Polycomb Target Research Articles

Abstract IA025: A DNA methylation atlas of normal human cell types

Abstract DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies. Citation Format: Tommy Kaplan. A DNA methylation atlas of normal human cell types [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA025.

Cancer-associated DNA hypermethylation of Polycomb targets requires DNMT3A dual recognition of histone H2AK119 ubiquitination and the nucleosome acidic patch.

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

Dual roles of histone H3 lysine-4 in antagonizing Polycomb group function and promoting target gene expression.

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the ON/OFF state of targets like the Hox genes. Trithorax encodes a methyltransferase specific to histone H3 lysine-4 (H3K4). However, there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo . Here, we demonstrate two key roles for replication-dependent histone H3.2K4 in target control. We find that H3.2K4 antagonizes Polycomb group catalytic activity and that it is required for proper target gene activation. We conclude that H3.2K4 directly regulates expression of Polycomb targets.

Homeobox and Polycomb target gene methylation in human solid tumors

DNA methylation is an epigenetic mark that plays an important role in defining cancer phenotypes, with global hypomethylation and focal hypermethylation at CpG islands observed in tumors. These methylation marks can also be used to define tumor types and provide an avenue for biomarker identification. The homeobox gene class is one that has potential for this use, as well as other genes that are Polycomb Repressive Complex 2 targets. To begin to unravel this relationship, we performed a pan-cancer DNA methylation analysis using sixteen Illumina HM450k array datasets from TCGA, delving into cancer-specific qualities and commonalities between tumor types with a focus on homeobox genes. Our comparisons of tumor to normal samples suggest that homeobox genes commonly harbor significant hypermethylated differentially methylated regions. We identified two homeobox genes, HOXA3 and HOXD10, that are hypermethylated in all 16 cancer types. Furthermore, we identified several potential homeobox gene biomarkers from our analysis that are uniquely methylated in only one tumor type and that could be used as screening tools in the future. Overall, our study demonstrates unique patterns of DNA methylation in multiple tumor types and expands on the interplay between the homeobox gene class and oncogenesis.

Abstract 2848: MYC and TGFβ promote group 3 medulloblastoma tumor resistance through deregulation of Polycomb targets

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at <50%, few recurrent mutations, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some MB tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We hypothesize that the TGFβ pathway and MYC contribute to the intrinsic resistance of G3MB through deregulation of key genes and pathways. We previously established humanized models for SHHMB by introducing MYCN or PTCH1 deletions into neuroepithelial stem cells (NESC) derived from normal human induced pluripotent stem cells (hIPSCs). In this study, we transduced NESCs with TGFb effectors activated in G3MB alone and/or in combination with MYC, prioritizing combinations observed in patients. Excitingly, both MYC and TGFβ effectors drove tumor formation in vivo with the combination of TGFβ effectors with MYC leading to more aggressive tumors. Moreover, they clustered with human G3/G4MB samples, indicating they phenocopy the relevant tumor of interest. We next found that NESCs expressing MYC with either TGFβR1 or TGFβ1 showed resistance to clinical TGFβR1 inhibitors, compared to cells driven by either TGFβR1 or TGFβ1 alone. To decipher mechanisms of resistance, we integrated CUT&RUN to probe for MYC genomic localization and relevant histone PTMs with RNA-seq analysis and discovered a subset of genes upregulated in MYC and TGFb-driven lines that are targets of the histone demethylase KDM2B. Loss of function mutations in KDM2B occur in G3MB patients, indicating that KDM2B deregulated genes are critical in G3MB. We postulate that epigenetic remodeling via MYC and recruitment of other MYC-interacting cofactors to PRC targets culminates in transcriptional changes that lead to aggressive disease. Work from others suggests that PRC-mediated chromatin regulation is critical in many adult and pediatric brain tumors. Overall, our studies provide important insight on identifying new therapeutic avenues for patients with MYC and TGFβ driven G3MB. Citation Format: Zulekha A. Qadeer, Samanta Westelman, Mackenzie O. Johnson, Elise Hou, Shane Grele, Kyle Smith, Liam Hendrikse, Linyu Wang, Sarah Husain, Mary Clare Beytagh, Christin Schmidt, Miller Huang, Michael D. Taylor, Paul Northcott, William A. Weiss. MYC and TGFβ promote group 3 medulloblastoma tumor resistance through deregulation of Polycomb targets [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 2848.

Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase transition.

Polycomb group proteins have a critical role in silencing transcription during development. It is commonly proposed that Polycomb-dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking the binding of activating complexes. Recently, it has also been argued that liquid-liquid demixing of Polycomb proteins facilitates this compaction and repression by phase-separating target genes into a membraneless compartment. To test these models, we used Optical Reconstruction of Chromatin Architecture to trace the Hoxa gene cluster, a canonical Polycomb target, in thousands of single cells. Across multiple cell types, we find that Polycomb-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction or phase-separation models. Using polymer simulations, we show that these observed flexible ensembles can be explained by 'spatial feedback'-transient contacts that contribute to the propagation of the epigenetic state (epigenetic memory), without inducing a globular organization.

Abstract B033: Mapping subclonal epigenetic evolution in colorectal cancer by spatial analysis of DNA hypermethylation

Abstract Aberrant patterns of DNA methylation (DNAme) have long been noted in colorectal cancer (CRC). Early studies found overall hypomethylation of the cancer genome, while subsequent work revealed focal hypermethylation at specific loci, especially CpG-rich promoters. Loci occupied by polycomb repressive complex 2 in stem cells are remarkably enriched in cancer-specific hypermethylation events. These Polycomb Target Genes (PTGs) include important transcriptional regulators of lineage specification and differentiation, suggesting that hypermethylation-mediated silencing of PTGs could play an active role in CRC initiation or progression. Despite these findings, we have limited knowledge of when, where, and how this hypermethylation arises. Preliminary evidence from bulk tumor samples in the Cancer Genome Atlas suggests that many of these PTG hypermethylation events may be almost completely clonal. Additionally, we have noted rare, densely methylated reads covering PTGs in cancer-adjacent, histologically normal tissue. These observations suggest that PTG hypermethylation may be a very early event in carcinogenesis and may play a permissive or even active role in that process. To better understand the timing and evolution of PTG hypermethylation events in CRC, we have developed a protocol that allows us to extract dozens of small (20µL), spatially annotated mini-bulk samples from live tumors. These mini-bulks may be frozen or dissociated to viable single-cell suspensions. Remaining tumor tissue is preserved in the form of spatially annotated FFPE blocks, which can be used for analyses such as high-plex immunofluorescence or spatial transcriptomics. Using this process, we have collected over 500 spatially annotated samples from 6 CRC cases, including 196 micro-bulk samples profiled using the Infinium MethylationEPIC array. Additionally, we have profiled DNAme in 147 single cells using a dramatically improved single-cell whole-genome bisulfite sequencing protocol developed in our lab. Analysis of EPIC array data reveals wide variation in DNAme heterogeneity between tumors. While in some tumors, the vast majority of DNA hypermethylation events are clonal, other tumors reveal significant intra-tumor heterogeneity, including two or more prominent DNAme subclones as well as a substantial number of ‘private’ hypermethylation events that occur in only one sample. Mapping DNAme information back onto our spatial data reveals that in at least one case, DNAme clones are spatially partitioned and are associated with distinct histology. Ongoing work includes inference of phylogenetic trees from DNA hypermethylation events using non-reversible models of methylation accumulation, allowing us to measure the relative timing of hypermethylation events, as well as integration of array, whole-genome, and single-cell methylation data to more completely characterize DNAme subclones, characterize cellular DNAme heterogeneity at variably methylated sites, and shed further light on the evolution of cancer-related DNA hypermethylation. Citation Format: Nathan J. Spix, Hsiao-yun Milliron, Manpreet Kalkat, Emily Eugster, David W. Chesla, David Sokol, Emily Jung, Paula Nolte, Kelly K. Krzyzanowski, Toshinori Hinoue, Hui Shen, Peter W. Laird. Mapping subclonal epigenetic evolution in colorectal cancer by spatial analysis of DNA hypermethylation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B033.

Abstract C171: Immune desert microenvironment is associated with poor outcome in Wilms tumors

Abstract Background: Presence of anaplasia is a potent marker for adverse outcome in patients with Wilms tumors with diffuse anaplasia (DAWT). Patients with WT with focal anaplasia (FAWT) are considered as intermediate risk. Besides TP53 mutations thought to arise in anaplastic components, the crosstalk between genomic alterations and immune landscape remains unknown. Methods: We performed whole-exome and/or RNA sequencing on genomic DNA derived from 12 matched tumor-normal WTs and extended analysis in an independent dataset of 9 cases. TP53 mutations and tumor infiltrating lymphocytes (TILs: CD3, CD4 and CD8) were also assessed. Validation was performed using TARGET-WT (n=96) and GEO-WT (n=12) cohorts. Prognostic role of TILs was investigated in another 55 pre-treated WTs. Results: TP53 (50%) and DROSHA (33.3%) were the sole recurrent mutations identified. TP53 mutations occurred in mutually exclusive manner with chromatin-modifying genes which were altered in both FAWT (100%) and DAWT (33.3%). Microenvironment analysis revealed a reproducible immune desert WTs with poor outcome that enriched in DAWT cases and TP53 mutation, which mirrored the inversion of CD4:CD8 ratio in TP53-mutated comparing to TP53-wild tumors (P=0.003). Immune desert WTs were also characterized by overexpression of DNA repair pathways, PRC2, HDCA, and polycomb targets of EZH2, leading to higher sensitivity to HDCA inhibitor of vorinostat. Additionally, the immunophenotypes were tightly associated with replication stress subtypes; WTs with high replication stress were susceptible to ATR and WEE1 inhibitors. High ratio of CD4/CD8 was considered as independent recurrence-free and overall survival factors for WTs (both P<0.05). Conclusions: Our results reveal that WT with anaplastic features are characterized by either TP53 alterations or chromatin-modifying genes. These data implicate TP53 as a tumor cell-intrinsic driver of an immune desert phenotype, in contrast with adult TP53 tumors. TP53 inactivation might therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy. Citation Format: Xiaoping Su, Gabriel Malouf. Immune desert microenvironment is associated with poor outcome in Wilms tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C171.

Silencing of genes by promoter hypermethylation shapes tumor microenvironment and resistance to immunotherapy in clear-cell renal cell carcinomas

The efficacy of immune checkpoint inhibitors varies in clear-cell renal cell carcinoma (ccRCC), with notable primary resistance among patients. Here, we integrate epigenetic (DNA methylation) and transcriptome data to identify a ccRCC subtype characterized by cancer-specific promoter hypermethylation and epigenetic silencing of Polycomb targets. We develop and validate an index of methylation-based epigenetic silencing (iMES) that predicts primary resistance to immune checkpoint inhibition (ICI) in the BIONIKK trial. High iMES is associated with VEGF pathway silencing, endothelial cell depletion, immune activation/suppression, EZH2 activation, BAP1/SETD2 deficiency, and resistance to ICI. Combination therapy with hypomethylating agents or tyrosine kinase inhibitors may benefit patients with high iMES. Intriguingly, tumors with low iMES exhibit increased endothelial cells and improved ICI response, suggesting the importance of angiogenesis in ICI treatment. We also develop a transcriptome-based analogous system for extended applicability of iMES. Our study underscores the interplay between epigenetic alterations and tumor microenvironment in determining immunotherapy response.

Epigenetic regulation limits competence of pluripotent stem cell-derived oocytes.

Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro-generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development invitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro-derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5-hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.

Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

Polycomb repressive complex 1 (PRC1) undergoes phase separation to form Polycomb condensates that are multi-component hubs for silencing Polycomb target genes. In this study, we demonstrate that formation and regulation of PRC1 condensates are consistent with the scaffold-client model, where the Chromobox 2 (CBX2) protein behaves as the scaffold while the other PRC1 proteins are clients. Such clients induce a re-entrantphase transition of CBX2 condensates. The composition of the multi-component PRC1 condensates (1) determines the dynamic properties of the scaffold protein; (2) selectively promotes the formation of CBX4-PRC1 condensates while dissolving condensates of CBX6-, CBX7-, and CBX8-PRC1; and (3) controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 in CBX2-PRC1 condensates and the exclusion of CBX6-PRC1 from CBX2-PRC1 condensates. Our findings uncover how multi-component PRC1 condensates are assembled via an intricate scaffold-client mechanism whereby the properties of the PRC1 condensates are sensitively regulated by its composition and stoichiometry.

Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1.

Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.

Reduced histone gene copy number disrupts Drosophila Polycomb function.

The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

PRC2.1- and PRC2.2-specific accessory proteins drive recruitment of different forms of canonical PRC1

Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout (KO) and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyzes the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1 but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalyzing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1- and PRC2.2-specific accessory proteins to Polycomb-mediated repression and uncover a new mechanism for cPRC1 recruitment.

SENP3 and USP7 regulate Polycomb-rixosome interactions and silencing functions

The rixosome and PRC1 silencing complexes are associated with deSUMOylating and deubiquitinating enzymes, SENP3 and USP7, respectively. How deSUMOylation and deubiquitylation contribute to rixosome- and Polycomb-mediated silencing is not fully understood. Here, we show that the enzymatic activities of SENP3 and USP7 are required for silencing of Polycomb target genes. SENP3 deSUMOylates several rixosome subunits, and this activity is required for association of the rixosome with PRC1. USP7 associates with canonical PRC1 (cPRC1) and deubiquitinates the chromodomain subunits CBX2 and CBX4, and inhibition of USP activity results in disassembly of cPRC1. Finally, both SENP3 and USP7 are required for Polycomb- and rixosome-dependent silencing at an ectopic reporter locus. These findings demonstrate that SUMOylation and ubiquitination regulate the assembly and activities of the rixosome and Polycomb complexes and raise the possibility that these modifications provide regulatory mechanisms that may be utilized during development or in response to environmental challenges.

A DNA methylation atlas of normal human cell types

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2–5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

EZH1-PRC2 Identifies a Potential Reservoir for Disease Relapse in Quiescent Cancer Cells Refractory to EZH2 Enzymatic Inhibition

Recurrent disease relapse and remission is a hallmark of indolent non-Hodgkin lymphoma (NHL). Remissions are often characterised by long periods of clinical inactivity without detectable disease. Furthermore, it is now standard practice to clinically monitor carefully selected asymptomatic patients with indolent lymphoma, often for years, reserving therapy for symptomatic disease progression, without adversely affecting the patient's prognosis. Identification and targeting of the putative cellular reservoir recurrently seeding disease relapse and progression in NHL has long tantalised researchers and would represent a significant advance in the field given the hypothetical prospect of deferring or preventing impending relapse. The oncogene EZH2 is upregulated in the lymphoid germinal center and forms the enzymatic core of Polycomb Repressive Complex 2 (PRC2) alongside essential core components EED and SUZ12, writing all methylation of H3K27, with H3K27me3-mediated silencing of the B-cell terminal differentiation programme. The EZH2 small molecule enzymatic inhibitor Tazemetostat has been approved as monotherapy beyond second line for relapsed/refractory EZH2-mutant and wild-type follicular lymphoma, with responders benefiting for a modest median duration of 10.9 months (Morschauser et al., Lancet Oncology, 2020). The mechanism(s) for disease progression in patients have not yet been published. Using TIG3-T human diploid fibroblasts as a model of cellular quiescence, we discover that a novel form of PRC2 predominates in non-dividing cells, which contains EZH1, EED, SUZ12 and the polycomb-like protein, PHF1, but crucially lacks EZH2. RNA-Seq and Western blot analysis revealed reciprocal expression patterns of EZH1 and EZH2 in serum-starved, quiescent (non-dividing) cells compared to asynchronous cycling cells. Using EED immunoprecipitation with subsequent mass spectrometry, we demonstrated that EZH1 replaces EZH2 as the predominant H3K27 methyltransferase in quiescent cells. Quantitative ChIP-sequencing with exogenous reference genome spike-in (ChIP-Rx) confirms that EHZ1-PRC2 largely replaces EZH2-PRC2 at polycomb target genes. While the EZH2 enzymatic inhibitor GSK343 and the EED allosteric inhibitor A395 significantly reduced H3K27me2 and H3K27me3 in cycling cells, those markes are unchanged in quiescent cells. This alludes to a function of EZH1-PRC2 in quiescent cells that is independent of its function as a H3K27 methyltransferase, and therefore not targetable by PRC2 enzymatic inhibitors. The EED PROTAC UNC6852 degrades EED (Figure 1), thereby destabilising both EZH1-PRC2 and EZH2-PRC2, as confirmed by Western blot and ChIP-Rx. Strikingly, this molecule resulted in a marked reduction of H3K27me2 and H3K27me3 in proliferating cells, but had no such effect in treated quiescent cells. Degradation of PRC2, however, did result in a modest reduction in protein levels and chromatin occupancy of the canonical PRC1 component CBX7, suggesting an interplay between EZH1-PRC2 occupancy and local cPRC1-mediated chromatin of compaction of Polycomb target genes. EZH2 and Ki-67 immunohistochemistry and EZH1 RNA in situ hybridization (RNA-ISH) were performed on lymph node biopsy specimens from two well-matched patients with histological grade 2 follicular lymphoma (Figure 2). Patient 1 has multiply relapsed follicular lymphoma, rapidly requiring therapy following this biopsy. Patient 2 presented with a right inguinal mass and stage 3A disease, though did not require therapy for 14 years from the time of this biopsy. Interestingly, while both patients harbour abundant EZH1-positive cells (approximately 60% of cells within neoplastic follicle), only patient 1 whose tumour expressed abundant EZH2 and Ki-67 had clinically active disease requiring therapy. Although this requires validation in a larger series, these cases illustrate the presence of lymphoma cells in both patients likely to be refractory to PRC2 enzymatic inhibitors in vivo. In summary, we propose that EZH1-PRC2 delineates a non-proliferating, PRC2 enzymatic inhibitor-resistant population of cells that could serve as a disease reservoir in indolent NHL. These cells are targetable by PROTAC-mediated EZH1-PRC2 degradation, with the resultant disruption reflecting the non-enzymatic roles of EZH1-PRC2. EZH1-PRC2 should be addressed as a therapeutic target to treat NHL in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction.

The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.

CRABP2 – A novel biomarker for high-risk endometrial cancer

ObjectiveInvestigate the clinical and functional implications of elevated CRABP2 expression in endometrial cancer (EC) patients. MethodsPatients were stratified into high and low CRABP2 expression groups using a decision tree classifier. Univariate and multivariate statistical analyses determined the prognostic and clinicopathological consequences of increased CRABP2 expression. A CRABP2 gene signature was generated using differential expression analysis, and analyzed using network-based approaches. The findings were validated in The Clinical Proteomic Tumor Analysis Consortium (CPTAC), a newly generated cohort of 120 endometrial tissues, and The Cancer Dependency Map (DepMap). Results60 (11%) patients in TCGA had high CRABP2 expression, whilst 468 (89%) had low expression. High expression was associated with serous EC, reduced overall survival, advanced stage and grade. Downstream retinoic acid receptors (RARG and RARA) were correlated with CRABP2 expression and were associated with worse prognosis in serous EC. The CRABP2 gene signature was enriched for Polycomb target gene sets, and was regulated by ELP3 and BMP7. BMP7 expression was increased in the CRABP2-high group, was associated with worse prognosis, and CRISPR-Cas9 screens revealed correlations in its cell-fitness score with CRABP2 following gene knockout. The opposite was true for ELP3, suggesting opposing effects from both master regulators. ConclusionsCRABP2 expression is associated with poor prognosis and advanced EC. The expression of RARA and RARG correlates with CRABP2 and are associated with worse prognosis in advanced histological subtypes. Polycomb target gene sets and two master regulators, ELP3 and BMP7, were identified as functionally relevant mechanisms driving aberrant CRABP2 expression.

Abstract A011: Emergence of persister cells following bromodomain inhibition in Ewing sarcoma

Abstract Ewing sarcoma (ES) is driven by the tumor initiating fusion EWS::FLI1 that causes widespread transcriptional dysregulation via epigenetic reprogramming. ES cells are highly plastic and heterogenous and have features of both neural crest and mesenchymal lineages. Epigenetic plasticity is well established as a mediator of disease progression and drug resistance, leading to the creation of heterogeneous tumor cell subpopulations that can initiate metastasis and relapse. Successful therapeutic strategies for metastatic or relapsed ES patients are limited and unchanged in the last three decades. Identification of new therapeutic strategies, and defining the effect of new agents on ES biology, is critical to improving patient outcomes. Given the epigenetic dependencies of ES, epigenetic modifying agents are an important class of drugs to investigate. The class of bromodomain and extra-terminal domain protein inhibitors (BETi) have shown preclinical efficacy in ES models but drug combinations will be needed. We are testing the hypothesis that transcriptional rewiring downstream of BETi will create new dependencies in ES cells that could be therapeutically exploited with biologically-defined combinations. Our studies show that the BETi, BMS-986158, the first BETi to be tested in pediatric patients, has profound cytostatic effects in preclinical ES models in vitro and in vivo. Our data further reveal that long term exposure to BMS-986158 creates a persister population of cells that reacquires proliferative capacity. We performed RNA-seq studies on parent, acutely treated (72 hours), and persister populations from three ES cell lines to define the effects of short and long term BET inhibition. Exposure to BMS-986158 led to strong inhibition of the EWS::FLI1-activated gene signature that was evident both acutely and in persister cells. Thus, despite continued inhibition of EWS::FLI1 activity, persister cells restored their ability to survive and proliferate. Gene Set Enrichment Analysis of transcripts that were significantly altered in persister cells identified gene programs involved in development and lineage commitment, including polycomb targets, suggesting that persister cells may have been rewired to more stem-like states. In addition, in two of the three cell lines, persister cells demonstrated upregulation of MYC target genes and cell cycle programs that were initially repressed by BET inhibition. Ongoing studies are investigating the molecular mechanisms by which MYC programs are reactivated in persister cells. We anticipate that epigenetic reprogramming mediates the transcriptional rewiring of MYC programs in persister cells, thereby escaping the cytostatic effects of continued BET inihbition. By defining these mechanisms we aim to identify new vulnerabilities that can be exploited in the context of BETi combination trials in the future. Citation Format: Shireen S. Ganapathi, Nicolas M. Garcia, Veerin R. Sirihorachai, Elizabeth R. Lawlor. Emergence of persister cells following bromodomain inhibition in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A011.