An Allosteric PRC2 Inhibitor Targeting EED Suppresses Tumor

Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0428

Cancer Tumor Biology and Immunology Research

An Allosteric PRC2 Inhibitor Targeting EED Suppresses Tumor Progression by Modulating the Immune Response Hongping Dong, Shaojun Liu, Xuejie Zhang, Sheng Chen, Lijing Kang, Yanni Chen, Shichao Ma, Xianlei Fu, Yanchao Liu, Hailong Zhang, and Bin Zou

Abstract

Aberrant activity of polycomb repressive complex 2 (PRC2) led to 59.3% tumor growth suppression and increased fre- þ is involved in a wide range of human cancer progression. The quency of effector CD8 T-cell infiltrates in tumors. Pharma- WD40 repeat-containing protein EED is a core component of codynamic analysis revealed that CXCL10 was highly upregu- þ PRC2 and enhances PRC2 activity through interaction with lated, suggesting that CXCL10 triggers the trafficking of CD8 H3K27me3. In this study, we report the discovery of a class of T cells toward tumor sites. Our results demonstrate for the first pyrimidone compounds, represented by BR-001, as potent time that inhibition of EED modulates the tumor immune allosteric inhibitors of PRC2. X-ray co-crystallography showed microenvironment to induce regression of colon tumors and that BR-001 directly binds EED in the H3K27me3-binding therefore has the potential to be used in combination with pocket. BR-001 displayed antitumor potency in vitro and immune-oncology therapy. in vivo. In Karpas422 and Pfeiffer xenograft mouse models, twice daily oral dosing with BR-001 resulted in robust anti- Significance: BR-001, a potent inhibitor of the EED subunit tumor activity. BR-001 was also efficacious in syngeneic CT26 of the PRC2 complex, suppresses tumor progression by mod- colon tumor-bearing mice; oral dosing of 30 mg/kg of BR-001 ulating the tumor microenvironment.

Introduction stabilization of the PRC2 complex (5, 10–12) and synergistic stimulation of the PRC2 activity (5, 12–14). Polycomb repressive complex 2 (PRC2) plays important roles PRC2 is the only known methyltransferase that can specifically in regulating gene expression involved in cell differentiation and mono-, di-, or trimethylate H3 at lysine 27. Only trimethylated development (1). PRC2 consists of 4 core components: enhancer H3K27 (H3K27me3) is tightly linked to gene silencing at specific of zeste 2 or 1 (EZH2/1), embryonic ectoderm development loci through chromatin compaction (6, 7, 10, 15). Global (EED), suppressor of zeste 12 (SUZ12), and retinoblastoma- H3K27me3 levels are mediated by 2 conceptually separate associated proteins 46 and 48 (RBAP46/RBAP48; refs. 2, 3). EZH2 mechanisms___PRC2 enzymatic activity and PRC2 recruit- is the methyltransferase catalytic subunit responsible for H3K27 ment (16). PRC2 activity is regulated by multiple factors, including methylation. EED enhances the enzymatic activity of PRC2 via allosteric activators described above, various histone-modifiers binding with H3K27me3. SUZ12 interacts with all other subunits (H3K4me3, H3K36me2/3; refs. 17–19) and PRC2-interacting and contributes to the stability of the complex (4, 5). RBAP46/ partners (DNA and RNA; refs. 16, 20, 21). PRC2 recruitment to RBAP48 (also known as RBBP4/7) recognizes histone H3 and chromatin was suggested to be governed by a combination of H4 (6–8), and might regulate the substrate specificity of PRC2 (9). several molecules and gene-specific transcription factors, resulting Besides the canonical PRC2 core members, several cofactors were in PRC2 enrichment in transcriptionally inactive loci. identified to modulate the PRC2 activity. The best characterized EED harbors both a scaffold and an H3K27me3-binding func- cofactors are Jumonji and AT-rich interaction domain 2 (JARID2) tions. As a scaffolding protein, EED assembles and stabilizes the and adipocyte enhancer-binding protein 2 (AEBP2). Structural PRC2 complex. EED binding of H3K27me3 allosterically activates and functional analysis implicates that both JARID2 and AEBP2 PRC2 and propagates H3K27 methylation in repressive chroma- mimic histone H3 tails to bind with PRC2, resulting in global tin for gene silencing (22, 23). Structurally, the interaction stimulates the folding of an unstructured region of EZH2 into an alpha helix. The newly formed EZH2 helix in turn stabilizes the Shanghai Blueray Biopharma Co. Ltd., Shanghai, China. SET-I helix, which is part of the substrate binding site of the EZH2 Note: Supplementary data for this article are available at Cancer Research SET domain (4, 22, 24). Furthermore, recent studies indicated Online (http://cancerres.aacrjournals.org/). EED not only functions as a stimulator to enhance the activity of Corresponding Authors: Hongping Dong, Shanghai Blueray Biopharma Co. Ltd., PRC2 itself, but also serves as an epigenetic exchange factor to 576 Libing Road, Building 3, Pudong New Area, Shanghai 201301. China. Phone: coordinate the activities of PRC1 and PRC2. EED recruits PRC1 to 86-21-61160506, ext. 6135; E-mail: [email protected]; and Bin Zou, [email protected] H3K27me3 loci and enhances PRC1-mediated H2A ubiquitin E3 ligase activity to further maintain gene silence (25–27). – Cancer Res 2019;79:5587 96 Dysregulation of PRC2 function is broadly associated with doi: 10.1158/0008-5472.CAN-19-0428 various cancers. Increased activity of PRC2 results in high global 2019 American Association for Cancer Research. levels of H3K27me3, which are linked to different types of cancer,

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including lymphoma, prostate, breast, and myeloma cancers (28). the dark at room temperature for 1 hour and was read on The importance of PRC2 linked to tumorigenesis has been val- Spectramax i3 (Molecular Devices) at emission 570 nm and idated by disrupting the PRC2 enzymatic activity using a number excitation 680 nm. The signal at 680 nm was used to quantify of EZH2 inhibitors (29–31). Several SAM-competitive EZH2 compound inhibition and normalized with DMSO and negative inhibitors are currently in clinical trials (32, 33, NCT02860286, control (no EED). The data were then fit to a dose–response NCT03480646, NCT03603951, NCT02732275, NCT03460977). equation using the GraphPad Prism6 to get the IC50 values. However, these inhibitors have imperfect pharmacological prop- erties, such as short half-life, moderate to high clearance rate, Differential scanning fluorimetry assay and low permeability in cell-based assays (34). Furthermore, Thermal shift assay was performed using a real-time PCR drug resistance is probably another issue. Thus, development instrument from Bio-Rad. The 25 mL of reaction mixture contain- of novel inhibitors against other components of PRC2 has ing 2.5 mg EED full-length protein, 50 to 100 mmol/L BR-001 and gained attention, as demonstrated by the development of EED 1:1000 diluted SyproOrange dye in buffer (0.1 M HEPES, pH 7.5, inhibitors (33, 35–38). One of EED inhibitors is currently in and 150 mmol/L NaCl; triplicate per sample) was incubated at clinical trial (NCT02000651). room temperature for 30 minutes. SyproOrange was purchased Here we report the discovery of a potent EED inhibitor, BR-001, from Invitrogen and 5X working concentration in the reaction. which disrupts EED-H3K27me3 interaction. Co-crystal crystal- Melting temperature (Tm) of EED protein was measured using the lography shows that BR-001 binds to the same pocket in EED as following program: 25C for 2 minutes, 25C for 30 seconds, and H3K27me3. BR-001 decreases H3K27 methylation and has increase 1 C every 30 seconds till to 90 C. The Tm values were potent antiproliferative activity in vitro. BR-001 exhibited excel- obtained from the midpoint of the transition. lent efficacy against Karpas422 and Pfeiffer xenograft models, efficiently repressed H3K27me3 in tumors. Additionally, BR-001 H3K27me3 ELISA assay also showed anti-tumor effect in the CT26 syngeneic mouse The global H3K27me3 levels in cultured cells or tumor nodules þ model. The antitumor efficacy mainly relied on CD8 T-cell– were detected by ELISA assay. Briefly, 5,000 cells were seeded in mediated immunity. Collectively, BR-001 exerts its antitumor each well of 96-well plates and treated with the compounds activity through 2 mechanisms: direct inhibition of the activity immediately. DMSO was used as a control. After 72 hours treat- of PRC2 and modulation of immune response in tumor micro- ment, PBS-washed cells were lysed in 100 mL 0.4 N HCl buffer for environment. Of note, modulation of the tumor immune micro- 2 hours at 4C with gently shaking. The cell lysate was then environment by EED inhibitor BR-001 probably depends on neutralized by adding 80 mL neutralization buffer consisting of specific types of cancer. 0.5 M Na2HPO4 (pH 12.5), protease inhibitor cocktail (1:100), and 2.5 mmol/L DTT, mixed well by agitating. Ten microliters (for H3K27me3 analysis) or 2.5 mL (for H3 analysis) of cell lysate was Materials and Methods transferred to 2 Optiplate-384 HB plates (PerkinElmer), respec- All cell lines used in the experiments were purchased from tively. The final volume was adjusted to 50 mL with PBS. The assay Cobioer. All cells have been conducted Mycoplasma testing plates were incubated at 4C overnight with gently shaking for using MycoAlert Mycoplasma Detection Kit from Lonza and ELISA detection (triplicate per sample). are grown in a 37 C incubator at 5% CO2. Karpas 422 (from Tumor samples were homogenized in 0.4N HCl buffer. 20 to ECACC), WSU-DLCL2 and HPB-ALL (from DSMZ), Pfeiffer, 30 mg tumor tissues in 400 mL of 0.4N HCl were smashed by SNU-16 (from ATCC), and CT26 cells (from KCLB) are cultured JXFSTPRP-64 (JingXin) under conditions of 68 Hz, 120 seconds. in RPMI1640 medium with 10% FBS. KATO III (from ATCC) After 1 hour incubation at 4C, the tumor tissue homogenate was and 697 cells (from DSMZ) are cultured in medium IMDM centrifuged for 15 minutes. One hundred microliters of super- with 20% and 10% FBS, respectively. U87MG cells (from natant was transferred to a new tube and neutralized with 80 mL ATCC) are cultured in MEM medium with 10% FBS and NEAA neutralizing buffer as described above but without DTT. Total plus NaP. LN229 cells (from ATCC) are cultured in medium protein in supernatant was quantified by Pierce BCA protein Assay DMEM with 10% FBS. Kit (Thermo Fisher Scientific). 30 ng protein/well (for H3K27me3 analysis) or 7.5 ng protein/well (for H3 analysis) was transferred AlphaScreen competition binding assay to 2 Optiplate-384 HB plates (PerkinElmer), respectively. The The potency of compounds was tested using AlphaScreen final volume was adjusted to 50 mL with PBS. The assay plates were competition assay. Briefly, the compounds were 3-fold serial incubated at 4C overnight with gently shaking for ELISA detec- dilutions in DMSO and further diluted 133.3 folds using assay tion (triplicate per sample). buffer (25 mmol/L HEPES, pH 8.0, 0.5% BSA, 0.02% Tween-20 To perform ELISA assay, the assay plates were washed with TBST and 50 mmol/L NaCl). Five microliters of the serially diluted buffer for 5 times, blocked with TBST containing 5% BSA at room compounds was transferred to each well of a 384-well plate temperature for 1 to 2 hours. Then 30 mL per well primary (ProxiPlate 384 plus plate; PerkinElmer) containing 10 mLof antibody (Supplementary Table S1) was added to the plates and 30 nmol/L His-tagged EED (1–441aa) protein and 37.5 nmol/L incubated at room temperature for 1 hour. After 5 times of wash biotinylated H3K27me3 peptide (19–33aa). The reaction mixture with TBST, 30 mL of the secondary antibody (Supplementary was incubated at room temperature for 30 minutes. The 4 Table S1) was added to each well and incubated at room working solution (20 mg/mL) of AlphaScreen detection beads temperature for another 1 hour. After wash, 30 mL of enhanced mix (PerkinElmer) was freshly prepared by mixing nickel chelate chemiluminescence substrate (ECL; Yeason) was added to each acceptor beads and streptavidin donor beads in a 1:1 ratio into the well. The signal was detected using SpectraMax i3x (Molecular buffer described above. Five microliters of beads mixture was Devices). The data were then fit to a dose–response equation using immediately added to the plate. The plate was then incubated in the GraphPad Prism6 to get the IC50 values.

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Cell proliferation assay structures were checked using the MolProbity server. The statistics A total of 10,000 cells per well were plated onto 96-well round of structure refinement and the quality of the final model were bottom plates (Corning, #3799), and treated with BR-001 at the summarized in Supplementary Table S2. Protein structure figures indicated concentrations. Every 3 to 4 days, viable cell number in were presented with PyMol. DMSO control was detected using Vi-CELL (Beckman Coulter). The same density of cells was seeded back to a new plate. The split RNA extraction and qRT-PCR cell number for BR-001 treated groups was determined by the RNA was isolated from the cultured cells using E.Z.N.A. Total ratio calculated from DMSO control. At day 13, 40 mL of CellTiter- RNA Kit I (Omega Bio-Tek, Cat# R6834-02) according to man- Glo reagent from Promega Corporation was added to the plates. ufacturer's instructions. The cells were treated with DMSO or BR- The cell viability was detected by read luminescence signal via 001 at various concentrations for 2 days before total RNA extrac- SpectraMax i3x (Molecular Devices). The IC50 was calculated tion. The tumor tissues were homogenized in the buffer from kit using GraphPad Prism6. using JXFSTPRP-64 under the same conditions as described above, then centrifuged at 4C for 15min. Isolated RNA was Crystallization and structure determination quantified using the NanoDrop One (Thermo Scientific). Gene Hanging drop vapor diffusion method was used for crystalli- expression was analyzed using One Step RT-PCR Kit (Bio-Rad, zation, with the crystallization well containing 3.6 M NaCOOH, Cat# 172551). 200 500 ng total RNA was added to the reaction 16% glycerol, and 0.1M Bis-Tris, pH 6.0, and a drop with a 1:1 mix following the product protocol. Quantitative real-time PCR volume of EED protein and crystallization solution. The crystal (qRT-PCR) was performed in a CFX384 Touch Real-Time Detec- was soaked with crystallization solution containing 0.5 mmol/L tion System (Bio-Rad). The qRT-PCR primers were listed in BR-001 for 24 hours. 30% glycerol was used as cryoprotectant for Supplementary Table S2. Relative quantified RNA was normal- flash frozen in liquid nitrogen. Diffraction data were collected at ized with USF1 or GAPDH housekeeping gene. beamline BL17U in Shanghai Synchrotron Radiation Facility (SSRF). The data set was processed using HKL3000. Protein extraction and Western blotting The protein structure of EED in complex with ligand EED226 Total protein from the cultured cells or tumor samples were (PDB: 5WUK) was used as a search model for molecular replace- extracted and subject to process western blotting. Isolation of ment using Phaser. The ligand BR-001 were manually built in protein from the cultured cells was done by lysing the cells using COOT and the structures was further refined by Phenix. The final RIPA buffer (Thermo Fisher Scientific; Catalog No. 89901)

Figure 1. BR-001 from medical design is a potent allosteric inhibitor of PRC2. A, Structure of BR-001. B, Biochemical activity of BR-001 inhibition of EED–H3K27me3 interaction (n ¼ 6; mean SEM). C, DSF analysis for EED in the absence of compound (DMSO control), in the presence of BR-001 with 50 or 100 mmol/L. D, Effect of BR-001 on H3K27me3 levels in Karpas 422 cells measured by 3-d ELISA assay. E, Effect of BR-001 on proliferation of Karpas422 measured by CTG at day 13. All experiments were repeated at least three times and representative data are shown (n ¼ 6; mean SEM).

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supplemented with a protease inhibitor cocktail. Protein in tumor Tumor-infiltrating lymphocytes analysis tissues was extracted by homogenizing. Briefly, 30 to 50 mg tumor Flow cytometry single-cell suspensions of tumors were pre- tissue in 240 to 400 mL RIPA buffer was smashed using JXFSTPRP- pared and analyzed by Pharmaron. Additional information is 64 under the same conditions as described previously. After the available in the Supplementary Materials and Methods. centrifuge, protein concentration in the supernatant was determined using a Pierce BCA Kit (Thermo Fisher Scientific). Results Western blot analysis BR-001 is a potent EED inhibitor in vitro Thirty micrograms of total protein was separated on 10% to To inhibit the PRC2 activity, we designed EED-binding ligands 12% SDS-PAGE, then transferred to PVDF membrane (Bio-Rad, that can disrupt the EED–H3K27me3 interaction. Using a recently Catalog No. 1620239). The membrane was incubated with published allosteric PRC2 inhibitor EED226 as a starting primary antibody (Supplementary Table S1) in blocking buffer point (35), we performed scaffold hopping of the core structure (TBST with 5% non-fat powdered milk) at 4C for overnight. Then followed by side chains optimization. Such medicinal chemistry the membrane was incubated with the secondary antibody (Sup- effort led to a new class of pyrimidone compounds, exemplified plementary Table S1) for 1 hour at room temperature after wash. by BR-001 (Fig. 1A). In a competition binding assay, BR-001 The protein was visualized by enhanced chemiluminescence showed a dose-dependent displacement of H3K27me3 from EED (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific) with an IC50 value of 4.5 nmol/L. As a negative control, an EZH2 and detected using ChemiDoc Imaging Systems (Bio-Rad). inhibitor EPZ6438 did not show any displacement activity (Fig. 1B). Furthermore, BR-001 significantly increased the thermal Tumor xenografts stability of EED, as evidenced by 6 to 10C increase in melting The study is compliant with all relevant ethical regulations temperature (Fig. 1C) in thermal shift assay. BR-001 had no regarding animal research. The efficacy studies shown in Fig. 4D activity against 371 wild-type kinases (Supplementary Table S3). and E was conducted in Crown Bioscience Inc., and the efficacy These results suggest that BR-001 is a selective EED binder to study shown in Fig. 5D was conducted in Pharmaron. All experi- compete against H3K27me3 binding. ments conducted were performed in female NOD/SCID mice in EED is a core component of PRC2, which catalyzes H3K27me3 the Association for Assessment and Accreditation of Laboratory formation. Targeting EED or EZH2 inhibition can cause similar Animal Care certificated facility. All the procedures and protocols effect on H3K27 methylation (36). We evaluated the effect of BR- related to animal handling, care, and the treatment in the study 001 on the cellular level of H3K27me3 using a cell-free ELISA were approved by the Institutional Animal Care and Use Com- assay. Karpas422 cells were treated with BR-001 for 72 hours and mittee of Crown Bioscience or Pharmaron. lysed in HCl. The levels of H3K27me3 in the cell lysate were

Figure 2. Co-crystal structure of EED with BR-001. A, Overall structure of EED in complex with BR-001. BR-001 is shown in sticks mode colored in yellow with highlighting view. B, Details of intermolecular interactions between EED residues and ligand BR-001 shown in sticks mode. Hydrogen bond is indicated by orange dashes and multipolar interaction between ﬂuorine and amide side chain of N194 is indicated by blue dashes. C, Superimposition of EED apo in salmon-wheat and EED-BR-001 colored as in B.

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measured. As shown in Fig. 1D, BR-001 significantly reduced the Resistance to BR-001 is associated with H3K27 acetylation cellular H3K27me3 level and also exhibited a strong antiproli- Most DLBCL cell lines were reported to carry gain-of-function ferative effect on the cells (Fig. 1E). Our results suggest that BR- mutations of EZH2 and were phenotypically more sensitive to 001 effectively inhibits H3K27 methylation and proliferation of EZH2 inhibitors than the cells with wild-type EZH2. We tested BR- Karpas 422 cells. 001 activity in 3 DLBCL cells with EZH2 Y641 mutations. In line with previous reports, BR-001 exhibited good potency against 3 Structure of EED in complex with BR-001 cell lines (Fig. 3A and B). To assess whether BR-001 also has We solved the co-crystal structure of EED in complex with antiproliferation effects in cells with high-level expression of BR-001 at a 2.0-Å resolution (Fig. 2A; Supplementary Table S4). EZH2, we evaluated BR-001 activity against gastric, glioma, and Hydrogens were not added during the structure refinement and lymphoblastic leukemia cell lines with high or low expression of deuterium of BR-001 were not distinguishable in the density EZH2. Most of the cell lines did not significantly respond to PRC2 map. BR-001 bound to the induced aromatic cage of EED. inhibition, indicating no correlation between cellular EZH2 Multiple interactions between EED and BR-001 are observed: expression level and their phenotypic sensitivity to EED inhibitor, R365 forms pi-cation interaction with benzene ring; the amide BR-001 in the tested cells. side chain of N194 forms multipolar interaction with fluorine A recent report demonstrated that MLL1 expression level and and Y365, Y148, F97 form pi-pi stack with the central ring H3K27ac upregulation are positively correlation with resistance (Fig.2B).Comparedwiththeapostructure,cageresidues to EZH2 inhibition (39). In agreement with the report, our results undergo conformation change upon binding with BR-001, showed that the cells that were sensitive to BR-001 expressed low including Y365 and R367 (Fig. 2C). Together, BR-001 binds level of MLL-1, whereas BR-001-resistant cells had relative high with EED in the aromatic cage by inducing local conformation level of MLL-1 and H3K27Ac (Fig. 3C). KATO III was an excep- changes. tional cell line, which expressed MLL-1 at a high level and

A 125 Karpas422 SNU-16 Pfeiffer HPB-ALL WSU-DLBCL LN229 KATOIII U87MG

100

50 Normalized cell viability (%) 25

0 0 0.0015 0.0045 0.014 0.041 0.123 0.37 1.11 3.33 10 Compound concentration (µmol/L)

BCL D C -16 -ALL Karpas422 KATOIII SNU-16 HPB-ALL 697 Pfeiffer HPB KATOIII U87MG 100 Karpas422 LN229 SNU

697 BR-001 (100 nmol/L) LN229

HPB-ALL U87MG - + - + - + - + SNU-16 KATOIII Pfeiffer Karpas422 WSU-DLB 180kDa MLL-1 10 MLL-1 180kDa 17kDa 1 H3K27Ac 17kDa H3K27Ac

( µ mol/L) 0.1 H3K27me3 17kDa H3K27me3 17kDa 50

IC 0.01 EZH2 98kDa EZH2 98kDa

0.001 Histone 3 17kDa Histone 3 17kDa MLL-1(Low) MLL-1(high) GAPDH 37kDa GAPDH 37kDa MLL-1(middle) MLL-1(very high)

Figure 3. The effect of BR-001 on the proliferation and gene expression of various tumor cells. A, Dose-dependent effect of BR-001 on the proliferation of different types of cells with or without EZH2 mutations. Representative data from three biological replicates. B, IC50 values were calculated at 13-day treatment in different cell types. The experiments were repeated three times and representative data were shown. C, Protein expression levels of MLL1 and H3K27me3 in the sensitive cells and insensitive cells were examined by Western blot analysis. D, The changes of H3K27ac and H3K27me3 in the cells exposed to BR-001. Indicated cells were treated with BR-001 for 72 hours. Levels of H3K27ac and H3K27me3 were examined by Western blot analysis.

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ABCKarpas 422 cell line Pfeiffer cell line 1.2 Karpas 422 8 1,500 DMSO DMSO Pfeiffer BR−001 (0.11 µmol/L) − µ BR−001 (0.33 µmol/L) BR 001 (0.11 mol/L) 900 − µ BR−001 (0.33 µmol/L) ) 0.9 BR 001 (1 mol/L) BR−001 (1 µmol/L) BR−001 (3 µmol/L) n 6 − µ

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1,800 T 2,400 Vehicle po bid T

BR-001(40mpk po bid) u umo BR-001(100mpk po bid) 0 m BR-001(40mpk po bid) 2.0 1,500 or 2,000 BR-001(100mpk po bid) 0 r g gr r

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Tumor volume (mm 1 58 121519222629333640 Tumor volume (mm 0 3 7 10 1417 2124 2831 35 39 Vehicle 40 mg/kg 100 mg/kg Days posttreatment Days on treatment BR-001

H3K27me3 levels in Pfeiffer tumors Derepression of PRC2 target genes I Derepression of PRC2 target genes GHin Karpas422 tumors in Pfeiffer tumors 2.0 8 150 Vehicle Vehicle BR-001(40mpk) 120 BR-001(40mpk) 1.5 BR-001(100mpk) BR-001(100mpk) 3 6 ression cle) i p

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0.5 old ivegeneex t (Folds of vehicle) (F 2 30 Rela 0.0 Relative gene expression Vehicle 40 mg/kg 100 mg/kg 0 0 BR-001 RTP4 CASP1 SPP1 CTSO CD86 P16 RTP4 CASP1 SPP1 CTSO CD86 P16

Figure 4. BR-001 inhibited proliferation of DLBCL cells and tumor growth, and upregulated target genes expression. A, Antiproliferation activity against Karpas422 and Pfeiffer cells. B and C, qRT-PCR analysis of gene expression level in Karpas422 (B) and Pfeiffer (C) after BR-001 treatment. RNA was extracted from the cells treated with BR-001 at 48 hours at indicated concentrations (n ¼ 3; mean SEM). D, Dose-dependent efﬁcacy following oral administration of BR-001 twice daily in Karpas 422 xenograft model. TGI at day 36 treated with 40 and 100 mg/kg of BR-001 is shown (75% and 85%, respectively). E, Dose-dependent efﬁcacy following oral administration of BR-001 twice daily in Pfeiffer xenograft model. TGI at day 36 treated with 40 and 100 mg/kg of BR-001 were shown (83% and 96, respectively). F, H3K27me3 levels in response to BR-001 treatment were assessed in Karpas 422 xenografts collected from D. G, H3K27me3 levels in response to BR-001 treatment were assessed in Pfeiffer xenografts collected from G. H, PRC2 target genes in tumors collected from D were analyzed by quantitative RT-PCR. I, PRC2 target genes in tumors collected from G were analyzed by qRT-PCR. , P < 0.01; , P < 0.001; , P < 0.0001 by one-way ANOVA.

H3K27ac at a low or undetectable level, was sensitive to BR001. that the global loss of H3K27me3 following inhibition of PRC2 Moreover, no increase of H3K27ac was observed when KATO by BR-001 is associated with transcriptional activation of PRC2 III and sensitive cells were treated with BR-001. In contrast, target genes. H3K27ac level was increased in the insensitive cells tested (Fig. 3D). The levels of H3K27me3 in all cell lines tested were BR-001 has potent antitumor efficacy in vivo decreased upon BR-001 treatment (Fig. 3D; Supplementary PRC2 inactivation effectively inhibited DLBCL cells with EZH2 Fig. S1A and S1B). Taken together, the results suggest that Y641 mutation in vitro and markedly suppressed the tumor H3K27ac upregulation mainly contribute to the resistance to growth of EZH2 mutant DLBCL xenografts in immunocompro- PRC2 inhibition. mised mice (35, 36). We chose 2 sensitive cell lines (Karpas422 We further examined the gene expression changes in DLBCL and Pfeiffer) to establish xenograft models. 40 or 100 mg/kg twice cells. Six known PRC2 target genes were evaluated in both Karpas daily of BR-001 were orally dosed to mice at day 14 postimplan- 422 and Pfeiffer cells that are sensitive to BR-001 (Fig. 4A). Dose- tation. The tumor volume was measured every 3 or 4 days. BR-001 dependent induction of gene expression was observed in both cell showed a dose-dependent efficacy in both xenografts. 100 mg/kg lines (Fig. 4B and C). Overall, the changes of gene expression in doses of BR-001 induced 85% tumor growth inhibition (TGI) in Karpas 422 is much larger than that in Pfeiffer cells. The mech- Karpas 422 and 96% TGI in Pfeiffer models at day 36 posttreat- anism of variation of expression level of genes in the 2 cell lines ment. The body weight in both models was not significantly will warrant further investigations. The results demonstrate affected (Fig. 4D and E; Supplementary Fig. S2A and S2B). Levels

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of H3K27me3 and PRC2 target genes in tumors were analyzed at BR-001 modulates immune response to suppress syngeneic the last day of treatment. BR-001 caused substantial decreases of CT26 colon tumor H3K27me3 in dose-dependent manner (Fig. 4F and G) and It has been reported that disrupting PRC2 function has immu- upregulated the PRC2 target genes (Fig. 4H and I). nomodulatory activity. Highly expressed PRC2 components was

Figure 5. BR-001 suppressed CT26 tumor growth in syngeneic model through modulating immune response. A, Antiproliferation activity of BR-001 against murine CT26 colon carcinoma cells. Cell viability was determined after 13-day treatment. 5-FU was used as a positive control. B, qRT-PCR analysis of the PRC target genes associated with immunogenicity in the CT26 cells. The CT26 cells were incubated with 10 mmol/L BR-001 for 48 hours, total RNA was extracted and quantiﬁed for qRT-PCR. C, Stimulation of CXCL10 expression by cytokines in the CT26 cells. The CT26 cells were treated with 10 mmol/L BR001, 2000 U/mL INFg,or 2000 U/mL TNFa for 48 hours; total RNA was isolated for qRT-PCR analysis. D, Dose-dependent responses of CT26 tumor-bearing mouse model to BR-001 treatment. E, qRT-PCR analysis of immune genes in CT26 tumors collected from 12-day treatment in D. F, Western blot detection of their protein levels in CT26 tumors collected from 12-day treatment in D. G, Flow cytometry analysis of TILs. H and I, The percentage of CD8þ T cells and regulatory T cells. J, The þ þ þ þ corresponding ratios of CD8 T cells versus CD4 CD25 Foxp3 Tregs in the CT26 tumor tissues. , P < 0.05; , P < 0.01; , P < 0.001 by one-way ANOVA. The CT26 tumors were collected from 12-day treatment in D.

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inversely associated with the expression of CD4, CD8, and affect by BR-001 treatment either (Supplementary Fig. S3E and subsequent production of Th1-type chemokines in human S3F). Collectively, these results indicated that PRC2 inhibition by colon cancer (40). In melanoma cancer cells, PRC2 inhibition BR-001 has an immunomodulatory effect in CT26 colon carci- þ promoted expression of PRC2 target genes associated with noma. Highly expressed CXCL-10 effectively recruited CD8 T þ tumor immunogenicity (41). To investigate whether BR-001 cells to tumor positions, resulting in increase of the ratio of CD8 was able to upregulate immunogenicity-related genes in colo- T cells to Treg cells. It should be noted that all tumor samples for rectal cancer cells, we examined the expression of the target gene, protein and TILs analyses were performed after the last day genes and cell proliferation ability in murine CT26 cells that of BR-001 dosing. were exposed to BR-001. BR-001 had no antiproliferative effect on the CT26 cells, while a reference compound, 5-FU (princi- pally as a thymidylate synthase inhibitor) showed activity Discussion against the cells (Fig. 5A). Next, we evaluated mRNA level of Epigenetic deregulations are associated with aberrant transcrip- immune-related genes including the member of the major tion and gene function and consequently drive cancer develop- histocompatibility complex, chemokine and receptor. Interest- ment. Increased methylation activity of PRC2 has been frequently ingly, we found only the mRNA level of CXCL10 was greatly found in a wide variety of cancerous tissue types. In addition, increased in treated cells compared with the DMSO-treated accumulated evidence suggests that PRC2 also plays a somewhat control cells (Fig. 5B). The fold change of CXCL10 gene expres- antagonistic role in the antitumor immune response. PRC2 þ sion in the 10 mmol/L BR-001 treated CT26 cells was similar to silences Th1-type chemokines to suppress CD8 T cell migration. that in 2000 U IFNg-stimulated cells (Fig. 5C). Upregulation of EZH2 or other PRC2 components confers resis- CXCL10 is a chemoattractant for activated T cells and plays an tance to immunotherapy (40, 41, 43–46). Therefore inhibition of important role in recruitment of effector T cells into tumor PRC2 in combination with checkpoint inhibitors might be an sites (41, 42). The above in vitro result suggests a hypothesis that attractive strategy for cancer therapy. BR-001 might be able to repress CT26 tumor growth in vivo Here we reported the discovery of a potent, selective EED through stimulating CXCL10 expression. To further test this inhibitor, BR-001. BR-001 has good phenotypic activities hypothesis, we assessed antitumor efficacy of the EED inhibitor in vitro and in vivo, and also exhibits anti-tumor efficacy in the BR-001 in a CT26 syngeneic model. Treatment of the xenograft CT26 syngeneic model. BR-001 treatment leads to higher with BR-001 showed significant tumor repression with TGI value expression levels of CXCL10 in CT26 cells and tumors. In of 59.3% at a 30 mg/kg dose (Fig. 5D). The body weight was not support our studies, previous reports show that PRC2 compo- significantly affected (Supplementary Fig. S2C). BR-001 caused nents and H3K27me3-mediated the TH1-type chemokine gene substantial increase of CXCL10 at both mRNA and protein levels silencing in ovarian and colon cells (40, 43). Thus removal of in the CT26 tumor, whereas EZH2 level remained the same H3K27me3 increases CXCL-10 expression. Cytokine TNFa in (Fig. 5E and F). These results correlate well with our in vitro data. CT26 tumor is also moderately upregulated upon BR-001 Furthermore, we found that the TNFa level was moderately treatment. TNFa is a multifunctional cytokine playing key roles increased in the BR-001-treated CT26 tumors, but decreased in in inflammation and immunity. One function is to remodel the the CT26 cells (Fig. 5E and B). The discrepancy between the CT26 tumor microenvironment by promoting the activation of cyto- þ tumor and CT26 cells is likely due to the difference between in vitro toxic CD8 T cells (47, 48). Our data show that TNFa strongly and in vivo environment. stimulates CXCL10 expression in CT26 cells (Fig. 5C), suggest- Inhibition of PRC2 by BR-001 was sufficient to cause the CT26 ing that TNFa plays a role in modulating immune response tumor suppression. To investigate whether the combination of throughtriggeringCXCL10expressioninCT26carcinoma BR-001 with anti-PD-1 could further improve antitumor activity, tumor. we conducted the combination therapy in the CT26 xenograft The syngeneic study is encouraging and may provide an model. Co-administration of 30 mg/kg (twice daily) of BR-001 opportunity for immune-oncology therapy. In our study, BR- and 10 mg/kg (twice a week) of anti-PD-1 did not further reduced 001 and anti-PD-1 combination did not provide any advantage the tumor size when compared with treatment with BR-001 as a over BR-001 alone in the CT26 xenograft (Fig. 5D). This is single agent at the dose of 30 mg/kg (Fig. 5D). Thus, no synergy another evidence to show that PRC2 inhibition plus immuno- effect was observed in BR-001 and anti-PD-1 combination. The therapy did not benefit for colon carcinoma. Synergistic anti- result is consistent with a previous report that EZH2 inhibition tumor activity is only observed in tumors that PRC2 members plus immunotherapy did not provide any advantage in MC-38 are upregulated upon immunotherapy. In contrast, the tumors colon tumor (41). that are bare or absent upregulation of PRC2 does not benefit To assess the mode of action of PRC2 inactivation-caused TGI, from the addition of PRC2 blockade to immunothera- we analyzed tumor-infiltrating lymphocytes (TIL) from fresh py (41, 49). Hence, the cancers characterized by increased tumor samples. Compared with vehicle control, BR-001 treat- PRC2 members after immunotherapy could be suitable for þ ment significantly increased the counts of CD8 T cells in TILs combination of EED/PRC2 inhibitors with immune checkpoint (Fig. 5G and H). Treg cell counts in TILs remained the same inhibitors. between treatment and control groups. Therefore, the ratio of Overall, our data suggested that BR-001 is a potent EED þ CD8 T cells to Treg cells in BR-001 treated groups was increased inhibitor with 2 antitumor mechanisms for therapy: direct inhi- þ þ (Fig. 5I and J). The level of CD11b Gr-1 MDSCs was also higher bition by inactivation of PRC2 and modulation of the tumor þ in the BR-001–treated tumor tissues (Supplementary Fig. S3A). As microenvironment by increasing CD8 T-cell tumor infiltration. controls, natural killer cells and tumor-associated microphage and white blood cells (Supplementary Fig. S3B, S3C, and S3D) Disclosure of Potential Conflicts of Interest þ did not change. Total T cells and CD4 T cells did not significantly No potential conflicts of interest were disclosed.

5594 Cancer Res; 79(21) November 1, 2019 Cancer Research

Inhibition of PRC2 Activity Induces Antitumor Effects

Authors' Contributions Acknowledgments Conception and design: H. Dong, Y. Chen, B. Zou We would like to thank staff at SSRF for X-ray diffraction data collection. We Development of methodology: S. Liu, X. Zhang, Y. Chen are grateful to researchers at Crown Bioscience Inc. and Pharmaron who support Acquisition of data (provided animals, acquired and managed patients, our in vivo pharmacology studies and FACS analysis. We thank Joseph provided facilities, etc.): X. Zhang, S. Chen, L. Kang, Y. Chen, Y. Liu M. Salvino and reviewers for critical and constructive suggestions of the article. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Dong, S. Liu, X. Zhang, S. Chen, L. Kang, Y. Chen, The costs of publication of this article were defrayed in part by the payment of Y. Liu, B. Zou page charges. This article must therefore be hereby marked advertisement in Writing, review, and/or revision of the manuscript: H. Dong, S. Liu, Y. Chen, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. H. Zhang, B. Zou Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Chen, Y. Chen, S. Ma, X. Fu Received February 3, 2019; revised April 11, 2019; accepted August 1, 2019; Study supervision: H. Dong, B. Zou published ﬁrst August 8, 2019.

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5596 Cancer Res; 79(21) November 1, 2019 Cancer Research

An Allosteric PRC2 Inhibitor Targeting EED Suppresses Tumor Progression by Modulating the Immune Response

Hongping Dong, Shaojun Liu, Xuejie Zhang, et al.

Cancer Res 2019;79:5587-5596. Published OnlineFirst August 8, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0428

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