPR)

148 TRIP13 protein was prepared in 10mM sodium acetate (PH = 5.5) and then covalently

149 immobilized on CM5 sensor chip xia amine-coupling procedure. The rest binding sites

150 of the sensor chip were blocked by ethanolamine. The kinetic measurements of

151 compounds were performed at 25 °C with Biacore T2000 (GE Healthcare). In this step,

152 compounds were diluted at different concentrations in PBS buffer (10mM HEPES PH

153 = 7.4, 150 mM NaCl, 3 mM EDTA), and were flowed over the chip at rate of 30

154 ml/min. The combining time and dissociation time was set at 120 s and 150 s,

155 respectively. Data analysis was finished via the state model of T2000 evaluation

156 software (GE Healthcare).

157 Cell viability assay

158 Cell viability assay was performed as previously described (13). Briefly, cells were

159 seeded in triplicate in 96-well plates and then treated with DCZ0415. Cell viability

160 was measured using the Cell Counting Kit (CCK)-8 assays.

161 Apoptosis assay

162 Apoptosis assay was performed as previously described (14). Briefly, cells were

163 treated with or without DCZ0415. Then, cells were collected and stained with

164 Annexin-V for 15 min and then PI for 5 min at room temperature. Stained cells were

165 detected via using flow cytometry.

166 Crystallization, data collection, and structural determination

167 Wild type TRIP13 protein was mixed with AMP-PNP at a molar ratio of 1:2 and

168 incubated on ice for 1 h to allow complex formation. Crystallization was achieved by

169 sitting-drop vapor diffusion at 4 °C with the well solution containing 0.1 M bicine, pH

170 9.0, and 10% (v/v) (+/-)-2-Methyl-2,4-pentanediol. Crystals were gradually transferred

171 to a harvesting solution containing the precipitant solution and 25% glycerol, prior to

172 flash-freezing them in liquid nitrogen for storage. Native and Se-Met-SAD datasets

173 were collected under cryogenic conditions (100 K) at the beamlines BL18U1 and

174 BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF), and were processed

175 using the program HKL3000 (15). The Single-wavelength Anomalous Diffraction

176 (SAD) data phases were calculated using the CCP4i (16) suite and four selenium atoms

177 were located and refined. The initial SAD map was significantly improved by solvent

178 flattening. A model was built into the experimental electron density using the programs

179 CCP4i and Coot (16) and further refined in the program Phenix (17). The native

180 structure was determined by molecular replacement using the crystal structure of

181 Se-Met TRIP13 as the initial model, and further refined in Coot and Phenix (17).

182 Figures of the crystal structures were generated with the program PyMOL

183 (Schrodinger L. The PyMOL Molecular Graphics System, Version 1.8. 2015).

184 Plasmids for TRIP13 WT and TRIP13 MT expression

185 The oligonucleotide sequence specific for TRIP13 silencing (sgRNA) were designed.

186 The packaging plasmids VSVG and psPAX2 were used to produce recombinant

187 lentivirus by transfecting HEK293T cells. Lentiviral transduction of myeloma cell lines

188 was performed using polybrene. Stable cell lines were selected with puromycin (2.5

189 ug/ml). The efficiency of viral transduction was > 95%. Then, PCDH constructs were

190 used to generate human TRIP13 WT and TRIP13 MT overexpression plasmids for

191 transfection into sgTRIP13 cells.

192 DNA double-strand break (DSB) repair assay

193 DNA double-strand break (DSB) repair assay was performed as previously described

194 (18). Briefly, the efficiency of nonhomologous end joining (NHEJ), homologous

195 recombination (HR) was measured using a GFP-based reporter system

196 Tumor xenograft models

197 (1) Nude mice (6-weeks-old) were purchased from Shanghai SLAC Laboratory

198 Animal Co., Ltd. (Shanghai, China) Human H929 cells (1×106) in 100L of serum-free

199 culture medium were subcutaneously injected into the upper flank region of the nude

200 mice. After the tumor growth of mice, mice were randomly assigned to 2 groups: the

201 control group (DMSO, Tween-80 and saline), 50 mg/kg DCZ0415-treated group

202 (dissolved in DMSO, Tween-80 and saline solution).

203 (2) BALB/c mice (6-weeks-old) were injected subcutaneously in the right flank with or

204 without 5 × 105 MOPC-315 cells in a volume of 0.1 mL. After tumor growth of mice,

205 mice were randomly assigned to 2 groups: the control group (DMSO, Tween-80 and

206 saline), 25 mg/kg DCZ0415-treated group (dissolved in DMSO, Tween-80 and saline

207 solution). Mice were then administered with or without 25 mg/kg DCZ0415 via

208 intraperitoneal injection every day for 15 days. All mice were euthanized at the end of

209 the experiment and tumors were photographed. Tumor volumes were measured using a

210 vernier caliper and calculated using the formula: tumor volume (mm3) = 1/2

211 ×(relatively shorter diameter)2×(relatively longer diameter). All animal studies were

212 approved by the Institutional Review Board of Shanghai Tenth People’s Hospital (ID:

213 SYXK 2011-0111).

214 Statistical analysis

215 Statistical analyses were performed using Prism software (GraphPad). Data are

216 expressed as means ± standard deviation (SD). Data were considered statistically 8

217 significant at p < 0.05. The Student’s t-test was used to compare two groups. The

218 log-rank test was used for survival curves. The combination index (CI) values were

219 calculated by median dose effect analysis using commercially available software

220 (CalcuSyn; Biosoft). All tests of statistical significance were two sided.

221 Results

222 Determination of the crystal structure of wild-type human TRIP13

223 We set out to study the crystal structure of wild-type human TRIP13 (wt_hTRIP13) to

224 aid the discovery and rational design of TRIP13 inhibitors for MM therapeutics.

225 Wild-type human TRIP13 protein exists as a mixture of monomer and oligomers in

226 solution (Fig. S1A). To obtain homogeneous sample for crystallization, we further

227 purified wt_hTRIP13 monomer using mono-Q ion exchange chromatography and gel

228 filtration chromatography. We used AMP-PNP (a non-hydrolysable analogue of ATP)

229 to co-crystallize with wt_hTRIP13. AMP-PNP has a binding affinity of 49 µM to

230 wt_hTRIP13 (Fig. S1B). We determined the crystal structure of wt_hTRIP13 by

231 single-wavelength anomalous dispersion at a resolution of 2.6 Å (Table S1). There is

232 one copy of wt_hTRIP13 per asymmetric unit and the crystal packing belongs to the

233 space group P65. Similar to recently reported mutant structure of human TRIP13

234 (TRIP13E253Q) (8), wt_hTRIP13 assembled into a helical filament instead of the classic

235 hexamer ring of the AAA+ ATPase family, probably due to the crystal packing (Fig.

236 S1C).

237 The wt_hTRIP13 monomer comprises three domains: an N-terminal domain (NTD)

238 that is involved in substrate recognition, and the large and small AAA domains that

239 form the catalytic site for ATP hydrolysis (Fig. 1A and 1B). Positive electron densities

240 could be observed inside the ATP-binding cleft of TRIP13 in the Fo-Fc map of the

241 wt_hTRIP13 structure, but we failed to fit the AMP-PNP molecule into this density.

242 Superposition of the wt_hTRIP13 structure with the structure of TRIP13E253Q mutant in

243 complex with ATP (PDB: 5VQA) revealed that the base and phosphate groups of ATP

244 happens to occupy the positive electron densities (Fig. 1C). The key residues that

245 participate in coordinating and hydrolyzing ATP, such as K185/T186 of the Walker A

246 motif, N300 of the Sensor 1 motif, and R386 of the Sensor 2 motif, adopt almost

247 identical conformations between the two structures (Fig. 1C). However, as for the

248 residue E253 of the Walker B motif, its side chain sticks outward from the ATP-binding

249 cleft and seems not to participate in ATP-binding as the residue Q253 in the

250 TRIP13E253Q mutant (Fig. 1C). Residue E253 has a higher b factor compared with the

251 other parts of the structure, indicating that its conformation might be dynamic and

252 prone to alternate.

253 TRIP13 inhibitor DCZ0415 binds to TRIP13

254 Based on our wt_hTRIP13 structure, structure-based molecular docking was applied to

255 virtually screen our in-house compound library with 8,000 compounds using Smina, a

256 fork of AutoDock Vina, with default parameters (19,20). Of the compounds identified,

257 76 were selected for biological testing (Table S2). Viability test of MM cells revealed

258 some active compounds, of which DCZ0415 was the most promising inhibitor based

259 on biology screening (Fig. 2A).

260 To further validate whether DCZ0415 targeted TRIP13, a series of assays were

261 performed. We employed an affinity pull-down target verification system, in which

262 DCZ0415 was conjugated with a biotin. Compared with the unconjugated biotin, the

263 addition of DCZ0415-biotin to the cell lysate brought down endogenous TRIP13 (Fig.

264 2B). And a 2% input of total cell lysate was tested by immunoblotting analyses (right)

265 (Fig. 2B). In this study, we measured the interaction between DCZ0415 and TRIP13

266 using nuclear magnetic resonance (NMR). We observed that the CPMG spectra of 200M

267 DCZ0415 with the addition of 5, 8, 10M TRIP13 (Fig. 2C) and the STD spectrum of

268 200 M DCZ0415 with the addition of 5 M TRIP13 both showed the interaction (Fig.

269 2D). In our study, we measured the interaction between TRIP13 and DCZ0415 using

270 SPR assay. The binding affinity (KD) was calculated from SPR data. The

271 measurements displayed a strong affinity with a KD value of 2.42±1.26 M (Fig. 2E

272 and F).

273 DCZ0415 inhibits MM cell growth and induces apoptosis 10

274 To evaluate the inhibitory effect of DCZ0415, MM cells were treated with DCZ0415

275 for 72 h and cell viability was assessed. Data showed that DCZ0415 induced a

276 significant dose-dependent decrease in viability (Fig. 3A). Using CCK-8 assay, the

277 half-maximal inhibitory concentration (IC50) of DCZ0415 was 1.0–10M calculated

278 by CalcuSyn in MM cell lines. To further investigate the anti-myeloma activity of

279 DCZ0415, two representative cell lines were treated with DCZ0415 (0–40 M) for 24,

280 48, and 72 h. We observed that DCZ0415 decreased cell viability in a time- and

281 dose-dependent manner (Fig. S2A). To examine the effect of DCZ0415 on the colony

282 formation of MM cells, soft agar clonogenic assays were performed. MM cells treated

283 with DCZ0415 showed a significant decrease in colony formation, indicating that this

284 compound inhibits cell proliferation (Fig. 3B and S2B). EdU assays were employed to

285 examine if DCZ0415 affects DNA synthesis. Compared to that in the control group,

286 the percentage of EdU-positive cells was significantly decreased with DCZ0415

287 treatment, indicating that DCZ0415 exerts cytotoxic effects by inhibiting DNA

288 synthesis in MM cells (Fig. S2C).

289 Bone marrow stromal cells (BMSCs) mediated the paracrine growth of MM cells

290 and protect against the cytotoxicity of anti-myeloma agents via cytokine secretion (21).

291 To determine whether DCZ0415 could overcome the protective effects of the bone

292 marrow microenvironment, MM cells were cultured with or without HS-5 BMSCs in

293 the presence or absence of DCZ0415 (Fig. 3C and S2D). As a positive control, cells

294 were treated with melphalan with or without BMSCs for the same period of time.

295 Results revealed that DCZ0415 treatment significantly inhibited MM cell viability in

296 the presence and absence of BMSCs, but melphalan-induced cytotoxicity could be

297 abrogated by BMSCs (Fig. S2E).

298 Cytokines interleukin-6 (IL-6) and insulin growth factor-1 (IGF-1), which are

299 secreted by MM cells and BMSCs, have been reported to promote MM cell

300 proliferation, migration, and drug resistance (22). We therefore examined the effect of

301 DCZ0415 on IL-6- or IGF-1-induced MM cell growth. MM cells were cultured either

302 alone or with IL-6 or IGF-1. Significant inhibition of IL-6- or IGF-1-induced MM cell

303 growth was observed with DCZ0415 treatment (Fig. 3D and S2F), suggesting that this 11

304 compound not only directly targets MM cells, but can also overcome the cytoprotective

305 effects of the host bone marrow microenvironment.

306 We next examined whether DCZ0415 functioned by inducing apoptotic cell death

307 (23). MM cells were exposed to various concentrations of DCZ0415, leading to

308 significant increases in the proportion of early- (Annexin-V+/PI−) and late-stage

309 (Annexin-V+/PI+) apoptotic cells compared to that in control cells exposed to DMSO

310 (Fig. 3E and S3A). To determine whether DCZ0415 could induce apoptosis in patient

311 MM cells, purified CD138+ cells were examined from six newly diagnosed and four

312 refractory/relapsed MM patients who were refractory to bortezomib. Patient cells

313 showed a dose-dependent relationship between DCZ0415 treatment and apoptotic cell

314 death. This confirmed that DCZ0415 can trigger cytotoxicity in bortezomib-resistant

315 primary myeloma cells. In contrast, DCZ0415 does not significantly induce normal

316 peripheral blood mononuclear cells (PBMCs) apoptosis (Fig. 3F). And we tested the

317 sensitivity of cells of PBMCs and patients to TRIP13 expression. The results showed

318 that cells with high expression of TRIP13 were more sensitive to DCZ0415 than those

319 with low expression of TRIP13 (Fig. 3F). Because BCL2 family proteins affect

320 apoptosis via the regulation of cytochrome C release, which then mediates caspase

321 activation (24), the effects of DCZ0415 on the expression of caspase enzymes,

322 anti-apoptotic BCL2, and pro-apoptotic protein BAX were evaluated.Caspase-8 and 9

323 activities increased in a dose-dependent manner in MM cells treated with DCZ0415 for

324 48 h. Furthermore, DCZ0415 treatment decreased the expression of BCL2 in a

325 dose-dependent manner but increased the expression of BAX (Fig. S3B). Also

326 reduction of TRIP13 induced similar increase in BAX and decrease in BCL2 (Fig.

327 S3C). To determine the dependence of DCZ0415-induced apoptosis on the caspase

328 pathway, we assessed the ability of the pan-caspase inhibitor Z-VAD-FMK to protect

329 against cell apoptosis. As shown in Figure 3G, Z-VAD-FMK partially blocked

330 DCZ0415-induced cell apoptosis as determined by Annexin V-PI staining. These data

331 demonstrate that DCZ0415 triggers caspase-dependent apoptosis in MM cells. In

332 addition to apoptosis, effects on cell cycle progression might be important for the

333 action of anti-cancer drugs. We therefore evaluated the effects of DCZ0415 on cell 12

334 cycle progression by flow cytometry analysis. Treatment induced a significant

335 accumulation in G0/G1 MM cells (Fig.3H and S3D). As essential components of the

336 cell cycle machinery, cyclins function to bind and activate specific cyclin dependent

337 kinase (CDK) partners. Thus, protein levels of CDK4, CDK6, and Cyclin D were

338 evaluated. In agreement with flow cytometry data, we observed a marked

339 dose-dependent decrease in Cyclin D, CDK4, and CDK6 after DCZ0415

340 administration (Fig. S3E).

341 The anti-myeloma activity of DCZ0415 depends on TRIP13

342 To determine if the anti-myeloma activity of DCZ0415 is dependent on TRIP13,

343 TRIP13-sgRNA and point mutation plasmids were designed. Treatment of sgTRIP13

344 cells with DCZ0415 led to the loss of sensitivity compared to that of sgControl

345 transfected wild-type cells (Fig. 4A). And sgTRIP13 and sgControl cells were

346 separately treated with melphalan or left untreated. The results showed that sgTRIP13

347 and sgControl cells were both sensitive to melphalan (Fig.S4A and S4B). This finding

348 suggested that cells with deleted TRIP13 were specifically resistant to DCZ0415.

349 We evaluated the effects of DCZ0415 on cell cycle progression in sgControl and

350 sgTRIP13 cells by using flow cytometry analysis. The sgControl cells were blocked at

351 G0/G1 stage under DCZ0415 treatment. However, compared with in sgControl cells,

352 sgTRIP13 cells showed no significant changes in the cell cycle under DCZ0415

353 treatment (Fig. S4C). These results suggested that the anti-myeloma activity of

354 DCZ0415 depends on TRIP13. Based on the structure of TRIP13 and DCZ0415, we

355 speculated that valine (V140), serine (S187), and arginine (R386) of TRIP13 are

356 essential for DCZ0415 binding. We thus mutated TRIP13 V140/S187/R386 (TRIP13

357 WT) to TRIP13 alanine (A) 140/187/386 (TRIP13 MT); we then down-regulated

358 endogenous TRIP13 expression using the sgTRIP13 target intron sequence and

359 overexpressed TRIP13 WT or TRIP13 MT. Pull-down assay shown that DCZ0145

360 bound to TRIP13 WT cells, but not TRIP13 MT cells (Fig. 4B). Cell viability of was

361 compared among the groups (TRIP13 WT and TRIP13 MT) with DCZ0415 treatment.

362 We found that DCZ0415 decreased cell viability in the TRIP13 WT group, whereas

363 TRIP13 MT cells were resistant to DCZ0415, as compared to TRIP13 WT cells. The

364 protein expression of TRIP13 was also examined among these groups (Fig. 4C). This

365 suggests that DCZ0415-TRIP13 binding is essential for the anti-myeloma activity of

366 DCZ0415. Our findings suggested that TRIP13 is essential for the anti-myeloma

367 activity of DCZ0415.

368 DCZ0415 impaired DNA repair and inhibited NF-κB activity in MM cells

369 Some anti-cancer agents sensitize cancer cells by inducing DNA DSBs and DNA

370 damage responses (25). In the response of mammalian cells to DNA DSBs,

371 phosphorylation of histone H2AX (γ-H2AX) at sites proximal to the DNA breaks has

372 been reported (26). In this study, γ-H2AX levels were evaluated in MM cells following

373 DCZ0415 treatment by immunofluorescence analysis. The results revealed that

374 γ-H2AX levels were higher in MM cells treated with DCZ0415 than baseline γ-H2AX

375 levels (untreated control), which reflected ongoing DNA damage (Fig. 5A). Ataxia

376 telangiectasia mutated (ATM) protein kinase is a key mediator of this DNA damage

377 response, which induces cell cycle arrest and facilitates DNA repair by activating

378 downstream targets such as the cell cycle checkpoint kinase (CHK2) (27,28). The

379 protein levels of phosphorylated (p)-ATM and phosphorylated (p)-CHK2 were found

380 to be increased in a dose-dependent manner in DCZ0415-treated MM cells compared

381 with the untreated control cells (Fig. S5A). A DNA damage response accompanied by

382 efficient and appropriate repair of DSBs is essential for the preservation of genomic

383 integrity. However, in cancer, the repair of anti-cancer agent-induced DSBs by the

384 NHEJ or HR repair pathways promotes treatment resistance and subsequent relapse in

385 patients (26). TRIP13 also enhanced NHEJ repair and induced treatment resistance via

386 binding to NHEJ proteins KU70/KU80/DNA-PKcs in head and neck cancer (10). Thus,

387 we evaluated whether DCZ0415 impaired DNA repair via the NHEJ repair pathways

388 using GFP-based reporter assay, which was excellent tool to measure the efficiency of

389 NHEJ repair. The results indicated that DCZ0415 suppressed the NHEJ repair pathway

390 (Fig. 5B), which was consistent with TRIP13 promoting DSB-induced NHEJ repair.

391 And we confirmed that TRIP13 interacted with the NHEJ key regulator KU70/KU80

392 (Fig. S5B). Besides, our results shown that 10 M DCZ0415 had a little effect on HR

393 repair pathway, but 20 M DCZ0415 could significantly inhibit the HR repair (Fig.

394 S5C). With DCZ0415 treatment, the protein level of γ-H2AX increased in TRIP13 WT

395 cells. However, the protein level of γ-H2AX in TRIP13 MT cells, which did not bind

396 DCZ0415, showed no significant change under DCZ0415 treatment (Fig. 5C). We

397 studied NHEJ repair in TRIP13 MT and WT cells using a GFP-based reporter assay.

398 Following DCZ0415 treatment, greater levels of NHEJ repair were detected in TRIP13

399 MT cells compared with TRIP13 WT cells (Fig. 5D). This suggested that TRIP13 MT

400 cells were resistant to DCZ0415, associated with diminished DNA damage and greater

401 NHEJ repair compared to TRIP13 WT cells. Besides, to address the impact of

402 DCZ0415 on mitotic spindles, α-tubulin/DAPI staining was performed. As shown in

403 Figure S5D, DCZ0415 induced spindle multipolarity, suggesting that DCZ0415 acted

404 as a spindle poison, which supported the involvement of TRIP13 in regulation of the

405 mitotic spindle.

406 NF-κB is aberrantly activated in MM and promotes cell survival and malignancy

407 by upregulating anti-apoptotic genes (29). To investigate the possible contribution of

408 the NF-κB signaling pathway to pathogenesis, we investigated whether iκBα and

409 NF-κB p65 phosphorylation were decreased by DCZ0415 treatment. Compared with

410 the untreated control cells, the protein levels of phosphorylated (p)-iκBα and

411 phosphorylated (p)-NF-κB p65 were decreased in MM cells treated with DCZ0415

412 (Fig. 5E). And DCZ0415 showed a strong inhibitory effect on NF-κB–promoter

413 luciferase activity (Fig. 5F). As shown in Figure 5G, DCZ0415-induced cell apoptosis

414 could be partially rescued by Tumor Necrosis Factor (TNF)-α. These results suggested

415 that cell death induced by DCZ0415 may be mediated via the inhibition of the NF-κB

416 signaling pathway. Importantly, TRIP13 increased NF-κB–promoter luciferase activity

417 (Fig. S5E).

418 Combined treatment with DCZ0415 and melphalan or HDAC inhibitor

419 panobinostat induces synergistic anti-myeloma activity

420 To further evaluate its preclinical efficacy, we investigated the effects of DCZ0415 on

421 MM cell growth when used in combination with other anti-myeloma agents. The

422 cytotoxicity of combined DCZ0415 and melphalan (30) was examined in MM cells.

423 Melphalan-induced growth inhibition was enhanced with increasing concentrations of

424 DCZ0415. Calculation of the CI values using CalcuSyn software revealed synergistic

425 effects of DCZ0415 on melphalan against MM cells (Fig. 6A and B). Then we tested

426 DCZ0415 in combination with HDAC inhibitor panobinostat (31) in MM cells.

427 Isobologram analysis revealed synergy between DCZ0415 and panobinostat with a CI

428 less than 1 (Fig. 6C and D). This provided evidence for the beneficial effects of

429 combination therapy, which could effectively reduce the required concentration of

430 other anti-myeloma agents, thereby reducing the potential side effects.

431 MM xenografts are sensitive to DCZ0415

432 To investigate the therapeutic potential of DCZ0415 in vivo, an MM mouse xenograft

433 model was employed. The administration of DCZ0415 significantly reduced the

434 growth of MM cells-induced tumors in immune-deficient mice compared with control

435 mice (Fig. 7A). There were no significant differences in the body weight of nude mice

436 in each group, suggesting that DCZ0415 was well tolerated (Fig. 7B). The toxicity of

437 DCZ0415 was also examined by hematoxylin and eosin (HE) staining of major organs

438 and no significant histological changes were observed in the liver and kidney of the

439 mice (Fig. S6A), indicating that the side effects of DCZ0415 were minimal.

440 Significantly, treatment with DCZ0415 resulted in a significant prolongation in overall

441 survival compared to vehicle-treated animals (Fig. 7C).

442 Furthermore, we performed a pharmacodynamic study whereby the harvested

443 tumors were analyzed for anti-proliferation and apoptosis markers. DCZ0415-treated

444 mice exhibited decreased tumor Ki-67 and p-p65 levels compared with control mice.

445 However, we observed an increase in cleaved Caspase-3, TUNEL (apoptosis markers)

446 and γ-H2AX following DCZ0415 treatment of MM cells compared with control cells

447 (Fig. 7D).

448 In order to examine DCZ0415 effect on immunocompetent mice, we injected

449 MOPC-315 cells subcutaneously into BALB/c mouse，and treated with 25 mg/kg

450 DCZ0415 or not. With DCZ0415 treatment, the growth of MM cells-induced tumors

451 significantly was reduced compared with control mice (Fig. 7E). There were no

452 significant changes in the body weight of BALB/c mice in each group (Fig. 7F).

453 Immunohistochemistry (IHC) studies for CD4, CD3 and CD8 expression from mice

454 post sacrifice shown that treatment with DCZ0415 significantly increased immune

455 effect cells infiltration, as evidenced by much greater CD4, CD3 and CD8 staining

456 surrounding MOPC-315 cells remains (Fig. 7G). There was no significant histological

457 change in the liver and kidneys of mice, as detected by HE staining, indicating that the

458 side effects of DCZ0415 were minimal (Fig. S6B).

459 These findings suggest that DCZ0415 yields potent anti-myeloma responses in

460 mice, prolonging their survival times. These data indicate that TRIP13 inhibitors

461 developed using this approach may provide a roadmap for candidate therapeutic agents

462 in MM.

463 Discussion

464 TRIP13 is thought to function as an oncogene based on meta-analysis of gene

465 expression datasets from various cell lines, and is significantly amplified in high-risk

466 MM patients (32). We previously showed that knockdown of TRIP13 inhibited the

467 growth of MM both in vitro and in vivo (11). We found that TRIP13 was

468 overexpressed in human MM cell lines, further supporting a role for TRIP13 as an

469 oncogene in MM. Therefore, we suggested that TRIP13 might represent an

470 anti-myeloma target and inhibition of TRIP13 could be a promising strategy in MM

471 therapy.

472 Protein crystal structures can offer invaluable insight into the molecular

473 mechanisms of action of specific proteins. The mechanisms of substrate recognition

474 and remodeling of TRIP13 were revealed by the crystal structure of human

475 TRIP13E253Q (8). However, it remained necessary to analyze the full-length, wild-type,

476 human TRIP13. In this study, the crystal structure of the wild-type TRIP13 protein was

477 resolved, which not only contributed towards our understanding of the molecular

478 mechanisms of TRIP13 activity, but also helped to identify new inhibitors against 17

479 TRIP13 activity.

480 DCZ0415 was identified via structure-based molecular docking and cellular

481 screening from in-house compound library. Our proof-of-concept experiments, which

482 include pull-down, NMR, and SPR assays, provide evidence that DCZ0415 binds to

483 TRIP13. Our studies employed MM cell lines, patient MM cells and xenograft models,

484 along with biochemical and genetic models, to demonstrate the anti-myeloma activity

485 of the TRIP13 inhibitor DCZ0415. DCZ0415 displayed highly potent anti-myeloma

486 activity against a large panel of MM cell lines. Furthermore, DCZ0415 results in a

487 significant reduction of the proliferation rate in MM cells as evidenced by colony

488 formation and EdU expression assays. The DCZ0415-induced reduction in

489 proliferation was associated with inducing apoptosis, arresting the cell cycle.

490 We previously reported that TRIP13 was an oncogene in MM; however, the

491 detailed mechanism by which TRIP13 promotes cell growth was not fully explained. In

492 this study, we found that DCZ0415 (a TRIP13 inhibitor) induced cell death via

493 inhibition of the NF-κB signaling pathway. Our results suggest that TRIP13 acts as an

494 oncogene by activating the NF-κB signaling pathway in MM. Using a cellular assay to

495 mimic MM in its microenvironment, we observed that DCZ0415 inhibited growth

496 inhibition of MM cells even in the presence of BMSCs and the cytokines IL-6 and

497 IGF-1. That suggested that besides its cytotoxicity on MM cells, DCZ0415 also targets

498 the BM microenvironment and overcomes the proliferative effects of BMSCs (Fig. 3C).

499 In BM microenvironment, MM cell adhesion to BMSCs triggers the NF-κB-dependent

500 transcription and secretion of cytokines such as IL-6 in BMSCs, which further

501 stimulate MM cell growth, survival, drug resistance (22,33,34). Moreover, activation

502 of NF-κB by cell adhesion and cytokines augments the binding of multiple myeloma

503 cells to BMSCs, which in turn induces IL-6 transcription and secretion in BMSCs.

504 Conversely, the inhibition of NF-κB activity abrogates this response (22,33,34). Our

505 data demonstrate that DCZ0415 inhibits NF-κB activity (Fig. 5E and 5F) and TRIP13

506 activated NF-κB pathway (Fig. S5E). Thus, DCZ0415 might prevent MM progression

507 by targeting TRIP13 and inhibiting NF-κB-dependent transcription and secretion of

508 cytokines in BMSCs and the expression of many cell adhesion molecules on both MM 18

509 cells and BMSCs, thereby disrupting the tumor-BM microenvironment interactions

510 that contribute to MM progression (33). Our results suggested that treatment of cells

511 with DCZ0415 was correlated with inhibition of NF-κB activity, cell cycle blockade

512 and apoptosis, although further in-depth study is required. In the future, we will

513 identify the mechanism underlying DCZ0415 treatment by using genetic tools.

514 Some anti-cancer agents induce apoptosis by enhancing DNA damage; however,

515 this is amended by DNA repair, which increases cell survival and induces drug

516 resistance (35,36). Thus, DNA repair inhibitors have received increased attention in

517 recent years. For example, it was reported that SCR7 was a putative inhibitor of NHEJ,

518 blocking end-joining by interfering with ligase IV binding to DNA, thereby leading to

519 accumulation of DSBs within the cells and culminating in cytotoxicity (36). DCZ0415

520 not only enhanced DNA damage, but also impeded DNA repair. As an inhibitor of

521 TRIP13, the impediment of DNA repair by DCZ0415 is understandable, which is

522 consistent with previous data that TRIP13 is essential for DSB repair (10). For

523 example, it was revealed that instead of having a checkpoint role, TRIP13 was required

524 for one of the two major classes of recombination in meiosis that was required for

525 repairing DNA breaks. TRIP13 was also shown to be involved in DNA repair induced

526 by programmed DSBs in meiotic recombination (37). Our study indicated that

527 DCZ0415 impaired NHEJ, as shown in Figure 5B. NHEJ is the major DSB repair

528 pathway in mammalian cells and is activated by DSB ends being recognized by the KU

529 (KU70 and KU80) heterodimer (38). It was recently reported that TRIP13 promoted

530 NHEJ repair in head and neck cancer via binding of KU70 and KU80 (10). Consistent

531 with this, we observed endogenous binding between TRIP13 and KU70/KU80 in MM

532 cells, which also indicated that TRIP13 played a key role in NHEJ repair.

533 Resistance mechanisms present the largest hurdle to the cure of MM and it can

534 arise initially or emerge during the course of treatment (39). Thus, to examine the

535 synergistic effects of DCZ0415 and other anti-myeloma agents, we tested the effects of

536 DCZ0415 in combination with melphalan and panobinostat. Promisingly, DCZ0415

537 was able to enhance the cytotoxic effects of both melphalan and panobinostat in MM

538 cells. This suggests that DCZ0415 may have promising anti-myeloma activity both 19

539 alone and combined with other anti-myeloma agents. In summary, our studies

540 presented crystal structure of the wild-type human TRIP13 and identified an inhibitor

541 of TRIP13. The inhibitor, named as DCZ0415, effectively induced anti-myeloma

542 activity in MM, both in vitro and in vivo by impairing DSB repair and inhibiting

543 NF-κB pathway. Our present data suggests that TRIP13 could be an important target

544 for refractory/relapsed MM.

545 Acknowledgments

546 This work was supported by Grants from the National Natural Science Foundation of

547 China (Grant No. 81570190, 81870158, 31570766, U1632130, 81602515, 81529001,

548 and 81670194), National Key R&D Program of China (Grant No. 2016YFA0501803,

549 2017YFA0504504, and 2016YFA0502301), and Chinese Pharmaceutical Association -

550 Yiling Biopharmaceutical Innovation Project (CPAYLJ201908).

551 We thank D Yao, L Wu, W Qin, and R Zhang from the beamlines BL18U1 and

552 BL19U1 at National Center for Protein Science Shanghai (NCPSS) and Shanghai

553 Synchrotron Radiation Facility (SSRF) for help with crystal data collection.

554 References

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671

672

673

674

675

676

677

678

679

680 Figure Legends

681 Figure 1. Crystal structure of wt_hTRIP13

682 (A) Domain organization of the wt_hTRIP13 monomer. The N-terminal domain (NTD)

683 is shown in yellow, the large AAA domain in blue, the small AAA domain in green and

684 other regions in grey. (B) Crystal structure of wt_hTRIP13 monomer at 2.6 Å. The

685 N-terminal domain (NTD) is shown in yellow, the large AAA domain in blue, and the

686 small AAA domain in green. The ATP-binding cleft is shown in the box. (C) Enlarged

687 view of the ATP-binding cleft as boxed in Figure 1B. The structure (colored as in

688 Figure 1B) is superposed onto the structure of TRIP13E253Q-ATP (PDB: 5VQA).

689 Positive electron density of the Fo-Fc map inside the ATP-binding cleft of wt_hTRIP13

690 is contoured at 3.0 σ and colored in grey. See also Figure S1.

691 Figure 2. Binding of DCZ0415 to TRIP13

692 (A) The structure of DCZ0415 (upper panel) and its binding mode to TRIP13 as

693 determined by molecular docking (lower panel). Three residues forming hydrogen

694 bonds are shown as gray sticks, DCZ0415 as yellow sticks, and hydrogen bonds as

695 yellow dashes. (B) A pull-down assay was used to detect the binding of

696 DCZ0415-biotin to TRIP13. Immunoblotting analyses of the input proteins (right

697 panels). (C) CPMG spectra was acquired using 200 M of DCZ0415 alone (colored in

698 red) and 200 M of DCZ0415 with the addition of 5, 8 or 10 M of TRIP13 (colored

699 in cyan, green, and blue, respectively). (D) The STD spectrum was acquired using 200

700 M of DCZ0415 with the addition of 5 M of TRIP13. (E and F) SPR biosensor was

701 used to detect the binding of DCZ0415-biotin to TRIP13. Apparent KD value is

702 calculated by SPR the data. The fitted KD is 2.42±1.26 M.

703 Figure 3. DCZ0415 inhibits the proliferation of MM cell lines and induces

704 apoptosis

705 (A) Cell viability of MM cells with DCZ0415 treatment for 72 h at the indicated

706 concentrations. IC50 values were the means from three independent experiments. (B)

707 Soft agar colony formation of ARP-1 cells with DMSO or DCZ0415 treatment at the

708 indicated concentrations. Representative images of colonies are shown in the left

709 panels. Quantification of colony numbers is shown in the right panel. The y-axis

710 represents the percentage of colonies relative to the number of DMSO-treated cells.

711 Statistical evaluation was performed using the Student’s t tests. (C) Activity of

712 DCZ0415 against ARP-1 cell lines cultured in the presence or absence of the HS-5

713 stromal cell line for 48 h. The result was expressed as means ± SD of 3 independent

714 experiments. (D) Activity of DCZ0415 against ARP-1 cell lines cultured in the

715 presence or absence of IL-6 and IGF-1 for 48 h. Error bars represent standard deviation

716 (SD). The result was expressed as means ± SD of 3 independent experiments. (E) Flow

717 cytometry evaluation of Annexin-V-positive apoptotic cells in DCZ0415-treated ARP-1

718 cells. (F) Flow cytometry evaluation of apoptosis in patient MM cells after DCZ0415

719 treatment for 48 h. Normal PBMCs from healthy donors (PBMCs#1–PBMCs#3) was

720 treated with the indicated concentrations of DCZ0415 for 48 h, and then apoptosis was

721 analyzed. Protein levels of TRIP13 were evaluated in PBMCs#1, PBMCs#2, Pt#2,

722 Pt#3, Pt#9 and Pt#10 cells. (G) ARP-1 cells were incubated with or without

723 pan-caspase inhibitor Z-VAD-FMK for 1 h and then treated with DCZ0415 (0 or 10

724 μM) for 48 h, followed by assessment of cell apoptosis using Annexin V/PI staining.

725 Columns (right panel) represent the average percent of Annexin V positive cells from

726 three independent experiments, which are shown as the mean ± SD. (H) Cell cycle

727 analysis of DCZ0415 (0, 10 and 20 μM, 24h)-treated ARP-1 cells. P values were

728 calculated using the Student’s t tests (*p < 0.05, **p < 0.01, ***p < 0.001). See also

729 Figure S2 and Figure S3.

730 Figure 4. The anti-MM activity of DCZ0415 depends on TRIP13

731 (A) The viability of MM cells transfected with empty vector or TRIP13-sgRNA with

732 DCZ0415 treatment (0, 5, 10, 20 and 40 μM, 48 h) was analyzed by a CCK-8 assay.

733 SgControl represents non-target scramble-transfected cells. TRIP13 sgRNA represents

734 TRIP13-silenced cells. The result was expressed as means ± SD of 3 independent

735 experiments. (B) A pull-down assay was used to test the binding of DCZ0415-biotin

736 with TRIP13 WT cells or TRIP13 MT cells. TRIP13 WT represents TRIP13 wild type

737 overexpression in TRIP13-silenced cells, whereas TRIP13 MT represents TRIP13

738 V140/S187/R386A overexpression in TRIP13-silence cells. (C) Cell viability in

739 TRIP13 WT, and TRIP13 MT groups treated with DCZ0415, as analyzed by a CCK-8

740 assay. The result was expressed as means ± SD of 3 independent experiments.

741 Figure 5. DCZ0415 impaired DNA repair and inhibited NF-κB activity in MM

742 cells

743 (A) Immunofluorescence staining of cellular γ-H2AX in ARP-1 and H929 cells with

744 or without DCZ0415 treatment for 24 h. (B) NHEJ was quantified by GFP and DsRed

745 expression as analyzed by flow cytometry. Error bars indicated SD. The result was

746 expressed as means ± SD of 3 independent experiments. (C) TRIP13 WT and TRIP13

747 MT cells were separately treated with DCZ0415 (0 and 10 μM) for 24 h. TRIP13 WT

748 represents TRIP13 wild type overexpression in TRIP13-silenced cells, whereas

749 TRIP13 MT represents TRIP13 V140/S187/R386A overexpression in TRIP13-silence

750 cells. Expression of γ-H2AX was tested by immunoblotting analyses. (D) TRIP13 WT

751 and TRIP13 MT cells were separately treated with or without DCZ0415 for 48 h.

752 NHEJ was quantified by GFP and DsRed expression as analyzed by flow cytometry.

753 Error bars indicated SD. The result was expressed as means ± SD of 3 independent

754 experiments. (E) Protein level of p-iκBα, iκBα, p-p65 and p65 were evaluated in whole

755 cell lysates from ARP-1 and OCI-MY5 cells after treatment with or without 10μM

756 DCZ0415 for 48 h. (F) NF-κB luciferase reporter was transfected in ARP-1 cells. The

757 cells were treated with or without 10 μM DCZ0415 for 24 h, and then the relative

758 luciferase activity was analyzed. The result was expressed as means ± SD of 3

759 independent experiments. (G) ARP-1 cells were treated with or without 20 μM

760 DCZ0415 for 24 h, with or without 20 ng/ml TNF-α for 1 h, and then analyzed by flow

761 cytometry.

762 Figure 6. DCZ0415 in combination with melphalan or panobinostat functions

763 synergistically to exert cytotoxicity

764 (A and B) ARP-1 (A) and OCI-MY5 (B) cells were treated with DCZ0415 (10-80 μM)

765 plus melphalan (2.5-20 μM) for 48 h, which was followed by a CCK-8 assay to

766 determine cell viability. The synergistic cytotoxic effects of DCZ0415 and melphalan

767 are shown. CI < 1 indicated synergistic activity as determined using CalcuSyn software.

768 The Fa fraction represented the cells affected. (C and D) ARP-1 (C) and OCI-MY5 (D)

769 cells were treated with DCZ0415 (10-80 μM) plus panobinostat (2.5-20 nM) for 48 h,

770 which was followed by a CCK-8 assay to determine cell viability. Synergistic

771 anti-myeloma activity was analyzed. Error bars denote SD. All results were expressed

772 as means ± SD of 3 independent experiments.

773 Figure 7. Anti-tumorigenic effects of DCZ0415 in a xenograft model of MM

774 (A) Average and standard deviation of the tumor volumes (cm3) versus time. H929

775 cells were injected subcutaneously into mice (n = 7/group) and then mice were treated

776 with or without 50 mg/kg DCZ0415 every day for 14 days. Tumor sizes were measured

777 every two days. (B) Averages and standard deviations of nude mouse weights versus

778 the time (mean weight ± SD, 7/group). (C) Graphs of % survival over time (until the

779 tumor volume reached 2,000 mm3) for the duration of the experiment. ‘‘Control’’ and

780 ‘‘DCZ0415’’ represent mice bearing tumors that were treated with the vehicle or

781 DCZ0415, respectively. Kaplan-Meier plots of mice treated with the vehicle or

782 DCZ0415. Survival was significantly increased in DCZ0415-treated mice compared

783 with the control group (n = 9/group). p < 0.001 versus the control group. (D)

784 Representative images of Ki-67, Caspase-3, TUNEL, p-p65 and γ-H2AX

785 immunohistochemical staining of tumor tissues after 14 days of treatment with vehicle

786 or DCZ0415. (E) Average and standard deviation of the tumor volumes (cm3) versus

787 time. MOPC-315 cells were injected subcutaneously into BALB/c mice (n = 5/group)

788 and then mice were treated with or without 25 mg/kg DCZ0415 every day for 15 days.

789 Tumor sizes were measured every two days. (F) Averages and standard deviations of

790 BALB/c mice weights versus the time (mean weight ± SD, 5/group). (G)

791 Representative images of CD3, CD4, and CD8 immunohistochemical staining of tumor

792 tissues after 15 days of treatment with vehicle or DCZ0415.

A Small Molecule Inhibitor Targeting TRIP13 suppresses multiple myeloma progression

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

Cancer Res Published OnlineFirst November 15, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-3987

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