PRC2 Inhibitor Research Articles

Abstract 6586: ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models

Abstract Polycomb repressive complex 2 (PRC2) dysregulation occurs in multiple tumor types and is associated with poor prognosis in patients with prostate cancer. In patients treated with androgen receptor pathway inhibitors (ARPI), emergence of tumor cell plasticity is associated with resistance to therapy. Dysregulation of epigenetic reprogramming factors, including PRC2, creates an environment that is permissive for such lineage plasticity. Inhibition of the PRC2 complex therefore may provide an opportunity to overcome or prevent the emergence of plasticity in prostate cancer. Notably, emerging clinical trial data has demonstrated the potential of ARPI and PRC2 inhibitor combination therapy to improve outcomes in patients with metastatic prostate cancer. PRC2 tri-methylates histone H3 at lysine 27 (H3K27me3), leading to long-term transcriptional silencing, a key mechanism regulating cellular functions such as cell growth and differentiation. Three core subunits comprise PRC2: the catalytic subunit enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). EED is essential for the histone methyltransferase activity of PRC2. First-generation PRC2 inhibitors, which target EZH2, exhibit poor drug properties, drug-drug interaction liabilities and short half-life requiring twice daily dosing at high doses in patients. We developed a second generation PRC2 inhibitor, ORIC-944, an allosteric inhibitor of PRC2 that binds the EED subunit. ORIC-944 is a potent, highly selective, orally bioavailable inhibitor with best-in-class properties. ORIC-944 demonstrated single agent tumor growth inhibition in a spectrum of in vivo prostate cancer models, including AR-positive, AR-mutant, ARv7, ARPI-responsive and ARPI-resistant models. In vitro analysis of the combination of ARPIs and ORIC-944 showed synergy in multiple prostate cancer cells, utilizing BLISS and other quantitative methods. In vivo studies revealed that ORIC-944 demonstrated increased efficacy in combination with ARPI, consistent with the in vitro synergy. RNA-seq analysis of transcriptional changes induced by ORIC-944 provided mechanistic insight into the role of PRC2 in prostate cancer lineage plasticity and combination response. These results position ORIC-944 as a best-in-class PRC2 inhibitor for evaluation in combination with ARPI in patients with metastatic prostate cancer. A phase 1b trial of ORIC-944 is ongoing (NCT05413421). Citation Format: Anneleen Daemen, Natalie Yuen, Amber Wang, Aleksandr Pankov, Livia Ulicna, Chelsea Chen, Frank L. Duong, Jason Long, Matthew A. Marx, James G. Christensen, Lori S. Friedman, Melissa R. Junttila. ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6586.

The Cellular Impact, Immediate Targets and Mechanism of Action of NSD2 in t(4;14) Multiple Myeloma

INTRODUCTION: NSD2 is a potential therapeutic target in the 15% of cases of multiple myeloma that carry a t(4;14) translocation. As a consequence of the translocation, strong Ig enhancers are placed in proximity to NSD2 and FGFR3. FGFR3 is deleted at a subsequent time point in 25% of cases, while all cases retain NSD2 expression consistent with it likely being the key oncogenic driver. NSD2 is an H3K36 dimethyl transferase, a histone modification mark associated with active transcription, but the molecular targets and the mechanism by which NSD2 regulates its targets is unknown. The cellular function of NSD2 is also largely unknown, but previous shRNA knockdown studies have suggested it is involved in cell proliferation. The major limitation to understanding the role of NSD2 is the lack of an appropriate experimental system with which to investigate its molecular and cellular function. To address this deficit and to identify the primary direct targets of NSD2 as a means to elucidate its mechanism of action we have generated NSD2 dTAG and NSD2 KO cells together with NSD2 reconstitution systems. METHODS: We have utilized CRISPR gene editing to knock-in a dTAG in NSD2 in the t(4;14) KMS-11 cell line. This system enables the inducible and rapid degradation of NSD2 following the addition of a small-molecule degrader which is otherwise inert. We have also generated clonal NSD2 KO KMS-11 cells that allow us to observe and study the stable phenotypes of NSD2 KO. Because H3K36me2 deposited by NSD2 leads to induction of DNMT3A-mediated DNA methylation and suppression of PRC2-mediated H3K27me3, we have generated DNMT3A KO on top of NSD2dTAG/KO and have used two different PRC2 inhibitors to dissect the dependency of NSD2 function on such secondary modifications. To systematically dissect the dependency on NSD2 isoforms and its methyltransferase activity, we have reconstituted NSD2 in both NSD2 dTAG and NSD2 KO KMS-11 cells, including WT NSD2I WT, NSD2II WT, NSD2 Y1179A (LOF), NSD2 E1099K (GOF). To maximally match the timing of NSD2 perturbation, NSD2 reconstitution was generated in NSD2 dTAG and NSD2 KO cells in a Tet-ON and Tet-OFF expression system, respectively. Further we have generated KMS-11 cells overexpressing Dox-inducible H3.3K36M to investigate the consequences of abrogation of H3K36me2 by this dominant negative mutant of H3.3. We have done SLAM-seq to assess the transcriptional targets of NSD2 and will extend this with ATAC-seq and CUT&Tag to identify the molecular targets of NSD2. RESULTS: We show that NSD2 is rapidly degraded within 1 h when a degrader is added and subsequently a significant reduction of H3K36me2 is seen by 24 hours although it takes more than 3 days to see its deep depletion. We observed a reciprocal increase of H3K27me3 as previously noted. Depletion of H3K36me2 and increase in H3K27me3 was also seen in the NSD2 KO KMS-11 cells. Paradoxically we didn't see an induction of apoptosis in response to the degradation of NSD2 or its KO. In sharp contrast to the previously published shRNA results only a mild inhibition of cell proliferation was observed in NSD2 KO cells and over an extended period of 3 weeks in the dTAG system. We didn't observe an effect of H3.3 K36M overexpression on cell proliferation although it abolished H3K36me2 very efficiently. These experiments demonstrate that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth, which is likely independent of its H3K36me2 methyltransferase activity. SLAMseq results show that NSD2 degradation in the dTAG system causes transcriptional changes of a specific set of newly synthesized mRNAs. ATAC-seq and CUT&Tag experiments are being run and will be superimposed on the SLAM-seq data to identify immediate targets of NSD2. Subsequently, use of PRC2 inhibitors and DNMT3A KO cells in the dTAG cells and the NSD2 reconstitution experiments will uncover the mechanisms of how NSD2 controls the transcription of its targets. CONCLUSION: We show that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth and that NSD2 controls the transcription of a specific set of genes and does not exert a global impact on the transcriptome. We will further define the immediate targets of NSD2 by leveraging our SLAM-seq, ATAC-seq, and CUT&Tag data in NSD2 dTAG/KO and NSD2-reconstituted cells and by blocking the crosstalk with other epigenetic marks (i.e. H3K27me3 and DNAme).

Endogenous bioluminescent reporters reveal a sustained increase in utrophin gene expression upon EZH2 and ERK1/2 inhibition

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.

Abstract B013: Suppression of TET1 expression down-regulates p16 and contributes to the resistance of SWI/SNF LOF tumors to PRC2 inhibition

Abstract Polycomb repressive complex 2 (PRC2) drives the expansion of H3K27me3 epigenetic marker and suppress the expression of target genes including tumor suppressors. Activation mutations of EZH2, the catalytic subunit of the PRC2 complex, are thought to be the driver for certain malignancies such as diffused large B cell lymphoma (DLBCL). The SWI/SNF complex remodels nucleosomes to modulate transcription, they are generally considered as tumor suppressors and loss-of-function (LOF) mutations are frequently found across a wide variety of cancers. Many studies suggest that PRC2 is important for the proliferation of tumor cells with SWI/SNF LOF mutations, indeed, EZH2 inhibitor tazemetostat was approved by FDA for epithelioid sarcoma with SWI/SNF subunit SMARCB1 deletion. While not all SWI/SNF LOF tumors respond to PRC2 inhibition, we would like to know what subset of these tumors are sensitive to PRC2 inhibition. In this study, we have developed a potent and selective PRC2 inhibitor BR1763 which blocks the interaction of EED with tri-methylated histone H3 at Lys 27 (H3K27me3) and inhibits cellular H3K27me3 levels. By analyzing the gene expression patterns of BR1763 treated cells, we found that up-regulation of CDKN2A/p16 and down-regulation of E2F downstream gene are essential for cellular response to PRC2 inhibition. Further studies also confirmed that PRC2 inhibition caused P16 up-regulation and induced cell senescence for the sensitive cells. Importantly, suppression of TET1 expression contributes to the resistance to PRC2 inhibition in SWI/SNF LOF tumors, which may be caused by PcG hypermethylation on p16 promoters. TET1 expression, together with SWI/SNF LOF mutation status, can largely predict the sensitivity of tumor cells to PRC2 inhibition. Citation Format: Hailong Zhang. Suppression of TET1 expression down-regulates p16 and contributes to the resistance of SWI/SNF LOF tumors to PRC2 inhibition. [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 B013.

Genome-wide CRISPR screen identifies PRC2 and KMT2D-COMPASS as regulators of distinct EMT trajectories that contribute differentially to metastasis.

Epithelial-mesenchymal transition (EMT) programs operate within carcinoma cells in which they generate phenotypes associated with malignant progression. In their various manifestations, EMT programs enable epithelial cells to enter into a series of intermediate states arrayed along the E-M phenotypic spectrum. At present, we lack a coherent understanding of how carcinoma cells control their entrance into and continued residence in these various states, and which of these states favor the process of metastasis. Here, we characterize a layer of EMT-regulating machinery that governs E-M plasticity (EMP). This machinery consists of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, that operate as critical regulators to maintain a stable epithelial state. Interestingly, loss of these two complexes unlocks two distinct EMT trajectories. Dysfunction of PRC2, but not KMT2D-COMPASS, yields a quasi-mesenchymal state that is associated with highly metastatic capabilities and poor survival of breast cancer patients, suggesting great caution should be applied when PRC2 inhibitors are evaluated clinically in certain patient cohorts. These observations identify epigenetic factors that regulate E-M plasticity, determine specific intermediate EMT states and, as a direct consequence, govern the metastatic ability of carcinoma cells.

Identification of SET/EED Dual Binders As Innovative PRC2 Inhibitors

Background: The inhibition of PRC2, implicated in the pathogenesis of several tumors, can be a useful therapeutic strategy for cancer treatment. In the literature, two types of PRC2 modulators are reported: competitive inhibitors of S-adenosyl methionine binding to the catalytic subunit EZH2; and allosteric ligands that prevent the interaction of the trimethylated H3K27 lysine in histone 3 to the EED subunit. The lack of dual EZH2/EED modulators drove us to search for compounds capable of recognizing both domains. Materials & methods: This goal was pursued by combining pharmacophore- and docking-based virtual screening of the Multi-Target Ligand Chemotheca database. Prediction tools for absorption, distribution, metabolism and excretion and pan-assay interference compounds were also applied. Results: Finally, five 1,2,3-triazole derivatives were identified as promising dual EZH2/EED modulators. Conclusion: Our multistage screening protocol highlighted the great potential of Chemotheca for identifying polypharmacological agents.

The transcriptional elongation factor CTR9 demarcates PRC2-mediated H3K27me3 domains by altering PRC2 subtype equilibrium.

CTR9 is the scaffold subunit in polymerase-associated factor complex (PAFc), a multifunctional complex employed in multiple steps of RNA Polymerase II (RNAPII)-mediated transcription. CTR9/PAFc is well known as an evolutionarily conserved elongation factor that regulates gene activation via coupling with histone modifications enzymes. However, little is known about its function to restrain repressive histone markers. Using inducible and stable CTR9 knockdown breast cancer cell lines, we discovered that the H3K27me3 levels are strictly controlled by CTR9. Quantitative profiling of histone modifications revealed a striking increase of H3K27me3 levels upon loss of CTR9. Moreover, loss of CTR9 leads to genome-wide expansion of H3K27me3, as well as increased recruitment of PRC2 on chromatin, which can be reversed by CTR9 restoration. Further, CTR9 depletion triggers a PRC2 subtype switch from the less active PRC2.2, to the more active PRC2.1 with higher methyltransferase activity. As a consequence, CTR9 depletion generates vulnerability that renders breast cancer cells hypersensitive to PRC2 inhibitors. Our findings that CTR9 demarcates PRC2-mediated H3K27me3 levels and genomic distribution provide a unique mechanism that explains the transition from transcriptionally active chromatin states to repressive chromatin states and sheds light on the biological functions of CTR9 in development and cancer.

PRC2 Inhibitors Overcome Glucocorticoid Resistance Driven by NSD2 Mutation in Pediatric Acute Lymphoblastic Leukemia.

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.

1525MO Phase I/II study of MAK683 in patients (pts) with advanced malignancies including epithelioid sarcoma

EED is a core component of PRC2 which modifies the epigenetic status of target genes, including cell cycle control genes. Loss of SNF5 function in epithelioid sarcoma drives oncogenesis by imbalanced PRC2 activity. MAK683 is a potent, S-adenosyl-l-methionine, non-competitive PRC2 inhibitor that impairs EED binding to tri-methylated lysine 27 on histone H3 (H3K27me3), preventing allosteric activation of this complex. This is a phase I/II study of MAK683, in adult pts with advanced malignancies who have exhausted or have no effective standard treatment.

Independent domains for recruitment of PRC1 and PRC2 by human XIST.

XIST establishes inactivation across its chromosome of origin, even when expressed from autosomal transgenes. To identify the regions of human XIST essential for recruiting heterochromatic marks we generated a series of overlapping deletions in an autosomal inducible XIST transgene present in 8p of the HT1080 male fibrosarcoma cell line. We examined the ability of each construct to enrich its unified XIST territory with the histone marks established by PRC1 and PRC2 as well as the heterochromatin factors MacroH2A and SMCHD1. Chromatin enrichment of ubH2A by PRC1 required four distinct regions of XIST, and these were completely distinct from the two domains crucial for enrichment of H3K27me3 by PRC2. Both the domains required, as well as the impact of PRC1 and PRC2 inhibitors, suggest that PRC1 is required for SMCHD1 while PRC2 function is necessary for MacroH2A recruitment, although incomplete overlap of regions implicates roles for additional factors. This cooperativity between factors contributes to the requirement for multiple separate domains being required for each feature examined. The independence of the PRC1/PRC2 pathways was observed when XIST was expressed both autosomally or from the X chromosome suggesting that these observations are not purely a result of the context in which XIST operates. Although independent domains were required for the PRC1 and PRC2 pathways overall all regions tested were important for some aspect of XIST functionality, demonstrating both modularity and cooperativity across the XIST lncRNA.

Pathogenic Impacts of Dysregulated Polycomb Repressive Complex Function in Hematological Malignancies.

Polycomb repressive complexes (PRCs) are epigenetic regulators that mediate repressive histone modifications. PRCs play a pivotal role in the maintenance of hematopoietic stem cells through repression of target genes involved in cell proliferation and differentiation. Next-generation sequencing technologies have revealed that various hematologic malignancies harbor mutations in PRC2 genes, such as EZH2, EED, and SUZ12, and PRC1.1 genes, such as BCOR and BCORL1. Except for the activating EZH2 mutations detected in lymphoma, most of these mutations compromise PRC function and are frequently associated with resistance to chemotherapeutic agents and poor prognosis. Recent studies have shown that mutations in PRC genes are druggable targets. Several PRC2 inhibitors, including EZH2-specific inhibitors and EZH1 and EZH2 dual inhibitors have shown therapeutic efficacy for tumors with and without activating EZH2 mutations. Moreover, EZH2 loss-of-function mutations appear to be attractive therapeutic targets for implementing the concept of synthetic lethality. Further understanding of the epigenetic dysregulation associated with PRCs in hematological malignancies should improve treatment outcomes.

Computer Designed PRC2 Inhibitor, EBdCas9, Reveals Functional TATA Boxes in Distal Promoter Regions

The critical process in development, bifurcation of cellular fates, requires epigenetic H3K27me3 marks propagated by PRC2 complex. However, the precise chromatin loci of functional H3K27me3 marks are not yet known. Here we identify critical PRC2 functional sites at a single nucleosome resolution. We fused a computationally designed protein, EED binder (EB) that competes with EZH2 and thereby disrupts PRC2 function, to dCas9 (EBdCas9) to direct PRC2 inhibition at a precise locus using gRNA. We targeted EBdCas9 to 4 different genes (TBX18, p16, CDX2 and GATA3) and observed epigenetic remodeling at a single nucleosome resolution resulting in gene activation. Remarkably, while traditional TATA box is located 30bp upstream of TSS, we identified a functional TATA box, >500bp of TSS, normally repressed by PRC2 complex. Deletion of this TATA box eliminates EBdCas9 dependent TBP recruitment and transcriptional activation. Targeting EBdCas9 to CDX2 and GATA3 results in trophoblast trans-differentiation. EBdCas9 technology is broadly applicable for epigenetic regulation at a single locus to control gene expression.

PRC2 engages a bivalent H3K27M-H3K27me3 dinucleosome inhibitor

A lysine-to-methionine mutation at lysine 27 of histone 3 (H3K27M) has been shown to promote oncogenesis in a subset of pediatric gliomas. While there is evidence that this "oncohistone" mutation acts by inhibiting the histone methyltransferase PRC2, the details of this proposed mechanism nevertheless continue to be debated. Recent evidence suggests that PRC2 must simultaneously bind both H3K27M and H3K27me3 to experience competitive inhibition of its methyltransferase activity. In this work, we used PRC2 inhibitor treatments in a transgenic H3K27M cell line to validate this dependence in a cellular context. We further used designer chromatin inhibitors to probe the geometric constraints of PRC2 engagement of H3K27M and H3K27me3 in a biochemical setting. We found that PRC2 binds to a bivalent inhibitor unit consisting of an H3K27M and an H3K27me3 nucleosome and exhibits a distance dependence in its affinity for such an inhibitor, which favors closer proximity of the 2 nucleosomes within a chromatin array. Together, our data precisely delineate fundamental aspects of the H3K27M inhibitor and support a model wherein PRC2 becomes trapped at H3K27M-H3K27me3 boundaries.

PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP ‘oncohistone-mimic’, that dysregulate gene silencing to promote tumorigenesis.

Abstract LB-288: An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED

Abstract Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12 and plays pivotal roles in transcriptional regulation through its histone H3K27 methyltransferase activity. Dysregulation of PRC2 is observed in multiple human cancers, for example, the catalytic subunit EZH2 is overexpressed in a wide range of human cancers and gain-of-function mutations of EZH2 within its catalytic site have been reported in human B-cell lymphoma, parathyroid carcinoma and melanoma. Small molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported and showed anti-lymphoma efficacy in pre-clinical studies. EED within the PRC2 complex allosterically activate the enzymatic activity by binding to tri-methylated H3K27 (H3K27me3). Here we report the discovery of EED226, a potent and selective PRC2 inhibitor directly binding to the H3K27me3 binding pocket of EED. EED226 induces conformational change upon binding EED leading to loss of PRC2 activity. EED226 shows similar activity as SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show EED226 inhibits PRC2 activity via an allosteric mechanism and offers opportunity for treatment of PRC2-dependent cancers. Citation Format: Wei Qi, Kehao Zhao, Justin Gu, Ying Huang, Youzhen Wang, Hailong Zhang, Man Zhang, Jeff Zhang, Zhengtian Yu, Ling Li, Lin Teng, Shannon Chuai, Chao Zhang, Mengxi Zhao, HoMan Chan, Zijun Chen, Douglas Fang, Fei Qi, Leying Feng, Lijian Feng, Yuan Gao, Hui Ge, Xinjian Ge, Andreas Lingel, Guobin Li, Ying Lin, Yueqin Liu, Fangjun Luo, Minlong Shi, Long Wang, Zhaofu Wang, Yanyan Yu, Jue Zeng, Chenhui Zeng, Lijun Zhang, Qiong Zhang, Shaolian Zhou, Counde Oyang, Peter Atadja, En Li. An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-288. doi:10.1158/1538-7445.AM2017-LB-288

Abstract 3091: Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction

Abstract Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.

Abstract A32: Aberrant gene repression with H3K27me3 modification in human small cell lung cancer

Abstract Small cell lung cancer (SCLC), approximately 15% of all the lung cancer cases, is a subtype of lung cancer with poor prognosis. Expression profiling analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq analysis for H3K27me3 in 3 SCLC cell lines, Lu130, H209 and DMS53, showed that genes enriched with H3K27me3 mark in SCLC were not only PRC2-target genes in ES cells, but also other genes, such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were significantly repressed in expression, and introduction of them into SCLC cell lines lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of those H3K27me3(+) genes, including PRC target genes. Treatment with a PRC2 inhibitor resulted in growth inhibition of SCLC cell lines, indicating that high expression of PRC2 would contribute to gene repression in SCLC, and may play a role in genesis of SCLC. Citation Format: Atsushi Kaneda, Teruyuki Sato, Shingo Tsuji, Takayuki Isagawa, Toshihiro Nukiwa, Yuichi Ishikawa, Hiroyuki Aburatani. Aberrant gene repression with H3K27me3 modification in human small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A32.

EZH2 overexpression in natural killer/T-cell lymphoma confers growth advantage independently of histone methyltransferase activity

AbstractThe role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.