Component Of Polycomb Repressive Complex Research Articles

The zinc-finger protein POGZ associates with Polycomb repressive complex 1 to regulate bone morphogenetic protein signaling during neuronal differentiation.

Polycomb Repressive Complex 1 (PRC1) is a family of epigenetic regulators critical for mammalian development. Elucidating PRC1 composition and function across cell types and developmental stages is key to understanding the epigenetic regulation of cell fate determination. In this study, we discovered POGZ, a prominent Autism Spectrum Disorder (ASD) risk factor, as a novel component of PRC1.6, forming the PRC1.6-POGZ complex. Functional assays revealed that POGZ elicits transcriptional repression that is dependent on RING1B expression. Analysis of publicly available ChIP-Seq data showed that POGZ highly colocalizes with RING1B and HP1γ, two PRC1.6 components, at genes involved in multiple aspects of transcriptional regulation in the embryonic mouse cortex. Although Pogz knockout (KO) does not compromise stem cell pluripotency, Pogz ablation in neuronal progenitor cells (NPCs) led to widespread transcriptomic dysregulation with failed activation of key neuronal genes. Finally, we demonstrated that PRC1.6-POGZ regulates neuronal differentiation by repressing the bone morphogenetic protein (BMP) signaling pathway. These findings reveal a mechanism by which PRC1 and POGZ coordinate transcriptional regulation during neuronal differentiation, which offers insights into how disruptions in this pathway may contribute to neurodevelopmental disorders such as ASD.

Exploring oncogenic roles and clinical significance of EZH2: focus on non-canonical activities.

The enhancer of zeste homolog 2 (EZH2) is a catalytic component of Polycomb repressive complex 2 (PRC2) mediating the methylation of histone 3 lysine 27 (H3K27me3) and hence the epigenetic repression of target genes, known as canonical function. Growing evidence indicates that EZH2 has non-canonical roles that are exerted as PRC2-dependent and PRC2-independent methylation of non-histone proteins, and methyltransferase-independent interactions of EZH2 with various proteins contributing to gene expression regulation and alterations in the protein stability. EZH2 is frequently mutated and/or its expression is deregulated in various cancer types. The cancer sensitivity to inhibitors of EZH2 enzymatic activity and state-of-the-art approaches to deplete EZH2 with chemical degraders are discussed. This review also presents the clinical trials in various phases that evaluate the use of EZH2 inhibitors, both as monotherapy and in combination with other agents for the treatment of patients with diverse types of cancers.

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (H. Kasai, K. Mochizuki, T. Tanaka, A. Yamashita, et al., J Virol 95:e01784-20, 2021, https://doi.org/10.1128/jvi.01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28γ-binding protein. The proteasome activator PA28γ destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28γ knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28γ, while PA28γ knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28γ at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28γ-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28γ mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28γ and the lysine acetyltransferase CBP/p300 and then accelerated PA28γ acetylation and heptazmerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28γ to increase the proteasomal targeting of RNF2.IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma. PA28γ, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28γ-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28γ acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

BMI-1 in Breast Cancer - Biological Role and Clinical Implications.

Breast cancer is the most frequent cancer among women. Despite extensive research in recent years the molecular basis of breast cancer development, growth and metastasis remains unclear. Numerous studies highlight the involvement of BMI-1 in tumorigenesis. BMI-1 protein is a key component of Polycomb Repressive Complex 1, which by ubiquitinylation of histone H2A, regulates expression of genes involved in various cellular processes including cell cycle, proliferation and programmed cell death. Overexpression of BMI-1 has been often associated with breast cancer development and progression. This review summarizes the current state of knowledge of BMI-1's role in breast cancer biology and its potential significance as prognostic marker and potential target of new anticancer therapy.

THE EPIGENETIC REGULATORY PROTEIN CBX2 AS A NOVEL THERAPEUTIC TARGET FOR GLIOBLASTOMA

Abstract AIMS Glioblastoma (GBM) is the deadliest and most common form of brain tumour. The average length of survival for GBM patients remains low, in part, due to a lack of targeted therapeutics. Identification and validation of novel therapeutic targets is therefore crucial. Chomobox 2 (CBX2; a component of polycomb repressive complex 1 (PRC1)), represses gene expression and has a pro-oncogenic role in several aggressive cancer subtypes. CBX2 expression is elevated in GBM, however, very little is known about its role in this deadly disease. We aim to assess the phenotypic and gene expression effects of preventing CBX2-chromatin interaction in patient-derived GBM cell models, and to determine the functional mechanisms by which CBX2 promotes GBM progression; thereby determining its potential as a novel therapeutic target. METHOD Crucial to impactful target validation is the use of translationally relevant models, this study utilises a 3D spheroid model, using patient-derived cells, which more closely recapitulates the in vivo tumour microenvironment. GBM cell monolayers and spheroids were treated with SW2_152F, a selective CBX2 inhibitor. The phenotypic effects of CBX2 inhibition were assessed by cell count, CellTiter-Glo viability assays, fluorescence microscopy, and flow cytometry. Genes and biological pathways differentially regulated following CBX2 inhibition were assessed by RNA-sequencing and Gene Set Enrichment Analysis (GSEA). RESULTS Inhibition of CBX2 induced cell death and reduced cell viability in both 2D and 3D GBM cultures. RNA-seq and GSEA of SW2_152F-treated cells identified dysregulation of gene signatures involved in pro-oncogenic MYC and E2F signalling pathways, and dysregulation of genes associated with G2/M cell cycle progression. CONCLUSION Preventing CBX2-chromatin interaction induces GBM cell death. Further research will identify CBX2- chromatin binding sites to characterise the activity and direct regulatory role of CBX2 in GBM. Furthermore, we will assess the phenotypic and gene expression effects of CBX2 inhibition and depletion on ex vivo maintained GBM tissue biopsies.

Nucleocytoplasmic β-catenin expression contributes to neuroendocrine differentiation in muscle invasive bladder cancer.

Bladder cancers are heterogeneous in nature, showing diverse molecular profiles and histopathological characteristics, which pose challenges for diagnosis and treatment. However, understanding the molecular basis of such heterogeneity has remained elusive. This study aimed to elucidate the molecular landscape of neuroendocrine-like bladder tumors, focusing on the involvement of β-catenin localization. Analyzing the transcriptome data and benefiting from the molecular classification tool, we undertook an in-depth analysis of muscle-invasive bladder cancers to uncover the molecular characteristics of the neuroendocrine-like differentiation. The study explored the contribution of transcription factors and chromatin remodeling complexes to neuroendocrine differentiation in bladder cancer. The study revealed a significant correlation between β-catenin localization and neuroendocrine differentiation in muscle-invasive bladder tumors, highlighting the molecular complexity of neuroendocrine-like tumors. Enrichment of YY1 transcription factor, E2F family members, and Polycomb repressive complex components in β-catenin-positive tumors suggest their potential contribution to neuroendocrine phenotypes. Our findings contribute valuable insights into the molecular complexity of neuroendocrine-like bladder tumors. By identifying potential therapeutic targets and refining diagnostic strategies, this study advances our understanding of endocrinology in the context of bladder cancer. Further investigations into the functional implications of these molecular relationships are warranted to enhance our knowledge and guide future therapeutic interventions.

Discovery and Characterization of a Novel Cereblon-Recruiting PRC1 Bridged PROTAC Degrader.

Bridged PROTAC is a novel protein complex degrader strategy that exploits the target protein's binding partner to degrade undruggable proteins by inducing proximity to an E3 ubiquitin ligase. In this study, we discovered for the first time that cereblon (CRBN) can be employed for the bridged PROTAC approach and report the first-in-class CRBN-recruiting and EED-binding polycomb repressive complex 1 (PRC1) degrader, compound 1 (MS181). We show that 1 induces preferential degradation of PRC1 components, BMI1 and RING1B, in an EED-, CRBN-, and ubiquitin-proteosome system (UPS)-dependent manner. Compound 1 also has superior antiproliferative activity in multiple metastatic cancer cell lines over EED-binding PRC2 degraders and can be efficacious in VHL-defective cancer cells. Altogether, compound 1 is a valuable chemical biology tool to study the role of PRC1 in cancer. Importantly, we show that CRBN can be utilized to develop bridged PROTACs, expanding the bridged PROTAC technology for degrading undruggable proteins.

Abstract 3223: Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa)

Abstract Advanced PCa, especially castration-resistant PCa, represents an urgent unmet treatment need. Dysregulation of the polycomb repressive complex 2 (PRC2), which mediates trimethylation of histone H3 at lysine 27 (H3K27me3) and leads to gene silencing, is common in PCa and correlates with poor prognosis. EED, a core PRC2 component, is crucial for histone methyltransferase activity. MDM2, a negative regulator of p53, is often amplified or overexpressed in PCa and associated with metastasis and poor clinical outcomes. We evaluated antitumor activity and molecular mechanisms of EED inhibitor APG-5918/EEDi-5273 and MDM2 selective inhibitor alrizomadlin in preclinical PCa models. Human castration-resistant (22Rv1) and androgen-dependent (LNCaP) PCa cell lines were used in antiproliferation assays. Cell viability was determined by CellTiter-Glo luminescent assays. Western blotting was used to explore mechanisms of action. Subcutaneous 22Rv1 and LNCaP cell line-derived xenograft (CDX) models were established to evaluate antitumor effects of APG-5918 and alrizomadlin in vivo. Single-agent APG-5918 or alrizomadlin had limited inhibition on PCa cell growth; combined, they demonstrated synergistic antitumor activity. In the 22Rv1 CDX model, single-agent alrizomadlin (100 mg/kg) or APG-5918 (100 mg/kg) administered for 26 days exerted no/limited antitumor activity, with treatment-to-control (T/C) values of 93.39% and 49.89%, respectively. In contrast, the doublet enhanced antitumor activity (T/C value, 33.22%), with a synergistic index of 1.40. In the LNCaP CDX model, APG-5918 (100 mg/kg) administered for 28 days exhibited minimal antitumor effects (T/C value on last day of dosing, 80.32%). Alrizomadlin (100 or 50 mg/kg) showed limited antitumor activity (T/C value, 47.61%). Combined treatment significantly enhanced antitumor activity (T/C value, 15.69%), with a synergistic index of 2.44. Regarding putative mechanisms, western blot analyses showed that APG-5918 downregulated oncogenic drivers and DNA methylation factors (UHRF1, DNMT1) and on-target-related pharmacodynamic markers (H3K27me3, EED, EZH2). Alrizomadlin markedly downregulated UHRF1 and DNMT1, and upregulated p53 and p21 expression. Combined treatment further enhanced downregulation of DNMT1, UHRF1, cell cycle pathway proteins (pRb, CDK6), antiapoptotic protein MCL-1, and synergistically increased cleavage of PARP-1, a marker of apoptosis. In summary, we show that targeting EED and MDM2 synergistically enhances antitumor activity in PCa cell lines and CDX models. Mechanistically, this combination likely modulated pathways related to DNA methylation, cell cycle, and apoptosis. The findings provide a scientific rationale for future clinical development of APG-5918 and alrizomadlin for PCa patients. Citation Format: Yan Yin, Yun Yang, Eric Liang, Baisong Li, Hengbang Wang, Dajun Yang, Yifan Zhai. Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa) [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 3223.

Liquid–liquid phase separation of H3K27me3 reader BP1 regulates transcriptional repression

BackgroundBromo-adjacent homology-plant homeodomain domain containing protein 1 (BP1) is a reader of histone post-translational modifications in fungi. BP1 recognizes trimethylation of lysine 27 in histone H3 (H3K27me3), an epigenetic hallmark of gene silencing. However, whether and how BP1 participates in transcriptional repression remains poorly understood.ResultsWe report that BP1 forms phase-separated liquid condensates to modulate its biological function in Fusarium graminearum. Deletion assays reveal that intrinsically disordered region 2 (IDR2) of BP1 mediates its liquid–liquid phase separation. The phase separation of BP1 is indispensable for its interaction with suppressor of Zeste 12, a component of polycomb repressive complex 2. Furthermore, IDR2 deletion abolishes BP1-H3K27me3 binding and alleviates the transcriptional repression of secondary metabolism-related genes, especially deoxynivalenol mycotoxin biosynthesis genes.ConclusionsBP1 maintains transcriptional repression by forming liquid–liquid phase-separated condensates, expanding our understanding of the relationship between post-translational modifications and liquid–liquid phase separation.

Abstract A081: Chromobox 2 (CBX2) drives an immunosuppressive tumor microenvironment in high grade serous carcinoma (HGSC)

Abstract Introduction: Epigenetic reader and component of Polycomb Repressor Complex 1 (PRC1), Chromobox 2 (CBX2) is expressed in greater than 75% of high-grade serous carcinoma (HGSC) tumors. Increased CBX2 expression conveys a poorer disease-free and overall survival. CBX2 knockdown leads to increased chemotherapy sensitivity and attenuates stem-like properties, while CBX2 overexpression is sufficient to promote chemoresistance. In an HGSC immune competent murine model, knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME), understanding that TIME plays a critical role in disease progression and development of therapy resistance. Methods: We performed an exploration of existing data sets, including The Cancer Genome Atlas (TCGA), TIMERv2, and Carcinoma EcoTyper. We used HGSC cell lines (OVCAR4 and PEO1) with knockdown of CBX2 and RNA-sequencing. KEGG pathway analysis performed. A syngeneic ID8 mouse model was completed with shCBX2 compared to shControl. Nanostring Immuno-Oncology Panel and Multispectral Immunohistochemistry (mIHC) were completed to evaluate the TIME. Cell lines with genetic CBX2 modulation were utilized to create spheroids to recapitulate HGSC growth and confocal microscopy was employed to evaluate macrophage recruitment. Monocyte recruitment and macrophage polarization were evaluated using flow cytometry. Results: We found that existing data sets demonstrate a shift in TIME dependent on up or downregulation of CBX2. For instance, high CBX2 is associated with decreased CD8+ T cells (14.69% vs. 4.89%, p=0.0032) and increased undifferentiated macrophages (14.77 vs. 29.93%, p=0.006). Also, CBX2 is a defining feature of the Cell State Type 6 Carcinoma EcoType, which is associated with poor prognosis. RNA sequencing identified an association between CBX2 and CCL2, a cytokine known to recruit immunosuppressive macrophages. Pathway analysis of differentially expressed genes demonstrates an enrichment of several immune signatures. CBX2 knockdown in the ID8 syngeneic mouse model led to decreased tumor progression compared to control (11.50 mg. vs 51.63 mg, p=0.0001). NanoString analysis of the specimens suggest a shift in immune cell composition, with an increase in macrophages. mIHC defined the spatial relationships in this composition and confirmed increase in macrophage infiltration (28.64% vs. 34.60%, p=0.0231). Confocal microscopy and co-culture experiments to explore monocyte recruitment, found that both knockdown and overexpression of CBX2 lead to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2 overexpressing cells showed an increase in M2-like (CD206+) macrophages and decreased phagocytosis activity. Conclusion: Increased CBX2 expression leads to increased recruitment and polarization of M2-like macrophages leading to an immunosuppressive TIME. Targeting of CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies. Citation Format: Lindsay W. Brubaker, Ritsuko Iwanaga, Tomomi M Yamamoto, Elizabeth R. Woodruff, Nicole Marjon, Siddhartha Mitra, Benjamin G. Bitler. Chromobox 2 (CBX2) drives an immunosuppressive tumor microenvironment in high grade serous carcinoma (HGSC) [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A081.

The competitive mechanism of EZH1 and EZH2 in promoting oral squamous cell carcinoma

Enhancer of Zeste Homolog 1 (EZH1) and Enhancer of Zeste Homolog 2 (EZH2) are the key components of polycomb repressive complex 2 (PRC2); however, the roles of these proteins in oral squamous cell carcinoma (OSCC) have yet to be elucidated. In this study, we aimed to determine the respective roles of these proteins in OSCC by investigating the expression levels of EZH1 and EZH2 in OSCC tissues (N = 63) by immunohistochemistry. In addition, we used lentiviruses to construct stable OSCC cell lines that overexpressed EZH1 and EZH2. Then, we investigated these cell lines for cell viability, colony formation capacity, stemness, and epithelial-mesenchymal transition (EMT). Binding competition between EZH1 and EZH2 with PRC2 was further evaluated using Co-immunoprecipitation (Co-IP). Compared with normal tissues, the expression levels of EZH2 in OSCC tissues was up-regulated, while the expression of EZH1 was down-regulated. EZH2 enhanced cell viability, colony formation capacity, stemness, and EMT, while EZH1 did not. Furthermore, analysis indicated that EZH1 and EZH2 bound competitively to PRC2 and influenced the methylation status of H3K27. In conclusion, our findings verified that EZH1 and EZH2 play opposing roles in OSCC and that EZH1 and EZH2 compete as the key component of PRC2, thus affecting the characteristics of OSCC via the methylation of H3K27.

A mouse model of Weaver syndrome displays overgrowth and excess osteogenesis reversible with KDM6A/6B inhibition.

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters, indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-Seq comparing osteoblasts differentiated from Ezh2R684C/+, and Ezh2+/+ BM-mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A and KDM6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels, and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

Augmenting L3MBTL2-induced condensates suppresses tumor growth in osteosarcoma.

Osteosarcoma is a highly aggressive cancer and lacks effective therapeutic targets. We found that L3MBTL2 acts as a tumor suppressor by transcriptionally repressing IFIT2 in osteosarcoma. L3MBTL2 recruits the components of Polycomb repressive complex 1.6 to form condensates via both Pho-binding pockets and polybasic regions within carboxyl-terminal intrinsically disordered regions; the L3MBTL2-induced condensates are required for its tumor suppression. Multi-monoubiquitination of L3MBTL2 by UBE2O results in its proteasomal degradation, and the UBE2O/L3MBTL2 axis was crucial for osteosarcoma growth. There is a reverse correlation between L3MBTL2 and UBE2O in osteosarcoma tissues, and higher UBE2O and lower L3MBTL2 are associated with poorer prognosis in osteosarcoma. Pharmacological blockage of UBE2O by arsenic trioxide can enhance L3MBTL2-induced condensates and consequently suppress osteosarcoma growth. Our findings unveil a crucial biological function of L3MBTL2-induced condensates in mediating tumor suppression, proposing the UBE2O-L3MBTL2 axis as a potential cancer therapeutic target in osteosarcoma.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

HbF Inducing Eed Inhibitor Ftx-6058 Selectively and Reversibly Impacts Bone Marrow in Wild-Type Mice

Sickle cell disease (SCD) is a genetic disorder characterized by red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications resulting from a mutation in the HBB gene. Increasing fetal hemoglobin (HbF) levels has shown promise in mitigating disease-related pathophysiology. In individuals with SCD who exhibit hereditary persistence of fetal hemoglobin (HPFH) and have HbF levels ranging from 20% to 30%, a remarkable reduction in symptoms is observed, highlighting the protective effect of increased HbF in SCD. Preclinical and clinical findings indicate that compounds inhibiting the embryonic ectoderm development (EED) component of polycomb repressive complex 2 (PRC2) leads to elevations in HbF to therapeutic levels. These results suggest the potential for a substantial benefit for individuals living with SCD. To further understand the effects of PRC2 inhibition, this study aims to characterize the onset and reversibility of EED inhibition by FTX-6058. Results from in vivo studies in wild-type mice demonstrated upregulation of the mouse fetal hemoglobin ortholog mRNA (Hbb-bh1) as well as target engagement in bone marrow though quantification of H3K27 trimethylation during seven days of active dosing of FTX-6058. Target engagement and differential gene expression profiles in the bone marrow subsequently returned to baseline shortly after the dosing period. These findings demonstrate that inhibition of EED through FTX-6058 leads to a minimal, transient impact on the bone marrow in mice, and any transcriptional changes are rapidly reversible following cessation of dosing. A small molecule EED inhibitor has the potential to provide a new treatment for sickle cell disease without irreversibly altering the bone marrow.

Functional analyses of the polycomb-group genes in sea lamprey embryos undergoing programmed DNA loss.

During early development, sea lamprey embryos undergo programmatic elimination of DNA from somatic progenitor cells in a process termed programmed genome rearrangement (PGR). Eliminated DNA eventually becomes condensed into micronuclei, which are then physically degraded and permanently lost from the cell. Previous studies indicated that many of the genes eliminated during PGR have mammalian homologs that are bound by polycomb repressive complex (PRC) in embryonic stem cells. To test whether PRC components play a role in the faithful elimination of germline-specific sequences, we used a combination of CRISPR/Cas9 and lightsheet microscopy to investigate the impact of gene knockouts on early development and the progression through stages of DNA elimination. Analysis of knockout embryos for the core PRC2 subunits EZH, SUZ12, and EED show that disruption of all three genes results in an increase in micronucleus number, altered distribution of micronuclei within embryos, and an increase in micronucleus volume in mutant embryos. While the upstream events of DNA elimination are not strongly impacted by loss of PRC2 components, this study suggests that PRC2 plays a role in the later stages of elimination related to micronucleus condensation and degradation. These findings also suggest that other genes/epigenetic pathways may work in parallel during DNA elimination to mediate chromatin structure, accessibility, and the ultimate loss of germline-specific DNA.

SUZ12 inhibition attenuates cell proliferation of glioblastoma via post-translational regulation of CDKN1B.

Human gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. Differential expression of Polycomb repressive complex 2 (PRC2) has been reported in various subtypes of glioma. However, the role of PRC2 in uncontrolled growth in glioma and its underlying molecular mechanisms remain to be elucidated. We aimed to investigate the functional role of PRC2 in human glioblastoma cell growth by silencing SUZ12, the non-catalytic core component of PRC2. Knockdown of SUZ12 was achieved by infecting T98G cells with lentivirus carrying sequences specifically targeting SUZ12 (shSUZ12). Gene expression was examined by quantitative PCR and western analysis. The impact of shSUZ12 on cell growth was assessed using a cell proliferation assay. Cell cycle distribution was analyzed by flow cytometry, and protein stability was evaluated in cycloheximide-treated cells. Subcellular localization was examined through immunofluorescence staining and biochemical cytoplasmic-nuclear fractionation. Gene expression analysis was also performed on human specimens from normal brain and glioblastoma patients. SUZ12 knockdown (SUZ12 KD) led to widespread decrease in the PRC2-specific histone mark, accompanied by a slowdown of cell proliferation through G1 arrest. In SUZ12 KD cells, the degradation of CDKN1B protein was reduced, resulting from alterations in the MYC-SKP2-CDKN1B axis. Furthermore, nuclear localization of CDKN1B was enhanced in SUZ12 KD cells. Analysis of human glioblastoma samples yielded increased expression of EZH2 and MYC along with reduced CDKN1B compared to normal human brain tissue. Our findings suggest a novel role for SUZ12 in cell proliferation through post-translational regulation of CDKN1B in glioblastoma.

Recognition of H2AK119ub plays an important role in RSF1-regulated early Xenopus development.

Polycomb group (PcG) proteins are key regulators of gene expression and developmental programs via covalent modification of histones, but the factors that interpret histone modification marks to regulate embryogenesis are less studied. We previously identified Remodeling and Spacing Factor 1 (RSF1) as a reader of histone H2A lysine 119 ubiquitination (H2AK119ub), the histone mark deposited by Polycomb Repressive Complex 1 (PRC1). In the current study, we used Xenopus laevis as a model to investigate how RSF1 affects early embryonic development and whether recognition of H2AK119ub is important for the function of RSF1. We showed that knockdown of Xenopus RSF1, rsf1, not only induced gastrulation defects as reported previously, but specific targeted knockdown in prospective neural precursors induced neural and neural crest defects, with reductions of marker genes. In addition, similar to knockdown of PRC1 components in Xenopus, the anterior-posterior neural patterning was affected in rsf1 knockdown embryos. Binding of H2AK119ub appeared to be crucial for rsf1 function, as a construct with deletion of the UAB domain, which is required for RSF1 to recognize the H2AK119ub nucleosomes, failed to rescue rsf1 morphant embryos and was less effective in interfering with early Xenopus development when ectopically expressed. Furthermore, ectopic deposition of H2AK119ub on the Smad2 target gene gsc using a ring1a-smad2 fusion protein led to ectopic recruitment of RSF1. The fusion protein was inefficient in inducing mesodermal markers in the animal region or a secondary axis when expressed in the ventral tissues. Taken together, our results reveal that rsf1 modulates similar developmental processes in early Xenopus embryos as components of PRC1 do, and that RSF1 acts at least partially through binding to the H2AK119ub mark via the UAB domain during development.