Polycomb Repressive Complex 2 Genes 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.

Exploring the Association Between PRC2 Genes Variants and Lung Cancer Risk in Chinese Han Population.

Genetic susceptibilities play a large role in the pathogenesis of lung cancer (LC). The polycomb repressive complex 2 (PRC2) is a conserved chromatin-associated complex that represses gene expression and is crucial for proper organismal development and gene expression patterns. Despite PRC2 dysregulation has been observed in various human cancers, the relationship between PRC2 genes variants and lung cancer risk remains largely unexplored. To investigate the association between single nucleotide polymorphisms (SNPs) in PRC2 genes and the risk of developing LC, we genotyped blood genomic DNA from 270 LC patients and 452 healthy individuals of Chinese Han ethnicity using the TaqMan™ genotyping technique. We found that rs17171119T>G(adjusted odds ratio (OR) = 0.662, 95% CI: 0.467-0.938, P < 0.05), rs10898459 T>C(adjusted OR = 0.615, 95% CI: 0.4-0.947, P < 0.05), and rs1136258 C>T(adjusted OR = 0.273, 95% CI: 0.186-0.401, P < 0.001) were significantly associated with a reduced risk of LC. Stratified analysis revealed a protective effect of rs17171119 in both male and female patients, specifically those with lung adenocarcinoma (LUAD). Additionally, rs1391221 showed a protective effect in both the LUAD and lung squamous cell carcinoma (LUSC) groups, while rs1136258 exhibited a protective effect in both females and males, as well as in both LUAD and LUSC groups. Furthermore, analysis of The Cancer Genome Atlas (TCGA) dataset revealed expression levels of EED and RBBP4 in both LUAD and LUSC. This study provides evidence that allelic variants in EZH2, EED, and RBBP4 may act as protective factors against LC development and could serve as genetic markers associated with susceptibility to LC.

Genomic Patterns of Malignant Peripheral Nerve Sheath Tumor (MPNST) Evolution Correlate with Clinical Outcome and Are Detectable in Cell-Free DNA.

MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups. This article is highlighted in the In This Issue feature, p. 517.

Abstract 1614: Creation of malignant peripheral nerve sheath tumor models deficient for polycomb repressive complex 2 and identification of therapeutic vulnerabilities

Abstract We constructed models of nerve sheath tumors that arise in Neurofibromatosis Type 1 (NF1) patients and conducted therapeutics discovery using synthetic lethal pharmacogenomic screens. Given both plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) arise within the Schwann cell lineage, we developed a drug discovery pipeline to identify targeted therapeutics for treating NF1-related neoplasia. Using CRISPR/Cas9, immortalized human Schwann cell lines were created lacking the NF1 gene or NF1 and components of Polycomb Repressive Complex 2 (PRC2), such as SUZ12. ~80% of all MPNST harbor loss of function mutations in PRC2 genes, which is highly suggestive that perturbation of epigenetic homeostasis plays a role in malignant transformation of neurofibromas. Our models mimic the genetics of MPNSTs and allowed us to identify selectively lethal drugs which exploit vulnerabilities specific to these genetic drivers. We describe the identification of these novel drugs, extensive in vivo testing in models of MPNST, and mechanistic studies to explain why they act specifically on MPNSTs. We identified compounds showing selective lethality towards NF1/SUZ12 double mutant cells. These include drugs affecting epigenetic homeostasis, such as HDAC inhibitors (HDACi). Moreover, many of these drugs showed strong synergy when tested in combination with a MEK inhibitor (MEKi) against NF1/SUZ12 deficient human Schwann and MPNST cell lines. We investigated the mechanism of this synergy using genetics, transcriptome, and proteome analysis. Interestingly, these mechanistic studies revealed possible emerging avenues of resistance the cells are utilizing to compensate for and survive therapeutic intervention. For instance, proteomics analysis of PRC2 deficient MPNST cell lines treated with combination therapy of MEKi and HDACi indicates activation of specific survival and stress response pathways. Targeting these pathways in conjunction with the therapeutics we have identified could prevent emergence of resistance and tumor escape. Clinically interesting drug candidates were advanced and tested as single agents and in combination in multiple in vivo models of MPNST (including patient derived and cell line xenografts). The FDA approved drugs vorinostat (HDACi) and selumetinib (MEKi) have shown dramatic efficacy, with combination therapy exhibiting strong synergy in vivo. This includes durable responses in survival studies and the ability to dramatically shrink established tumors, with treated tumors showing markers of apoptosis. The discovery of novel agents effective against several models of MPNST is exciting as there are currently no approved targeted therapies for MPNSTs. Our results implicate targeting of epigenetic homeostasis, in combination with MEKi, as a major vulnerability of MPNSTs deficient for PRC2 activity. Citation Format: Kyle B. Williams, Alex Larsson, Justin Tibbits, Christopher L. Moertel, David A. Largaespada. Creation of malignant peripheral nerve sheath tumor models deficient for polycomb repressive complex 2 and identification of therapeutic vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1614.

JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain gene repression and is essential for cell differentiation. In low-grade endometrial stromal sarcoma (LG-ESS), the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. We show that JAZF1-SUZ12 dysregulates PRC2 composition, genome occupancy, histone modification, gene expression, and cell differentiation. Loss of the SUZ12N terminus in the fusion protein abrogates interaction with specific PRC2 accessory factors, reduces occupancy at PRC2 target genes, and diminishes H3K27me3. Fusion to JAZF1 increases H4Kac at PRC2 target genes and triggers recruitment to JAZF1 binding sites during cell differentiation. In human endometrial stromal cells, JAZF1-SUZ12 upregulated PRC2 target genes normally activated during decidualization while repressing genes associated with immune clearance, and JAZF1-SUZ12-induced genes were also overexpressed in LG-ESS. These results reveal defects in chromatin regulation, gene expression, and cell differentiation caused by JAZF1-SUZ12 that may underlie its role in oncogenesis.

Polycomb repressive complex 2 mutations predict survival benefit in advanced cancer patients treated with immune checkpoint inhibitors.

BackgroundNumerous biomarkers are being tested to enhance the ability of clinicians to predict responses and prognosis after treatment with immune checkpoint inhibitors (ICIs). Polycomb repressive complex 2 (PRC2) is a histone methyltransferase family that plays a major role in chromatin silencing. Preclinical evidence implicates PRC2 components such as enhancer of zeste homolog 2 (EZH2) in immune resistance. This study aimed to assess the clinical relevance of PRC2 mutations in the clinical outcome of ICI-treated patients.Materials and methodsNext-generation sequencing (NGS) data from tumor samples of patients treated with ICIs (anti-PD-1/PD-L1, anti-CTLA-4 or both) were interrogated for alterations in PRC2-related genes. The Kaplan–Meier method was used to assess the association between altered and unaltered PRC2-related genes with overall survival.ResultsSomatic NGS data from 1662 advanced-stage, ICI-treated patients with various primaries (lung, melanoma, bladder, kidney, head neck, esophagogastric, glioma, colorectal, breast, unknown primary) were examined. Seventy patients (4%) harbored truncating or missense mutations or fusions in EZH2 (2.4%), EZH1 (1.2%), SUZ12 (0.9%) or EED (0.7%) genes. Patients carrying alterations in PRC2 genes had significantly longer median overall survival (44 months) compared with those with unaltered tumors (18 months, log-rank P=0.0174). These findings were validated in two additional cohorts of patients (n=313) with various primaries (melanoma, lung, bladder, head neck, anal, sarcoma) who were treated with ICIs.ConclusionsInactivating mutations in the PRC2 chromatin silencing machinery, although rare, may predict favorable outcomes in ICI-treated patients with metastatic cancers. This warrants prospective confirmation, and suggests that epigenetic regulators could serve as surrogate markers to guide ICI treatment decisions.

Loss of PRC2 Enforces a Mesenchymal Neural Crest Stem Cell Phenotype in NF1-Deficient Cancer Through Activation of Core Transcription Factors

Genetic alterations that result in the dysregulation of polycomb repressive complex 2 (PRC2) are common features of multiple cancer types. Loss-of-function mutations in the PRC2 genes, SUZ12 or EED, occur at high frequency in malignant peripheral nerve sheath tumors (MPNST), a clinically aggressive sarcoma that arises from NF1-deficient Schwann cells. To define the oncogenic mechanisms mediated by PRC2 loss, we used engineered MPNST model systems that dynamically reassemble a competent PRC2 coupled with single cell sequencing from clinical samples to evaluate the transcriptomic and epigenetic consequences of PRC2 loss. We discovered a two-pronged oncogenic process; first, PRC2 loss leads to remodeling of bivalent chromatin and the enhancer landscape of the tumor cell. These epigenetic changes result in the upregulation of developmentally regulated transcription factors, including FOXC1, which enforce a transcriptional circuit that is a core vulnerability of the cell. Second, PRC2 loss reduces type I interferon signaling and antigen presentation as a downstream consequence of hyperactivated Ras signaling and its crosstalk with STAT and IRF transcription factors. Mapping of the transcriptional program of these PRC2-deficient tumor cells onto a constructed developmental trajectory of the developing neural crest stem cell revealed that changes induced by PRC2 loss enforced a cellular profile characteristic of a primitive mesenchymal neural crest stem cell.

Ezh2 loss propagates hypermethylation at T cell differentiation-regulating genes to promote leukemic transformation.

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a new pathological entity with poor outcomes in T cell ALL (T-ALL) that is characterized by a high incidence of loss-of-function mutations in polycomb repressive complex 2 (PRC2) genes. We generated a mouse model of ETP-ALL by deleting Ezh2, one of the PRC2 genes, in p53-null hematopoietic cells. The loss of Ezh2 in p53-null hematopoietic cells impeded the differentiation of ETPs and eventually induced ETP-ALL-like disease in mice, indicating that PRC2 functions as a bona fide tumor suppressor in ETPs. A large portion of PRC2 target genes acquired DNA hypermethylation of their promoters following reductions in H3K27me3 levels upon the loss of Ezh2, which included pivotal T cell differentiation-regulating genes. The reactivation of a set of regulators by a DNA-demethylating agent, but not the transduction of single regulator genes, effectively induced the differentiation of ETP-ALL cells. Thus, PRC2 protects key T cell developmental regulators from DNA hypermethylation in order to keep them primed for activation upon subsequent differentiation phases, while its insufficiency predisposes ETPs to leukemic transformation. These results revealed a previously unrecognized epigenetic switch in response to PRC2 dysfunction and provide the basis for specific rational epigenetic therapy for ETP-ALL with PRC2 insufficiency.

Molecular Mechanism behind the Synergistic Activity of Proteasome Inhibition and PRC2 Inhibition in the Treatment of Multiple Myeloma

Proteasome inhibitors (PIs) such as bortezomib and carfilzomib play a central role in the treatment of multiple myeloma (MM). However, the almost inevitable resistance to PIs necessitates the search for novel strategies to improve patient outcome. The methyltransferase EZH2 and its homolog EZH1 are components of polycomb repressive complex 2 (PRC2), inducing H3K27me3 and repressing the transcription of target genes. Recent studies have linked EZH2 to tumorigenesis including MM. In this study, we investigated the molecular mechanism of PRC2 inhibition as a partner of PIs for the treatment of MM.We first examined the impact of proteasome inhibition on EZH2. Bortezomib as well as carfilzomib remarkably decreased EZH2 protein, and downregulated its mRNA in dose- and time-dependent manners. As EZH2 is a downstream target of E2F1, the effects of bortezomib on RB-E2F pathway were investigated. Bortezomib downregulated E2F1 protein and mRNA with notable decrease of phosphorylated RB protein, due to accumulation of cyclin-dependent kinase inhibitors such as p21 and p27. ChIP assay revealed that bortezomib significantly inhibited the binding of E2F1 to EZH2 promoter, and E2F1 overexpression resulted in upregulation of EZH2 in MM cells. These data suggest that bortezomib transcriptionally downregulates EZH2 via modulating RB-E2F pathway.Next we used lentiviral vectors to overexpress EZH2 in RPMI8226 cells and observed diminished sensitivity to bortezomib in EZH2-overexpressing cells compared to cells transduced with an empty vector. Remarkably, the combined treatment of bortezomib and UNC1999, a dual inhibitor of EZH2 and EZH1, restored the sensitivity of MM cells to bortezomib. Notably, UNC1999 enhanced the cytotoxicity induced by bortezomib in vitro and in vivo partly through enhanced apoptosis. Carfilzomib also demonstrated strong synergy with UNC1999 in vitro, suggesting broad application of this strategy.To characterize the mechanism of action of PRC2 inhibition alone and in combination with proteasome inhibition, we performed RNA sequencing (RNA-seq) of MM.1S cells treated with UNC1999, bortezomib or the combination of both agents versus DMSO-treated cells and chromatin immunoprecipitation sequencing (ChIP-seq) for H3K27me3 of UNC1999 versus DMSO-treated MM.1S cells. Importantly, we identified the direct targets of UNC1999 as those with significantly enhanced expression (>1.5 fold UNC1999/Control) and remarkable reduction of H3K27me3 (≥ 2-fold). These genes included NR4A1, EGR1 and LTB. EGR1 and LTB are known tumor suppressor candidates in MM, while NR4A1 is implicated in myeloid and lymphoid malignancies. Upregulation of NR4A1 and reduction of H3K27me3 at the NR4A1promotor were confirmed using manual RT-PCR and ChIP, respectively. Notably, overexpression of NR4A1 significantly inhibited the growth of MM cells, suggesting a tumor suppressive role for NR4A1 in MM. Notably, MYC (c-Myc), a major contributor to the pathogenesis of MM, was greatly downregulated in NR4A1-overexpressing cells. MYC is reportedly a direct target of NR4A1 that suppresses its expression. We found that UNC1999 downregulated MYC mRNA and protein. Moreover, the combination of UNC1999 and bortezomib remarkably suppressed MYC-related gene sets.Gene set enrichment analysis (GSEA) showed that while PRC2 genes were positively enriched in UNC1999- and combination-treated cells, they were not significantly enriched in bortezomib-treated cells. In addition, although bortezomib downregulated EZH2, EZH1 and H3K27me3 mark were not affected in bortezomib-treated cells. This suggested that inhibition of EZH2 alone is not enough to completely suppress PRC2 function. Therefore, we compared the combination of bortezomib and UNC1999 with that of bortezomib and a specific EZH2 inhibitor, GSK126. UNC1999 induced much better synergistic activity with bortezomib than GSK126 as evidenced by the combination index, associated with further reduction of the levels of H3K27me3. This underlines the importance of dual inhibition of EZH2 and EZH1 to fully block PRC2 activity.In conclusion, our findings demonstrate that the combination of dual inhibition of EZH2 and EZH1 together with proteasome inhibition cooperatively blocks PRC2 function, resulting in derepression of tumor suppressors such as NR4A1 and inhibition of MYC. Thus, this combination is a promising new therapeutic option for the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.

Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.

Leukemia-Associated MLL-AF10 Fusion Maintains an Active State of Chromatin on Its Target Genes By Recruiting Tip60

Chromosome translocation involving the mixed lineage leukemia (MLL) gene which generates an in-frame fusion gene of the MLL 5′-region and partner genes, is a common rearrangement in acute myeloid and lymphoid leukemia that is associated with poor prognosis. Knock-in and retroviral transduction studies show that MLL-fusion results in constitutive activation of the transcription of target genes such as Hoxa9 and Meis1 during the development of leukemia. Recent studies show that several transcription regulators, such as Dot1L, Cbx8, PAF1, and AEP/EAP complexes, are required for the leukemogenic activity of MLL-fusion; however, the underlying mechanisms remain elusive. To clarify the mechanism of epigenetic regulation by MLL-fusions, we established a novel leukemia model by generating a conditional MLL-AF10 fusion gene, MLL-AF10 flox, in which the 3′-AF10 region is deleted by 4-OHT-activated Cre-ERT recombinase, resulting in inactivation of MLL-AF10 flox (Figure 1A). Mouse hematopoietic stem/progenitor cells (c-kit+) were immortalized by retroviral transduction of MLL-AF10 flox and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. Treatment of MLL-AF10 flox cells with 4-OHT in vitro to inactivate MLL-AF10 flox downregulated Hoxa9 expression and markedly decreased colony-forming ability. In addition, the inactivation of MLL-AF10 flox rapidly decreased the acetylation level of the histone H2A variant H2A.Z on the Hoxa9 locus. These results suggest that MLL-AF10, possibly together with a histone acetyltransferase (HAT), regulates the acetylation of H2A.Z on the Hoxa9 locus. To identify the HAT responsible for H2A.Z acetylation induced by MLL-AF10, protein complexes associated with H2A.Z-containing nucleosomes were purified, resulting in the identification of Tip60, a MYST-type HAT in a complex with H2A.Z. MLL-AF10 physically interacted with Tip60 via the AF10 C-terminal portion of MLL-AF10 (Figure 1B). ChIP analysis showed that MLL-AF10 and Tip60 co-localize on the Hoxa9 locus in MLL-AF10-transformed cells (MLL-AF10 cells). Furthermore, conditional deletion of Tip60 in MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) cells dramatically downregulated Hoxa9 expression and resulted in the accumulation of unacetylated H2A.Z on the Hoxa9 locus. Consistent with these data, in vitro acetylation analysis showed that Tip60 directly acetylates H2A.Z. To assess the role of Tip60 in leukemia development in vivo, MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) leukemia cells were injected into recipient mice. Animals receiving intraperitoneal injection of tamoxifen to delete Tip60 failed to develop MLL-AF10 leukemia (Figure 1C). These data indicate that Tip60 is required for the development of MLL-AF10 leukemia and suggest that MLL-AF10 recruits Tip60 to acetylate H2A.Z on the Hoxa9 locus. The effect of H2A.Z acetylation on Hoxa9 expression was examined by purifying nucleosomes containing acetylation-deficient 3KR H2A.Z (which mimics unacetylated H2A.Z), in which lysines 4, 7, and 11 were substituted by arginine. 3KR H2A.Z preferentially formed nucleosomes with histone H3 trimethylation at lysine 27, which is catalyzed by polycomb repressive complex 2 (PRC2). This finding suggests that nucleosomes including unacetylated H2A.Z are the preferential targets of PRC2. Loss of Tip60 in MLL-AF10 cells resulted in decreased levels of acetylated H2A.Z on the Hoxa9 locus and the recruitment of Ezh2 (a catalytic subunit of PRC2) and increased histone H3 K27 trimethylation. Taken together, these data indicate that Tip60 is a critical factor in the development of MLL-AF10 leukemia. MLL-AF10 may maintain an active chromatin state on its target genesby recruiting Tip60, which acetylates H2A.Z to prevent PRC2 recruitment and gene silencing. On the other hand, unacetylated H2A.Z may be a signal for PRC2 recruitment, which would be induced as a result of Tip60 loss or inactivation of MLL-AF10. [Display omitted] DisclosuresKitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

An Epigenetic Role for Disrupted Paternal Gene Expression in Postzygotic Seed Abortion in Arabidopsis Interspecific Hybrids

Interspecific hybrids often increase the levels of heterozygosity and hybrid vigor, but some interspecific hybrid seeds are aborted shortly after fertilization. The mechanism behind this postzygotic seed abortion is poorly understood. Here, we report genome-wide analysis of allelic expression changes in developing siliques and seeds in three F1 interspecific crosses between Arabidopsis thaliana (Col, Ler, or C24) and Arabidopsis arenosa. The majority of maternally expressed genes (MEGs) were shared among all three F1 interspecific crosses, whereas ∼90% of 272 paternally expressed genes (PEGs) were found only in one or two F1 crosses, suggesting a role for disrupted paternal gene expression in seed abortion that varies in different crosses. Consistent with this notion, 12 PEGs in the infertile interspecific hybrids matched MEGs in fertile intraspecific hybrids. This disruption of PEGs in the interspecific hybrids was consistent with the upregulation of the genes in the paternal-excess interploidy cross (2X6) between a diploid mother and a hexaploid father, leading to the seed abortion. Moreover, a subset of PEGs in the interspecific crosses were also upregulated in the intraspecific hybrid met1XWT or meaXWT, in which the mutant of MET1 (DNA METHYLTRANSFERASE1) or MEDEA, a Polycomb Repressive Complex2 gene, was used as the maternal parent. These data suggest that maternal epigenetic factors and paternal gene expression play important roles in the postzygotic seed abortion in interspecific hybrids or neo-allopolyploids.

Possible roles for polycomb repressive complex 2 in cereal endosperm.

The polycomb repressive complex 2 (PRC2) is an evolutionarily conserved multimeric protein complex in both plants and animals. In contrast to animals, plants have evolved a range of different components of PRC2 and form diverse complexes that act in the control of key regulatory genes at many stages of development during the life cycle. A number of studies, particularly in the model species Arabidopsis thaliana, have highlighted the role of PRC2 and of epigenetic controls via parent-of-origin specific gene expression for endosperm development. However, recent research in cereal plants has revealed that although some components of PRC2 show evolutionary conservation with respect to parent-of-origin specific gene expression patterns, the identity of the imprinted genes encoding PRC2 components is not conserved. This disparity may reflect the facts that cereal plant genomes have undergone different patterns of duplication during evolution compared to A. thaliana and that the endosperm development program is not identical in monocots and eudicots. In this context, we focus this review on the expression of imprinted PRC2 genes and their roles in endosperm development in cereals.

Concurrent Loss Of Ezh2 and Tet2 Cooperates In The Pathogenesis Of Myelodysplastic Disorders,

Inactivating somatic mutations in polycomb-group (PcG) genes such as EZH2 and ASXL1occur frequently in patients with myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN overlap disorders. While these mutations suggest a tumor suppressor function of polycomb repressive complex 2 (PRC2)-related genes in these diseases, both the impact of each PcG mutation and its interplay with coinciding mutations remain largely unknown. To understand the contribution of inactivating PcG mutations to the development of myeloid malignancies, genomic DNA from 119 patients with MDS and related neoplasms were analyzed for mutations in EZH2, ASXL1 and TET2 by high-throughput sequencing. Inactivating mutations in EZH2 and ASXL1 were detected in 8.4 and 16.8 % of patients, respectively. Moreover, 3.4 % of patients had deletion of EZH2 (located at 7q36) associated with -7 and 7q- chromosomal abnormalities. Notably, 57.1 % of these EZH2 mutations coexisted with TET2 mutations. Conversely, 34.8 % of patients with TET2 mutations had coexisting EZH2mutations. In order to understand the impact of inactivating EZH2 mutations and concurrent EZH2 and TET2 mutations on hematopoiesis, we crossed Cre-ERT;Ezh2fl/fl mice and Tet2 gene trap mice (Tet2KD/KD). Due to the early time of death in Tet2KD/KD mice and a necessity to exclude the influence of the loss of Tet2 and Ezh2 in BM niche cells, we transplanted E14.5 fetal liver cells from Cre-ERT control (WT), Cre-ERT;Tet2KD/KD, Cre-ERT;Ezh2fl/fl and Cre-ERT;Tet2KD/KDEzh2fl/fl CD45.2 mice into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen at 4 weeks post-transplantation. During a long observation period, we found that Ezh2Δ/Δ mice developed MDS/MPN and half of the mice died by 10 months post-transplantation. They showed myeloproliferative features characterized by extramedullary hematopoiesis in the spleen as evident from splenomegaly with a marked increase in LSK cells. They were anemic and showed increased apoptosis in Ter119highCD71high erythroblasts in the BM, suggesting ineffective erythropoiesis, a feature compatible with myelodysplastic disorders. Ezh2Δ/Δ mice also showed dysplasia of myeloid cells, including a pseudo Pelger-Huët anomaly. To our surprise, concurrent deletion of Tet2 and Ezh2 significantly shortened the latency of disease development of not only MDS/MPN but also MDS, and all of the compound mice died of pneumonia by 10 months. Tet2KD/KDEzh2Δ/Δ MDS/MPN mice showed myeloproliferative features, including monocytosis and/or splenomegaly with extramedullary hematopoiesis. In contrast, Tet2KD/KDEzh2Δ/Δ MDS mice did not show obvious myeloproliferative features, but showed a trend of pancytopenia. The proportion of Annexin V+ cells in CD71highTer119high erythroblasts was significantly higher in both MDS/MPN and MDS mice compared to their WT counterparts, implicating enhanced apoptosis as a cause of anemia. Furthermore, myeloid dysplasia was more pronounced in these mice compared to Ezh2Δ/Δmice. Gene set enrichment analysis with microarray data showed that the Myc module was significantly enriched in Ezh2Δ/Δ LSK cells and became highly enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of MDS/MPN and MDS in Tet2KD/KDEzh2Δ/Δ mice. As expected, all of the PRC2 gene sets (Ezh2 targets and Ezh1 targets) showed a trend of positive enrichment in Ezh2Δ/Δ and Tet2KD/KDEzh2Δ/Δ LSK cells. Notably, however, Ezh1 targets became negatively enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of myelodysplastic disorders. ChIP-seq and microarray analysis data showed that upon deletion of Ezh2, a series of potential PcG related target oncogenes, such as Hmga2 and Pbx3, became derepressed in LSK cells. On the other hand, key developmental regulator genes, such as genes encoding homeobox, paired-box, T-box, forkhead and Gata family transcription factors and zinc finger DNA-binding proteins, were kept transcriptionally repressed by the compensatory action of Ezh1. Our findings provide the first evidence of the tumor suppressor function of EZH2 and demonstrate the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders. These two models represent novel, genetically accurate models of myelodysplastic disorders amenable to epigenomic as well as preclinical therapeutic studies. Disclosures: No relevant conflicts of interest to declare.

Abstract IA08: Targeting histone methylation in leukemia

Abstract Leukemias harboring rearrangements of the MLL gene carry a poor prognosis. Over the past 6 years, it has become increasingly clear that fusions of MLL induce widespread epigenetic deregulation that may mediate much of their transforming activity. The histone methyltransferase DOT1L, which methylates histone 3 on lysine 79 (H3K79), has received particular attention. Genome-wide H3K79 methylation profiles in MLL-rearranged leukemias are abnormal, and can serve to distinguish MLL-rearranged from other types of leukemias. Loss of H3K79 methylation affects expression of MLL-target loci and is detrimental to the leukemogenic activity of MLL-rearranged cells, suggesting that a DOT1L dependent, aberrant epigenetic program drives transformation in these leukemias. Small molecule DOT1L inhibitors have been developed and selectively inhibit proliferation of MLL-rearranged leukemia cells. These molecules are now in clinical trials for patients with relapsed refractory leukemia. Also, the Polycomb Repressive Complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers, but its role in cancer development and progression remains unclear. We have used conditional alleles for the PRC2 components Enhancer of Zeste 2 (Ezh2) and Embryonic Ectoderm Development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared to wildtype leukemia, Ezh2-null MLL-AF9-mediated acute myeloid leukemia (AML) show decreased organ infiltration and failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML. In contrast, inactivation of the essential PRC2 gene, Eed led to complete ablation of PRC2-function, which is incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared to Ezh2-null leukemic cells, including downregulation of Myc target genes and upregulation of PRC2 targets. These data show that histone-modifying enzymes play critical roles in leukemia and may be relevant therapeutic targets in these diseases. Citation Format: Scott A. Armstrong. Targeting histone methylation in leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA08.

Polycomb repressive complex 2 is required for MLL-AF9 leukemia

A growing body of data suggests the importance of epigenetic mechanisms in cancer. Polycomb repressive complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers. However, its role in cancer development and progression remains unclear. We used conditional alleles for the PRC2 components enhancer of zeste 2 (Ezh2) and embryonic ectoderm development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared with wild-type leukemia, Ezh2-null MLL-AF9-mediated acute myeloid leukemia (AML) failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML, but plays a role in leukemia progression. Genome-wide analyses of PRC2-mediated trimethylation of histone 3 demonstrated locus-specific persistence of H3K27me3 despite inactivation of Ezh2, suggesting partial compensation by Ezh1. In contrast, inactivation of the essential PRC2 gene, Eed, led to complete ablation of PRC2 function, which was incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared with Ezh2-null leukemic cells, including down-regulation of Myc target genes and up-regulation of PRC2 targets. Manipulating PRC2 function may be of therapeutic benefit in AML.

Myc Regulates the Transcription of the PRC2 Gene To Control the Expression of Developmental Genes in Embryonic Stem Cells

Myc family members are critical to maintain embryonic stem cells (ESC) in the undifferentiated state. However, the mechanism by which they perform this task has not yet been elucidated. Here we show that Myc directly upregulates the transcription of all core components of the Polycomb repressive complex 2 (PRC2) as well as the ESC-specific PRC2-associated factors. By expressing Myc protein fused with the estrogen receptor (Myc-ER) in fibroblasts, we observed that Myc, binding to the regulatory elements of Suz12, Ezh2, and Eed, induces the acetylation of histones H3 and H4 and the recruitment of elongating RNA polymerase II at their promoters. The silencing of both c-Myc and N-Myc in ESC results in reduced expression of PRC2 and H3K27me3 at Polycomb target developmental regulators and upregulation of genes involved in primitive endoderm differentiation. The ectopic expression of PRC2 in ESC, either silenced for c-Myc and N-Myc or induced to differentiate by leukemia inhibitory factor (LIF) withdrawal, is sufficient to maintain the H3K27me3 mark at genes with bivalent histone modifications and keep repressed the genes involved in ESC differentiation. Thus, Myc proteins control the expression of developmental regulators via the upregulation of the Polycomb PRC2 complex.

Abstract 4707: Discovery and characterization of PCAT-1, a novel lincRNA implicated in prostate cancer tumorigenesis

Abstract High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel disease-specific markers for clinical uses and uncharacterized aspects of tumor biology, particularly non-coding RNA (ncRNA) species. To illustrate this approach, we employed RNA-Seq on a cohort of 102 prostate tissues and cells lines. We found that aberrant expression profiles of novel tissue-specific ncRNAs distinguished benign, cancerous, and metastatic tumors, and we defined a core set of 121 novel ncRNAs whose dysregulation characterizes prostate cancer. Among these, a novel prostate-cancer specific ncRNA (termed PCAT-1) defined a subset of aggressive cancers with low expression of the epigenetic regulator EZH2, a component of the Polycomb Repressive Complex 2 (PRC2) commonly upregulated in metastatic cancers. In vitro chromatin immunoprecipitation, RNA immunoprecipitation, and drug treatment assays for core PRC2 genes indicated that the PRC2 complex directly binds and represses PCAT-1, and that PCAT-1 transcript reciprocally binds PRC2. By contrast, in vitro models with high levels of endogenous PCAT-1 transcript did not recapitulate PRC2-mediated repression, and in these cells siRNA-mediated knockdown of PCAT-1 showed a 25 – 50% decrease in cell proliferation. Using gene expression arrays, we determined that PCAT-1 contributes to the transcriptional regulation of genes in several key biological processes, including cell cycle. These data suggest that PCAT-1 exhibits two biological states: a PRC2-repressed state and an active state that promotes proliferation. Next, we showed that novel ncRNAs may serve a clinical purpose for the non-invasive detection and stratification of prostate cancer patients. We performed qPCR on patient urine samples (n=108) and found that a custom ncRNA expression signature, which includes PCAT-1, both diagnosed prostate cancer effectively and yielded prognostic information. Indeed, a high ncRNA expression signature value correlated with high-grade histology (Gleason score &amp;gt;=7 vs. Gleason score =6; p = 0.01). Taken together, the findings presented herein establish the utility of RNA-Seq to comprehensively identify unannotated ncRNAs, such as PCAT-1, implicated in cancer. Our data suggest that PCAT-1 promotes cell proliferation, that in its inactive state PCAT-1 is mechanistically repressed by PRC2, and that PCAT-1 may serve as a candidate biomarker for non-invasive clinical tests. We further speculate that applying these methodologies to other diseases may reveal key aspects of disease biology and clinically important biomarkers, particularly for diseases that currently lack good non-invasive tests in fluids such as blood serum or urine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2011-4707

Dosage-Sensitive Function of RETINOBLASTOMA RELATED and Convergent Epigenetic Control Are Required during the Arabidopsis Life Cycle

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.

A polycomb repressive complex 2 gene regulates apogamy and gives evolutionary insights into early land plant evolution

Land plants have distinct developmental programs in haploid (gametophyte) and diploid (sporophyte) generations. Although usually the two programs strictly alternate at fertilization and meiosis, one program can be induced during the other program. In a process called apogamy, cells of the gametophyte other than the egg cell initiate sporophyte development. Here, we report for the moss Physcomitrella patens that apogamy resulted from deletion of the gene orthologous to the Arabidopsis thaliana CURLY LEAF (PpCLF), which encodes a component of polycomb repressive complex 2 (PRC2). In the deletion lines, a gametophytic vegetative cell frequently gave rise to a sporophyte-like body. This body grew indeterminately from an apical cell with the character of a sporophytic pluripotent stem cell but did not form a sporangium. Furthermore, with continued culture, the sporophyte-like body branched. Sporophyte branching is almost unknown among extant bryophytes. When PpCLF was expressed in the deletion lines once the sporophyte-like bodies had formed, pluripotent stem cell activity was arrested and a sporangium-like organ formed. Supported by the observed pattern of PpCLF expression, these results demonstrate that, in the gametophyte, PpCLF represses initiation of a sporophytic pluripotent stem cell and, in the sporophyte, represses that stem cell activity and induces reproductive organ development. In land plants, branching, along with indeterminate apical growth and delayed initiation of spore-bearing reproductive organs, were conspicuous innovations for the evolution of a dominant sporophyte plant body. Our study provides insights into the role of PRC2 gene regulation for sustaining evolutionary innovation in land plants.