BI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer Cells

Published OnlineFirst March 29, 2019; DOI: 10.1158/1535-7163.MCT-18-0918

Small Molecule Therapeutics Molecular Cancer Therapeutics BI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer Cells by Activating the Nur77-Bcl-2 Apoptotic Pathway Xiaohui Chen1, Xihua Cao2, Xuhuang Tu1, Gulimiran Alitongbieke1, Zebin Xia2, Xiaotong Li1, Ziwen Chen1,MeimeiYin,DanXu1, Shangjie Guo1, Zongxi Li1, Liqun Chen1,XindaoZhang1,DingyuXu1, Meichun Gao1,JieLiu1, Zhiping Zeng1, Hu Zhou1,YingSu2, and Xiao-kun Zhang1,2

Abstract

Nur77 (also called TR3 or NGFI-B), an orphan member of indole-3-carbinol metabolite, as a modulator of the Nur77- the nuclear receptor superfamily, induces apoptosis by trans- Bcl-2 apoptotic pathway. BI1071 binds Nur77 with high locating to mitochondria where it interacts with Bcl-2 to affinity, promotes Nur77 mitochondrial targeting and inter- convert Bcl-2 from an antiapoptotic to a pro-apoptotic mol- action with Bcl-2, and effectively induces apoptosis of cancer ecule. Nur77 posttranslational modification such as phos- cells in a Nur77- and Bcl-2–dependent manner. Studies with phorylation has been shown to induce Nur77 translocation animal model showed that BI1071 potently inhibited the from the nucleus to mitochondria. However, small molecules growth of tumor cells in animals through its induction of that can bind directly to Nur77 to trigger its mitochondrial apoptosis. Our results identify BI1071 as a novel Nur77- localization and Bcl-2 interaction remain to be explored. Here, binding modulator of the Nur77-Bcl-2 apoptotic pathway, we report our identification and characterization of DIM-C- which may serve as a promising lead for treating cancers with þ pPhCF3 MeSO3 (BI1071), an oxidized product derived from overexpression of Bcl-2.

Introduction Bcl-2 in regulating the apoptosis of cancer cells and in the resistance of cancer cells to a variety of radio- and chemother- Nur77 (NR4A1;alsoknownasNGFI-BandTR3)isperhaps apeutic agents, understanding how the Nur77-Bcl-2 apoptotic the most potent apoptotic member of the nuclear receptor pathway is regulated and discovering its small-molecule mod- superfamily (1–8). The death effect of Nur77 was initially ulators may offer new strategies to develop effective cancer recognized during studying the apoptosis of immature thymo- therapeutics. However, small molecules that can activate the cytes, T-cell hybridomas (9, 10). Later, we found that Nur77 Nur77-Bcl-2 apoptotic pathway by binding to Nur77 to trigger mediates the death effect of the retinoid-related molecule Nur77 mitochondrial translocation and interaction with Bcl-2 AHPN (also called CD437) in cancer cells (11). Furthermore, have not been reported. we discovered a nongenomic action of Nur77, in which Nur77 As an orphan nuclear receptor, Nur77 lacks a canonical migrates from the nucleus to the cytoplasm, where it targets ligand-binding pocket (LBP; refs. 16, 17), which excludes small mitochondria to trigger cytochrome c release and apoptosis in molecules from binding to Nur77 to regulate Nur77 functions cancer cells (12–14). Further studies demonstrated in various via the canonical LBP-binding mechanism. Recent advance has cancer types that such a Nur77 mitochondrial apoptotic path- revealed the existence of alternate small-molecule binding way is characterized by its interaction with Bcl-2 and the regions on the surface of nuclear receptors, and compounds conversion of Bcl-2 from an antiapoptotic molecule to a that bind to alternate sites other than LBP have been identified pro-apoptotic molecule (6, 15). Given the pivotal role of for some nuclear receptors (18, 19), including Nur77 (20–24). These developments inspire us to discover Nur77-binding compounds that can regulate the Nur77-Bcl-2 apoptotic path- 1 School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Inno- way.Here,wereportthatasaltformofa3,30-diindolymethane vative Drug Target Research, Xiamen University, Xiamen, China. 2Cancer Center, (DIM) derivative (di(1H-indol-3-yl)(4-(trifluoromethyl)phe- Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. nyl)methane; named BI1071 here) can bind to Nur77 to induce Note: Supplementary data for this article are available at Molecular Cancer apoptosis of cancer cells through the Nur77-Bcl-2 apoptotic Therapeutics Online (http://mct.aacrjournals.org/). pathway. BI1071 binds to Nur77 at submicromolar concentra- X. Chen and X. Cao contributed equally to this article. tion and induces apoptosis that is dependent on the expression Corresponding Authors: Ying Su, Sanford Burnham Prebys Medical Discovery of both Nur77 and Bcl-2. BI1071 also effectively inhibits the Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037; E-maill: growth of tumor cells in animals. Moreover, BI1071 binding to [email protected] and Xiao-kun Zhang, Xiamen University, Xiamen, China; Nur77 induces not only its mitochondrial targeting but also its E-mail: [email protected] interaction with Bcl-2. Our results therefore identify BI1071 as doi: 10.1158/1535-7163.MCT-18-0918 the first Nur77-binding small molecule that promotes the 2019 American Association for Cancer Research. Nur77-Bcl-2 apoptotic pathway.

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A New Activator of the Nur77-Bcl-2 Apoptotic Pathway

Materials and Methods Generation of Nur77 and Bcl-2 knockout cells by CRISPR/Cas9 system Cell culture Knocking out Nur77 and Bcl-2 from HeLa cells used the The following cell lines are used in our study. HCT116 colon CRISPR/Cas9 system. gRNA targeting sequence of Nur77 (50- cancer, MDA-MB-231, HS578T, BT549, MCF-7, and T47D breast ACCTTCATGGACGGCTACAC-30) and Bcl-2 (50-GAGAACAGGG- cancer, breast epithelial cell line MCF-10A, HeLa ovarian cancer, TACGATAACC-30) was cloned into gRNA cloning vector Px330 mouse embryonic fibroblast (MEF) cells and HEK293T embryonic (Addgene, 71707) and confirmed by sequencing. The accession cells were cultured in DMEM, whereas ZR-75-1, HCC1937 breast numbers of Nur77 and Bcl-2 are NM 001202233 and cancer and SW480 colon cancer were cultured in RPIM-1640 - NM 000633.2 respectively. To screen for cells lacking Nur77 or medium containing 10% FBS. Human colonic epithelial cells - Bcl-2, HeLa cells were transfected with control vector and gRNA (HCoEpiC) were cultured in colonic epithelial cell medium (CoE- expression vectors, followed by G418 selection (0.5 mg/mL). piCM, Cat. #2951). Cell lines HCT116 (ATCC, CCL-247), SW480 Single colonies were subjected to Western blotting using anti- (ATCC, CCL-228), and HEK293T (ATCC, CRL-11268) were Nur77 and anti–Bcl-2 antibody to select knockout cells. obtained from the ATCC. Cell lines MCF-10A (SCSP-660), MDA-MB-231 (SCSP-5043), HeLa (TCHu187), HS578T Cell viability determination and cell death assay (TCHu127), BT549 (TCHu 93), MCF-7 (SCSP-531), ZR-75-1 Cell viability was analyzed by using colorimetric 3-(4,5- (TCHu126), T47D (TCHu 87), and HCC1937 (TCHu148) were dimethylthiazol-dimethylthiazol-2-yl)-2,5-diphenyletetrazolium obtained from Chinese Academy of Science Shanghai Cell Bank on Bromide (MTT) assay as described previously (12–14, 24). December 09, 2016. Cell line HCoEpiC was obtained from Scien- ceCell (Cat. #2950) on October 05, 2018. MEF cells were isolated Mammalian one hybrid assay from embryonic day 13 wild-type (WT) and Nur77 knock out (KO) HEK293T cells were co-transfected with pG5 Luciferase reporter mice. The cells were grown in the cell incubator with 5% CO2 at together with the plasmid encoding RXRa-LBD fused with the 37 C. Sub-confluent cells with exponential growth were used Gal4 DNA-binding domain and other expression plasmids as throughout the experiments. Cells plated onto cell culture dishes described previously (18, 25). After transfection, cells were treated and kept in 10% FBS for 24 hours were treated with compounds or with DMSO or BI1071, and assayed by using the Dual-Luciferase transfected with plasmids. Cell transfection was carried out by Reporter Assay System (Promega). Transfection efficiency was using Lipofectamin 2000 according to the manufacturer's instruc- normalized to Renilla luciferase activity. tion. The cells were tested by using Mycoplasma Hoechst Stain Assay kit (Beyotime, C0296) every 6 months. We added the Hoechest Cell fractionation solution to stain the cells with 50% density at room temperature for For cellular fractionation (12–14, 24), cells were lysed in cold 30 minutes and then used the confocal microscope to observe the buffer A (10 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl , cells. In the cells without Mycoplasma infection, only the blue 2 10 mmol/L KCl, 0.5 mmol/L dithiothreitol) with a cocktail of fluorescence of the nucleus was observed. Filamentous blue fluo- proteinase inhibitors on ice for 10 minutes as described previ- rescence can be observed around the nucleus in Mycoplasma con- ously. Cytoplasmic fraction was collected by centrifuging at 6,000 taminated cell samples. The cells were prevented from Mycoplasma rpm for 10 minutes. Pellets containing nuclei were resuspended in infection by using plasmocin (Invivogen, ant-mpt). No positive cold high-salt buffer C (20 mmol/L HEPES-KOH (pH 7.9), 25% Mycoplasma tests was observed during the time of our experiments. glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol) with a cocktail of proteinase Plasmids inhibitors on ice for 30 minutes. Plasmids pcmv-myc-Nur77, GFP-Nur77, GFP-Nur77/LBD, GST-Bcl-2, pcmv-myc-Bcl-2, Flag-cmv-Bcl-2 were constructed as GST-pull down – described (12 14, 24). Plasmids pcmv-myc-Nur77/H372D, GST or GST-Bcl-2 fusion protein (0.5 mg) was immobilized on pcmv-myc-Nur77/H372A, pcmv-myc-Nur77/Y453L, pcmv-myc- glutathione-Sepharose beads and incubated with purified His- Nur77/C566K were constructed by using the PCR and Quick- Nur77-LBD (0.2 mg) in the presence of different concentration of Chang mutagenesis kit. BI1071 as described previously (12–14, 24). Bound Nur77-LBD was analyzed by Western blotting. Antibodies and reagents Anti-Ki67 (Cat. ab15580) and anti-Hsp60 (Cat. ab46798) Western blotting and immunoprecipitation antibodies were purchased from Abcam (UK); anti–b-actin (Cat. Western blotting and co-immunoprecipitation (co-IP) were 4970S), anti-Cleaved caspase-3 (Cat. 9661S), and anti-Nur77 performed as described (12–14, 24). (Cat. 3960S) antibodies were purchased from Cell Signal Tech- nology; anti-Myc (9E10; Cat. Sc-40), anti-Nur77 (M-210; Cat. Generation of Nur77 and Bcl-2 knockout cells by CRISPR/Cas9 sc-5569), anti-PCNA (Santa Cruz Biotechnology sc-7907), anti–a- system tubulin (Santa Cruz Biotechnology sc-8035), anti-Bcl-2 (Santa Knocking out Nur77 and Bcl-2 from HeLa cells used the Cruz Biotechnology sc-783), anti-PARP (Santa Cruz Biotechnol- CRISPR/Cas9 system. gRNA targeting sequence of Nur77 (50- ogy sc-7150), and anti-GST (sc-138) antibodies were purchased ACCTTCATGGACGGCTACAC-30) and Bcl-2 (50-GAGAACAGGG- from Santa Cruz Biotechnology (Santa Cruz Biotechnology); TACGATAACC-30) was cloned into gRNA cloning vector Px330 anti-Flag (Cat.F1804) antibody was purchased from Sigma; (Addgene, 71707) and confirmed by sequencing. To screen for Mito-tracker deep red (Cat. M22426), JC-1 Probe (Cat. T3168) cells lacking Nur77 or Bcl-2, HeLa cells were transfected with and mitoSOX Red Mitochondrial Superoxide Indicator (Cat. control vector and gRNA expression vectors, followed by G418 M36008) were purchased from Thermo Fisher. selection (0.5 mg/mL). Single colonies were subjected to Western

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blotting using anti-Nur77 and anti–Bcl-2 antibody to select 75%, 50%), followed by washing in PBS. Antigen retrieval was knockout cells. performed in 10 mmol/L sodium citrate buffer (pH 6.0), which was microwaved at 100C for 20 minutes. After rinsed twice in Apoptosis assay PBS, sections were blocked at room temperature for 1 hour by Cells were placed on 6-well plates with a density of 1 106 per using 10% normal goat serum, followed by incubation with anti- well. After 24 hours, the cells treated with different concentration ki67, anti-cleaved caspase 3 overnight at 4C. Colors were devel- of BI1071 for 6 hours, and then the suspended and the adherent oped with a DAB horseradish peroxidase color development kit. cells were collected, stained with Annexin V-FITC for 10 minutes and with propidium iodide for 5 minutes, and analyzed imme- Docking experiments diately by cytoFLEX Flow Cytometry System (Beckman-Coulter) Schrodinger's (www.schrodinger.com) Glide (27), a grid-based using FITC and PC5.5. docking program was used for the docking study of BI1071 to the protein. The crystal structure of Nur77-LBD in complex with a Determination of Dcm and ROS cytosporone B analog (Protein Data Bank code 3V3Q) was used. The determination of Dym and ROS was performed as previ- Docking was performed with the implemented standard routine ously described elsewhere (26). JC-1 probe was used to measure in Glide. The Glide GScore was used as docking score to rank the mitochondrial depolarization in cells. Cells were first treated with docking results. Poses were further visually investigated to check different concentration of BI1071 for 6 hours and then followed for their interactions with the protein in the docking site. with the addition of JC-1 staining solution (5 mg/mL) for 20 Schrodinger's€ Maestro was used as the primary graphical user minutes at 37C. After washing with PBS twice, mitochondrial interfaces for the visualization of the crystal structure and docking membrane potentials were analyzed immediately by cytoFLEX results. Flow Cytometry System using FITC and PE. Mitochondrial depolarization was measured by a change in the ratio of green/red Surface plasmon resonance fluorescence intensity. ROS was monitored with the mitoSOX Red The binding kinetics between Nur77-LBD and compounds Mitochondrial Superoxide Indicator and analyzed by cytoFLEX were performed on a BIAcore T200 instrument (GE Healthcare) Flow Cytometry System using PE. at 25C (24). Nur77-LBD were diluted to 0.05 mg/mL in 50 mmol/L NaOAc (pH 5.0) and immobilized on a CM5 sensor chip Immunostaining (GE Healthcare) by amine coupling at densities approximately Cells were fixed in 4% paraformaldehyde. For mitochondrial 10,000 RU according to the manufacturer's instructions. Gradient staining, cells were incubated with anti-Hsp60 goat immuno- concentrations of compounds were injected into the flow cells in globulin G (IgG; Santa Cruz Biotechnology), followed by anti- running buffer (PBS, 0.4% DMSO) at a flow rate of 30 mL/min for goat IgG conjugated with Cy3. The nuclei were visualized by DAPI 150 seconds of association phase followed by a 420 seconds staining. Fluorescent images were collected and analyzed by using dissociation phase and a 30 seconds regeneration phase a fluorescence microscopy or MRC-1024 MP laser-scanning con- (10 mmol/L Glycin-HCl, pH 2.5). The data were analyzed using focal microscope (Bio-Rad). BIAcore T200 Evaluation Software 2.0 and referenced for blank injections and reference Surface. The dissociation constant (Kd) Animal studies was fitted to the standard 1:1 interaction model and calculated The protocols for animal studies were approved by the Animal using the global fitting of the kinetic data from gradient Care and Use Committee of Xiamen University, and all mice were concentrations. handled in accordance with the "Guide for the Care and Use of Laboratory Animals" and the "Principles for the Utilization and Statistical analysis Care of Vertebrate Animals." For MMTV–PyMT mice breast cancer Data were expressed as mean SD. Each assay was repeated in model, female MMTV-PyMT mice of 12-weeks-old were randomly triplicate in three independent experiments. The statistical signif- divided into two groups (n ¼ 7 each), treated with a daily oral dose icance of the differences among the means of several groups was of BI1071 (5 mg/kg) for 18 days. Standard histopathological determined using the Student t test. analysis of tumor tissue was performed. BI1071 was dissolved in DMSO and diluted with normal saline containing 5.0% (V/V) Tween-80 to a final concentration 0.5 mg/mL. Normal saline with Results DMSO and 5.0% Tween-80 was used as the vehicle control. For A salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect xenograft nude mouse study, male BALB/c nude mice (6-weeks- In our effort to identify small molecules that modulate the old) were subcutaneously injected with log growth-phase of Nur77/Bcl-2 apoptotic pathway, we evaluated an in-house com- SW620 cells (1 106 cells in 0.1 mL PBS). Mice were treated pound library, which includes di(1H-indol-3-yl)(4-(trifluoro- orally after 7 days of transplantation with BI1071 once a day. Body methyl)phenyl)methane (DIM-C-pPhCF3, Fig. 1A; ref. 28). We weight and tumor size were measured every 3 days. Tumors were surprisingly observed that the freshly prepared DIM-C-pPhCF3 measured and weighted. Tissues isolated from the nude mice were solution was not as active as the aged solution in apoptosis fixed with 4% paraformaldehyde. TdT-mediated dUTP nick end induction. In addition, the freshly made DIM-C-pPhCF3 solution labeling assay was performed according to the manufacturer's is colorless, however, it turns reddish when it is aged or exposed to instructions (In situ Cell Death Detection Kit; Roche). air at room temperature. Thus, we presumed that the compound underwent oxidization and the oxidized DIM-C-pPhCF3 was Immunohistochemistry more active than DIM-C-pPhCF3. To test our hypothesis, DIM- Four-mm-thick sections were deparaffinized and rehydrated C-pPhCF3 was oxidized in the presence of methanesulfonic using xylene and a graded series of ethanol (100%, 95%, 85%, acid, and the products were subsequently purified to obtain the

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Figure 1. þ A salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect. A, Structure of DIM-C-pPhCF3 and DIM-C-pPhCF3 MeSO3 (BI1071). B, HCT116 cells treated with the indicated concentration of BI1071 or DIM-C-pPhCF3 (labeled as CF3) for 3 days were assessed by MTT assay. Data are shown as mean SD (n ¼ 6). C, HCT116 cells treated with the indicated concentration of BI1071 for 6 hours were analyzed for PARP cleavage by Western blotting. D, HCT116 cells treated with 0.5 mmol/L BI1071 for 6 hours were visualized by DAPI staining. Apoptotic cells were counted in 200 cells. E, Level of PARP cleavage and cleaved caspase 3 in MDA-MB-231 cells treated with the indicated concentration of BI1071 for 6 hours was determined by Western blotting. F, Annexin V/PI staining of MDA-MB-231 cells treated with the indicated concentration of BI1071 for 6 hours was analyzed by flow cytometry. G, MDA-MB-231 cells treated with the indicated concentration of BI1071 for 6 hours were stained with JC-1. Aggregated JC-1, red fluorescence (PE), and monomeric JC-1, green fluorescence (FITC), were measured by flow cytometry. Statistical data were mean SEM of five independent images. , P < 0.1; , P < 0.001 (Student t test). H, Mitochondrial ROS production in MDA-MB-231 treated with the indicated concentration of BI1071 for 6 hours was analyzed by flow cytometry. For Western blots and flow cytometry experiments, one of three similar experiments is shown.

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oxidized DIM-C-pPhCF3:di((1H-indol-3-yl)(4-trifluoromethyl- used to evaluate the anticancer effect of BI1071. Administration of þ phenyl)methylium methanesulfonate (DIM-C-pPhCF3 MeSO3 ; the MMTV-PyMT mice with BI1071 (5 mg/kg) potently inhibited BI1071, Fig. 1A; Supplementary Methods for synthesis and puri- the growth of PyMT mammary tumor (Fig. 2E and F). Western fication). BI1071 was then tested in comparison with DIM-C- blotting of tumor tissues prepared from treated and non-treated pPhCF3 for growth inhibition and apoptosis induction. Figure mice revealed that the expression levels of two proliferation 1B showed that BI1071 inhibited the growth of HCT116 colon markers, PCNA and Ki67, were markedly reduced by BI1071 cancer cells with an IC50 of 0.06 mmol/L, which is about 25-fold (Fig. 2G). Immunostaining also showed a reduced expression of more active than DIM-C-pPhCF3 (IC50 ¼ 1.5 mmol/L). Treatment Ki67 and enhanced expression of cleaved caspase 3 in tumor of MDA-MB-231 cells with 0.5 mmol/L BI1071 for 6 hours effec- tissue specimens prepared from mice treated with BI1071 tively induced PARP cleavage, an indication of apoptosis, while (Fig. 2H). There was not significant difference in the body weight DIM-C-pPhCF3 had no effect under the same condition (Supple- (without tumor weight) between the control mice and the mentary Fig. S1A). Dose-dependent study demonstrated that BI1071-treated mice in both animal models (Supplementary Fig. BI1071 could induce PARP cleavage at submicromolar concen- S4A and S4B). These data demonstrated that BI1071 potently trations in HCT116 cells (Fig. 1C) and other cancer cell lines inhibited the growth of tumor cells in animals through its induc- (Supplementary Fig. S1B). tion of apoptosis. Interestingly, BI1071 was effective in various breast cancer cell lines analyzed regardless of its hormone dependency (Supple- BI1071 induces Nur77-dependent apoptosis and Nur77 mentary Fig. S2). Furthermore, BI1071 did not display apoptotic mitochondrial targeting effect in the non-transformed mammary and normal colon cells We next determined whether BI1071-induced apoptosis (Supplementary Fig. S3A and S3B), indicating that BI1071 selec- was Nur77-dependent by examining its apoptotic effect in tively induces apoptosis in cancer cells. The apoptotic effect of mouse embryonic fibroblast (MEF) and MEF lacking Nur77 BI1071 was also confirmed by its induction of extensive nuclear (Nur77 / MEF). BI1071 dose-dependently inhibited the growth condensation and fragmentation revealed by DAPI staining in of MEFs, but such an inhibitory effect was significantly dimin- cells treated with 0.5 mmol/L BI1071 for 6 hours (Fig. 1D; Sup- ished in Nur77 / MEFs (Fig. 3A). Induction of PARP cleavage in plementary Fig. S1C) and confirmed as well by PARP and caspase MEFs by BI1071 was also attenuated in Nur77 / MEFs (Fig. 3B). 3 cleavage assays (Fig. 1E). The effect of BI1071 on cell death was The death effect of BI1071 was also evaluated in Nur77 genome further assessed using flow cytometry-based Annexin V/ Propi- knockout HeLa cells generated by CRISPR/Cas9 technology. dium iodide (PI) apoptosis assay. Dose-dependent study showed Induction of PARP cleavage and caspase 3 activation by BI1071 that about 31.63% of MDA-MB-231 cells were apoptotic when were strongly suppressed in Nur77 / HeLa cells (Fig. 3C). treated with 1 mmol/L of BI1071 for 6 hours, whereas only 1.31% Annexin V/PI staining revealed a reduced apoptotic effect of of cells were apoptotic in vehicle control cells (Fig. 1F). BI1071 in Nur77 / HeLa cells than in the parental HeLa cells Loss of mitochondrial membrane potential (Dcm) represents (from 35.55% to 3.25%; Fig. 3D). Furthermore, unlike the paren- one of the hallmarks of apoptosis. To assess whether the BI1071- tal HeLa cells, Nur77 / HeLa cells did not display BI1071- induced apoptosis was related to the intrinsic mitochondrial induced mitochondrial membrane potential loss measured by pathway, we used JC-1, the mitochondrial-specific dye, to mon- JC-1 staining (Fig. 3E) or BI1071-induced release of mitochon- itor the changes of mitochondrial membrane potential (29). drial ROS (Fig. 3F). To further address the role of Nur77, we MDA-MB-231 breast cancer cells treated with BI1071 were stained transfected the ligand-binding domain (LBD) of Nur77, Nur77- with JC-1. JC-1 dye accumulation in mitochondria is dependent LBD, into HEK293T cells and asked whether the overexpression of of mitochondrial membrane potential, accompanied by a shift of Nur77-LBD could influence the effect of BI1071. Indeed, trans- JC-1 fluorescence emission from green to red. In comparison with fection of Nur77-LBD enhanced the killing effect of BI1071, with healthy cells, apoptotic cells display an increase in the green/red 36% of the transfected HEK293T cells undergoing apoptosis, fluorescence intensity ratio. Analysis of both red and green fluo- while 4.5% of the non-transfected cells were apoptotic (Supple- rescence emissions by flow cytometry revealed a dose-dependent mentary Fig. S5). Together, these results demonstrated that BI1071 induction of mitochondrial membrane dysfunction. After BI1071 targets Nur77 to induce cancer cell apoptosis. treatment with 1 mmol/L of BI1071 for 6 hours, the green to red Our observation that BI1071 induced mitochondria-depen- ratio increased from 100% to 345% (Fig. 1G). Mitochondrial dent apoptosis prompted us to determine whether BI1071 exerted dysfunction was also revealed by marked increase in intracellular its Nur77-dependent apoptosis by promoting Nur77 mitochon- mitochondrial reactive oxygen species (mito-ROS) in MDA-MB- drial targeting. Immunostaining showed that Nur77 was mainly 231 cells exposed to BI1071 in a dose-dependent manner localized in the nucleus of HCT116 cells. However, it was pre- (Fig. 1H). Collectively, these data suggested that BI1071 induced dominantly cytoplasmic when cells were treated with 0.5 mmol/L mitochondrion-related apoptosis in cancer cells. of BI1071 for 2 hours (Fig. 3G). To confirm the effect of BI1071 on Nur77 cytoplasmic localization, HEK293T cells were transfected BI1071 inhibits the growth of tumor cells in vivo with GFP-Nur77 and subsequently treated with 0.5 mmol/L To assess the apoptotic effect of BI1071 in animals, SW620 BI1071. Transfected GFP-Nur77 resided in the nucleus, however colon cancer cells were inoculated subcutaneously in the right it was diffusely distributed in both the cytoplasm and nucleus and left hind-side flank of nude mice. Administration of tumor- upon BI1071 treatment (Fig. 3H). Cells transfected with GFP- bearing nude mice with BI1071 inhibited the growth of SW620 Nur77-LBD also responded well to BI1071. Only for cells treated xenograft tumor in a dose- and time-dependent manner (Fig. 2A– with BI1071, GFP-Nur77-LBD colocalized extensively with the C). TUNEL assay revealed extensive apoptosis in BI1071-treated mitochondria-specific Hsp60 protein revealed by confocal tumor specimens as compared with control tumor (Fig. 2D). microscopy (Fig. 3I) and co-accumulated with the Hsp60 protein MMTV-PyMT-transgenic mouse model of breast cancer was also in the heavy membrane fraction shown by cellular fractionation

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Figure 2. Effect of BI1071 on tumor growth and apoptosis in animals. A, Nude mice (n ¼ 6) injected with SW620 (2 106 cells) were administered with the indicated dose of BI1071 once a day, and tumors were measured every 3 days. B and C, 12 days after administration of BI1071, nude mice bearing SW620 tumor were sacriﬁced and tumors were removed, weighted, and showed (, P < 0.001, Student t test). D, Representative TUNEL staining images illustrating the apoptotic effect of BI1071. The apoptotic cells were detected by TUNEL assay in specimens of xenograft tumors. E, Representative images of MMTV–PyMT mammary tumor model mice and tumors from mice administered with or without BI1071. For MMTV–PyMT mammary tumor model, female wild-type MMTV-PyMT mice that were 12 weeks old were randomly divided into two groups (n ¼ 7), treated with daily oral doses of BI1071 (5 mg/kg) for 18 days. F, Inhibition of PyMT tumor growth by BI1071. Mice treated with BI1071 as in E, and tumors were weighted (n ¼ 7). G, Western blot analysis of the expression of PARP, Ki67, and PCNA in tumor tissues prepared from 3 MMTV-PyMT mice treated with or without BI1071 (5 mg/kg) for 18 days. H, Representative immunocytochemistry staining showing the expression of Ki67 and cleaved caspase 3 in tumor tissues prepared from MMTV-PyMT mice treated with or without BI1071 (5 mg/kg) for 18 days.

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Figure 3. BI1071 induces Nur77-dependent apoptosis and Nur77 mitochondrial targeting. A, MEFs and Nur77/ MEFs treated with the indicated concentration of BI1071 for 6 hours were assessed by MTT assay. (, P < 0.01 and , P < 0.001, Student t test). B, PARP cleavage in MEFs or Nur77/ MEFs treated with 0.5 mmol/L BI1071 for 6 hours was determined by Western blotting. C–F, HeLa or Nur77/HeLa cells were treated with 0.5 mmol/L BI1071 for 6 hours. Cells were then subjected to Western blotting for PARP cleavage and cleaved caspase 3 detecting (C), Annexin V/PI staining for apoptosis measurement (D), JC-1 staining for measuring mitochondrial membrane potential (E) or mito-SOX staining for determining the production of mitochondrial ROS (F). One of three similar experiments is shown. NS, not signiﬁcant; , P < 0.001 (Student t test). G, Subcellular localization of endogenous Nur77 in HCT116 cells treated with 0.5 mmol/L BI1071 for 2 hours was analyzed by confocal microscopy after immunostained with anti-Nur77 antibody. Nuclei were visualized by DAPI staining. H, HeLa cells transfected with GFP-Nur77 were treated with 0.5 mmol/L BI1071 for 2 hours and visualized by confocal microscopy. I, HeLa cells transfected with GFP-Nur77- LBD were treated with BI1071 (0.5 mmol/L) for 2 hours were immunostained with anti-Hsp60 antibody and visualized by confocal microscopy. J, HEK293T cells were transfected with GFP-Nur77-LBD and treated with 0.5 mmol/L BI1071 for 2 hours. Cytosolic (Cyt) and HM fractions were then prepared and analyzed by Western blotting. Expression of cytoplasmic IkBa and mitochondrial Hsp60 was shown to indicate the purity of cytosolic and mitochondrial fractions, respectively. K, HEK293T cells were transfected with Myc-Nur77. Whole cell lysate (WCL) and mitochondria-enriched heavy membrane (HM) fractions were prepared from HEK293T cells treated with 0.5 mmol/L BI1071 for 2 hours and analyzed by Western blotting. Expression of nuclear PARP and mitochondrial Hsp60 was shown to ensure the purity of HM fraction. For cellular fractionation experiments, one of three similar experiments is shown.

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A New Activator of the Nur77-Bcl-2 Apoptotic Pathway

(Fig. 3J). The effect of BI1071 on inducing Nur77 mitochondrial binding region (20–24). BI1071 failed to induce PARP cleavage targeting was further illustrated by cellular fractionation expe- in cells transfected with Nur77/H372D or Nur77/Y453L, whereas riments showing that a significant amount of transfected it strongly induced PARP cleavage in cells transfected with Nur77 Myc-Nur77 accumulated in the mitochondria-enriched heavy or Nur77/C566K (Fig. 4G). Similarly, in cells transfected with membrane (HM) fraction when cells were treated with BI1071 Nur77/H372D or Nur77/Y453L, the effect of BI1071 on inducing (Fig. 3K). Taken together, these data demonstrated that BI1071 Mito-ROS generation (Fig. 4H), on apoptosis analyzed using dual exerted its Nur77-dependent apoptotic effect by promoting staining with fluorescent Annexin V and PI (Fig. 4I), or on loss of Nur77 mitochondrial targeting. mitochondrial membrane potential (Fig. 4J) was much attenuated when compared with cells transfected with Nur77 or BI1071 binds Nur77 to induce its mitochondrial targeting and Nur77/C566K. Taken together, these data demonstrated that apoptosis BI1071 exerted its Nur77-dependent apoptotic effect by a direct Although Nur77 lacks a LBP and no endogenous ligands have Nur77-binding mechanism. yet been identified (16, 17), recent crystallographic studies have identified several regions on the surface of the Nur77 protein as BI1071-induced Nur77 mitochondrial targeting and apoptosis small-molecule binding regions (20, 22, 30). We therefore deter- is Bcl-2 dependent mined whether BI1071 binds directly to Nur77 to induce its We previously showed that the Nur77 mitochondria-depen- mitochondrial targeting and apoptosis. Surface plasmon reso- dent apoptotic pathway involved Nur77 interaction with Bcl- nance (SPR) analyses revealed that DIM-C-pPhCF3 bound to 2 (14). We therefore asked whether Bcl-2 plays a role in Nur77-LBD with a Kd of 3.0 mmol/L (Fig. 4A) and that BI1071 BI1071-induced apoptosis. Thus, the apoptotic effect of BI1071 / bound to Nur77-LBD protein with a Kd of 0.17 mmol/L (Fig. 4B), was evaluated in MEFs and MEFs lacking Bcl-2 (Bcl-2 MEF). which demonstrated that BI1071 binding to Nur77-LBD was 18- BI1071 at 0.5 mmol/L effectively induced PARP cleavage in fold stronger than DIM-C-pPhCF3. We also evaluated the effect of MEFs, whereas it had no apparent effect on PARP cleavage in BI1071 on the transcriptional activity of Nur77/RXRa-LBD het- Bcl-2 / MEFs (Fig. 5A). This was confirmed by DAPI staining erodimer. Co-transfection of pBind-RXRa-LBD and Nur77 showing that Bcl-2 / MEF cells were much more resistant than strongly activate the reporter transcriptional activity when cells MEFs to the apoptotic effect of BI1071 (Fig. 5B). In addition, the were treated with 9-cis-RA, a RXRa ligand (Fig. 4C). BI1071 further impaired effect of BI1071 in Bcl-2 / MEFs could be rescued by re- dose-dependently induced the reporter activity (Fig. 4C), likely expression of Bcl-2 (Supplementary Fig. S7). In response to due to its binding to Nur77. To exclude the possibility that BI1071 0.5 mmol/L of BI1071, 40% of MEFs displayed chromatin con- acted on RXRa, Glu453 and Glu456 in the activation function 2 densation and nuclear fragmentation, whereas only 14% of (AF2) region of RXRa (31) were substituted with Ala and the Bcl-2 / MEF cells exhibited similar apoptotic features. We also resulting mutant, RXRa-LBD/E453,6A, was used to repeat the used the CRISPR/Cas9 technology to generate Bcl-2 knockout reporter assay. As expected, 9-cis-RA failed to induce the reporter HeLa cells and showed that the effect of BI1071 on inducing PARP activity in cells transfected with Nur77 and pBind-RXRa-LBD- cleavage was almost completely suppressed in Bcl-2 / HeLa cells E453,6A. However, BI1071 could still activate the reporter gene (Fig. 5C). The role of Bcl-2 in mediating the death effect of BI1071 transcription (Fig. 4C), demonstrating that BI1071 induced was also illustrated by Annexin V/PI staining showing a reduced reporter gene transcription through Nur77 binding but not RXRa. apoptotic effect of BI1071 in Bcl-2 / HeLa cells compared with its We also excluded the possibility of BI1071 binding to other effect in the parental HeLa cells (from 24.59% to 7.95%), and in nuclear receptors using reporter assays (Supplementary Fig. Bcl-2 / MEF cells compared with the parental MEF cells (from S6A). We next employed molecular docking approach to study 76.65% to 10.27%; Fig. 5D). Induction of the mitochondrial how BI1071 bound to Nur77-LBD. Our docking studies showed membrane potential loss by BI1071 was also suppressed in that BI1071 docked better to a binding region formed by helices Bcl-2 / MEFs and Bcl-2 / HeLa cells (Fig. 5E). Furthermore, H1, H5, H7 and H8, and loops H1-H2, H5-B1 and H7-H8. The BI1071-induced release of mitochondrial ROS was compromised docked mode also suggested that the indole ring of BI1071 made by loss of Bcl-2 (Fig. 5F). These results revealed a crucial role of key interaction with the side chains of H372 and Y453 located Bcl-2 in mediating the Nur77-dependent apoptotic effect of in H1 and H5, respectively (Fig. 4D). For comparison, we also B1071, demonstrating that the compound acts through the docked DIM-C-pPhCF3 to the same region. As shown in Fig. 4D, Nur77-Bcl-2 apoptotic pathway. BI1071 fit better to the binding groove with the bis-indolyl rings embedded deeper in the groove. The bis-indolyl rings of BI1071 BI1071 promotes Nur77 interaction with Bcl-2 also was positioned to form p-p interaction with Y453 and to In this study, we have showed that BI1071 can bind to Nur77 to make stronger interaction with H372. To test this binding mode, induce its migration from the nucleus to mitochondria, where it H372 was mutated into either Ala or Asp. When tested in the Gal4 interacts with Bcl-2 and triggers Bcl-2–dependent apoptosis. reporter assay for its response to BI1071, Nur77/H372D (Fig. 4E) However, if the binding of BI1071 to Nur77 promotes the or Nur77/H372A (Supplementary Fig. S6B) could not induce the interaction between Nur77 and Bcl-2 is not clear. Therefore, we reporter gene transcription in response to BI1071 treatment, investigated whether BI1071 binding to Nur77 enhanced the revealing a critical role of H372 in BI1071 binding. Nur77/ Nur77 interaction with Bcl-2. In vitro GST-pull down assays H372A also failed to respond to BI1071 to accumulate in the showed that Nur77-LBD was pulled down by GST-Bcl-2 in a heavy membrane fraction in the cellular fractionation assay BI1071 concentration-dependent manner (Fig. 6A). Cell-based (Fig. 4F). To further characterize the binding of BI1071 and its Co-IP showed that Nur77 (Fig. 6B) or Nur77-LBD (Fig. 6C) apoptotic effect, we made 2 more mutants: mutant Nur77/Y453L, transfected in HEK293T cells interacted with Bcl-2 when cells another key residue suggested by the docking studies, and mutant were treated with BI1071. Endogenous Nur77 could be specifi- Nur77/C566K, a residue located in another reported ligand- cally immunoprecipitated together with endogenous Bcl-2 by

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Figure 4. BI1071 binds directly to Nur77. A and B, Binding of DIM-C-pPhCF3 (A) or BI1071 (B) to puriﬁed Nur77-LBD by SPR. C, HEK293T cells were transfected with Gal-4 reporter plasmid and Gal-4-RXRa-LBD or Gal-4-RXRa-LBD/E453,6A together with Myc-Nur77, and treated with the indicated concentration of BI1071 or

9-cis-RA for 12 hours. Reporter activities were measured. D, Molecular modeling of the binding of BI1071 (in yellow sticks) and DIM-C-pPhCF3 (in green sticks) to Nur77-LBD. E. HEK293T cells transfected with Gal-4 reporter plasmid and Gal-4-RXRa-LBD together with Myc-Nur77 or Myc-Nur77/H372D were treated with the indicated concentration of BI1071, and reporter activities were measured. , P < 0.01; , P < 0.001 (Student t test). F, HEK293T cells were transfected with Myc-Nur77, Myc-Nur77/H372A, and treated with BI1071 (0.5 mmol/L) for 2 hours. WCL and HM fractions were then prepared and analyzed by Western blotting. G and J, Nur77/HeLa cells were transfected with the indicated Nur77 and mutant plasmids and treated with 0.5 mmol/L BI1071 for 6 hours. Cells were then subjected to Western blotting for PARP cleavage (G), mito-SOX staining for determining the production of mitochondrial ROS (H), Annexin V/PI staining for apoptosis (I), or JC-1 staining for measuring mitochondrial membrane potential (J). One of three similar experiments is shown. NS, not signiﬁcant; , P < 0.001 (Student t test).

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A New Activator of the Nur77-Bcl-2 Apoptotic Pathway

Figure 5. Bcl-2–dependent induction of apoptosis by BI1071. A and B, MEFs or Bcl-2/ MEFs were treated with 0.5 mmol/L BI1071 for 6 hours. Cells were then subjected to Western blot analysis for PARP cleavage (A) or DAPI staining for apoptosis (B). Apoptotic cells were counted in 200 cells. , P < 0.001 (Student t test). C, PARP cleavage in HeLa or Bcl-2/ HeLa cells treated with 0.5 mmol/L BI1071 for 6 hours was analyzed by Western blotting. D–F, MEFs or Bcl-2/ MEFs, HeLa cells or Bcl-2/ HeLa cells were treated with 0.5 mmol/L BI1071 for 6 hours. Cells were subjected to Annexin V/PI staining for apoptosis measurement (D), JC-1 staining for measuring mitochondrial membrane potential (E), or mito-SOX staining for determining the production of mitochondrial ROS (F). One of three similar experiments is shown; , P < 0.1; , P < 0.001 (Student t test). anti-Bcl-2 antibody only when cells were treated with BI1071 GFP-Nur77-LBD (Fig. 6F; Supplementary Fig. S8B) with Bcl-2 in (Fig. 6D). Moreover, confocal microscopy analysis revealed that cells. To further address the role of BI1071 on inducing Nur77 BI1071 promoted extensive mitochondrial colocalization of interaction with Bcl-2, the aforementioned Nur77 mutants were transfected GFP-Nur77 (Fig. 6E; Supplementary Fig. S8A) or analyzed. Fig. 6G showed that Nur77/C566K like the wild-type

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Figure 6. BI1071 promotes Nur77 interaction with Bcl-2. A, GST-pull down. Puriﬁed Nur77-LBD incubated with or without the indicated concentration of BI1071 was pulled down by GST or GST-Bcl-2 protein and analyzed by Western blotting. B and C, Co-immunoprecipitation assay. HEK293T transfected with Myc-Bcl-2 together with GFP-Nur77 (B) or GFP-Nur77-LBD (C) was treated with or without 0.5 mmol/L BI1071 and analyzed by co-immunoprecipitation assays using anti-Myc antibody. D, Interaction of endogenous Nur77 and Bcl-2 in MDA-MB-231 cells treated with or without 0.5 mmol/L BI1071 for 2 hours was analyzed by co-immunoprecipitation assay using anti-Bcl-2 antibody. E and F, Colocalization of Nur77 with Bcl-2. HEK293T cells were transfected with Myc-Bcl-2 together with GFP-Nur77 (E)orGFP-Nur77-LBD(F), treated with or without 0.5 mmol/L BI1071 for 2 hours, stained with anti-Myc antibody, and visualized using confocal microscopy. G, HEK293T cells transfected with the indicated expression plasmids were treated with 0.5 mmol/L BI1071 for 2 hours and analyzed by co-immunoprecipitation assays using anti-Flag antibody. For Co-IP experiments, one of three similar experiments is shown.

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A New Activator of the Nur77-Bcl-2 Apoptotic Pathway

Nur77 interacted strongly with Bcl-2 in a BI1071-dependent cancer cells in vitro and in animals (12). NuBCP-9 is located at the manner. In contrast, Nur77/H372D and Nur77/Y453L failed C-terminal portion of H7. Interestingly, our docking studies to interact with Bcl-2 in the presence of BI1071. The importance suggested that H7 was part of the BI1071-binding region and of the binding of BI1071 on inducing Nur77 interaction with BI1071 could potentially interact directly with amino acids D499 Bcl-2 was also illustrated by immunostaining showing extensive and A450, structurally flanking the residues from which NuBCP-9 colocalization of transfected Flag Bcl-2 with the wild-type Nur77 is derived. Therefore, it is conceivable that the BI1071-bound LBD, but not with Nur77 LBD H372A (Supplementary Fig. S8C). Nur77 offers a more suitable Bcl-2–interacting interface that Thus, binding of BI1071 to Nur77 promotes Nur77 interaction promotes the formation of Nur77/Bcl-2 complex, and thus aug- with Bcl-2 and mitochondrial localization. ments the biological effect of BI1071. A critical step in the Nur77 mitochondrial apoptotic pathway is the interaction of Nur77 with Bcl-2, which induces a conforma- Discussion tion change in Bcl-2 and converts Bcl-2 from a pro-survival to a BI1071 is a salt form of DIM-C-pPhCF3 (Fig. 1A) previously killer (12, 14). Members of the Bcl-2 family are critical regulators reported to induce Nur77-dependent apoptosis (30, 32). How- of apoptosis. As the funding member of the Bcl-2 family, Bcl-2 acts ever, relative high concentrations (around 10 mmol/L) of as a survival molecule to protect cells from programmed cell DIM-C-pPhCF3 are required for its induction of apoptosis death. Bcl-2 overexpression is often observed in cancer cells and is and activation of Nur77. To our surprise, we observed that aged associated with cancer treatment resistance and poor progno- DIM-C-pPhCF3. was generally more active than the freshly pre- sis (33–36). Thus, Bcl-2 has been an important drug target pared one in apoptosis induction. This led to our synthesis of the (37, 38). Two strategies are commonly used to develop therapeu- oxidized product of DIM-C-pPhCF3, methanesulfonate salt of tic agents targeting Bcl-2: The first relies on making use of anti- DIM-C-pPhCF3 (BI1071). Our evaluation of BI1071 revealed its sense oligonucleotides to block Bcl-2 expression and the second superior death effect in cancer cells. BI1071 was also very effective relies on designing and optimizing BH3 small-molecule or pep- in other cancer cell lines and in animal tumor models. The tide mimetics that bind the Bcl-2 BH3-binding cleft, antagonizing apoptotic effect of several DIM derivatives has been shown to its antiapoptotic activity (37, 38). Like Bcl-2, Nur77 is overex- be Nur77-dependent and various pathways were proposed to pressed in a variety of cancer cells and plays a dual role in account for their apoptotic effect (2). However, the mechanism mediating apoptosis and survival of cancer cells (39, 40). by which Nur77 mediates their death effect remains elusive, Although the growth promoting effect of Nur77 appears to be which is conceivably due to the activation of multiple pathways dependent on its nuclear action, the death effect of Nur77 involves bythehighcompoundconcentrationusedinthestudies.For its translocation from nucleus to cytoplasm (41–43). Our results instance, both transcriptional agonist such as DIM-C-pPhOCH3 showed that cancer cells are sensitive to the treatment of BI1071 as and transcriptional antagonist such as DIM-C-pPhOH were compared with normal cells, consisting with the fact that the level shown to induce Nur77-dependent apoptosis (28). Our finding of Nur77 is elevated in cancer cells. Thus, targeting Nur77 by that oxidization of DIM-C-pPhCF3 could augment its death BI1071 will have less effect on normal cells, and therefore likely effect offered an opportunity to delineate the mechanism by offer a high therapeutic index. The ability of Nur77 to interact with which Nur77 mediates the apoptotic effect of DIM-related Bcl-2 to not only suppress its antiapoptotic function but also small molecules. To this end, our studies showed that the convert Bcl-2 into a pro-apoptotic molecule (12, 14) provides a potent apoptotic effect of BI1071 was Nur-77 dependent promising strategy to target both Nur77 and Bcl-2 for cancer (Fig. 3) and was a result of its induction of Nur77 mitochon- therapy. Agents that can bind directly to Nur77 to promote Nur77 drial targeting via a direct Nur77-binding mechanism. Further- translocation and interaction with Bcl-2 are unique in that they more, our results revealed that the death effect of BI1071 was can simultaneously target both Nur77 and Bcl-2. The reported also dependent of Bcl-2 expression and that BI1071 could Nur77-derived peptide with 9 amino acids (NuBCP-9) and its induce Nur77 interaction with Bcl-2 leading to Nur77 coloca- enantiomer as Bcl-2–converting peptides has demonstrated such lization with Bcl-2 at mitochondria and apoptosis. a potential (12, 44, 45). In this regard, our identification of Binding studies showed that BI1071 could bind to Nur77 better small molecules that can directly bind Nur77 to activate the than DIM-C-pPhCF3. This is likely due to the difference in the Nur77-Bcl-2 apoptotic pathway is significant and BI1071 repre- structural conformations between the oxidized and the unoxi- sents the first lead of this class of small molecules, which warrants dized forms of DIM-C-pPhCF3, perhaps resulted from different further evaluation. atomic orbital hybridization of the central C atom. In the oxidized form the central C is sp2-hybridized, positively charged and Disclosure of Potential Conflicts of Interest bonded to 3 atoms with a co-planner arrangement, whereas in No potential conflicts of interest were disclosed. the unoxidized form, C is sp3-hybridized and bonded to 4 atoms with a tetrahedral arrangement. Differences in structural confor- Authors' Contributions mation and charge distributions can affect how molecules bind to Conception and design: X. Chen, X. Cao, G. Alitongbieke, Z. Xia, Y. Su, proteins. Our docking results suggested that BI1071 could interact X.-K. Zhang more strongly with Nur77-LBD than DIM-C-pPhCF3 due to the Development of methodology: X. Chen, X. Cao, X. Tu, G. Alitongbieke, M. Yin, different conformations adopted by the compounds. Mutagenesis D. Xu, S. Guo, Y. Su studies confirmed that H372 and Y453 were 2 key residues Acquisition of data (provided animals, acquired and managed patients, involving in the binding of BI1071 as suggested by the docking provided facilities, etc.): X. Chen, X. Cao, X. Tu, G. Alitongbieke, X. Li, D. Xu, S. Guo, Z. Li, L. Chen, X. Zhang studies. Previously, we located the critical region in Nur77 respon- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, sible for its interaction with Bcl-2 and identified a peptide NuBCP- computational analysis): X. Chen, X. Cao, X. Tu, G. Alitongbieke, Z. Xia, X. Li, 9 as Bcl-2-converting peptide, capable of inducing apoptosis of Z. Chen, D. Xu, S. Guo, Z. Li, H. Zhou, Y. Su, X.-K. Zhang

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Writing, review, and/or revision of the manuscript: X. Chen, X. Cao, M. Gao, Demonstration of Marine Economy Innovative Development Project Y. Su, X.-K. Zhang (16PYY007SF1; to X.-k. Zhang), the Fujian Provincial Science and Technology Administrative, technical, or material support (i.e., reporting or organizing Department (2017YZ0002-1; to X.-k. Zhang), and the National Institutes of data, constructing databases): X. Chen, X. Cao, X. Tu, G. Alitongbieke, Z. Xia, Health (R01 CA198982; to X.-k. Zhang). X. Li, M. Yin, D. Xu, S. Guo, Z. Li, L. Chen, D. Xu, J. Liu, Z. Zeng, H. Zhou, Y. Su Study supervision: X. Cao, X. Li, H. Zhou, Y. Su, X.-K. Zhang The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Acknowledgments advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate The authors thank Dr. Marcia Dawson for her contributions in chemistry this fact. (including conception and design) to this work, and to her memory this article is dedicated. We also thank Dr. Lin Li for her critical reading of this article. This study was supported in part by grants from the Natural Science Foundation of Received August 13, 2018; revised December 28, 2018; accepted March 14, China (U1405229, 81672749, 31271453, 31471318; to X.-k. Zhang), Regional 2019; published ﬁrst March 29, 2019.

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BI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer Cells by Activating the Nur77-Bcl-2 Apoptotic Pathway

Xiaohui Chen, Xihua Cao, Xuhuang Tu, et al.

Mol Cancer Ther 2019;18:886-899. Published OnlineFirst March 29, 2019.

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