A cryptic transactivation domain of EZH2 binds AR and AR’s splice variant, promoting oncogene activation and tumorous transformation

Abstract Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2’s multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of both EZH2’s canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2TAD for mediating EZH2’s non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancer that rely on the circuits wired by EZH2 and AR.


INTRODUCTION
Prostate cancer is the most frequently diagnosed, noncutaneous malignancy in men, causing ∼30 000 deaths annually in the USA (1). Androgen deprivation therapy (ADT) is the standard treatment for prostate cancer (2), yet it is almost inevitable that patients receiving ADT become refractory and eventually develop castration-resistant prostate cancer (CRPC) (3,4). Both genetic and epigenetic deregulations play critical roles during the development of prostate cancer and its stepwise progression into more ad-vanced and aggressive forms, notably CRPC. Androgen receptor (AR) and Enhancer of Zeste Homolog 2 (EZH2) are among the most relevant prostate cancer-promoting oncoproteins. Amplification of the AR gene, mutation of AR's ligand-binding domain (LBD) and/or AR cofactordirected mechanisms can lead to a hyperactive AR signaling during prostate tumorigenesis (5,6); in addition, the AR gene undergoes alternative splicing with inclusion of cryptic exons in CRPC, giving rise to the truncated, hormoneindependent, constitutively active AR variants such as AR splice variant 7 (AR-V7) (7,8).
EZH2 is widely known as a catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which induces trimethylation of histone H3 lysine 27 (H3K27me3) to maintain repression of a battery of tumor-suppressive, differentiationpromoting and immunity-related transcripts (9)(10)(11)(12)(13)(14). Overexpression of EZH2 due to genomic loss of EZH2-targeting microRNA is associated with prostate cancer progression, correlating to poor clinical outcomes (15). Increasing evidence demonstrated that, in prostate cancers including CRPC, EZH2 also has non-conventional functions in binding to non-PRC2 partners (16), such as AR and fibrillarin for potentiating transcriptional activation (17)(18)(19)(20) and translation (21), respectively. Studies of other types of cancer such as breast cancer also showed EZH2 overexpression not correlated with the increase of H3K27me3 (22), consistent with a PRC2-independent role for EZH2. Indeed, a recent study of MLL-rearranged acute myeloid leukemia (AML) (23) demonstrated that EZH2 directly binds cMyc and p300 through a cryptic transactivation domain (TAD), independently of its PRC2 function, to promote oncogenesis (23,24). However, the role of the TAD of EZH2 (EZH2 TAD ) in prostate cancer remains unexplored to date.
Previously, it has been shown that knockdown (KD) or knockout (KO) of EZH2 in various prostate tumor models suppressed cancer cell proliferation (15,(17)(18)(19)21), laying a strong foundation for therapeutically targeting EZH2 as an attractive strategy for the treatment of prostate cancer. Small-molecule inhibitors that selectively target the methyltransferase activity harbored within EZH2's Su(var)3-9, Enhancer-of-zeste and Trithorax (SET) domain have been developed, some of which are currently under clinical development (15,25). However, it is most likely that these EZH2 enzymatic inhibitors cannot block EZH2's methyltransferase-independent functions such as those related to gene activation and/or scaffolding (e.g. recruiting/binding non-PRC2 factors), some of which have been suggested to be equally critical for oncogenesis (18,21,23). Strategies for completely blocking the multilevel activities of EZH2 in prostate cancer need to be developed.
Next, the cell:bead sample was incubated with antibodies against the protein target (1:100 dilution) and then permeabilized in digitonin-containing buffer, which was followed by washing in digitonin buffer, incubation with pAGmicrococcal nuclease (MNase) and another wash in digitonin buffer to remove unbound pAG-MNase. After the final wash, cells were subjected to digestion following the pAG-MNase activation by addition of pAG-MNase digestion buffer, followed by incubation on a rotator for 2 h at 4 • C. Solubilized chromatin was then released using the CUT&RUN stop buffer, in which an equal amount of Drosophila spike-in chromatin (0.5 ng/sample) was added across all samples for spike-in normalization, and DNA purification was carried out with a polymerase chain reaction (PCR) cleanup kit (Qiagen). About 5 ng of the purified CUT&RUN DNA was used for preparation of multiplexed libraries with the NEB Ultra II DNA Library Prep Kit as per the manufacturer's instruction. Deep sequencing was conducted using an Illumina NextSeq 500 Sequencing System (available at the core facility of the Department of Pharmacology, University of North Carolina).

Chromatin immunoprecipitation (ChIP) followed by quantitative PCR (ChIP-qPCR)
ChIP-qPCR was performed as previously described (23,28,29). Information of primers used for ChIP-qPCR is given in Supplementary Table S4.

ChIP sequencing (ChIP-seq) and CUT&RUN data analysis
ChIP-seq datasets downloaded from the NCBI Gene Expression Omnibus (GEO) were re-analyzed as previously described (23). AR CUT&RUN was conducted with a pan-AR antibody, which recognizes an N-terminal region of AR-FL and AR-V7 (Cell Signaling Technology, Cat # 5153). CUT&RUN data analysis including spike-in normalization was conducted as before (23). In brief, raw reads were mapped to the reference genome (hg19) by using bowtie v2.3.5. The non-primary alignment, PCR duplicates and blacklist regions were removed from aligned data by using Samtools (v1.9), Picard 'MarkDuplicates' function (v2.20.4) and bedtools (v2.28.0), respectively. Peak calling was performed by using MACS2 (macs2 callpeak -f BAMPE -g hs/mm -keep-dup 1). Deeptools (v3.3.0) was used to generate bigwig files. Genomic binding profiles were generated by using the deepTools 'bam-Compare' functions. Profiles of CUT&RUN read densities were displayed in Integrative Genomics Viewer (IGV, Broad Institute). Heatmaps of ChIP-seq signals were generated by using the deepTools 'computeMatrix' and 'plotHeatmap' functions. Distribution of peaks was analyzed by the 'an-notatePeaks.pl' function of HOMER (Hypergeometric Optimization of Motif Enrichment) (30). Motif analysis was performed by the SeqPos tool in Cistrome to uncover the motifs that are enriched close to peak centers by taking peak locations as the input (31).

RNA sequencing (RNA-seq) and data analysis
RNA-seq was performed as described (23,28). Total RNA was first purified by using the RNeasy Plus Mini Kit (Qi-agen, #74136) and then treated with the Turbo DNAfree kit (Thermo, #AM1907) to remove genomic DNA. Multiplexed RNA-seq libraries were generated and subjected to deep sequencing. Reads were mapped to the reference genome followed by analysis of differentially expressed genes (DEGs) as before (28,32). Fastq files were aligned to the GRCh38 human genome (GRCh38.d1.vd1.fa) using STAR v2.4.2 (33) with parameters: -outSAMtype BAM Unsorted -quantMode TranscriptomeSAM. Transcript abundance was estimated with salmon v0.1.19 (34) to quantify the transcriptome defined by Gencode v22. Gene level counts were summed across isoforms, and genes with low counts (maximum expression <10) were filtered for downstream analyses. Raw read counts were used for DEG analysis by DESeq2 v1. 38.2 (35) where the size normalization factor was estimated based on median-of-ratios. GSEA (36) was performed as described (23,32). Expression heatmaps were generated using mean-centered log 2converted TPM (transcripts per million) sorted in descending order based on expression values in R's package 'gplots' v3.0.3 with either no clustering or column hierarchical clustering by average linkage. Volcano plots visualizing DEGs were produced by using R's package 'EnhancedVolcano' v3.11. Annotation of DEGs was conducted by using Metascape (37). DisGeNET (38) was used to annotate genes associated with human disease.

Plasmid construction
Wild-type (WT) or the serially deleted version of AR was fused in-frame with an N-terminal Flag tag and subsequently cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). GST fusion constructs with full-length EZH2 or only EZH2 TAD , either WT or the TAD transactivation-dead mutant (23) [including F145A + F171A (FA) or F145K + F171K (FK)], and the pCDH-EF1a-neo-based lentiviral constructs containing HA-tagged EZH2 (WT or TAD transactivation-dead mutant) were previously described and used as before (23). EZH2 SET domain deletion was generated by using a sitedirected mutagenesis kit (Agilent, 200521). All plasmids were verified by direct Sanger sequencing. Information on primers used for plasmid construction is provided in Supplementary Table S4.

Transient transfection
The 293T cells were seeded in a 100 mm dish with fresh medium, and then transfected on the next day following a previously described protocol using polyethylenimine (PEI; Sigma, # 408727) (39). Cells were harvested 48 h after transfection for downstream analysis such as co-IP or immunoblotting.
The siRNA-or shRNA-mediated gene knockdown (KD) Cells were transfected with small interfering RNA (siRNA) using the Lipofectamine RNAi MAX reagent according to the manufacturer's instruction. The MISSION® esiRNA for human CRBN (Sigma, # EHU047571) was ordered and used as per the vendor's guideline. The pLKO.1-based lentiviral short hairpin RNA (shRNA) plasmids for KD of EZH2 (23), AR or ARV7 (28) or a scrambled control (40) were described and used as before.

Viral production and stable cell line generation
Lentivirus was prepared with the packaging system in 293T cells. In brief, 293T cells were co-transfected with lentiviral vector and the packaging plasmids (psPAX2 and pMD2.5G), and supernatant containing virus was harvested at 48 and 72 h post-transfection. After filtration with a 0.45 M filter, virus-containing supernatant was used to infect cells in the presence of 8 g/ml polybrene. At 48 h post-infection, cells were selected with either 1 g/ml puromycin (Gibco) or 1 mg/ml geneticin (Gibco) for 7 days to establish stable expression cell lines.

Immunoblotting
Cells were collected and lysed in EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl, 0.5% NP-40, 0.1 mM EDTA and 10% glycerol) freshly supplemented with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitor (Roche). Protein concentration of the cell lysate was measured by Bradford assay (BioRad). Equal amounts of protein lysates were separated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Quantification of the protein band intensity was performed by ImageJ software after normalization to that of GAPDH.

Co-immunoprecipitation (co-IP) and GST pull-down
IP was performed as described previously (23,41). Cell pellets were lysed in EBC buffer (freshly supplemented with protease and phosphatase inhibitor cocktail) on ice for 30 min. After sonication, debris was removed by centrifugation at 12 000 g for 15 min at 4 • C. For IP of HA-tagged protein, HA-conjugated beads (Roche, # 11815016001) were incubated with lysate overnight at 4 • C. GST-tagged protein was purified as previously described (23). GST pull-down was conducted with cell lysate and 1 g of GST fusion recombinant protein as described (23,41).

Reverse transcription followed by qPCR (RT-qPCR)
RNA was isolated by using the RNeasy PLUS Mini Kit (Qiagen). Reverse transcription was performed with 1 g of total RNA using the cDNA Reverse Transcription kit according to the manufacturer's protocols (Invitrogen), followed by qPCR using SYBR Green Master Mix (BioRad) on a QuantStudio 6 Flex Real-Time PCR System (Thermo). Relative abundance of gene expression was calculated using the comparative CT method which compares Ct value of the target gene with that of GAPDH. Primers used for RT-qPCR are listed in Supplementary Table S4.

Cell proliferation assays
Cells were seeded at a density of 1000-2500 cells per well in a 96-well plate in triplicate and incubated with medium containing the tested compound. Medium with fresh compound was changed every 2 days. At each time point, MTT reagent (Promega) was added to the cell culture medium and incubated for 1-2 h before measurement of the absorbance at 490 nm by using the CYTATION-5 imaging reader (BioTek). The EC 50 (effective control to 50% growth inhibition) values were calculated by using a non-linear regression analysis of the mean ± standard deviation (SD) from at least triplicated datasets for each biological assay.

2D colony formation assay
The 2D colony formation assay was performed as previously described (23). Briefly, 5000 cells were plated in a 6well plate and incubated with medium containing the compound. After incubation for 2-3 weeks, cells were fixed by 100% methanol and stained with crystal violet.

Soft agar assay
Soft agar-based colony formation assay was performed as described previously (41). Briefly, 22Rv1 cells were plated at a density of 24 000 cells/ml in complete medium supplemented with 0.4% agarose, which was added onto a bottom layer composed of medium with 1% agarose. Every 4 days, 0.5 ml of fresh complete medium containing the tested compound was added onto cells. After incubation for 3-4 weeks, cell culture plates were stained with 100 g/ml iodonitrotetrazolium chloride solution (Sigma) and, after incubation overnight, the numbers of cell colonies were counted.

Cell fractionation
A total of 1 × 10 6 cells were harvested, washed with cold phospate-buffered saline (PBS) and resuspended in 200 l of CSK buffer (10 mM PIPES pH 7.0, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl 2 and 0.1% Triton X-100, freshly supplemented with protease/phosphatase inhibitor cocktail), followed by incubation on ice for 30 min, as described (32). Then, the sample was subject to centrifugation at 1300 g for 5 min at 4 • C to collect the supernatant (which refers to the soluble fraction) and pellet fraction (which refers to the chromatin fraction). Cell pellets were then dissolved in 1.5× SDS loading buffer. The same amounts of protein sample were used for immunoblotting.

Ubiquitination assay
The 22Rv1 cells treated with the tested compound were harvested and extracted in 100 l of EBC buffer containing 1% SDS. Cell extract was heat-denatured at 95 • C for 5 min and then diluted with 900 l of EBC buffer. After brief sonication and centrifugation, lysate was subjected to IP with antibody of the target protein, followed by anti-ubiquitin immunoblotting.

Tumor growth in xenografted animal models
All animal experiments were approved and performed in accord with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill. A total of 1 × 10 6 22Rv1 cells were suspended in 100 l of a 1:1 mixture of PBS and Matrigel (BD Biosciences) and subcutaneously (s.c.) injected into dorsal flanks of NOD/SCID/gammanull (NSG) male mice bilaterally (carried out by the Animal Studies Core, UNC Lineberger Comprehensive Cancer Center). Measurement of xenografted tumors was performed three times per week by using a caliper, and the tumor volume was calculated. Mice were sacrificed when tumors reached the maximum allowed size.

Statistics and reproducibility
Statistical analyses were performed by using GraphPad Prism (version 9). Unpaired two-tailed Student's t-test was used for comparing two sets of data with assumed normal distribution. Data are presented as the mean ± SD from at least three independent experiments. *, ** and *** denote P-values <0.05, 0.01 and 0.005, respectively. P <0.05 was considered to be statistically significant. NS denotes not significant. No statistical methods were used to pre-determine sample size. All data from representative experiments (such as imaging and micrograph) were repeated at least 2-3 times independently, which showed similar results.

EZH2 non-canonically binds genomic sites with the geneactive markers, RNA polymerase II (Pol II) and AR or AR-V7 in prostate cancer cells
We sought to systematically define genome-wide binding patterns of EZH2 in prostate cancer by performing CUT&RUN (42) for EZH2 and H3K27me3, a generepressive histone mark characteristic of the canonical EZH2:PRC2 complex, in 22Rv1 cells, a commonly used CRPC model. Replicated mapping profiles of EZH2 or H3K27me3 were highly correlated (Supplementary Figure  S1A), which were additionally correlated to the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) dataset of the same cells. A portion of the called EZH2 peaks (8436 or 34.5%) overlapped H3K27me3 and lacked chromatin accessibility ( Figure  1A, B; Supplementary Figure S1B); thus, we termed these EZH2 + /H3K27me3 + /low-accessibility sites as EZH2ensemble or canonical targets of EZH2:PRC2. Meanwhile, a larger proportion of EZH2-binding sites (15 987 or 65.5%) lacked H3K27me3 and, instead, were enriched for high ATAC-seq signals ( Figure 1A, B; Supplementary Figure S1B), indicating a feature of open chromatin and a non-canonical function of EZH2; thus, we defined these EZH2 + /H3K27me3 − /high-accessibility sites as EZH2solo targets. Next, we interrogated additional chromatin marks at EZH2-solo sites. Reminiscent of what was observed in MLL-rearranged AML (23), the EZH2-solo peaks identified from 22Rv1 CRPC cells were overwhelmingly enriched for a set of gene activation-related markers including H3K27ac, H3K4me2, H3K4me3, BRD4 (a histone acetylation reader and transcriptional coactivator) and RNA Pol II ( Figure 1C, bottom). Such a correlational pattern was in stark contrast to what was seen with the EZH2 + /H3K27me3 + -co-bound peaks ( Figure 1C, top).
Furthermore, overall expression of the genes associated with EZH2-solo sites was significantly higher than that with EZH2:PRC2-ensemble peaks, based on RNA-seq data of 22Rv1 cells (28) (Supplementary Figure S1C).
The above observations highlighted the existence of both classic EZH2:PRC2 sites and non-canonical EZH2-solo targets in prostate cancer, in agreement with previous reports that EZH2 forms interactions with AR, mediating target gene activation instead of repression (17,18,20,43). Consistently, a significant portion of EZH2-solo sites overlapped binding of AR and/or AR-V7 ( Figure 1D), both expressed in 22Rv1 cells (28), whereas AR/AR-V7 binding at those canonical PRC2 sites was found to be generally minimal ( Figure 1E). Notably, there was an elevation of AR/AR-V7 enrichment at EZH2-solo sites in the presence of dihydrotestosterone (DHT), compared with the ligand-deprived condition ( Figure 1D), presumably due to formation of AR:AR-V7 heterodimers that enable more robust binding of AR/AR-V7 to the genome (44). Approximately 2100 AR sites and 5500 AR-V7 sites were co-bound with EZH2 and lacking H3K27me3, thus termed as EZH2solo:AR/AR-V7 sites ( Figure 1F). In agreement with the notion that AR-V7 acts as a constitutively active form of AR, the majority of EZH2-solo:AR peaks overlapped EZH2-solo:AR-V7 peaks ( Figure 1G). Overall signals of AR/AR-V7 binding at EZH2 solo:AR-or EZH2 solo:AR-V7-co-bound peaks were found on the lower side among signals of all called peaks (Supplementary Figure S1D). Androgen-responsive element (ARE) and the binding motif of FOXA1, a known cofactor of AR, were most enriched at both EZH2-solo:AR and EZH2-solo:AR-V7 sites (Supplementary Figure S1E-F), indicating an AR/cofactor (FOXA1)-driven recruitment. Gene Ontology (GO) and DisGeNET analyses of the sites commonly bound by EZH2-solo, AR and AR-V7 uncovered the enrichment for genes involved in cell proliferation, tissue development, stress response and prostate cancer metastasis (Supplementary Figure S1G, H). Approximately 50% of EZH2solo:AR-V7 and 33% of EZH2-solo:AR sites were localized at gene promoters ( Figure 1H). To assess whether or not the aforementioned EZH2-solo:AR co-binding is conserved across different prostate cancer cell models, we next related those EZH2-solo:AR sites identified from 22Rv1 cells with the publicly available datasets of various traditional prostate cancer cell lines (18,45). While EZH2-solo:AR-cobound peaks showed similar co-occupancy by both oncoproteins in the two additional AR-positive prostate cancer lines, namely LNCaP-abl ( Figure 1I) and VCaP cells (Figure 1J), such binding of EZH2 to the same genomic sites was lacking in the two AR-negative cells, PC3 and DU145 (Figure 1K). These observations suggested potential recruitment of EZH2 by AR to a subset of AR-binding sites in a PRC2-independent fashion.

EZH2, AR and AR-V7 cooperate to activate transcription of a set of the clinically relevant oncogenes in prostate cancer
The above genome-wide profiling showed that EZH2-solo sites in prostate cancer exhibit co-localization with AR and/or AR-V7, RNA Pol II and prominent gene-active histone marks, which pointed to a gene activation-related role   for EZH2, differing from its canonical EZH2:PRC2 function seen at sites exhibiting high H3K27me3 and low chromatin accessibility. To further determine EZH2-mediated gene regulation in prostate cancer, we performed RNAseq after EZH2 KD in 22Rv1 cells (Supplementary Table S1; Figure S2A). As expected, depletion of EZH2 greatly inhibited 22Rv1 cell growth (Supplementary Figure  S2B). Analysis of DEGs showed more transcripts downregulated than up-regulated upon EZH2 KD relative to control (Supplementary Figure S2C), consistent with more EZH2-binding sites showing high chromatin accessibility in 22Rv1 cells ( Figure 1A-C). GO analysis of DEGs downregulated following EZH2 depletion showed the enrichment of pathways related to cell cycle, DNA replication and cell metabolism (Supplementary Figure S2D). Furthermore, we compared the transcriptomic profiles of 22Rv1 cells after EZH2 KD with those following either full-length AR-or AR-V7-specific KD (28). Here, we identified 130 genes to be co-activated by AR, AR-V7 and EZH2 ( Table  S2). Notably, 329 genes were co-activated by AR, AR-V7 and EZH2 with a relaxed cut-off (adjusted P or q value <0.05; Supplementary Figure S2F); here, about half of EZH2-activated genes (46.8%) overlapped those activated by AR, while around one-third of EZH2-activated genes (32.3%) overlapped those activated by AR-V7, suggesting substantial overlapping among EZH2-, AR-and AR-V7up-regulated transcripts. Conversely, only one gene was found co-repressed by AR, AR-V7 and EZH2 (Supplementary Figure S2G), indicating that the EZH2:AR/AR-V7 complex mainly functions to activate target gene expression. In addition, those EZH2:AR:AR-V7-co-up-regulated genes were generally bound directly by EZH2, AR, AR-V7, coactivators (BRD4), RNA Pol II and gene-active histone marks, but devoid of H3K27me3 ( Figure 2B), which again pointed to a gene activation effect by EZH2 and AR/AR-V7 at these targets. GO ( Figure 2C) and DisGeNET (Figure 2D) analyses also showed the EZH2-solo:AR:AR-V7co-activated genes enriched for the pathways associated with cell cycle, DNA repair, metabolism, and prostate cancer progression and metastasis. Through integration of genomic binding and RNA-seq profiles, we determined genes that were co-up-regulated by EZH2, AR and AR-V7 and also exhibited direct co-binding of the three ( Figure 2E, labeled on the right), as exemplified by PRC1, CDK2, MYBL2, HMGB1 and PDIA4 ( Figure 2F; Supplementary Figure S2H). High expression of EZH2-solo:AR:AR-V7co-activated transcripts was found to be correlated with poor prognosis of prostate cancer: the patients with higher expression of PRC1, CDK2 or MYBL2 in tumor cells exhibited significantly worse clinical outcomes ( Figure 2G, H); additionally, the expression of these genes was found to be positively associated with the EZH2 and AR levels in the TCGA prostate cancer patients (Supplementary Figure  S2I). RT . In stark contrast, expression of the same genes was significantly inhibited by treatment with the p300/CBP catalytic inhibitor A-485, which suppressed histone acetylation (47) ( Figure 2I; Supplementary Figure S2O). Compared with mock treatment, KD of AR or AR-V7 significantly reduced the direct binding of EZH2 onto the EZH2-solo:AR:AR-V7-co-targeted sites, but not canonical EZH2:PRC2 targets ( Figure 2J). As a control, AR or AR-V7 loss did not affect EZH2 protein levels in these cells (Supplementary Figure S2P). Thus, establishment of EZH2-solo binding relies on the presence of AR/AR-V7, in agreement with a lack of EZH2-solo:AR:AR-V7 sites in the AR-negative prostate cancer cells ( Figure 1K). Together, our results substantiated a PRC2-independent function of EZH2 at its solo targets in prostate cancer, which acts in concert with AR and/or AR-V7 for target gene activation, with p300/CBP and BRD4 potentially serving as co-activators.

Interaction between EZH2 TAD and AR is required for establishment of EZH2-solo binding at AR sites and for malignant growth of prostate cancer
EZH2 associates with AR (17,18,20,48). We validated interaction of EZH2 with AR and an AR variant by using both co-IP ( Figure 3A, lane 2 versus 1) and GST pulldown (Supplementary Figure S3A). The biochemical basis underlying EZH2:AR/variant interaction, however, remains murky. Recently, a cryptic transactivation domain of EZH2 (EZH2 TAD ) was reported to interact directly with cMyc and co-activators (such as p300) for promoting target gene activation (23,24,43). We thus queried whether or not EZH2 TAD also interacts with AR and AR variant in prostate cancer. First, GST pull-down using recombinant EZH2 TAD readily detected interaction with both AR and AR-V7, expressed either endogenously or exogenously ( Figure 3B; Supplementary Figure S3B; lane 2 versus 1). Such interaction with AR or AR-V7 was not observed using the EZH2 TAD mutant that carries substitution of the two key hydrophobic residues (Phe145 and Phe171) with either alanine or a charged lysine residue, which dramatically reduced the transactivation effect by EZH2 TAD (23,24) Figure S3B; see lanes of FA or FK versus WT). In agreement, full-length AR interacted with both WT EZH2 and its SET-domain-deleted form, but not the two EZH2 TAD transactivation-dead mutants, in co-IP ( Figure 3A; lanes 3 and 4 versus 2 and 5), supporting an essential requirement for EZH2 TAD for mediating EZH2:AR interaction.
We next queried what region within AR mediates EZH2 TAD interaction. Here, we found that GST-EZH2 TAD bound both full-length AR and its C-terminal-truncated form (Supplementary Figure S3C), showing that AR's Nterminal domain (amino acids 1-503) is sufficient to mediate EZH2 TAD interaction. Moreover, we constructed a set of AR serial deletion constructs for GST pull-down and further narrowed down the EZH2 TAD -interacting regions to two segments of AR: its amino acids 1-180, which is en-  TMPRSS2 The y-axis shows averaged signals after normalization to those of GAPDH and to mock-treated samples (n = 3; mean ± SD; unpaired two-tailed Student's t-test). (J) ChIP-qPCR for EZH2 binding at the promoter (pro) or enhancer (enh) of the indicated EZH2 target genes, either those EZH2-'solo' ones co-targeted by EZH2:AR:AR-V7 (left) or the canonical EZH2:PRC2 targets (right), in 22Rv1 cells after depletion of EZH2, AR or AR-V7, relative to mock (red). The y-axis shows averaged signals after normalization to those of input and then to controls (n = 3; mean ± SD; unpaired two-tailed Student's t-test). IgG [dark blue; using shRNA empty vector (shScr)-transduced samples] serves as a non-specific antibody control.   To discern the role of EZH2 TAD in mediating genomic recruitment of EZH2, we conducted CUT&RUN for HAtagged WT or TAD-mutated EZH2 following its stable expression into 22Rv1 cells. Compared with the WT, the TADmutated EZH2 exhibited significantly reduced genomic binding ( Figure 3E), as exemplified by what was seen at PRC1 and MYBL2 (Supplementary Figure S3D). Notably, there was a preferential decrease of binding of EZH2 at its solo target sites, compared with EZH2:PRC2 ensemble sites ( Figure 3F, right versus left). ChIP-qPCR corroborated that, relative to the WT, the EZH2 TAD mutant exhibited significantly reduced binding at the tested EZH2-solo targets, and not those canonical targets of EZH2:PRC2 ( Figure  3G). Lastly, we evaluated the requirement of EZH2 TAD for prostate cancer growth. By using 22Rv1 cells with endogenous EZH2 depleted for gene rescue ( Figure 3H), we found that, compared with WT EZH2, its TAD transactivationdead mutants failed to rescue the decreased expression of EZH2-solo target genes ( Figure 3I) and failed to rescue the EZH2 loss-related growth inhibition in vitro Overall, our observations demonstrated a previously unappreciated, indispensable requirement for EZH2 TAD for mediating EZH2 interaction with AR and AR variant, for EZH2's chromatin recruitment to EZH2-solo sites and for promoting prostate cancer cell growth in vitro and in vivo.

MS177, an EZH2 degrader, efficiently degrades both EZH2:PRC2 and EZH2:AR/AR-V7 complexes in prostate cancer cells
A handful of small molecules have been developed for blocking the catalytic activity harbored within EZH2's catalytic domain (SET), some of which are under clinical evaluation (49). However, we predicted efficacies of these EZH2 enzymatic inhibitors to be low due to failure in targeting non-canonical, SET(PRC2)-independent functions of EZH2 (23), such as EZH2:AR-mediated prostate oncogene activation, which we showed are TAD dependent and also clinically relevant. This notion was corroborated by a generally mild or a lack of effect by C24, a potent EZH2 SET inhibitor (51), on growth of prostate cancer cells in vitro (Supplementary Figure S4A). Using Proteolysis Targeting Chimera (PROTAC) technology, we recently developed MS177, an EZH2-targeting PROTAC/degrader, that can simultaneously deplete EZH2 and EZH2-associated factors in cells (23). Thus, we examined the EZH2-degrading effect by MS177 in prostate cancer cells. As expected, MS177 treatment in 22Rv1 cells dose-dependently depleted cellu-lar EZH2, regardless of being in the soluble or chromatinassociated fraction (Supplementary Figure S4B), and its PRC2 partners (EED and SUZ12), an effect not observed following comparable treatment with C24 or either of the two inactive analog compounds of MS177, namely MS177N1 (E3 binding-dead) and MS177N2 (C24 bindingdead) (23) ( Figure 4A). The half-maximal degradation concentration (DC 50 ) value of MS177 in 22Rv1 cells was measured to be 0.86 ± 0.12 M and maximum degradation (D max ) value 77% (Supplementary Figure S4C), supporting that MS177 is indeed a valuable tool for degrading EZH2 in prostate cancer. More excitingly, we observed that, unlike the enzymatic inhibitor C24 or PROTAC-inactive controls (MS177N1 or MS177N2), MS177 also concentrationdependently degraded AR and/or AR-V7 across multiple tested lines of prostate cancer including 22Rv1, C4-2 and LNCaP ( Figure 4B, C; Supplementary Figure S4D). The effects by MS177 on degradation of EZH2:PRC2 and AR/AR-V7 were found to be generally comparable ( Figure  4B, C; Supplementary Figure S4D). In contrast, the EED degrader UNC6852 efficiently depleted EED and EEDassociated EZH2:PRC2 but did not alter the AR and AR-V7 levels in 22Rv1 cells ( Figure 4D), consistent with a PRC2-independent association of EZH2 with AR and AR variant as we observed at EZH2-solo sites.
We next explored MS177's mechanism of action (MOA) for degradation of AR and AR variant. First, cellular AR was found to be ubiquitinated and then depleted posttreatment with MS177 but not C24, while the transcriptional levels of AR and AR-V7 remained unchanged (Figure 4E, F), suggesting a proteasome-dependent degradation mechanism underlying AR depletion. Additionally, MS177-induced EZH2 degradation was effectively blocked by pre-treatment of cells with pomalidomide (the E3 ligase ligand module of MS177; Supplementary Figure S4E) or MLN4924 (a NEDD8 neddylation inhibitor that suppresses assembly of Cullin-based E3 ligase; Supplementary Figure S4F). Depletion of CRBN, the E3 ligase that MS177 recruits, also almost completely abrogated MS177-induced depletion of EZH2, AR and AR variant ( Figure 4G versus B). Lastly, depletion of EZH2 also completely blocked the MS177-mediated degradation of AR and AR variant ( Figure 4H, right versus left), and, in 22Rv1 cells with endogenous EZH2 depleted, MS177 was able to degrade the TAD-mutated form of EZH2 (harboring the intact SET, to which MS177 binds) but not the AR or AR variant anymore ( Figure 4I), supporting that the AR and AR variant degradation by MS177 requires the presence of EZH2 and EZH2 TAD -directed binding of AR and AR variant.

Genomics profiling further substantiates the effect of MS177 on inhibiting both EZH2:PRC2-and AR/AR-V7-related oncogenic nodes
To further delineate the gene regulatory effect of MS177 in prostate cancer, we performed spike-in-controlled   CUT&RUN for EZH2, H3K27me3 and AR (by using an antibody that recognizes full-length AR and AR variants) after treatment of 22Rv1 cells with MS177. Compared with mock treatment, MS177 significantly decreased genomewide binding of EZH2 at both EZH2:PRC2-ensemble sites and EZH2-solo sites ( Figure 5A, B; Supplementary Figure  S5A), exemplified by what was seen at non-canonical EZH2-solo targets such as PRC1, MYBL2 and CDK2 ( Figure 5C; Supplementary Figure S5B) and classic EZH2:PRC2 targets such as CCND2, MYT1 and WNT2B ( Figure 5D; Supplementary Figure S5C). As expected, MS177 treatment also decreased the overall H3K27me3 level from EZH2:PRC2 sites ( Figure 5E, F; Supplementary Figure S5D; also see bottom panels of Figure 5D and Supplementary Figure S5C). Relative to mock, MS177 treatment caused the expected decrease of AR binding at EZH2-solo sites where AR co-bound ( Figure 5G, H); more interestingly, MS177's inhibitory effect on AR binding was found to be genome-wide and extended to those AR sites where EZH2 does not bind at all (Supplementary Figure S5E), as observed at classic targets of AR and AR variant such as KLK3, KLK2 and FKBP5 ( Figure  5I; Supplementary Figure S5F). Such a genome-wide decrease of AR binding is probably due to a 'drainage' effect by MS177 on global AR and AR variant proteins in cells (see also the Discussion), highlighting a potential advantage of EZH2-targeting PROTAC degrader (MS177) over conventional enzymatic inhibitors of EZH2. Next, we evaluated the transcriptome-modulatory effect of MS177, compared with its inactive control compounds. Here, we treated 22Rv1 cells with either dimethylsulfoxide (DMSO), MS177, C24 or MS177N1, followed by RNAseq (Supplementary Table S3). Dramatic transcriptomic changes were observed only after treatment with MS177, but not C24 or MS177N1 ( Figure 6A). MS177-activated DEGs only showed mild changes following comparable treatment with C24 or MS177N1 ( Figure 6B). Likewise, those EZH2-repressed genes, defined as DEGs re-activated upon EZH2 KD, were found to be de-repressed by treatment with MS177, and not C24 or MS177N1 ( Figure 6C), again demonstrating a superior and unique effect of MS177 on altering the transcriptome of prostate cancer cells. Gene Set Enrichment Analysis (GSEA) revealed treatment of MS177 associated with re-activation of known H3K27me3or PRC2-repressed genes, reminiscent of what was seen with RNA-seq after EZH2 KD ( Figure 6D; Supplementary Figure S6A). Meanwhile, the AR/AR-V7 signaling genes were found to be significantly down-regulated upon MS177 treatment relative to mock treatment ( Figure 6E-G; Supplementary Figure S6B). Moreover, those genes co-activated by EZH2, AR and AR-V7, as we defined by KD studies (Figure 2A), were significantly inhibited by treatment with MS177 and not C24 or MS177N1 ( Figure 6H; Supplementary Figure S6C). By RT-qPCR, we further confirmed the unique effect of MS177 on down-regulating select EZH2solo:AR/AR-V7 target genes in 22Rv1 cells ( Figure 6I).
Collectively, our integrated CUT&RUN and RNA-seq profiling following pharmacological treatment or genetic depletion of EZH2 lent strong support for an on-target effect of MS177, resulting in simultaneous suppression of both EZH2:PRC2-and AR/AR-V7-directed oncogenic circuitries in prostate cancer.

Compared with the EZH2 enzymatic inhibitors, MS177 elicits much more potent antitumor effects in prostate cancer cells
Next, we sought to evaluate the antitumor effect of MS177 in a panel of cell lines representing different stages of prostate cancer, and EC 50 values of MS177 were measured ( Figure 7A). First, MS177 had little growth-inhibitory effect in a non-malignant prostate epithelial line RWPE1 ( Figure 7A bottom; Supplementary Figure S7A Figure S7G). In addition, MS177 dose-dependently inhibited colony-forming capability ( Figure 7G) and induced apoptosis of 22Rv1 cells, as assayed by immunoblotting for apoptosis markers ( Figure 7H), whereas the non-PROTAC controls, MS177N1 or C24, had little effect ( Figure 7G, H). Together, the EZH2-targeting PROTAC MS177 robustly induces growth inhibition in a range of human prostate cancer cell lines and, importantly, its cancer-killing effects are superior to those of EZH2 enzymatic inhibitors.

DISCUSSION
Increasing evidence pointed to multifaceted functions of EZH2 in cancer. In particular, EZH2 forms the so-called EZH2-solo sites in a PRC2-independent fashion and binds AR and AR variant in prostate cancer, eliciting a noncanonical gene activation-related effect (18). In this report, we unveiled, for the first time, (i) that EZH2 utilizes a hidden EZH2 TAD to form an interaction with AR and its constitutively active variant, AR-V7; here, we further mapped the EZH2 TAD -interacting interfaces to at least two unstructured regions of AR and AR-V7: the intrinsically disorganized poly(Q) and poly(G) protein sequences (57)(58)(59). These results suggest a multivalent protein-protein interaction involving unstructured protein regions (TADs themselves often being unstructured)     and, possibly, liquid-liquid phase separation as seen with other transcription factors such as OCT4 and ER (60), which merits future studies. (ii) Integrated genomic profiling (CUT&RUN, ATAC-seq and RNA-seq), in combination with mutagenesis of EZH2 TAD , clearly demonstrated that EZH2 TAD -mediated interaction with AR and AR variant is crucial for establishment of the EZH2solo:AR/AR-V7-co-bound pattern, for transcriptional activation of their common targets, and for the potentiation of advanced CRPC cell growth in vitro and in xenografted animal models. (iii) Furthermore, we show the higher expression of EZH2:AR:AR-V7-co-upregulated transcripts to be correlated with poorer outcomes of prostate cancer patients, thus demonstrating a clinical relevance for the EZH2-mediated non-canonical pathway studied by this work. (iv) By using a battery of biochemical, CUT&RUN and RNA-seq assays, we also observed that MS177, an EZH2-targeting PROTAC/degrader, efficiently causes ontarget degradation of EZH2 and EZH2-associated canonical (EZH2:PRC2) and non-canonical (EZH2:AR:AR-V7) onco-complexes in prostate cancer cells ( Figure 7I); here, we conducted careful MOA studies to show that such MS177mediated degradation of AR and AR variant indeed relies on EZH2 binding by MS177 (as demonstrated by EZH2 depletion assay) and relies on EZH2 TAD -mediated AR interaction (demonstrated by using an AR interaction-defective mutant of EZH2 TAD ). (v) Lastly, MS177 is superior to existing catalytic inhibitors of EZH2 in suppressing the in vitro growth of prostate cancer cells. Please note that, in individual gene KD experiments, we only knocked down each one of them (EZH2 alone, AR alone or AR-V7 alone) to generate a list of commonly affected genes, which were also affected by treatment with MS177 but not its PROTACinactive analogs ( Figure 3I). However, the gene-modulatory effect of MS177 is far more dramatic, which is most probably due to MS177-induced simultaneous degradation of EZH2, AR and AR-V7 (as supported by our RNA-seq results, Supplementary Figure S6A, B; please also see the below Discussion section for a possible mechanism). We have also attempted to assess in vivo efficacy of MS177 in a 22Rv1 cell xenografted mouse model; however, we could not achieve sufficient exposure levels for MS177 in such an animal model (data not shown). Thus, we could not pursue this direction further with MS177. Optimization of MS177 into an improved EZH2 PROTAC/degrader with better in vivo pharmacokinetic properties is needed to demonstrate the in vivo efficacy of this therapeutic approach. Interplays between EZH2 and AR/AR variant can be complex. Previous studies also showed that EZH2 directly binds the AR gene promoter in a PRC2-independent manner to activate the transcription of AR and thus AR's downstream targets (such as PSA) and that EZH2 KD led to a significantly down-regulated AR protein level (20,24,48). In this study, we ruled out involvement of such a reported pathway in the MS177-induced degradation of AR and AR variant--indeed, within a short period of time of MS177 treatment (within 24 h), we found the AR and AR variant mRNA levels unaltered, and yet a vast majority of AR and AR variant proteins were already subjected to ubiquitination and proteasomal degradation. Given the existence of various cofactors of AR and AR variants, it is conceiv-able that only a subset of AR and AR variant proteins in cells forms interactions with EZH2 at a given time, a notion also supported by their common and distinctive genomebinding patterns ( Figure 1F). Thus, the observed dramatic and global effect of EZH2-targeting PROTAC (MS177) on degradation of AR and AR variant is most probably due to the fast-acting and recycling nature of MS177, which operates to essentially 'drain' the majority of cellular AR and AR variant proteins [which exhibit a half-life time of ∼6-7 h (61)] in repeated cycles of degrading the target (EZH2) and their partners from various AR-and AR variant-containing complexes in prostate cancer cells. A similar 'drainage' effect of MS177 was observed with cMyc, another EZH2 noncanonical partner, in AML cells (23). It appears that MS177 is more potent in the treatment of AR-positive prostate cancer cells than of AR-negative ones, pointing to the relevance of the AR-degrading effect of MS177. Please note that EZH2 TAD -mediated non-canonical gene activation functions may act in parallel with other EZH2-directed activities in prostate cancer, e.g. EZH2 was recently shown to regulate 2 -O-methylation of rRNA by directly interacting with fibrillarin, thereby promoting global protein translation of prostate cancer cells (21); moreover, EZH2 was also shown to bind to mutant p53 mRNA, increasing its stability and cap-independent protein translation in a methyltransferaseindependent manner (62). Further, EZH2 was reported to methylate FOXA1, leading to stabilization of FOXA1 and activation of cell cycle-related genes (63). In theory, EZH2targeting PROTACs represent a potentially attractive strategy for completely eliminating all of the multifaceted functions of EZH2 in prostate cancer, which awaits further investigation.