Subunit Of Polycomb Repressive Complex Research Articles

Abstract CT105: Preliminary results from the Phase I part of a first-in-human Phase I/II study of HH2853, an EZH1/2 inhibitor, in patients with relapsed/refractory non-Hodgkin lymphomas or advanced solid tumors

Abstract Background: The dysregulation of polycomb repressive complex 2 (PRC2) promotes tumorigenesis and progression. Two therapeutic agents targeting enhancer of zeste homolog (EZH) 2 or EZH1/2, the catalytic subunits of PRC2, have been approved in several cancer types. HH2853 is a novel selective EZH1/2 dual inhibitor, which has demonstrated superior anti-tumor efficacy to tazemetostat (EZH2 inhibitor approved by FDA) in various preclinical models. Methods: This is a first-in-human, open-label, multi-center, phase (Ph) I/II study of HH2853 in patients (pts) with relapsed/refractory (r/r) non-Hodgkin lymphomas (NHLs) or advanced solid tumors. HH2853 was administered orally twice daily (BID) on a continuous 28-day treatment cycle. Ph I consist of two parts: dose escalation part adopting accelerated titration followed by a Bayesian optimal interval design and dose extension part. Dose limiting toxicity (DLT) was evaluated during the 1st cycle in dose escalation. Safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary anti-tumor activity of HH2853 were explored in this Ph I study. Results: As of Oct 19, 2022, a total of 57 pts, including 50 pts with solid tumors and 7 pts with r/r follicular lymphoma (FL), were enrolled from 12 sites in China and the US. Twenty-five (43.9%) pts received ≥3 lines of prior systemic therapies. Six dose levels(50 mg, 100 mg, 200 mg, 400 mg, 600 mg and 800 mg)were evaluated. DLTs were observed in 2 of 8 DLT evaluable pts at 800 mg: one grade 3 platelet count decreased and one grade 3 diarrhea. A conclusive maximal tolerated dose was not reached. The most common treatment-related adverse events (TRAE) were diarrhea (45.6%), blood bilirubin increased (35.1%), white blood cell count (WBC) decreased (26.3%), platelet count decreased (26.3%), rash (24.6%) and anemia (22.8%). The most common ≥grade 3 TRAEs included anemia (8.8%), platelet count decreased (7.0%), WBC decreased (5.3%) and diarrhea (5.3%). TRAEs leading to dose interruption or reduction were reported in 17.5% and 8.8% pts respectively. No TRAE led to dose discontinuation or death. PK data indicated dose-related increase in exposure from 50 to 600 mg. PD data showed a significant inhibition (maximum reached >90%) of H3K27me3 in granulocytes and monocytes at 400-800 mg. Tumor responses were observed in 7 pts from 200 to 800 mg in multiple tumor types, including complete response in 1 patient with FL, partial response in 3 pts with epithelioid sarcoma, 2 pts with FL and 1 patient with malignant rhabdoid tumor of pancreas. Conclusions: This first-in-human study of HH2853 showed a manageable safety profile and promising anti-tumor activity in multiple tumor types, supporting further exploration in NHLs and solid tumors after recommended Ph II dose is determined.Clinical trial information: NCT04390737 Citation Format: Zhengfu Fan, Jin Wang, Meiyu Fang, Johnston Patrick, Tun Han, David Sommerhalder, Jifang Gong, Jilong Yang, Yun Yang, Javier Munoz, Yuqin Song, Zhiming Li, Xian'an Li, Qiuying Ma, Jinming Han, Lin Shen, Jun Zhu. Preliminary results from the Phase I part of a first-in-human Phase I/II study of HH2853, an EZH1/2 inhibitor, in patients with relapsed/refractory non-Hodgkin lymphomas or advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT105.

Abstract 6267: A novel and potent EZH1/2 dual inhibitor, HM97662 demonstrates antitumor activity in T-cell lymphoma

Abstract Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homology 2 (EZH2) and its homolog EZH1 are catalytic subunits of polycomb repressive complex 2 (PRC2), which trimethylate histone H3 at lysine 27 (H3K27me3) to repress transcription of its target genes. Although methlytransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. EZH1 also directly binds to chromatin and modulates its condensation. Recent studies have suggested that EZH1 as well as EZH2 played a critical role in T-cell lymphoma such as ATL/L and PTCL, which had high innate EZH1 and increased EZH2 expression upon acquisition of their malignancy. Consequently, dual inhibition of EZH1/2 might induce higher expression of downstream tumor suppressor genes than blocking EZH2 alone, expecting greater activity as an anti-cancer therapy. Herein, we presented a novel and potent EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of both EZH1 and EZH2 with 2.1 and 16 nM of IC50, respectively. Surface plasmon resonance (SPR) assay verified that HM97662 had great binding affinity on EZH1-EED-SUZ12 complex as well as EZH2-EED-SUZ12 complex than competitors. HM97662 also showed broad and strong antiproliferative activity against various T-cell lymphoma cell lines. Representatively in HH cells, HM97662 not only suppressed global tri-methylation of H3K27, but also dose-dependently increased protein expression levels that modulate cell cycle or cancer cell apoptosis. HM97662 exhibited increment of CDKN1A (p21) proteins involved in cell cycle arrest and induced protein levels of cleaved caspase-3 as well as PARP. Additionally, resulting induction of apoptosis by HM97662 in HH cells was confirmed by TUNEL staining. Mechanistically, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH cells. Two cell cycle repressor, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were dose-dependently increased by HM97662. We further conducted chromatin accessibility assay and identified that the chromatin structures of them were loosened and highly transcribed after the treatment of HM97662. Based our in vitro pharmacology data, we evaluated an antitumor activity of HM97662 in EZH1/2 co-expressed HuT-102 T-cell lymphoma cell mouse xenograft model, and daily oral dosing of HM97662 showed potent tumor growth inhibition. In conclusion, the present studies demonstrated that HM97662 has promising prospective for the treatment of patients with T-cell lymphoma. Given that the necessity of new treatment options on T-cell lymphoma, it is urgent to assess the effectiveness of HM97662 in further clinical trials. Citation Format: Jooyun Byun, Seung Hyun Jung, Yu-Yon Kim, Miyoung Lee, Gunwoo Lee, Heesun Moon, Eun Young Lee, Junghwa Park, Seon Yeong Han, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. A novel and potent EZH1/2 dual inhibitor, HM97662 demonstrates antitumor activity in T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6267.

Abstract 332: Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model

Abstract Triple negative breast cancer (TNBC) cells overexpress enhancer of zeste homolog 2 (EZH2) the catalytic subunit of polycomb repressive complex 2 (PRC2). EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Furthermore, several studies have also shown the involvement of the neurotransmitter, dopamine, in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast. Dopamine D1 receptor (D1R) activation in TNBC cell line induces apoptosis, autophagy, inhibit the invasion, and regress mammary tumors. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target D1R and EZH2 to completely ablate tumor growth and metastasis. Conventional in vitro models utilize monolayers to investigate the effect of drugs on cancer cells but do not accurately recapitulate the in vivo microenvironment as the cells are subjected to homogeneous growth conditions. Microgels are miniature compartmentalized hydrogels that yield isotropic cell culture conditions unbiased by gravitational or hydrodynamic forces. As such, microgels lend suitable microenvironments conducive to the growth of tumor spheroids, which impose radial-dependent growth conditions as observed in vivo. While non-transformed epithelial cells with reconstituted basement membrane form hollow, growth-arrested, polarized three-dimensional (3D) structures, tumorigenic cells form large, solid, proliferating and invasive structures characteristic of in vivo tumor, which are recapitulated by tumor spheroids. As such, tumor spheroids become an ideal model to study the sensitivity of drugs combination, which led us to investigate the efficacy of EZH2 inhibitor and D1R agonist on TNBC tumor spheroids. Tumor spheroids were formed in monodisperse calcium-alginate microgels generated via droplet-based microfluidics, which mimic the viscoelastic properties of decellularized ECM and induce aggregation by removing integrin-binding sites. Tumor spheroids formed after 2 days were subjected to single and combination therapies of GSK126, an EZH2 inhibitor, and A77636, a dopamine agonist over the span of 4 days. Single doses demonstrated a dose-dependent decrease in tumor spheroid area. Furthermore, the combination therapy demonstrated a regression in growth, synergistic decrease in area (CI < 0.6), and the induction of necrosis. Furthermore, knockout of EZH2 in the TNBC cells resulted in inability for tumor spheroid formation and a sensitivity to A77636. Our findings imply that EZH2 is critical for spheroid formation and growth in TNBC and suggest that targeting the EZH2 alongside D1R presents a novel strategy for enhancing EZH2 inhibition (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Xilal Y. Rima, Gautham Sarathy, Lara Rizzotto, Anagh Kulkarni, Dario Palmieri, Meisam Naeimi Kararoudi, Eduardo Reátegui, Bhuvaneswari Ramaswamy, Eswar Shankar. Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 332.

Abstract 6275: DW91170: A novel potent EZH1/2 dual inhibitor for lymphoma treatment

Abstract EZH2(Enhancer of zeste homolog2) is a catalytic subunit of PRC2 (polycomb repressive complex 2) for chromatin remodeling through methyl transfer activity. Previous studies have reported correlation between EZH2 and cancer progression with overexpression and mutations in various cancers. EZH1, homolog of EZH2, also has been known to role in development and progression of cancers by complementarily replacing EZH2. In recent researches, EZH1/2 dual inhibition showed global epigenetic modification on histone methylation and acetylation. Also, cancers associated with change in epigenetic factors such as mutations in subunit of SWI/SNF responded on EZH1/2 dual inhibition. Therefore, EZH1/2 dual inhibition is expected to be an effective strategy for cancer treatment. Herein, we present a novel potent EZH1/2 dual inhibitor, DW91170, as a promising candidate for treatment of lymphoma with improved anti-cancer activity. DW91170 selectively inhibited EZH1 and EZH2 enzyme activity. DW91170 brought about histone modification by reducing tri-methylation and enhancing acetylation of H3K27, which increased expression of target genes and cell cycle arrest was confirmed by treatment of DW91170. DW91170 showed effective tumor growth inhibition in two types of EZH2 hot spot mutation models. In Pfeiffer (A677 mutation) and WSU-DLCL2 (Y641 mutation) CDX models, DW91170 showed improved tumor regression activity with no significant abnormal signs such as weight loss. In order to verify the potential of DW91170 in R/R-lymphoma patients, the anti-cancer effect was evaluated in Rituximab-resistant cell line and R/R-DLBCL patient-derived PDX model. As a result, DW91170 efficiently inhibited Rituximab-resistant cell proliferation and suppressed tumor growth in R/R-DLBCL PDX model more significantly compared to other EZH inhibitors. Moreover, DW91170 has a good absorption profile that works in a physiological condition. Taken together, we demonstrated that DW91170, a dual epigenetic regulator of EZH1 and EZH2, showed great anti-tumor activities in various lymphoma cell lines and animal models such as CDX and PDX. We are currently trying to expand indication into solid tumor to find a targetable population in clinical trials. Citation Format: Doc-Gyun Jeong, Woon Heo, DongHyuk Shin, Yeonkyung Oh, Sangho Lee, Seongsu Jeong, Whuijung Park, Jiyoung Woo, Chanseok Jeon, Subin Kim, Jaekyung Lim, Yun-Ha Hwang. DW91170: A novel potent EZH1/2 dual inhibitor for lymphoma treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6275.

EZH2 Methyltransferase Regulates Neuroinflammation and Neuropathic Pain.

Recent studies by us and others have shown that enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, in glial cells regulates the genesis of neuropathic pain by modulating the production of proinflammatory cytokines and chemokines. In this review, we summarize recent advances in this research area. EZH2 is a subunit of polycomb repressive complex 2 (PRC2), which primarily serves as a histone methyltransferase to catalyze methylation of histone 3 on lysine 27 (H3K27), ultimately resulting in transcriptional repression. Animals with neuropathic pain exhibit increased EZH2 activity and neuroinflammation of the injured nerve, spinal cord, and anterior cingulate cortex. Inhibition of EZH2 with DZNep or GSK-126 ameliorates neuroinflammation and neuropathic pain. EZH2 protein expression increases upon activation of Toll-like receptor 4 and calcitonin gene-related peptide receptors, downregulation of miR-124-3p and miR-378 microRNAs, or upregulation of Lncenc1 and MALAT1 long noncoding RNAs. Genes suppressed by EZH2 include suppressor of cytokine signaling 3 (SOCS3), nuclear factor (erythroid-derived 2)-like-2 factor (NrF2), miR-29b-3p, miR-146a-5p, and brain-specific angiogenesis inhibitor 1 (BAI1). Pro-inflammatory mediators facilitate neuronal activation along pain-signaling pathways by sensitizing nociceptors in the periphery, as well as enhancing excitatory synaptic activities and suppressing inhibitory synaptic activities in the CNS. These studies collectively reveal that EZH2 is implicated in signaling pathways known to be key players in the process of neuroinflammation and genesis of neuropathic pain. Therefore, targeting the EZH2 signaling pathway may open a new avenue to mitigate neuroinflammation and neuropathic pain.

Loss of Ezh2 function remodels the DNA replication initiation landscape

In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication initiation. Most of these genes are bound and repressed by the Polycomb repressive complex-2 (PRC2) through the repressive H3K27me3 mark. This is the strongest overlap observed for a chromatin regulator with replication origin activity. Here, we asked whether Polycomb-mediated gene repression is functionally involved in recruiting DNA replication origins to transcriptionally silent genes. We show that the absence of EZH2, the catalytic subunit of PRC2, results in increased DNA replication initiation, specifically in the vicinity of EZH2 binding sites. The increase in DNA replication initiation does not correlate with transcriptional de-repression or the acquisition of activating histone marks but does correlate with loss of H3K27me3 from bivalent promoters.

EZH2 W113C is a gain-of-function mutation in B-cell lymphoma enabling both PRC2 methyltransferase activation and tazemetostat resistance

Polycomb Repressive Complex2 (PRC2) suppresses gene transcription by methylating lysine 27 of histone H3 (H3K27) and plays critical roles in embryonic development. Among the core PRC2 subunits, EZH2 is the catalytic subunit and EED allosterically activates EZH2 upon binding trimethylated H3K27 (H3K27me3). Activating mutations on Y641, A677, and A687 within the enzymatic SET domain of EZH2 have been associated with enhanced H3K27me3 and tumorigenicity of many cancers including B-cell lymphoma and melanoma. To tackle the critical residues outside the EZH2 SET domain, we examined EZH2 mutations in lymphoma from cancer genome databases and identified a novel gain-of-function (GoF) mutation W113C, which increases H3K27me3 in vitro and in vivo and promotes CDKN2A silencing to a similar level as the EZH2 Y641F. Different from other GoF mutations, this mutation is located in the SAL motif at the EZH2 N-terminus, which stabilizes the SET domain and facilitates substrate binding. This may explain how the W113C mutation increases PRC2 activity. Tazemetostat is an FDA-approved EZH2 binding inhibitor for follicular lymphoma treatment. Intriguingly, the W113C mutation leads to tazemetostat-resistance in cell and in vivo in both H3K27 methylation and tumor proliferation. Another class of allosteric PRC2 inhibitor binding EED overcomes the resistance, effectively decreases H3K27me3, and blocks tumor proliferation in cells expressing EZH2 W113C. As this mutation is originally identified from lymphoma samples, our results demonstrated its activating characteristic and the deleterious consequence, provide insights on PRC2 regulation, and support the continued exploration of treatment optimization for lymphoma patients.

Dissecting and targeting noncanonical functions of EZH2 in multiple myeloma via an EZH2 degrader.

Multiple myeloma (MM) is the second most common hematological malignancy with poor prognosis. Enhancer of zeste homolog 2 (EZH2) is the enzymatic subunit of polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) for transcriptional repression. EZH2 have been implicated in numerous hematological malignancies, including MM. However, noncanonical functions of EZH2 in MM tumorigenesis are not well understood. Here, we uncovered a noncanonical function of EZH2 in MM malignancy. In addition to the PRC2-mediated and H3K27me3-dependent canonical function, EZH2 interacts with cMyc and co-localizes with gene activation-related markers, promoting MM tumorigenesis in a PRC2- and H3K27me3-independent manner. Both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes can be effectively depleted in MM cells by MS177, an EZH2 degrader we reported previously, leading to profound activation of EZH2-PRC2-associated genes and simultaneous suppression of EZH2-cMyc oncogenic nodes. The MS177-induced degradation of both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes also reactivated immune response genes in MM cells. Phenotypically, targeting of EZH2's both canonical and noncanonical functions by MS177 effectively suppressed the proliferation of MM cells both in vitro and in vivo. Collectively, this study uncovers a new noncanonical function of EZH2 in MM tumorigenesis and provides a novel therapeutic strategy, pharmacological degradation of EZH2, for treating EZH2-dependent MM.

Epigenetic regulation of embryonic ectoderm development in stem cell differentiation and transformation during ontogenesis.

Dynamic chromatin accessibility regulates stem cell fate determination and tissue homeostasis via controlling gene expression. As a histone-modifying enzyme that predominantly mediates methylation of lysine 27 in histone H3 (H3K27me1/2/3), Polycomb repressive complex 2 (PRC2) plays the canonical role in targeting developmental regulators during stem cell differentiation and transformation. Embryonic ectoderm development (EED), the core scaffold subunit of PRC2 and as an H3K27me3-recognizing protein, has been broadly implicated with PRC2 stabilization and allosterically stimulated PRC2. Accumulating evidences from experimental data indicate that EED-associating epigenetic modifications are indispensable for stem cell maintenance and differentiation into specific cell lineages. In this review, we discuss the most updated advances to summarize the structural architecture of EED and its contributions and underlying mechanisms to mediating lineage differentiation of different stem cells during epigenetic modification to expand our understanding of PRC2.

Imagawa-Matsumoto syndrome: SUZ12-related overgrowth disorder.

The SUZ12 gene encodes a subunit of polycomb repressive complex 2 (PRC2) that is essential for development by silencing the expression of multiple genes. Germline heterozygous variants in SUZ12 have been found in Imagawa-Matsumoto syndrome (IMMAS) characterized by overgrowth and multiple dysmorphic features. Similarly, both EZH2 and EED also encode a subunit of PRC2 each and their pathogenic variants cause Weaver syndrome and Cohen-Gibson syndrome, respectively. Clinical manifestations of these syndromes significantly overlap, although their different prevalence rates have recently been noted: generalized overgrowth, intellectual disability, scoliosis, and excessive loose skin appear to be less prevalent in IMMAS than in the other two syndromes. We could not determine any apparent genotype-phenotype correlation in IMMAS. The phenotype of neurofibromatosis type 1 arising from NF1 deletion was also shown to be modified by the deletion of SUZ12, 560 kb away. This review deepens our understanding of the clinical and genetic characteristics of IMMAS together with other overgrowth syndromes related to PRC2. We also report on a novel IMMAS patient carrying a splicing variant (c.1023+1G>C) in SUZ12. This patient had a milder phenotype than other previously reported IMMAS cases, with no macrocephaly or overgrowth phenotypes, highlighting the clinical variation in IMMAS.

Discovery of a Novel Covalent EZH2 Inhibitor Based on Tazemetostat Scaffold for the Treatment of Ovarian Cancer.

Enhancer of zeste homologue 2 (EZH2) is the enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2), which plays an important role in post-translational modifications of histones. In this study, we designed and synthesized a new series EZH2 covalent inhibitors that have rarely been reported. Biochemical studies and mass spectrometry provide information that SKLB-03220 could covalently bind to the S-adenosylmethionine (SAM) pocket of EZH2. Besides, SKLB-03220 was highly potent for EZH2MUT, while exhibiting weak activities against other tested histone methyltransferases (HMTs) and kinases. Moreover, SKLB-03220 displayed noteworthy potency against ovarian cancer cell lines and continuously abolished H3K27me3 after washing out. Furthermore, oral administration of SKLB-03220 significantly inhibited tumor growth in PA-1 xenograft model without obvious adverse effects. Taken together, SKLB-03220 is a potent, selective EZH2 covalent inhibitor with noteworthy anticancer efficacy both in vitro and in vivo.

EZH inhibitors in lymphoma therapy

Enhancer of zeste homolog (EZH), a subunit of polycomb repressive complex 2 (PRC2), suppresses gene expression by methylation of H3K27. EZH is closely associated with B-cell development and pathogenesis of certain malignant lymphomas. In follicular lymphoma (FL), gain-of-function mutation and upregulation of EZH2 are observed in approximately 30% and 15% of cases, respectively. Moreover, one-third of diffuse large B-cell lymphomas carry an EZH2 mutation, mostly co-existing with translocation involving Bcl-2. Genome-wide trimethylation of H3K27 is a unique characteristic induced by upregulation of both EZH2 and EZH1, and is responsible for more than half of the gene suppression that occurs in adult T-cell leukemia/lymphoma (ATL). Inhibition of EZH can reduce H3K27 methylation and subsequently restore epigenetically suppressed genes. Currently, an EZH2 inhibitor and dual EZH1/2 inhibitor have been clinically used to treat relapsed/refractory FL and ATL, respectively. EZH-targeted treatment for lymphoma has only just begun, and further development of these drugs for various other malignancies, both alone and in combination with other therapeutics, is ongoing.

Polycomb Repressive Complex 2 in Oncology.

Dynamic regulation of the chromatin state by Polycomb Repressive Complex 2 (PRC2) provides an important mean for epigenetic gene control that can profoundly influence normal development and cell lineage specification. PRC2 and PRC2-induced methylation of histone H3 lysine 27 (H3K27) are critically involved in a wide range of DNA-templated processes, which at least include transcriptional repression and gene imprinting, organization of three-dimensional chromatin structure, DNA replication and DNA damage response and repair. PRC2-based genome regulation often goes wrong in diseases, notably cancer. This chapter discusses about different modes-of-action through which PRC2 and EZH2, a catalytic subunit of PRC2, mediate (epi)genomic and transcriptomic regulation. We will also discuss about how alteration or mutation of the PRC2 core or axillary component promotes oncogenesis, how post-translational modification regulates functionality of EZH2 and PRC2, and how PRC2 and other epigenetic pathways crosstalk. Lastly, we will briefly touch on advances in targeting EZH2 and PRC2 dependence as cancer therapeutics.

The role of EZH1 and EZH2 in development and cancer

Polycomb Repressive Complex 2 (PRC2) exhibits key roles in mammalian development through its temporospatial repression of gene expression. EZH1 or EZH2 is the catalytic subunit of PRC2 that mediates the mono-, di- and tri-methylation of histone H3 lysine 27 (H3K27me1/2/3), H3K27me2/me3 being a hallmark of facultative heterochromatin. PRC2 is a chromatinmodifying enzyme that is recruited to a limited number of "nucleation sites", spreads H3K27 methylation and fosters chromatin compaction. EZH1 and EZH2 exhibit differences in their expression patterns, levels of histone methyltransferase activity (HMT) in the context of PRC2, and DNA/nucleosome binding activity. This suggests that their roles in heterochromatin formation are disparate. Dysregulation of PRC2 activity leads to aberrant gene expression and is implicated in cancer and developmental diseases. In this review, we discuss the distinct function of PRC2/EZH1 and PRC2/EZH2 in the early and late developmental stages. We then discuss the cancers associated with PRC2/EZH1 and PRC2/EZH2. [BMB Reports 2022; 55(12): 595-601].

Abstract A025: Assessing the role of bivalency in MYCN-amplified neuroblastoma

Abstract Introduction: Neuroblastoma is an embryonic pediatric cancer with a broad spectrum of clinical behavior. In aggressive neuroblastoma tumors, we previously demonstrated enrichment of 5-hydroxymethylcytosine (5-hmC), a marker of transcriptional activation, on genes targeted by the repressive Polycomb Repressive Complex 2 (PRC2). PRC2 has been shown to be a dependency of MYCN-amplified neuroblastoma. In embryonic stem cells, 5-hmC is disproportionately enriched at the transcription start sites (TSS) of bivalent genes that are characterized by the presence of both repressive and activating histone modifications (H3K27me3 and H3K4me3). Bivalent genes are postulated to be poised for rapid activation during embryogenesis. We hypothesized that bivalent genes enriched for 5-hmC at the TSS play a key role in regulating the undifferentiated phenotype of MYCN-amplified neuroblastoma. Methods: Tumor derived 5-hmC profiles were obtained previously. Nano-hmC-seal was used to generate genome-wide 5-hmC profiles in the MYCN-amplified or overexpressed SK-N-BE2, LA1-55n, LA1-5s, NBLW-N, NBLW-S, NBL-S, and MYCN-non-amplified SH-SY5Y, and SHEP cell lines. H3K27me3, H3K4me3, and H3K27ac profiles were generated in these cell lines using ChIP-Seq. KAS-Seq, a chemical pulldown of ssDNA that marks active enhancers and transcribed genes, and RNA-Seq profiling were performed. MACS2 and deepTools were used to analyze ChIP-Seq, 5-hmC, and KAS-Seq data. Results: A comparison of 5-hmC profiles derived from diagnostic tumor biopsy samples with and without MYCN-amplification demonstrated increased 5-hmC deposition on SUZ12 and EZH2 (PRC2 subunits) target genes. MYCN-amplified and MYC overexpressing cell lines but not MYCN-non-amplified lines bore a TSS specific association between 5-hmC deposition and H3K27me3 enrichment. Using the SK-N-BE2 and NBLW-N cell lines, we identified 313 genes with both H3K4me3 and H3K27me3 deposition, which we defined as bivalent, and 806 genes with only H3K27me3 deposition. Both H3K27me3-only and bivalent genes had TSS specific 5-hmC deposition, were enriched in development and differentiation pathways, and had significantly lower expression in cell lines with high MYCN expression. However, unlike H3K27me3-only genes, bivalent genes were also enriched for transcription factor biding sites of AP-2 and E2F which co-regulate MYCN. Using a publicly available dataset of 498 tumors, we found that bivalent genes were transcriptionally repressed in MYCN-amplified tumor samples. Within the cohort of 175 high-risk patients, those with lower expression of bivalent genes (N=55) had worse overall survival (OS) from diagnosis than those (N=120) with high expression (5-year OS: 27.2% vs. 52.0%; 95% CI: 17.1%-43.3% vs 42.9%-63.1%, p=0.001 by log-rank test). Conclusion: The low-level expression of bivalent genes enriched for 5-hmC at the TSS in MYCN-amplified neuroblastoma cells may represent a novel mechanism by which dedifferentiated states and aggressive phenotype is maintained in MYCN-amplified neuroblastoma. Citation Format: Mohansrinivas Chennakesavalu, Gepoliano Chaves, Kelley Moore, Tong Wu, Ruitu Lyu, Rachel TerHaar, Alexandre Chlenski, Chuan He, Susan Cohn, Andrea Piunti, Mark Applebaum. Assessing the role of bivalency in MYCN-amplified neuroblastoma. [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 A025.

Abstract A024: PRC2 loss drives MPNST metastasis and matrix remodeling

Abstract Epigenetic reprogramming alters gene transcription in response to different cellular cues and can occur at both the DNA level as well as histone post-translational modifications. Polycomb repressive complex 2 (PRC2) is a common epigenetic modifier that is responsible for adding methyl groups to the twenty-seventh lysine of histone 3. This modification is associated with decreased gene transcription and recruits complexes that further compact chromatin. PRC2 mutations have been implicated in the tumorigenesis of many cancer types; however, the role that PRC2 plays in tumorigenesis is complex and not well understood. PRC2 loss-of-function mutations have been identified in malignant peripheral nerve sheath tumors (MPNST), an aggressive sarcoma. PRC2 mutations are found in the EED and SUZ12 subunits in 70-92% of tumors. There are currently no effective therapies for MPNSTs resulting in a poor prognosis for individuals with inoperable tumors. Using a CRISPR/Cas9 system to delete EED or SUZ12, we developed in vitro and in vivo models to further study the biology of PRC2 loss in MPNSTs. Our data suggests that loss of PRC2 subunits increases the expression of extra-cellular matrix (ECM) remodeling genes, promotes cell migration in vitro, and increases pulmonary metastasis in vivo. We also observed that loss of either EED or SUZ12 increases collagen handling and a clustering metastatic phenotype. Additionally, loss of H3K27me3 was correlated with increased ECM and ECM remodeling genes as well as metastasis and overall survival in patient RNA-seq and tumor microarray data, respectively. Further characterization of the underlying mechanisms of PRC2-dependent metastasis will allow us to better understand the role of PRC2 in MPNST biology and test novel therapies for these patients. Citation Format: Qierra R. Brockman, Amanda Scherer, Gavin R. McGivney, Wade R. Gutierrez, Andrew Voigt, Alexandra Isaacson, Emily A. Laverty, Grace Roughton, Vickie Knepper-Adrian, Benjamin Darbro, Munir R. Tanas, Christopher Stipp, Rebecca D. Dodd. PRC2 loss drives MPNST metastasis and matrix remodeling. [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 A024.

37. Epigenetic reprogramming of brain development pathways during non-small cell lung cancer metastasis to brain

Non-small cell lung cancer (NSCLC) is one of the most common and deadliest cancers, with roughly half of all patients initially presenting with both primary and metastatic disease. While the major events in the metastatic cascade have been identified, a mechanistic understanding of how NSCLC routinely, successfully colonizes the brain is largely unknown. To better understand this process, we profiled a combination of genomic, transcriptomic, and methylomic landscapes of 45 paired NSCLC primary and brain metastasis samples. 75 genes displayed recurrent metastasis enriched variants, largely implicated in focal adhesion and extracellular matrix receptor interactions. Variant allele frequencies over a wide range of epigenetic regulators displayed an increase in metastases, suggesting that epigenetic misregulation may be selected for and possibly contribute to NSCLC metastasis to brain. Consistent with these observations, we observed widespread changes in DNA methylation throughout disease progression, many found within brain-specific active enhancers and correlated with increased nearby gene expression. The greatest recurrent methylation changes during metastatic progression occurred over a subset of DNA methylation valleys (DMVs) enriched for H3K9me3 and bivalent marks H3K27me3 and H3K4me1 in normal lung. Mapping EZH2, the catalytic subunit of polycomb repressive complex 2 (PCR2), binding locations in H1299, a lymph node-derived lung cancer cell line, revealed a pervasive loss of EZH2 binding within DMVs accompanied by an increase in DNA methylation, exemplifying epigenetic switching. The vast majority of these DMR-associated DMVs harbor developmental genes, suggesting that altered epigenetic regulation of developmentally important genes may confer a selective advantage during metastatic progression.

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

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2's multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of bothEZH2's canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2TAD for mediating EZH2's non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancerthat rely on the circuits wired by EZH2 and AR.

89 (PB079) - Preliminary Clinical Data From Ongoing Phase 2 Study With Enhancer of Zeste Homolog 2 (EZH2) Inhibitor CPI-0209 in Patients With Advanced Solid Tumors or Hematologic Malignancies

Background: Enhancer of zeste homolog 2 (EZH2) is a histone-lysine N-methyltransferase enzyme and the catalytic subunit of polycomb repressive complex 2. ARID1A mutations have been linked to increased sensitivity to EZH2 inhibition. Inactivating BAP1 mutations in mesothelioma have been linked to dependency on EZH2 function. CPI-0209 is an investigative oral, small-molecule, second-generation, selective EZH2 inhibitor that shows durable and comprehensive target coverage based on findings in Phase 1 (NCT04104776; Lakhani, et al.

188 (PB068) - Updated Findings and Biomarker Analysis From the Ongoing Phase 1 Study of Enhancer of Zeste Homolog 2 (EZH2) Inhibitor CPI-0209 in Patients With Advanced Solid Tumors

188 (PB068) - Updated Findings and Biomarker Analysis From the Ongoing Phase 1 Study of Enhancer of Zeste Homolog 2 (EZH2) Inhibitor CPI-0209 in Patients With Advanced Solid Tumors