Polycomb Repressive Complex Research Articles

Polycomb repressive complex 2 is critical for mouse cortical glutamatergic neuron development.

The Polycomb Repressive Complex 2 (PRC2) regulates corticogenesis, yet the consequences of mutations to this epigenetic modifier in the mature brain are poorly defined. Importantly, PRC2 core genes are haploinsufficient and causative of several human neurodevelopmental disorders. To address the role of PRC2 in mature cortical structure and function, we conditionally deleted the PRC2 gene Eed from the developing mouse dorsal telencephalon. Adult homozygotes displayed smaller forebrain structures. Single-nucleus transcriptomics revealed that glutamatergic neurons were particularly affected, exhibiting dysregulated gene expression profiles, accompanied by aberrations in neuronal morphology and connectivity. Remarkably, homozygous mice performed well on challenging cognitive tasks. In contrast, while heterozygous mice did not exhibit clear anatomical or behavioral differences, they displayed dysregulation of neuronal genes and altered neuronal morphology that was strikingly different from homozygous phenotypes. Collectively, these data reveal how alterations to PRC2 function shape the mature brain and reveal a dose-specific role for PRC2 in determining glutamatergic neuron identity.

Mono-methylation of lysine 27 at histone 3 confers lifelong susceptibility to stress

Histone post-translational modifications are critical for mediating persistent alterations in gene expression. By combining unbiased proteomics profiling and genome-wide approaches, we uncovered a role for mono-methylation of lysine 27 at histone H3 (H3K27me1) in the enduring effects of stress. Specifically, mice susceptible to early life stress (ELS) or chronic social defeat stress (CSDS) displayed increased H3K27me1 enrichment in the nucleus accumbens (NAc), a key brain-reward region. Stress-induced H3K27me1 accumulation occurred at genes that control neuronal excitability and was mediated by the VEFS domain of SUZ12, a core subunit of the polycomb repressive complex-2, which controls H3K27 methylation patterns. Viral VEFS expression changed the transcriptional profile of the NAc, led to social, emotional, and cognitive abnormalities, and altered excitability and synaptic transmission of NAc D1-medium spiny neurons. Together, we describe a novel function of H3K27me1 in the brain and demonstrate its role as a "chromatin scar" that mediates lifelong stress susceptibility.

PRDM16-DT: A Brain and Astrocyte-Specific lncRNA Implicated in Alzheimer's Disease.

Astrocytes provide crucial support for neurons, contributing to synaptogenesis, synaptic maintenance, and neurotransmitter recycling. Under pathological conditions, deregulation of astrocytes contributes to neurodegenerative diseases such as Alzheimer's disease (AD), highlighting the growing interest in targeting astrocyte function to address early phases of AD pathogenesis. While most research in this field has focused on protein-coding genes, non-coding RNAs, particularly long non-coding RNAs (lncRNAs), have emerged as significant regulatory molecules. In this study, we identified the lncRNA PRDM16-DT as highly enriched in the human brain, where it is almost exclusively expressed in astrocytes. PRDM16-DT and its murine homolog, Prdm16os, are downregulated in the brains of AD patients and in AD models. In line with this, knockdown of PRDM16-DT and Prdm16os revealed its critical role in maintaining astrocyte homeostasis and supporting neuronal function by regulating genes essential for glutamate uptake, lactate release, and neuronal spine density through interactions with the RE1-Silencing Transcription factor (Rest) and Polycomb Repressive Complex 2 (PRC2). Notably, CRISPR-mediated overexpression of Prdm16os mitigated functional deficits in astrocytes induced by stimuli linked to AD pathogenesis. These findings underscore the importance of PRDM16-DT in astrocyte function and its potential as a novel therapeutic target for neurodegenerative disorders characterized by astrocyte dysfunction.

Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16.

Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16-H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A-H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.

HGG-40. UNRAVELING THE IMMUNOLOGIC VULNERABILITIES OF DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT

Abstract BACKGROUND Insights into the pathogenesis of diffuse hemispheric glioma, H3 G34-mutant (DHG-G34), an incurable high-grade glioma, have opened new therapeutic possibilities. The DHG-G34 oncohistone mutation impairs the epigenetic regulator SETD2, which normally orchestrates DNA-mismatch-repair and negatively regulates Polycomb Repressive Complex 2 (PRC2). In DHG-G34, a mismatch-repair-deficiency phenotype is predicted, but an anti-tumor immune response is notably absent, which we hypothesize is linked to the aberrant transcriptional repression by PRC2. Here, we investigate DHG-G34 immunosuppression mechanisms in a real-world multi-omics cohort and cell line model. METHODS Clinical, molecular, and immunologic characteristics of a DHG-G34 cohort, sequenced at Caris Life Sciences, are contrasted against diffuse midline glioma (DMG) and pediatric low-grade glioma (LGG). KNS-42 DHG-G34 cell line was exposed to valemetostat (a EZH1/2 inhibitor) or DMSO in-vitro. Flow cytometry and RNA-sequencing were utilized to determine differential immunologic characteristics. RESULTS 29 DHG-G34, 51 DMG, and 52 LGG were identified. DHG-G34 median OS was 15.4 mo., like DMG (HR=1.1, p=0.686). Common DHG-G34 alterations were TP53 (93.1%), ATRX (69.0%), PDGFRA (31.0%). Tumor mutational burden (TMB) was high, >=10mt/MB, in 10% of DHG-G34, with 0 DMG or LGG events (p<0.01, q>0.05), but innate and adaptive immune fractions and antigen-presenting genes were decreased (fold-change: 0.19–0.83, q<0.05). In KNS-42, PRC2 inhibition upregulated MHC class 1 (p<0.001). Of 402 missense variants, 43 were upregulated following EHZ2 inhibition, and 4 were downregulated (q<0.05). Among immunostimulatory genes, P2RX7, which engages T-cells, was downregulated in patient samples (q<0.001) and upregulated in EZH2 inhibited KNS-42 (q<0.001). CONCLUSIONS DHG-G34 has a propensity for high TMB but remains immunologically cold. Our pilot experiment suggests inhibiting PRC2’s unique role in DHG-G34 may sensitize DHG-G34 to an immune response by inducing MHC class 1 expression and transcriptional read-through of mutated transcripts. Understanding whether treatment induces TMB in DHG-G34 may affect timing of an immunotherapeutic intervention.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

EPEN-11. CHARACTERIZING EPIGENETIC MODULATIONS AND EZHIP DOWNSTREAM TARGETS IN PFA EPENDYMOMA

Abstract BACKGROUND Ependymomas can arise from all compartments of the central nervous system and ten distinct molecular groups have been identified in children and adults. In children, the most aggressive group is posterior fossa group A ependymoma (PFA). In PFA ependymomas, the aberrant overexpression of EZHIP (EZH inhibitory protein) has been implicated as a relevant driver of the disease. EZHIP suppresses the installing and spreading of the repressive epigenetic mark H3K27me3 via disturbing PRC2 (polycomb repressive complex 2) activity through inhibiting EZH2 (enhancer of zeste 2), which causes overall low levels of H3K27me3 in PFA. METHODS To get a better understanding how EZHIP may drive the disease, how it affects the epigenetic landscape and what the downstream targets of EZHIP are, we performed DNA sequencing, RNA expression profiling and ChIP-seq experiments of H3K27me3, H3K27ac, EZH2, and EZHIP in PFA, PFB, and ST-ZFTA ependymomas (n = 3 tumor cases each). RESULTS After validating the high levels of EZHIP and global reduction of H3K27me3 in PFA, we characterized the epigenetic landscape and EZHIP chromatin binding characteristics in PFA compared to PFB and ST-ZFTA ependymoma. Transcriptome analysis showed that overall more genes are up-regulated in PFA compared with PFB and ZFTA, which might be caused by its relatively high accessibility with a global loss of H3K27me3. EZHIP knockdown experiments confirmed direct changes on H3K27me3, H3K27ac and gene expression. We also found that PLAG1 (pleomorphic adenoma gene 1) expression linearly correlated with EZHIP expression in PFA tumors. ChIP-seq data indeed showed the PLAG1 locus to be bound by EZHIP and marked with increased H3K27ac and decreased H3K27me3 signals in PFA. CONCLUSIONS We identified PLAG1 as a potential downstream target of EZHIP in PFA tumors. Currently, more experiments on PLAG1 are being performed to further explore its role as a potential vulnerability in PFA ependymoma.

MDB-08. 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%, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We have generated new humanized models for G3MB by transforming neuroepithelial stem cells (NESC) derived from human induced pluripotent stem cells with TGFβ effectors 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 and MYC leading to more aggressive tumors. Moreover, NESC-derived tumors clustered with human G3/G4MB samples, indicating our models recapitulates human disease. 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 TGFβ-driven lines that are targets of the histone demethylase KDM2B and Polycomb Repressive Complex 2 (PRC2) member SUZ12. Moreover, we found that MYC is bound to regions normally regulated by PRC interactors. We postulate that chromatin 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, underscoring its significance in MB. Our studies provide important insight on identifying new therapeutic avenues with a focus on the epigenetic landscape for patients with MYC and TGFβ driven G3MB.

ETMR-19.BCOR/L1-ALTERATIONS REWIRE HISTONE REGULATION TO INDUCE ONCOGENIC PATHWAYS IN PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS

Abstract BACKGROUND Central nervous system (CNS) tumors with BCOR internal tandem duplication (ITD) or BCOR/L1 fusions are recently described tumor types with distinct molecular characteristics and poor clinical outcome. BCOR and its homologue BCORL1 are essential components of the non-canonical polycomb repressor complex (PRC) 1.1 thereby acting as epigenetic regulators exerting a profound impact on central cellular mechanisms. However, the influence of BCOR/L1-alterations on the functionality of PRC1.1 in pediatric CNS tumors remains understudied. METHODS We performed bulk as well as single-cell/nucleus RNA sequencing (scRNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) of CNS tumor tissue harboring BCOR-ITD or BCOR/L1 fusions. Additionally, BCOR/L1-altered, patient-derived cell models were used for downstream in vitro analyses. RESULTS ChIP-Seq analysis demonstrated binding of both KDM2B and mutant BCOR/L1 at PRC1.1 target genes whereas PRC1/2-mediated histone modifications H3K27me3 and H2AK119ub were absent. In addition, PRC1.1 target gene loci were characterized by high H3K27ac levels indicating increased transcriptional activity in BCOR/L1 mutant samples. Indeed, PRC1.1 targets such as FGFR1, GATA6, and ERBB3 were found to be upregulated in bulk transcriptome (n=37) analyses. Using scRNA-seq (n=10), we resolved a transcriptionally distinct cell population, specific for BCOR/L1-driven CNS tumors, which expressed high levels of PRC1.1 target genes. Besides PRC1.1 target genes, the targetable receptor tyrosine kinases PDGFR/FGFR(1/3), and IGF1R were found to be upregulated in pediatric BCOR/L1-altered CNS cancers. Accordingly, drug screens of patient-derived cell models revealed sensitivity towards small molecule inhibitors targeting the respective pathways. This translated well in anti-tumor effects of the FGFR/PDGFRA inhibitor nintedanib in BCORL1::NUTM2HP-fused patient-derived xenograft bearing mice. CONCLUSIONS Our data demonstrate for the first time that BCOR/L1-altered CNS tumors are driven by dysfunction of PRC1.1 histone modifications inducing profound transcriptomic changes ultimately activating oncogenic pathways. We further identified first feasible therapeutic targets including FGFR and PDGFR which may be leveraged for improved therapy of patients suffering from this aggressive tumor type.

ATRT-16. MAPPING THE DISORDERED METHYLOME IN ATRT

Abstract BACKGROUND Atypical Teratoid Rhabdoid Tumor (ATRT) is an aggressive pediatric brain tumor driven by inactivation of the chromatin regulator SMARCB1, with remarkably few other mutations. SMARCB1 loss leads to epigenetic dysregulation which plays a pivotal role in ATRT development and progression, and DNA methylation profiling has distinguished molecular subgroups of this disease. However, studies that provide a static view of the epigenome may not capture epigenetic variability that drives cellular plasticity and heterogeneity. Mapping epigenetic variability is crucial to understanding the phenotypic plasticity of this tumor and its ability to evade therapeutic interventions in the clinic. METHODS We performed whole genome bisulfite sequencing (WGBS) on 22 ATRT samples, 4 normal fetal brain controls, and 1 hindbrain neural stem cell control, and applied a novel analytic pipeline (InformME) to generate DNA methylation potential energy landscapes encapsulating epigenetic stochasticity. This characterizes mean methylation as well as methylation entropy. We identified key genes and regulatory regions with the most stochastic methylation signatures. RESULTS We find that ATRT exhibits a globally destabilized epigenome, with markedly elevated methylation entropy (a measure of variability/disorder). Methylation discordance mapped to key regulatory regions including bivalent promoters, quiescent chromatin regions, and enhancers, and specific genes, with an enrichment for targets of the Polycomb repressive complex 2 and regulators of pluripotency and differentiation. CONCLUSIONS The disordered methylome of ATRT could play a critical role in the tumor’s ability to evade therapies. Mapping the targets of epigenetic instability can identify targets for intervention. Moreover, strategies to constrain epigenetic variability may have therapeutic benefit by limiting the number of epigenetic states available to cancer cells.

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.

EZH2-associated tumor malignancy: A prominent target for cancer treatment.

The discussion in this review centers around the significant relationships between EZH2 and the initiation, progression, metastasis, metabolism, drug resistance, and immune regulation of cancer. Polycomb group (PcG) proteins, which encompass two primary Polycomb repressor complexes (PRC1 and PRC2), have been categorized. PRC2 consists mainly of four subunits, namely EZH2, EED, SUZ12, and RbAp46/48. As the crucial catalytic component within the PRC2 complex, EZH2 plays a pivotal role in controlling a wide range of biological processes. Overexpression/mutations of EZH2 have been detected in a wide variety of tumors. Several mechanisms of EZH regulation have been identified, including regulation EZH2 mRNA by miRNAs, LncRNAs, accessibility to DNA via DNA-binding proteins, post-translational modifications, and transcriptional regulation. EZH2 signaling triggers cancer progression and may intervene with anti-tumor immunity; therefore it has charmed attention as an effective therapeutic target in cancer therapy. Numerous nucleic acid-based therapies have been used in the modification of EZH2. In addition to gene therapy approaches, pharmaceutical compounds can be used to target the EZH2 signaling pathway in the treatment of cancer. EZH2-associated tumor cells and immune cells enhance the effects of the immune response in a variety of human malignancies. The combination of epigenetic modifying agents, such as anti-EZH2 compounds with immunotherapy, could potentially be efficacious even in the context of immunosuppressive tumors. Summary, understanding the mechanisms underlying resistance to EZH2 inhibitors may facilitate the development of novel drugs to prevent or treat relapse in treated patients.

Stat3-mediated Atg7 expression enhances anti-tumor immunity in melanoma.

Epigenetic modifications to DNA and chromatin control oncogenic and tumor suppressive mechanisms in melanoma. EZH2, the catalytic component of the Polycomb repressive complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant Ezh2 Y641F interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by EZH2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models we further investigated the role of pathways downstream of EZH2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of an Ezh2 Y641F epigenetic state. We found that expression of Atg7 is largely dependent on Stat3 expression and that deletion of Atg7 slows down melanoma cell growth in vivo, but not in vitro. Atg7 deletion also results in increased CD8+ T cells and reduced myelosuppressive cell infiltration in the tumor microenvironment, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.

Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time

Main conclusionIn Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression.CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.

A novel interplay between PRC2 and miR-3189 regulates epithelial-mesenchymal transition (EMT) via modulating COL6A2 in glioblastoma.

Recent studies have shed light on disrupted collagen signaling in Gliomas, yet the regulatory landscape remains largely unexplored. This study enquired into the role of polycomb repressive complex-2 (PRC2)-mediated H3K27me3 modification, a key epigenetic factor in glioma. Using in-house data, we identified miRNAs downregulated in glioblastoma (GBM) with the potential to regulate Collagen VI family genes. Notably, miR-3189 emerged as a prime PRC2 target. Its expression was significantly downregulated in Indian GBM patients as well as other glioma cohorts. Mechanistic insights, involving Luciferase assays, mutagenesis, and Western blot analysis, confirmed direct targeting of Collagen VI member COL6A2 by miR-3189-3p. Functional assays demonstrated that miR-3189-3p restrained GBM malignancy by inhibiting proliferation, migration, and epithelial-mesenchymal transition (EMT). Conversely, COL6A2 overexpressed in GBM patients, countered miR-3189, and promoted the malignant phenotype. Gene set enrichment analysis highlighted EMT enrichment in GBM patients with elevated COL6A2 expression, carrying prognostic implications. This study uncovers intricate interactions between two epigenetic regulators-H3K27me3 and miR-3189-working synergistically to modulate Collagen VI gene; thus, influencing the malignancy of GBM. Targeting this H3K27me3|miR-3189-3p|COL6A2 axis presents a potential therapeutic avenue against GBM.

Abstract IA023: Targeting epigenetic complexes in leukemia

Abstract Chromatin-associated multi-protein complexes, which often comprise proteins involved in epigenetic regulation, play an important role in various cancers, including leukemia. Thus, pharmacologic inhibition of proteins involved in these complexes can lead to new anti-cancer therapies. We have focused on therapeutic targeting of various chromatin-bound multi-protein complexes, including Mixed Lineage Leukemia 1 (MLL1) and Polycomb Repressive Complex 1 (PRC1) complexes, as a novel treatment for leukemia. These efforts led to the development of small molecule inhibitors targeting the menin-MLL1 interaction, which were translated to leukemia patients, demonstrating promising efficacy in Acute Myeloid Leukemia (AML) with NPM1 mutations or MLL1 translocations. However, clinical studies revealed resistance to the single agent menin inhibitor in a sub-set of AML patients, suggesting that combinatorial treatments or new generations of menin inhibitors might be required to overcome resistance. Our recent efforts to address resistance to menin inhibitors will be presented, including combinatorial studies with other targeted agents (e.g. kinases inhibitors), which resulted in synergistic effects. Furthermore, discovery of a new generation of menin inhibitors with promising activity against menin patient mutations will also be presented. Citation Format: Jolanta Grembecka. Targeting epigenetic complexes in leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA023.

Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer

BackgroundPlatinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers.MethodsProtein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing.ResultsCD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2.ConclusionsFor the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Proximal termination generates a transcriptional state that determines the rate of establishment of Polycomb silencing

The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.

Unraveling the immunologic vulnerabilities of diffuse hemispheric glioma, H3 G34-mutant.

2082 Background: Recent insights into the molecular basis of diffuse hemispheric glioma, H3 G34-mutant (DHG-G34), an incurable high-grade glioma, have opened new therapeutic possibilities. The oncohistone mutation in DHG-G34 impairs the epigenetic regulator SETD2, which normally orchestrates DNA-mismatch-repair and negatively regulates the transcriptional silencing activity of the Polycomb Repressive Complex 2 (PRC2). In DHG-G34, a mismatch-repair-deficiency phenotype is predicted, but an anti-tumor immune response is notably absent in clinical observations, which we posit is linked to the aberrant transcriptional repression by PRC2. Here, we investigate DHG-G34 immunosuppression mechanisms in a large real-world multi-omics cohort and human cell lines. Methods: Clinical, molecular, and immunologic characteristics of a DHG-G34 cohort, sequenced (DNA and RNA) at Caris Life Sciences (Phoenix, AZ), are contrasted against diffuse midline glioma (DMG) and pediatric low-grade glioma (LGG). A significant difference between genomic subgroups was defined as fold-change > 1.2. Statistical significance was determined using chi-square, Fishers-exact, and Mann Whitney U tests with corrections for multiple hypothesis testing (q < 0.05).Hazard ratio (HR) was calculated using the Cox proportional hazards model. KNS-42 DHG-G34 cell line was exposed in triplicate to 1 µM valemetostat (an EZH1/2 inhibitor) or DMSO in vitro. Flow cytometry and RNA-sequencing were utilized to determine differential immunologic characteristics. Results: 29 DHG-G34, 51 DMG, and 52 LGG were identified in Caris database. Median OS among DHG-G34 was 15.4 mo., similar to DMG (HR = 1.1, 95% CI: 0.70-1.73, p = 0.686). Most frequent alterations in DHG-G34 were TP53 (93.1%), ATRX (69.0%), PDGFRA (31.0%). Tumor mutational burden (TMB) was high, >=10mt/MB, in 10% of DHG-G34 and 0/51 in DMG and 0/52 LGG (p < 0.01, q > 0.05), but immune fractions including dendritic cells, T cells, macrophages, microglia, and antigen-presenting genes were decreased (fold-change: 0.19 – 0.83, q < 0.05). In KNS-42, PRC2 inhibition upregulated MHC class 1 (p < 0.001). Of 402 KNS-42 missense variants, transcription of 43 were upregulated following EHZ2 inhibition, and 4 were downregulated (q < 0.05). Among immunostimulatory genes, P2RX7, which engages T-cells and stimulates an inflammatory response, was downregulated in patient samples (q < 0.001) and upregulated in KNS-42 following EZH2 inhibition (q < 0.001). Conclusions: DHG-G34 has an increased propensity for high TMB but remains immunologically cold. Our pilot experiment suggests inhibiting PRC2’s unique role in DHG-G34 may sensitize DHG-G34 to an immune response by inducing MHC class 1 expression and transcriptional readthrough of mutated transcripts. Understanding whether treatment induces TMB in DHG-G34 may affect timing of an immunotherapeutic intervention.

Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis

Spatiotemporal regulation of gene expression by polycomb repressive complex 2 (PRC2) is critical for animal and plant development. The Arabidopsis fertilization independent seed (FIS)-PRC2 complex functions specifically during plant reproduction from gametogenesis to seed development. After a double fertilization event, triploid endosperm proliferates early, followed by the growth of a diploid embryo, which replaces the endosperm in Arabidopsis and many dicots. Key genes critical for endosperm proliferation such as IKU2 and MINI3 are activated after fertilization. Here we report that two MADS-box AGAMOUS-LIKE (AGL) proteins associate with the key endosperm proliferation loci and recruit the FIS-PRC2 repressive complex at 4–5 days after pollination (DAP). Interestingly, AGL9 and AGL15 only accumulate toward the end of endosperm proliferation at 4–5 DAP and promote the deposition of H3K27me3 marks at key endosperm proliferation loci. Disruption of AGL9 and AGL15 or overexpression of AGL9 or AGL15 significantly influence endosperm proliferation and cellularization. Genome-wide analysis with cleavage Under Targets and tagmentation (CUT&Tag) sequencing and RNA sequencing revealed the landscape of endosperm H3K27me3 marks and gene expression profiles in Col-0 and agl9 agl15. CUT&Tag qPCR also demonstrated the occupancy of the two MADS-box proteins and FIS-PRC2 on a few representative target loci. Our studies suggest that MADS-box proteins could potentially recruit PRC2 to regulate many other developmental processes in plants or even in fungi and animals.