Polycomb Repressive Complex 2 Inhibitors Research Articles

Abstract B031: PRC2 inhibitor suppresses angiogenesis in Hepatocellular carcinoma (HCC) by up-regulation of anti-angiogenic factors vasohibin1 (VASH1) and tissue inhibitor of metalloproteinases 3 (TIMP-3)

Abstract Tumor-associated angiogenesis plays a crucial role in the progression of tumor, especially in highly vascularized cancer, such as Liver hepatocellular carcinoma (LIHC), ovarian cancer, lung cancer, human head and neck cancers. Clinical research has shown that LIHC cells upregulate expression of vascular endothelial growth factor A (VEGFA), and induce proliferation and migration of epithelial cells under hypoxia condition, however, the mechanism of angiogenesis has not been completely elucidated in LIHC, especially in epigenetic regulation level. Polycomb repressive complex 2 (PRC2) is a multi-subunit protein complex that catalyzes the methylation of histone H3 at lysine 27, Enhancer of Zeste homolog 2 (EZH2) is the catalytic subunit, while embryonic ectoderm development (EED) recognizes H3K27me3 and enhance the catalytic activity of EZH2. A recent study suggests PRC2 promote angiogenesis in human ovarian carcinoma, while the detailed mechanism this process has not been illustrated. In this paper, we show BR1763, a potent, selective and orally bioavailable small-molecule inhibitor of EED, inhibits PRC2 activity, promote expression of vasohibin1 (VASH1) and tissue inhibitor of metalloproteinases 3 (TIMP3) in certain Hepatocellular Carcinoma (HCC) cells, and induces tumor growth inhibition by suppression of tumor-associated angiogenesis in vivo. Citation Format: Hailong Zhang. PRC2 inhibitor suppresses angiogenesis in Hepatocellular carcinoma (HCC) by up-regulation of anti-angiogenic factors vasohibin1 (VASH1) and tissue inhibitor of metalloproteinases 3 (TIMP-3). [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B031.

1258P HJM-353: A potent, selective and orally bioavailable EED inhibitor with robust anti-tumor activities

Polycomb Repressive Complex 2 (PRC2), a multimeric complex that catalyzes the methylation of histone H3 at lysine 27 with core subunits EZH2, EED and SUZ12, functions as an epigenetic modulator in regulating cell proliferation and development. Dysregulation of the PRC2 complex is implicated in hematological and solid malignancies and shown to correlate with poor prognosis in cancers. Recently EZH2 inhibitor Tazemetostat has been approved by FDA for the treatment of advanced epithelioid sarcoma and follicular lymphoma. However, acquired resistant mutations in EZH2 and complementary activation of EZH1 might impair the effectiveness of this class of PRC2 inhibitors. Targeting the allosteric subunit EED represents a new strategy to fully inhibit PRC2 activity and address the limitations of EZH2 inhibition. Herein, we report HJM-353 as an efficacious EED inhibitor for cancer therapy. In vitro activities of HJM-353 were assessed by CTG assay and Western blot in EZH2 mutant human cell lines.In vivo anti-tumor activities of HJM-353 were evaluated in DLBCL and gastric cancer xenograft models.Pharmacokinetic/pharmacodynamics correlation of HJM-353 was determined by LC-MS/MS, Western blot and Q-PCR. HJM-353 effectively reduced H3K27me3 level and inhibited cell proliferation with an IC50 of 33.8 nM in WSU-DLCL2 cells. It displayed favorable PK properties and induced complete tumor regression in KARPAS-422 derived DLBCL xenograft model. Plasma concentration of HJM-353 correlated with the inhibition of H3K27me3 and upregulation of PRC2 target genes in tumors, indicating a clear PK/PD relationship in the xenografts. In addition, HJM-353 caused tumor stasis as MAK683 did in a xKATO-III gastric xenograft model. In summary, HJM-353 is a potent, selective and orally bioavailable EED inhibitor with robust anti-tumor activities. We are developing HJM-353 for the treatment of hematological malignancies and solid tumors and expect to file an IND in the second half of 2022.

Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation.

Research progress on the function and mechanism of CXorf67 in PFA ependymoma

<p indent="0mm">CXorf6 (chromosome X open reading frame 67) protein contains 503 amino acids and is predicted <italic>in silico</italic> to be mostly disordered. <italic>CXorf6</italic>7 is barely expressed in most cell lines and human tissues. Therefore, for rather a long time, our understandings regarding functions of this protein are quite limited. In 2018, Pajtler et al. discovered a high expression of <italic>CXorf67</italic> specifically in posterior fossa A (PFA) ependymomas. After that, CXorf67 attracts increasing attention and efforts to explore its potential roles in PFA ependymomas. Based on DNA methylation profiling, ependymomas have been classified into 9 molecular subgroups. Of these 9 subgroups, PFA is one of the most malignant and aggressive ones with the highest incidence. PFA ependymomas mostly happen in infants and young children with a median age of <sc>~3 years old,</sc> and are generally refractory to chemotherapies. Current standard therapy for intracranial ependymomas is surgical resection combined with radiotherapy. However, the lateral localization of PFA ependymomas renders them hard to be removed completely, and together with a lack of chemotherapy options, they generally have a poor prognosis. Research from different labs has so far revealed two independent functions of CXorf67 in PFA ependymomas. One function of CXorf67 is related to the inhibition of PRC2 (Polycomb repressive complex 2) enzymatic activity and thus a global H3K27me3 reduction in PFA ependymomas. Results from several studies consistently suggested that CXorf67 functions as an inhibitor of PRC2 through an H3K27M-like mechanism. These findings implicate CXorf67 as a promising therapeutic target for PFA ependymomas, and efforts to develop drugs based on this PRC2-related function of CXorf67 remain on the way. On the other hand, a recent study revealed the other function of CXorf67 that is involved in DNA damage repair—CXorf67 inhibits DNA homologous recombination repair through blocking PALB2-BRCA2 interaction. This new function of CXorf67 renders it as a biomarker for PARP inhibitor treatment and provides a new treatment option for PFA patients with immediate clinical applicability. In this review, we summarize current findings about the functions and mechanisms of CXorf67, as well as the possible regulatory mechanisms of <italic>CXorf67</italic> expression in PFA ependymomas. We also discussed the clinical implications of the two functions of CXorf67 for PFA ependymomas. In addition to PFA ependymomas, <italic>CXorf67</italic> expression is also observed in diffuse intrinsic pontine gliomas (DIPG) and germ cell tumors. We believe that it will be of great significance to investigate whether <italic>CXorf67</italic> expression could also sensitize these two tumors to PARP inhibitors. Moreover, <italic>CXorf67</italic> is specifically unregulated in PFA, but not in any other ependymoma subgroup; and reasons for this remain currently unclear. Understanding of how <italic>CXorf67 </italic>expression is turned on in PFA ependymomas might enable a controllable expression of <italic>CXorf67</italic> in other types of tumors, thereby opening up new opportunities not only for PFA patients, but possibly also for other cancer patients.

Inhibition of polycomb repressive complex 2 by targeting EED protects against cisplatin-induced acute kidney injury.

Polycomb repressive complex 2 (PRC2) is a multicomponent complex with methyltransferase activity that catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3). Interaction of the epigenetic reader protein EED with EZH2, a catalytic unit of PRC, allosterically stimulates PRC2 activity. In this study, we investigated the role and underlying mechanism of the PRC2 in acute kidney injury (AKI) by using EED226, a highly selective PRC2 inhibitor, to target EED. Administration of EED226 improved renal function, attenuated renal pathological changes, and reduced renal tubular cell apoptosis in a murine model of cisplatin‐induced AKI. In cultured renal epithelial cells, treatment with either EED226 or EED siRNA also ameliorated cisplatin‐induced apoptosis. Mechanistically, EED226 treatment inhibited cisplatin‐induced phosphorylation of p53 and FOXO3a, two transcriptional factors contributing to apoptosis, and preserved expression of Sirtuin 3 and PGC1α, two proteins associated with mitochondrial protection in vivo and in vitro. EED226 was also effective in enhancing renal tubular cell proliferation, suppressing expression of multiple inflammatory cytokines, and reducing infiltration of macrophages to the injured kidney. These data suggest that inhibition of the PRC2 activity by targeting EED can protect against cisplatin‐induced AKI by promoting the survival and proliferation of renal tubular cells and inhibiting inflammatory response.

Abstract 5632: Combinatorial epigenetic targeting of PRC2 complexes upregulates MHC antigen presentation components in melanoma and gastrointestinal cancer cells

Abstract Low tumor immunogenicity is associated with immune escape, immunotherapy resistance, and poor patient survival [1-3]. Major histocompatibility complex (MHC) antigen presentation pathways (APPs) play a crucial role in promoting tumor T-cell recognition and initiating antitumoral immune responses. However, epigenetic silencing of APPs is a common mechanism in various cancers, including melanoma and gastrointestinal malignancies [3,4]. In the current investigation, mouse melanoma (B16/OVA) and human colon cancer (LOVO and HCT116), small intestine cancer (HUTU80), and normal colonic epithelial (CCD841) cell lines were treated with epigenetic drug candidates. As reported [5], a decrease in histone H3K27 methylation marks via polycomb repressive complex 2 (PRC2) inhibitors, as well as altered histone acetylation status from histone deacetylase 3 (HDAC3)-specific inhibition, increased the expression of APP components, based on immunoblotting and RT-qPCR experiments. Normal colonic epithelial cells showed resistance to the treatments, indicating cancer cell-specific effects of the test agents. In B16/OVA cells, flow cytometry and enzyme linked immunosorbent assays corroborated that HDAC3 and/or PRC2 inhibitor combinations increased cell surface occupancy of MHC-I complexes and increased CD8+ T-cell activation. These findings have implications for the clinical application of next-generation combinatorial epigenetic agents [6,7], targeted towards increased tumor immunogenicity and enhanced efficacy of new immunoepigenetic strategies. Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Discovery of the Clinical Candidate MAK683: An EED-Directed, Allosteric, and Selective PRC2 Inhibitor for the Treatment of Advanced Malignancies.

Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.

DCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

SUMMARYBifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Induction of senescence-associated secretory phenotype underlies the therapeutic efficacy of PRC2 inhibition in cancer

The methyltransferase Polycomb Repressive Complex 2 (PRC2), composed of EZH2, SUZ12, and EED subunits, is associated with transcriptional repression via tri-methylation of histone H3 on lysine 27 residue (H3K27me3). PRC2 is a valid drug target, as the EZH2 gain-of-function mutations identified in patient samples drive tumorigenesis. PRC2 inhibitors have been discovered and demonstrated anti-cancer efficacy in clinic. However, their pharmacological mechanisms are poorly understood. MAK683 is a potent EED inhibitor in clinical development. Focusing on MAK683-sensitive tumors with SMARCB1 or ARID1A loss, we identified a group of PRC2 target genes with high H3K27me3 signal through epigenomic and transcriptomic analysis. Multiple senescence-associated secretory phenotype (SASP) genes, such as GATA4, MMP2/10, ITGA2 and GBP1, are in this group besides previously identified CDKN2A/p16. Upon PRC2 inhibition, the de-repression of SASP genes is detected in multiple sensitive models and contributes to decreased Ki67+, extracellular matrix (ECM) reorganization, senescence associated inflammation and tumor regression even in CDKN2A/p16 knockout tumor. And the combination of PRC2 inhibitor and CDK4/6 inhibitor leads to better effect. The genes potential regulated by PRC2 in neuroblastoma samples exhibited significant enrichment of ECM and senescence associated inflammation, supporting the clinical relevance of our results. Altogether, our results unravel the pharmacological mechanism of PRC2 inhibitors and propose a combination strategy for MAK683 and other PRC2 drugs.

Simultaneous disruption of PRC2 and enhancer function underlies histone H3.3-K27M oncogenic activity in human hindbrain neural stem cells.

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.

Abstract 1131: ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models

Abstract The polycomb repressive complex 2 (PRC2) is responsible for the methylation of histone 3 at lysine 27 (H3K27) which leads to long-term transcriptional silencing. Through this epigenetic chromatin silencing mechanism, PRC2 plays a key role in regulating cellular functions such as cell growth and differentiation. PRC2 comprises three core subunits: the catalytic subunit enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12). EED directly interacts with histone H3K27me3 and is essential for the histone methyltransferase activity of PRC2. PRC2 dysregulation occurs in multiple solid tumors and hematological malignancies, resulting in elevated levels of PRC2 activity and H3K27 trimethylation, and has been linked to poor prognosis in patients with metastatic prostate cancer. First-generation PRC2 inhibitors which target EZH2 have demonstrated clinical activity in several cancers, yet the pharmacological and ADME properties of these compounds require high doses that only achieve partial target inhibition at clinically active levels and exhibit drug-drug interaction (DDI) liabilities. As an alternative strategy to fully inhibit the PRC2 complex, we developed ORIC-944, a potent and highly selective allosteric inhibitor of PRC2 via binding the EED subunit. This unique EED targeting strategy can more completely inhibit PRC2, for example, in the presence of innate or acquired resistance mutations in EZH2, and by addressing the potential compensatory escape mechanism of EZH1-driven tumor growth. ORIC-944 has potential best-in-class drug properties compared to first generation PRC2 inhibitors, including superior potency and improved DDI liabilities. In diffuse large B-cell lymphoma (DLBCL) xenografts in vivo, ORIC-944 significantly depleted H3K27 trimethylation and induced tumor regressions in a dose-dependent manner. ORIC-944 demonstrated strong tumor growth inhibition as a single agent with once daily oral dosing in both enzalutamide-responsive and enzalutamide-resistant prostate cancer models. ORIC-944 caused a significant reduction in tumor cell proliferation and anti-apoptotic signaling, as measured by Ki67 and survivin, respectively. Moreover, in PK/PD assessments, ORIC-944 strongly depleted H3K27me3 in treated tumors. These in vivo studies established that effective single agent inhibition of PRC2 via EED results in decreased tumor cell growth in PRC2-dependent prostate cancer models. In summary, ORIC-944 is a potent, highly selective, allosteric, orally bioavailable PRC2 inhibitor via the EED subunit that represents a differentiated strategy to block PRC2 activity in selected cancers. We are developing ORIC-944 as a best-in-class PRC2 inhibitor for the treatment of patients with advanced prostate cancer and expect to file an IND in the second half of 2021. Citation Format: Anneleen Daemen, Jessica D. Sun, Aleksandr Pankov, Frank L. Duong, Natalie Yuen, Shravani Barkund, Shelly Kaushik, Jae H. Chang, David M. Briere, Niranjan Sudhakar, Andrew Calinisan, Aaron Burns, John M. Ketcham, Matthew A. Marx, Peter Olson, James G. Christensen, Melissa R. Junttila, Lori S. Friedman. ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1131.

Genetic Impairments of PRC2 Activity in Oncology: Problems and Prospects

PRC2 (Polycomb repressive complex 2) is a conserved protein complex in multicellular organisms that is required to maintain gene repression. The catalytic subunit of PRC2, the EZH2 protein, provides the methylation of histone H3K27 (H3K27me1/2/3). It was demonstrated that a number of human cancers were associated with overexpression of PRC2 subunits, as well as with mutations that enhanced the EZH2 catalytic activity. At the same time, a group of cancers correlate with mutations that inhibit PRC2. A number of small molecule inhibitors to the PRC2 subunits have been developed, primarily to EZH2. One of these, tazemetostat, received approval in January 2020 in the United States for the treatment of epithelioid sarcoma. This review focuses on the role of PRC2 in cancer development and summarizes information on the designed PRC2 inhibitors.

New Framework for the Discovery of PRC2 Inhibitors: Epigenetic Drugs.

Over the past several years, remarkable progress towards the recognition of new therapeutic targets in tumor cells has led to the discovery and development of newer scaffolds of anti-tumor drugs. The exploration and exploitation of epigenetic regulation in tumor cells are of immense importance to both the pharmaceutical and academic biomedical literatures. Epigenetic mechanisms are indispensable for the normal development and maintenance of tissue-specific gene expression. Disruption of epigenetic processes to eradicate tumor cells is among the most promising intervention for cancer control. Polycomb repressive complex 2 (PRC2), a complex that methylates lysine 27 of histone H3 to promote transcriptional silencing, is involved in orchestrating significant pathways in a cell. Overexpression of PRC2 has been found in a number of cancerous malignancies, making it a major target for anti-cancer therapy. Despite its well-understood molecular mechanism, hyperactivation and drug resistance mutations in its subunits have become a matter of discussion. This review outlines the current understanding of the components of PRC2 in active complex formation and assesses their potential as a promising therapeutic target for cancer therapy. We also review the effects of mutations in the PRC2 components, in the purview of human cancers. Finally, we discuss some of the current challenges for therapeutic drug designs targeting the PRC2 complex.

H3.3-K27M drives neural stem cell-specific gliomagenesis in a human iPSC-derived model.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.

Abstract IA020: Chromatin regulation of the endometrium: What are our models telling us?

Abstract ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. ARID1A is a SWI/SNF chromatin-remodeling complex subunit. ARID1A loss in the mouse endometrial epithelium leads vaginal bleeding when combined with PI3K pathway activation. Sorted mutant endometrial epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT), inflammation and estrogen signaling in vivo. ARID1A loss leads to the expression of EMT genes, suggesting ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates. In contrast to its repressive roles, ARID1A is required for the activation estrogen responsive genes, including the progesterone receptor (PGR). Estrogen responsive genes are marked by bivalent chromatin domains containing both histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3). High H3K27me3 correlates with low PGR expression, suggesting Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 normally silences PGR. In support of this, pharmacological inhibition of PRC2 partially rescues PGR activation is estrogen treated, ARID1A-deficient cells. Given the known antagonistic relationship between SWI/SNF and PRC2 complexes, we propose that SWI/SNF normally opposes PRC2 mediated silencing of the PGR promoter in response to estrogen, leading to PGR expression. In the ARID1A mutant endometrium, PGR promoter silencing by PRC2 is maintained. In the absence of PGR, progesterone cannot oppose the actions estrogen in the endometrium, leading to unopposed estrogen signaling. Our data demonstrate ARID1A has both activating and repressive roles in the endometrium, and ARID1A loss contributes to multiple aspects of disease pathogenesis. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Chromatin regulation of the endometrium: What are our models telling us? [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA020.

PARP1 and PRC2 double deficiency promotes BRCA-proficient breast cancer growth by modification of the tumor microenvironment.

Poly (ADP-ribose) polymerase 1 (PARP1) and polycomb-repressive complex 2 (PRC2) are each known for their individual roles in cancer, but their cooperative roles have only been studied in the DNA damage repair process in the context of BRCA-mutant cancers. Here, we show that simultaneous inhibition of PARP1 and PRC2 in the MDA-MB-231 BRCA-proficient triple-negative breast cancer (TNBC) cell line leads to a synthetic viability independent of the mechanisms of DNA damage repair. Specifically, we find that either genetic depletion or pharmacological inhibition of both PARP1 and PRC2 can accelerate tumor growth rate. We attribute this to modifications in the tumor microenvironment (TME) that are induced by double-depleted breast cancer cells, such as promoting intratumoral angiogenesis and increasing the proportion of tumor-promoting type 2 (M2) macrophages. These changes subsequently inhibit cell death and promote proliferation. Mechanistically, we find that PARP1 and PRC2 double depletion induces not only a basal activation of the NF-κB pathway but also a maximal activation of NF-κB within the TME in response to external stimuli such as hypoxia and the presence of macrophages. In summary, our study reveals an unprecedented synthetic viable interaction between PARP1 and PRC2 in BRCA-proficient TNBC and identifies NF-κB as the downstream mediator. DATABASE: RNA-seq data are available in the GEO databases under the accession GSE142769.

Abstract PO-008: Bithioether Stapled Peptides as Allosteric Inhibitors of PRC2 function

Abstract Cancer is a genetic disease, but its development also involves multiple epigenetic alterations. Epigenetics regulates transcription by modulating chromatin architecture through different mechanisms. Hence, dysregulation of such mechanisms can result in aberrant gene expression or silencing, which in turn can lead to carcinogenesis. One of the most relevant epigenetic modifications is the methylation of lysine 27 at histone 3 (H3K27), a broadly known repressive histone mark. H3K27 methylation is incorporated by the polycomb repressive complex 2 (PRC2), a multimeric protein complex formed by four core components: EZH2, EED, SUZ12 and RbAp46/48, all of which are essential for its catalytic activity. Overexpression of PRC2 proteins, particularly of EZH2, results in hyperactivation of the complex and high levels of H3K27m3, which are associated to a myriad of human cancers. Hence, the discovery of PRC2 inhibitors is an area of active research for both the pharmaceutical industry and academic laboratories. The relevance of such compounds is highlighted by the recent accelerated approval of the first in class EZH2 inhibitor (tazemetostat, Epizyme), for the treatment of metastatic or locally advanced epithelioid sarcoma. Recent reports suggest that the interaction in between RbAp46/48 and Suz12 is crucial for both proper PRC2 assembly and function. Based on these structural considerations we have designed a series a stapled peptide inhibitors of the NBE domain of SUZ12, and thus of its association with RbAp46/48, as allosteric inhibitors of the catalytic activity of PRC2. Our lead cyclopeptide showed potent inhibition of H3K27 trimethylation in both in vitro and cellular assays, demonstrating that it is cell permeable and active in physiological conditions. Its inhibition of PRC2 catalytic activity produced a marked dose-dependent antiproliferative effect in metastatic Cakis-1 cells. Notably, in these experiments our compound was almost as effective as GSK126, a well-known orthosteric EZH2 inhibitor. We further present compelling evidences suggesting that our stapled peptide targets selectively PRC2, and more specifically, its catalytic subunit EZH2 in an allosteric fashion. To our knowledge, this bisthioether stapled peptide may well be the first example of such allosteric EZH2 inhibitors described to date. This class of compounds could be extremely valuable in addressing the resistance profiles recently reported in clinical trials with EZH2-SET domain inhibitors, in which extended dosing of these drugs have led to secondary EZH2 mutants resistant to treatment. Our compound’s unique mechanism of PRC2 inhibition, together with its potency, remarkable H3K27me3 inhibition selectivity, and low cytotoxicity to non-cancerous cells demonstrate this stapled peptide’s potential for future development of novel epigenetic cancer therapies. Citation Format: Gan Zhang, Adam Herskovits, Nissim Levy, Guillermo Gerona-Navarro. Bithioether Stapled Peptides as Allosteric Inhibitors of PRC2 function [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-008.

Dynamic-shared Pharmacophore Approach as Tool to Design New Allosteric PRC2 Inhibitors, Targeting EED Binding Pocket.

The Polycomb Repressive complex 2 (PRC2) maintains a repressive chromatin state and silences many genes, acting as methylase on histone tails. This enzyme was found overexpressed in many types of cancer. In this work, we have set up a Computer-Aided Drug Design approach based on the allosteric modulation of PRC2. In order to minimize the possible bias derived from using a single set of coordinates within the protein-ligand complex, a dynamic workflow was developed. In details, molecular dynamic was used as tool to identify the most significant ligand-protein interactions from several crystallized protein structures. The identified features were used for the creation of dynamic pharmacophore models and docking grid constraints for the design of new PRC2 allosteric modulators. Our protocol was retrospectively validated using a dataset of active and inactive compounds, and the results were compared to the classic approaches, through ROC curves and enrichment factor. Our approach suggested some important interaction features to be adopted for virtual screening performance improvement.

Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in EZH2 Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma

Simple SummaryTo overcome the potential threat of drug resistance or limit of potency, the combination treatment of drugs is a promising strategy. Around 22% of patients with GCB-DLBCL carry EZH2 gain-of-function mutations and several PRC2 inhibitors are under clinical trials. Herein, we demonstrate that combination of GSK126 with pomalidomide synergistically inhibit tumor growth through inducing B-cell maturation and apoptosis in EZH2 gain-of-function mutant DLBCL. Our study provides the molecular basis of the combination strategy of PRC2 inhibitors and IMiDs in DLBCLs harboring EZH2 hyperactive mutation.Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates genes involved in cell lineage and differentiation through methylating lysine 27 on histone H3 (H3K27me3). Recurrent gain-of-function mutations of EZH2 have been identified in various cancer types, in particular, diffuse large B-cell lymphoma (DLBCL), through large-scale genome-wide association studies and EZH2 depletion or pharmacological inhibition has been shown to exert an antiproliferative effect on cancer cells, both in vitro and in vivo. In the current study, a combination of pomalidomide and GSK126 synergistically inhibited the growth of EZH2 gain-of-function mutant Diffuse large B-cell lymphoma (DLBCL) cells. Furthermore, this synergistic effect appeared to be dependent on cereblon (CRBN), a cellular receptor of pomalidomide, but not degradation of IKAROS family zinc finger 1 (IKZF1) or IKAROS family zinc finger 3 (IKZF3). RNA sequencing analyses revealed that co-treatment with GSK126 and pomalidomide induced specific gene sets involved in B-cell differentiation and apoptosis. Synergistic growth inhibition and B-cell differentiation were further validated in xenograft mouse models. Our collective results provide a molecular basis for the mechanisms underlying the combined therapeutic effects of PRC2 inhibitors and pomalidomide on EZH2-mutated DLBCL.