Polycomb group proteins in cancer: multifaceted functions and strategies for modulation

Abstract Polycomb repressive complexes (PRCs) are a heterogenous collection of dozens, if not hundreds, of protein complexes composed of various combinations of subunits. PRCs are transcriptional repressors important for cell-type specificity during development, and as such, are commonly mis-regulated in cancer. PRCs are broadly characterized as PRC1 with histone ubiquitin ligase activity, or PRC2 with histone methyltransferase activity; however, the mechanism by which individual PRCs, particularly the highly diverse set of PRC1s, alter gene expression has not always been clear. Here we review the current understanding of how PRCs act, both individually and together, to establish and maintain gene repression, the biochemical contribution of individual PRC subunits, the mis-regulation of PRC function in different cancers, and the current strategies for modulating PRC activity. Increased mechanistic understanding of PRC function, as well as cancer-specific roles for individual PRC subunits, will uncover better targets and strategies for cancer therapies.


INTRODUCTION TO POLYCOMB GROUP PROTEINS
The proper regulation of chromatin structure during development is important for establishing and maintaining celltype specific transcriptional programs. Compared to unicellular organisms, metazoans have an increased number of chromatin regulators, which play essential roles in celltype specification. One such set of chromatin regulators are the Polycomb group (PcG) proteins, which were originally discovered in Drosophila and named for their roles in leg development (1). Subsequently, PcG proteins were identi-fied as crucial regulators of development in vertebrates, acting primarily to modify histones and reduce DNA accessibility (2,3). PcG proteins fall into two broad complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) ( Figure 1) (4). PRC1 is defined by a RING subunit, which is capable of H2AK119 monoubiquitination, while PRC2 is defined by an EZH subunit, which is capable of H3K27 methylation. Both complexes incorporate a variety of mutually exclusive subunits giving rise to an array of diverse complexes with distinct biochemical activities (5,6).

Polycomb propagation and chromatin compaction
What is sometimes referred to as 'canonical' Polycomb function is a mechanism by which Polycomb marks are propagated and maintained ( Figure 2B). This is initiated by the association of the PRC2 subunit EED with H3K27me3, which allosterically activates methyltransferase activity of EZH towards unmodified H3K27 at neighboring and nonneighboring nucleosomes (34,35). cPRC1 binds a subset of H3K27me3 via the N-terminal chromodomain of the CBX subunit, monoubiquitinates H2AK119 through the E3 lig- Initiation. ncPRC1 targets specific DNA sequences (hypomethylated CpG islands) and ubiquitinates H2AK119. H2AK119ub1 is recognized by JARID2 and AEBP2 of PRC2.2, leading to the recruitment of PRC2 and deposition of trimethylation on H3K27. PRC2.1 also associates with hypomethylated CpG islands via PCL-mediated DNA binding and deposits additional H3K27me3. This leads to an initial H3K27me3-mediated compaction, drawing nucleosomes closer for EED to perform allosteric activation of EZH activity. (B) Propagation/Maintenance. H3K27me3 binds to aromatic cage of EED, and allosterically activates EZH methyltransferase activity, leading to further trimethylation of nearby unmodified H3K27. H3K27me3 is recognized by the CBX subunit within cPRC1. cPRC1 recruitment leads to further chromatin compaction and transcriptional repression. ase domain of RING1A/B, and oligomerizes via the PHC and CBX subunits to physically compact chromatin (36) into large domains called Polycomb bodies (37). Formation of Polycomb bodies is dependent on PRC2 (38) and cPRC1, in particular the chromodomain of the CBX subunit (39,40) and the sterile alpha motif (SAM) oligomerization domain of PHC (41). These nuclear foci are spatially separated from regions of active transcription as well as regions of constitutive heterochromatin (42). Within these large 3D domains, PcG-bound regions make contacts with other PcGbound regions several megabases away, resulting in repression across the domain (33). In mammalian cells, Polycomb body distribution changes during differentiation (40,43), as well as transformation (42).

Maintenance of Polycomb sites
After Polycomb binding sites are established, PcG proteins maintain these sites during cell division. The loss of ncPRC1 (RYBP deletion) in mouse embryonic stem cells (mESCs) can be compensated for with elevated EED, H3K27me3, and CBX7 at shared target loci, indicating some redundancy for ncPRC1 by PRC2/cPRC1 in the maintenance of previously established Polycomb binding sites (21). While PRC2/cPRC1 is not required for initiating Polycomb binding sites, it is critical for transcriptional repression (17) and may be sufficient for Polycomb maintenance/memory, at least in some cell types (44). During differentiation, however, dynamic changes in PRC subunit composition and genome localization require establishment of new Polycomb sites (45).

Biochemical functions of individual PRC1 and PRC2 subunits
Advanced proteomic techniques have begun to identify the combinations of individual PRC subunits unique to particular cell types. While structural and functional studies have elucidated the mechanistic contribution of critical core subunits, the biochemical contribution of accessory and substoichiometric subunits, and the unique biochemical contribution of individual paralogs are not always clear. A summary of defined subunit domains and known biochemical functions is provided in Table 1.

PcG PROTEINS IN CANCER
Tumor genome sequencing has revealed frequent alteration of genes encoding chromatin regulators, including PcG proteins (109). PcG proteins have traditionally been viewed as oncogenic due to their ability to prevent differentiation (110) and bypass senescence (111,112), in part by repressing the INK/ARF tumor suppressor genes (113,114). This function is important for maintaining proliferation of both normal stem cells and cancer stem cells (CSCs) (11,115). Despite a generally oncogenic function for PcGs, the role for individual subunits can vary tremendously, with some subunits acting as both an oncogene and a tumor suppressor depending on the context. Predicting which cancers are dependent on PcG function and then determining the best subunit to target is still challenging, although several trends have emerged based on clinical and experimental data.

Genetic mutation of PcG genes in cancer
From the TCGA mutation data, it is clear that some cancers have a high incidence of PcG gene mutation ( Figure 3). The high mutation rates in many of these cancers, such as melanoma (SKCM), stomach cancer (STAD), colon cancer (COAD), uterine cancer (UCEC) and lung cancers (LUAD, LUSC) are more likely due to high mutational burden than any relevant function for PcG proteins (116). However, frequent mutations in some subunits, such as MGA and BCOR/BCORL1, as well as generally high mutation rates in cancers such as diffuse large B-cell lymphoma (DLBCL) and cholangiocarcinoma of the bile duct (CHOL) point to specific roles for Polycomb subunits in tumor suppression.
MGA. MGA mutations are frequently found in multiple cancer types (Figure 3). The oncogenic role of MGA mutation is likely not due to the loss of PRC1.6 function but instead due to the release of free MAX, which can associate with MYC, and facilitate MYC-mediated oncogenic transcription (117,118). Whether mutations in other PRC1.6 subunits affect MYC function in a similar manner has not been established.
DLBCL. Due to a prominent role for Polycomb in both Bcell and T-cell development, blood cancers have frequent genomic alterations in Polycomb genes (34,131). In most lymphoid malignant contexts, PRC subunits are overexpressed or activated; however, loss of function mutations are found in some specific types of lymphoid cancers, typically pediatric cancers, or cancers with mutations that activate Ras signaling (132).
TALL. Loss-of-function mutations in all three PRC2 subunits are found to be paired with Notch mutations in over 40% of early T-cell precursors, leading to T-cell acute lymphocytic leukemia (TALL) (133)(134)(135)(136)(137)(138). Loss of PRC2 subunits in this cell type prevents T-cell differentiation by allowing repressed, but primed, promoters marked by H3K27me3 to become fully methylated and constitutively repressed (139). In this setting, it is not the repressive function of PRC2 that is important for tumor suppression, but the reversible nature of PRC2-mediated repression.
MPNST. In conjunction with additional tumor suppressors, the PRC2 subunits EED and SUZ12 (but not EZH2) are mutated in 85% of malignant peripheral nerve sheath tumors (MPNST) that develop in patients with NF1 mutations (140). These cancers have loss of H3K27me2 in addition to H3K27me3, indicating different mechanistic outcomes compared to EZH2 mutant cancers (141).

Other genetic events leading to Polycomb loss-of-function
Oncohistone H3K27M. Lys27Met (K27M) mutations in histone H3.1 and H3.3 genes have been identified as genetic drivers in pediatric cancers (142)(143)(144)(145). Even though mutations in a single copy of H3 constitute only a small fraction (3.63%-17.61%) of total H3 expressed, H3K27me3 levels are globally reduced (146)(147)(148) due to increased affinity to, and inhibition of, EZH2 by H3K27M (147). The inhibition is specific, however, to allosterically activated EZH2 involved in the spread of H3K27me3 marks, and does not affect EZH2 activity at CpG islands (108). Instead, increased H3K27me3 is observed at this small subset of sites (149) leading to efforts to inhibit EZH2 (150,151) in addition to targeting aberrant H3K27Ac-mediated gene activation (152,153).

EZHIP overexpression.
Posterior fossa type A ependymomas have increased expression of EZHIP, which inhibits allosterically activated PRC2 through a motif similar to H3K27M (107,108,152,154). Similar to H3K27M cancers, a global decrease in H3K27me3 is observed in this cancer   (85) • The methyl binding function has been shown to be dispensable for repression (85) AUTS2 • Shown to be transcriptionally activating at target loci (28) • AUTS2 containing ncPRC1.5 has been shown to have reduced H2AK119 ubiquitination activity in vitro and AUTS2-RING1B co-bound genomic targets exhibit lower levels of H2AK119ub1 and H3K27me3 in mouse brain cells (28) • Catalytic subunit with SET domain-mediated H3K27 methylation activity using S-Adenosyl Methionine (SAM-e) as the methyl group donor (11) • H3K27 methylation activity is dependent on incorporation into a complex with EED and SUZ12 (11) • SET domain (SAM-e binding site) has been the target of the majority of EZH2 inhibitors • • AEBP2-null mESCs have been reported to show increased H3K27me3 levels due to formation of a hybrid PRC2 in these cells consisting of PRC2 core-JARID2-PCL2 (99) • Two isoforms identified in humanan adult specific isoform (51 kDa) and an embryo-specific smaller isoform (32 kDa), both containing the zinc fingers (100)  Figure 3. Heatmap of PcG subunit mutation frequency in cancer. The number of patient tumors with mutations in PcG genes was determined using TCGA datasets in cBioPortal, and the percentage in each cancer type is displayed as a heatmap. White boxes indicate that no mutational data was available.
type; however, therapeutic strategies based on this mechanism have not yet been investigated.

Genetic events leading to Polycomb dependency
Covered more extensively in (155).
EZH2 gain-of-function mutations. EZH2 is critical for the activation of resting B-cells and their entry into the cell cycle during germinal center (GC) development. As such, activation of PcGs through mutation or overexpression is commonly observed in B-cell leukemias and lymphomas. Heterozygous mutations of the Tyr641 (Y641F/N/S/H/C) are found in 22% of follicular lymphomas and GC type DL-BCLs (87,156,157) and mutations of A677 and A687 have been described in isolated cases of DLBCLs (88). These mutants are inactive as monomethyltransferases but efficient Somatic mutation of UTX. UTX is a member of the MLL4 COMPASS complex and is the demethylase responsible for removal of H3K27me3 marks, among others (161). Inactivating mutations in UTX occur in several types of human cancer (162,163) and result in the loss of the JmjC domain essential for demethylase activity (161). Many UTX mutant cancers have increased levels of H3K27me3 and are more sensitive to EZH2 inhibitors (163).

SWI/SNF alterations. Similar to what is observed in
Drosophila, Polycomb complexes and SWI/SNF chromatin remodelers are antagonistic in mammalian cells (164,165). As such, several cancers with SWI/SNF subunit alterations have increased H3K27me3 and an increased sensitivity to EZH2 deletion. In particular, pediatric rhabdoid tumors with loss of the SWI/SNF subunit SNF5 are dependent on EZH2 (166) and respond well to EZH2 inhibitors, which are now approved clinically for these cancers (167). Other SWI/SNF altered cancers that are dependent on EZH2 include SS18-SSX translocated synovial sarcoma (168), ARID1A-mutated ovarian cancer (169), SMARCA4mutated lung (170) and ovarian cancers (171), and PBRM1mutated renal cancers (172). Although the mechanism is not completely clear, some of the above cancers are dependent only on non-enzymatic functions of EZH2 (171).
MLL translocations. The fusion of the N-terminal portion of MLL with subunits of the super elongation complex, primarily ENL, AF9, or AF4, initiates leukemia through the constitutive activation of HOX and other hematopoietic stem cell maintenance genes. In MLL-rearranged leukemia, PRC2 promotes acute leukemogenesis through repression of general senescence regulators (173), and disruption of EED or both EZH paralogs inhibits growth of these leukemias (174).
In a more specific dependency, PRC1 subunit CBX8 interacts directly with AF9/ENL and is required for MLL-AF9 leukemogenesis in mice (67). Knockdown of CBX8 or inhibition of the CBX8 chromodomain significantly reduces viability and HOX gene expression in MLL-AF9 leukemia cell lines (175). In this context, CBX8 contributes to gene repression via RING1B association, and contributes to gene activation via AF9/ENL association (65,176). An activating role for CBX8 may be a result of CBX8 incorporation into other complexes (67) or a result of selective CBX8 antagonism by MLL-AF9 (62,65). The exact biochemical role of CBX8 in MLL-AF9 leukemia remains to be elucidated.

Cancers with general upregulation of Polycomb group genes
Comparing RNA-Seq data obtained from TCGA tumors to TCGA matched normal tissue, we defined differential gene regulation for all PcG genes across TCGA cancers ( Figure  4). Several cancers have increased transcription of almost all PcG genes; these cancers include cholangiocarcinoma of the bile duct (CHOL), diffuse large B-cell lymphoma (DL-BCL), pancreatic cancer (PAAD) and thymoma (THYM). The upregulation of Polycomb subunits has been confirmed in cholangiocarcinoma and pancreatic cancer (177,178) but has yet to be investigated in thymomas. While therapeutic targeting of PcG has not been extensively explored in any of these cancers, recent studies in pancreatic cancer indicate that targeting EZH2 or BMI1 may be effective in certain aggressive subtypes or cell populations (179,180).
DLBCL. The oncogenic role of PcG upregulation in Bcell lymphoma is well-established and is related to a requirement for Polycomb in germinal center activation (see Section 2.1) (181). While EZH2 inhibitors are not as cytotoxic in DLBCL with increased EZH2 as they are in DLBCL with activating mutations in EZH2 (see Section 2.4), they still inhibit growth and may be effective in combination therapies (182).
BMI1 has been studied extensively in the context of leukemia, and was one of the first PcG proteins identified as oncogenic in hematological diseases (183,184). Ectopic expression of BMI1 gives rise to B-and T-cell lymphomas (185,186), and deletion of BMI1 delays primary leukemia and blocks secondary leukemia (187)(188)(189). These results implicate cPRC1 as a potential target; however, the best therapeutic strategy to modulate cPRC1 function in this setting is unclear.
The upregulation of ncPRC1 subunits is also important in DLBCL, particularly subunits of ncPRC1.1, which is important for silencing B-cell differentiation genes during the development of DLBCL (190). This is likely related to the mechanism by which ncPRC1.1 restricts myeloid differentiation by cooperating with BCL6 and PRC2 to repress myeloid regulator genes (129,191). BCL6 inhibitors and degraders are under development (192)(193)(194), and ncPRC1.1specific inhibition may be similarly effective. KDM2B has two potentially targetable domains including the JmjC demethylase domain and the Zinc Finger (ZF)-C XX C DNA binding domain. Inhibitors targeting other JmjC domains have been reported; however, selectivity has been challeng- ing (195). If resolved, selective inhibitors to either domain could be extended to the development of KDM2B-specific inhibitors or degraders for DLBCL treatment.
EZH. EZH2 upregulation paired with EZH1 downregulation in tumors is likely related to the fact that EZH2 is expressed only in actively dividing cells and plays a vital role in cellular proliferation, while EZH1 is expressed in both dividing and differentiated cells. While some cancers are dependent on both paralogs, others are dependent only on EZH2, leading to the development of both selective and nonselective catalytic inhibitors (196). In some cancers with EZH2 upregulation, inhibition of the catalytic activity alone is insufficient to stall tumor growth, implicating catalytic-independent roles for EZH2 in cancer progression. This may include non-enzymatic roles for EZH2 in gene activation (197), as well as non-transcriptional functions, such as in cytosolic actin polymerization during metastasis (198). To modulate catalytic-independent functions of EZH2, selective degraders of EZH2 may be more generally effective as a therapeutic strategy (199).
RING. RING1B is the more commonly upregulated RING paralog in cancer, and as the catalytic subunit of PRC1, represents a good therapeutic target ( Figure 4); in contrast, RING1A is commonly downregulated in cancers and low expression correlates with poor prognosis, indicating that it may act as a tumor suppressor (200). Considering this, as well as the central importance of RING activity in many normal cells, the development of RING1B-specific inhibitors may prove more selective for cancer.
CBX. CBX paralogs are frequently misregulated in cancer, with CBX2 and CBX8 as the most commonly upregulated paralogs, and CBX7 and CBX6 as the most commonly downregulated (Figure 4). Supporting generally oncogenic roles, CBX2 and CBX8 have emerged as targets in lymphoma (190), hepatocellular carcinoma (201), breast cancer (202,203), prostate cancer (204) and ovarian cancer (205), while CBX6 and CBX7 have been implicated as tumor suppressors in thyroid cancer (206) lung cancer (207), glioblastoma (208), bladder cancer (209), cervical cancer (210) and breast cancer (211). There are exceptions, however; CBX7 acts as an oncogene in lymphoma and prostate cancer (212,213), and CBX6 acts as an oncogene in hepatocellular carcinoma (214). A correlation to any known role for CBX2 and CBX8 in development or cell cycle regulation is not apparent; however, the recently uncovered biochemical role for CBX2, and to a lesser degree for CBX8, in phase separation, may be related to oncogenic function (215,216). Whether selective inhibition of these paralogs has general utility for cancer therapy remains to be seen.

Cancer subpopulations with dependencies on PcG proteins
In many cancers, PcG proteins are not involved in the development of tumors but are instead involved in cancer progression or resistance to therapy, either through facilitating therapy-resistant subtypes or therapy-resistant cell populations.
Metastatic prostate cancer. Multiple PcG proteins are potential targets in prostate cancer, including EZH2, CBX2 and BMI1 (217)(218)(219)(220)(221)(222)(223). The most established target is the PRC2 subunit EZH2, which is overexpressed in metastatic prostate cancer and is strongly correlated with cell cycle and DNA repair genes. EZH2's role in prostate cancer may be at least partially separate from its function in PRC2, as it promotes androgen receptor (AR)-dependent gene activation and DNA damage repair in a manner independent of H3K27me3 (224,225). EZH2 is also upregulated in therapyresistant neuroendocrine prostate cancer, where it functions in conjunction with CBX2 to repress AR target gene expression and promote resistance to AR-targeted therapies (226,227). EZH2 inhibition across a variety of therapyresistant prostate cancers shows promising results, particularly to reverse chemotherapy and radiotherapy resistance (228,229). The oncogenic functions of RING1B and BMI1 along with the fusion BMI1-COMMD3 (230-233) may be additional targets for treating advanced prostate cancer.
Glioblastoma multiforme. Glioblastoma multiforme (GBM) is malignant Grade IV brain tumor, composed of heterogeneous cell populations, predominantly abnormal astrocytic cells. Within this population are glioma stem-like cell (GSC) with high expression of PcG genes (234). This population is responsible for resistance to radiation or chemotherapy and tumor repopulation after treatment (196,235,236). Short-term EZH2 depletion or inhibition stalls tumor growth, while prolonged EZH2 depletion drastically alters tumor cell identity and enhances tumor progression (196). The expression of other PcG subunits, such as BMI1, is also enriched in GBM cancer stem cells (237), although in a different GSC population, indicating that dual targeting of multiple PRC complexes may be necessary (196).
Colorectal cancer. Similar to glioblastoma, targeting cancer-initiating cells (CICs) in colorectal cancer is a promising strategy to prevent tumor recurrence after therapy. Human colorectal CIC function is dependent on BMI-1 (PCGF4), a subcomponent of PRC1 (238).

POLYCOMB GROUP PROTEIN MODULATORS: INHIBITORS/ DEGRADERS/ ACTIVATORS
Considering the importance of PcG proteins in gene regulation and cancer progression, many chemical approaches have been developed to target different aspects of Polycomb function. An overview of the different subunits targeted and the strategies for modulation are described in Figure 5 and summarized with chemical structures in Table 2. Additional derivatives of similar scaffolds are not all included.

PRC2 modulators
Overview of this section is depicted in Figure 5A.

EZH2 catalytic inhibitors. Given the central role for
H3K27me3 in Polycomb-mediated repression, most inhibitors to date target the catalytic activity of EZH2. Within this category, most developed inhibitors bind to the SET domain of EZH2 and compete with S-adenosyl-L-methionine (SAM-e) cofactor binding. EZH2 enzymatic inhibitors have impressive efficacy in certain preclinical and clinical cancer models. A more detailed description of the medicinal chemistry is highlighted in (239).

3-Deazaneplanocin A (DZNep).
DZNep is an inhibitor of the enzyme S-adenosylhomocysteine hydrolase. Inhibition of this hydrolase results in a reduction of cellular SAM-e, thereby inhibiting all SAM-e-dependent enzymes, including EZH2. DZNep effectively decreases the expression of PRC2 proteins EZH2, SUZ12, and EED, as well as global H3K27me3 levels, leading to the reactivation of the PRC2repressed genes and apoptosis in breast cancer cell line MCF-7 and colorectal cancer cell line HCT116, but not in normal cells (240). However, the lack of specificity for EZH2 inhibition makes DZNep unsuitable as a probe of EZH2 dependency, leading to further efforts to elucidate its contribution to EZH2-independent processes. GSK926 and GSK126. GSK926 was the first highly potent, SAM-e competitive and selective EZH2 inhibitor developed by GSK (241). GSK126 has improved properties and activity against cell migration and angiogenesis (159).

EPZ005687. EPZ005687 is a selective inhibitor of EZH2
(K i = 24 nM) and therefore blocks H3K27 methylation (158). EPZ005687 displays 50-fold selectivity for EZH2 over the closest methyltransferase EZH1, and >500-fold selectivity over 15 other methyltransferases. EPZ005687 inhibits the growth of EZH2-activating-mutant lymphoma cells, with lesser effect on wild-type lymphoma, making it the first-in-class chemical probe for evaluating the effect of inhibiting EZH2 enzymatic activity in cancer. EPZ005687 is also effective in inhibiting the proliferation and migration of synovial sarcoma cells driven by a translocation in SWI/SNF subunit SS18 (242). of EZH2. Although EPZ-6438 has similar MoA and selectivity in comparison to EPZ005678, EPZ-6438 has superior potency and drug-like properties. EPZ-6438 treatment of mice bearing EZH2-mutant non-Hodgkin lymphoma xenografts demonstrated complete and sustained tumor regressions with concurrent diminution of H3K27me3 levels in tumors. EPZ-6438 is currently FDA-approved for follicular lymphoma, B-cell non-Hodgkin lymphoma and epithelioid sarcoma (243)(244)(245)(246).

EPZ011989.
Optimized from EPZ-6438, EPZ011989 has a lower amine pK a , while the selectivity for EZH2 and potency against EZH2-mutant lymphoma are maintained (K i < 3 nM). In addition, it reduces H3K27me3 levels in tumors and exhibits good bioavailability, metabolic stability, PK/PD profile, and in vivo activity (247).

EI1.
The SAM-e competitive EZH2 inhibitor, EI1, was identified from a high-throughput screening (248). EI1 demonstrates potent inhibition of the enzymatic activity of both wildtype EZH2 and Y641F mutant with IC 50 of 15 nM and 13 nM, respectively. DLBCL cells treated with EI1 exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes, leading to decreased proliferation, increased cell cycle arrest, and apoptosis.

CPI-1205 and CPI-360.
CPI-360 is a highly potent (K i = 0.5 nM) EZH2 inhibitor developed at Constellation Pharmaceuticals from a pyridone-based high-throughput screen hit. It displays activity in both mutant EZH2-containing lymphomas as well as lymphomas with WT EZH2 (251). Optimized compound CPI-1205 is effective in vivo and welltolerated in clinical trials (252). Further modifications to improve residence time and metabolism are being pursued.

PF068721497.
A catalytic inhibitor developed at Pfizer from a focused library of dimethylpyridones. It is potent against both WT and mutant EZH2 and induces tumor regression in DLBCL mouse xenograft models (253).

Valemostat.
A catalytic inhibitor of both EZH1 and EZH2 developed by Daiichi Sankyo Co. (254) It is active against EZH2-mutant DLBCL as well as AML (255). It is in clinical trials for these cancers, as well as for TALL and urogenital cancers.

EZH2 degraders and downregulators.
MS1943. Although EZH2 inhibitors are effective in treating sarcoma and lymphoma, other cancers that are dependent on EZH2, such as triple negative breast cancer (TNBC), do not respond to catalytic inhibition. For these cancers, chemical approaches to degrade EZH2 are being explored. MS1943 is the first-in-class EZH2-selective degrader that effectively reduces EZH2 levels in cells (199). It is a bivalent compound consisting of an EZH2 inhibitor linked to an adamantane hydrophobic tag. The hydrophobic tag interacts with greasy core residues of the protein and destabilizes the protein folding, resulting in a partially folded protein that is subsequently degraded by the proteasome (256,257). MS1943 has profound cytotoxic effect in multiple TNBC cells in vitro and in vivo, with minimal effect on normal cells.
Ebastine. Ebastine, a marketed antihistamine drug, reduces EZH2 transcript levels, leading to decreased H3K27me3 levels in breast and prostate cancer cell lines. It decreases cancer cell growth and reduces tumor growth and progression in xenograft mice models (229). Ebastine demonstrates promise as an anticancer medication considering its potency as EZH2 inhibitor and its safety as an antihistamine that is already available on the market.

EZH2-EED interaction disruptors.
Since EZH2 enzymatic function depends on its interaction with EED, disruption of this interaction provides another route to modulate EZH2 activity. Disruption of EZH2-EED interaction also reduces the protein levels of EZH2 and may have broader therapeutic implications for PRC2-addicted cancers that are either dependent or independent of its HMT activity (172,199).
Astemizole. This antihistamine drug has been recently identified as a small molecule inhibitor of EZH2-EED interaction. It inhibits PRC2 activity by depleting the PRC2 subunits and decreasing H3K27me3 levels, resulting in antiproliferation activity in lymphomas (258).

SAH-EZH2.
SAH-EZH2 is a stabilized alpha-helix mimetic of the EZH2 peptide. It selectively inhibits H3K27 trimethylation of PRC2 in vitro and selectively suppresses the growth of leukemia and lymphoma cells by disrupting the EZH2-EED interaction (259). Stapled peptides have sub-optimal pharmacokinetics, reducing their utility as single dose agents; however, they may have therapeutic potential administered along with enzymatic inhibitors to boost efficacy and decrease resistance.

DC-PRC2in-01.
The co-crystal structure of astemizole in complex with EED was used for the structure-guided design of DC-PRC2in-01, a novel EZH2-EED interaction inhibitor (260). With a K d of 4.56 M, it causes degradation of PRC2 core subunits and a decrease in global H3K27me3 levels in lymphoma cancer cells.
EED inhibitors. The PRC2 subunit EED binds to the H3K27me3 mark and augments EZH2 enzymatic activity. As an alternative strategy to inhibiting the PRC2 methylation function, targeting EED can reduce PRC2 activity and prevent PRC2 binding to histones. EED inhibitors may also be advantageous in being capable of targeting both wildtype and mutant EZH2-dependent functions.
CBX8 UNC7040 Allosteric modulator of CBX8, abrogating its function as reader of H3K27me3 marks and increasing its non-specific binding to DNA, leading to inhibition of proliferation in lymphoma cells (290) 18 NAR Cancer, 2021, Vol. 3, No. 4

A-395.
A-395 binds to the WD40 domain of EED, competing with H3K27me3 in the aromatic binding pocket with an IC 50 of 7 nM. Inhibition of H3K27me3 binding prevents allosteric activation of PRC2; this resembles the results achieved with EZH2 inhibitors, but expands with activity against cell lines with acquired resistance to EZH2 inhibitors (261). Further structure-activity relationship studies on this compound found that 2,6-disubstitution of the N-benzyl improved the binding and cellular activities (262).
EED226. EED226 is a potent and selective PRC2 inhibitor developed from a high throughput screen hit that directly binds to the WD40 domain of EED. EED226 binding to EED induces a conformational change in the aromatic cage leading to the destabilization of EED-H3K27me3 interaction and a loss of PRC2 enzymatic activity. EED226 is effective in treating cancers susceptible to EZH2 inhibition, as well as cancers with a mutant EZH2 protein resistant to SAM-e competitive inhibitors (263). Several additional small molecules (EED666, EED162, EED210, and EED709) bind to EED in a similar manner, defining a common mechanism of inhibition and providing a starting point for further inhibitor development (263,264).

MAK683.
MAK683 is an allosteric inhibitor of PRC2 developed by Novartis (265). Similar to EED226, it binds EED and alters the conformation of the EED-H3K27me3 binding pocket, preventing EED-H3K27me3 interaction and EZH2 activation. This drug is being tested in clinical trials for patients with DLBCL, nasopharyngeal carcinoma, gastric cancer, ovarian cancer, prostate cancer and sarcomas.

EED proteolysis targeting chimeras (PROTACs).
UNC6852. An EED-targeted bivalent chemical degrader UNC6852 selectively degrades EED and other PRC2 components through recruitment of the VHL ubiquitin ligase. UNC6852 inhibits PRC2 catalytic activity, decreases H3K27me3 levels, and inhibits proliferation of DLBCL cell lines. PRC2-targeted degraders have the potential to overcome acquired resistance to EZH2 inhibitors. More importantly, UNC6852 provides a useful tool for identifying and treating cancers dependent on non-catalytic functions for PRC2 (268).

PROTAC 1 and PROTAC 2.
Simultaneously to UNC6852, a team at AstraZeneca reported different EED-based PROTAC molecules that utilize VHL recruitment to degrade PRC2 subunits and reduce PRC2 activity (269).

EED activators.
UNC5636. Loss-of-function (LOFs) mutations in cancer, unlike gain-of-function mutations, are bigger challenges for targeted therapeutics. A subset of myeloid disorders has a I363M LOF mutation of EED that leads to a loss in EZH2 enzymatic activity. UNC5636 was developed using computational simulations and structure-based design to selectively stimulate the catalytic activity of PRC2-EED-I363M and not WT PRC2. Although the compound lacks cell permeability, this work demonstrates the feasibility of developing targeted agonists for reversing the LOF mutant phenotype (270), which can also be a promising direction for other PcG LOF mutations.
Limitations of PRC2 inhibitors. Although EZH2 demonstrates a remarkable level of substrate specificity in catalyzing methylation of only one lysine (H3K27) among all histone peptide substrates, various in vitro and cell-based studies indicate that EZH2 has additional substrates such as JARID2, H2BK120, talin, and transcription factors such as STAT3, which may or may not be desirable targets for cancer (246,(271)(272)(273)(274). In addition, the cytotoxic effects of EZH2 inhibition on adult stem cells remain a major concern in therapeutical strategies.

PRC1 inhibitors
An overview of this section is summarized in Figure 5B.  (275). This molecule is the first chemical inhibitor of PRC1 E3 ubiquitin ligase activity, although its low potency and structural similarity to pan assay interference compounds raises concerns over cellular activity and selectivity.

RB-3.
Fragment-based screening and subsequent optimization of molecules that bind to the BMI1/RING1B dimer recently produced the first selective inhibitor of PRC1 RING ubiquitin ligase activity (276). This inhibitor binds RING1B with 3.6 M affinity and alters the conformation to block ubiquitination activity. It is selective for PRC1 and effectively blocks H2AK119ub1 in cells, but it is not selective for RING1B over RING1A or BMI1 over PCGF1, indicating that it likely blocks all PRC1-mediated ubiquitination. While complete inhibition of PRC1 may be toxic to some cell types, the reduction of PRC1 ubiquitin ligase activity, or subsequent development of inhibitors selective for individual PRC1 subcomplexes, may prove therapeutically useful.

BMI-1 inhibitors.
PTC209 and PTC596. PTC209 induces BMI-1 degradation and reduces PRC1 activity. Treatment of primary colorectal cancer xenografts with PTC-209 resulted in loss of colorectal CICs with irreversible tumor growth impairment and little cytotoxicity (238). Additional efforts from PTC Therapeutics resulted in PTC596, an orally bioavailable molecule currently in clinical trials (NCT02404480) for glioblastoma and fibrosarcoma. PTC596 inhibits APC/C CDC20 activity resulting in the persistent activation of CDK1 and CDK2, leading to hyperphosphorylation of BMI-1 and its degradation (277).
QW24. BMI-1 inhibitor QW24 decreases BMI-1 protein expression through the autophagy-lysosomal degradation pathway. Based on cell viability and protein regulation, QW24 is more potent than previously reported BMI-1 inhibitor PTC209. QW24 significantly inhibits stem-like properties in colorectal cancer cell lines, leading to attenuation of proliferation and metastasis. In animal studies, QW24 exhibits little toxicity in the subcutaneous xenograft model, with reduced tumor metastasis and increased mice survival (278).
CBX inhibitors. Five CBX proteins are incorporated into PRC1, which bind H3K27me3 via their chromodomains (ChDs). The flexibility of the ChD and high homology among the CBX ChD provide as major obstacles for developing potent, selective, and cell permeable CBX inhibitors.

CBX4/7.
Peptidic inhibitors. CBX7 promotes proliferation of prostate cancer cells (279) and malignant hematopoietic progenitor cells (280). The first CBX ChD ligands were peptidic in nature with the highest affinity for CBX7, with 0.28 ±0.05 M, and five-fold selectivity over CBX8 (281). UNC3866 was then developed with similar specificity for CBX7 and CBX4, but higher affinity and, most importantly, utilized a modification of the trimethyllysine to diethyllysine to allow for cell permeability. UNC3866 induces a senescence-like phenotype in PC3 prostate cancer cells and inhibits their proliferation at IC 50  Positive allosteric modulator UNC4976. UNC4976 is a modified version of UNC3866 with a large norcamphor group on the lysine instead of diethyls. The bulky modification does not change in vitro binding properties but does induce a conformational change that increases the affinity of CBX7 ChD to DNA/RNA. The enhanced efficacy of UNC4976 results from simultaneously antagonizing H3K27me3-specific binding while increasing non-specific DNA and RNA binding, shifting the equilibrium of CBX7containing PRC1 from H3K27me3 target regions (286).

CBX6.
Peptidic ligand 5. This inhibitor selectively inhibits the CBX6 ChD by occupying a small hydrophobic pocket adjacent to the aromatic cage. It displays good affinity (K d = 900 nM) with in vitro selectivity >5-fold over other CBX paralogs (287).

CBX6/CBX8.
Peptidic ligands 22 and 23. The first dual-selective inhibitors for CBX6 and CBX8 demonstrate high affinity to both CBX6 and CBX8 (288). Lead compounds from this study demonstrated efficacy in rhabdoid tumor cell line.

CBX8.
Peptidic inhibitor SW2 110A. The selection of directed DNA-encoded libraries (DELs) against multiple ChDs led to the development of SW2 110A, a selective, cellpermeable inhibitor of the CBX8 ChD with a K d of ∼800 nM, and minimal 5-fold selectivity (no binding to CBX4 and CBX6, 20-fold over CBX7, 5-fold over CBX2) for 20 NAR Cancer, 2021, Vol. 3, No. 4 CBX8 ChD over all other CBX paralogs in vitro. SW2 110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation (175,289).

Allosteric modulator UNC7040.
A potent positive allosteric modulator of CBX8, UNC7040, can antagonize H3K27me3 binding to CBX8 while increasing interactions with nucleic acids and participation in variant PRC1 (290). It has increased potency against CBX8 binding in DL-BCL cell compared to similar inhibitors that only inhibit H3K27me3 binding.

CBX2.
Peptidic inhibitor SW2 152F. The CBX2 ChD inhibitor SW2 152F has a K d of ∼80 nM, and 24 to 1000-fold selectivity for CBX2 over the other CBX paralogs in vitro. This inhibitor blocks CBX2-facilitated neuroendocrine differentiation in prostate cancer, mainly through de-repressing AR signaling in neuroendocrine differentiated prostate cancer cells (226).

CONCLUSIONS AND FUTURE OPPORTUNITIES
There are many cancers dependent on PcG proteins for initiation, progression, and chemotherapy resistance; however, the most effective method for targeting Polycomb function in a specific cancer is not always clear. Due to a central role for H3K27me3 in Polycomb-mediated repression and the relative homogeneity of PRC2 subunit composition, the majority of inhibitors developed to date target PRC2-mediated H3K27 trimethylation. Targeting the enzymatic SET domain of EZH1/2 is the most straightforward approach; however, drug-resistant mutant cancers, as well as cancers dependent on non-enzymatic PRC2 function, have necessitated alternate approaches to inhibit PRC2. Allosteric EED inhibitors, PRC2 disrupters, and EZH2/EED degraders can inhibit the growth of cancers insensitive to EZH2 enzymatic inhibitors, although the full utility of these compounds remains to be explored. Additional investigation of how this class of inhibitors affects both normal cells and cancers will reveal additional therapeutic utility. Targeting PRC2 is effective against many Polycombdependent cancers; however, the complete loss of H3K27me3 may be detrimental to many normal cells, particularly adult stem cells. In addition, it is likely that not all PRC2 function in a particular cancer is oncogenic, and some PRC2 activity may actually be tumor suppressive, as has been shown in certain cancer types. Therefore, inhibiting a subset of Polycomb targets or inhibiting cancer-specific PRCs may be more effective in some situations. For instance, in many cancers CBX2/CBX8 are oncogenic while CBX6/CBX7 are tumor suppressive. Inhibitors for cPRC1, specifically CBX ChD inhibitors, can define the utility of more specifically targeting the subset of oncogenic cPRC1s that act downstream of PRC2. Current CBX ChD inhibitors are limited by poor pharmacological properties; however, they are useful for defining individual CBX paralog(s) to focus on as drug targets. The development of more potent CBX inhibitors will potentially require novel strategies, such as allosteric modulation or even degradation.
Targeting ncPRC1s is another strategy that may be more selective for certain cancers. ncPRC1 has higher ubiquitin ligase activity than cPRC1; however, if nonspecific RING enzymatic inhibitors prove toxic, a better strategy may be to target specific ncPRC1 variant subunits. ncPRC1.1 and ncPRC1.6 bind different regions of the genome but are functionally redundant in ESCs. In contrast, ncPRC1.1 is essential in B-cell lymphomas, while ncPRC1.6 acts as a tumor suppressor. Degraders provide an opportunity to target subcomplex-specific ncPRC1 subunits containing nonessential enzymatic or binding domains; for instance, targeting ncPRC1.1 through KDM2B or targeting ncPRC1.6 through L3MBTL2.
The functional redundancy for many Polycomb subunits along with the cell-type specific expression of PcG genes indicates that dual inhibition of PRC subunits may be a more effective pharmacological strategy. In probe development, selectivity is the highest priority; however, for drug development targeting of multiple functionally redundant PcG targets might be more effective in increasing cancer death and decreasing resistance. Even so, identifying and inhibiting just the subset of PcG targets important in cancer would reduce toxicity to normal cells. Defining the redundant and specific biological functions for individual PcG proteins in cancer is required for this polypharmacology approach; however, using genetic screens for multiple targets is highly challenging. Instead, small molecule inhibitors can be rapidly screened in combination to identify dual targeting approaches that selectively inhibit Polycomb activity in a cancer-specific manner. Increasing the diversity of Polycomb subunit inhibitors will significantly facilitate this approach. An increased understanding of how specific PRCs/subunits biochemically contribute to Polycomb activity will help with prioritizing targets, as well as developing effective strategies for inhibition.