Ectodermal Research Articles

An EED/PRC2-H19 Loop Regulates Cerebellar Development.

EED (embryonic ectoderm development) is a core subunit of the polycomb repressive complex 2 (PRC2), which senses the trimethylation of histone H3 lysine 27 (H3K27). However, its biological function in cerebellar development remains unknown. Here, we show that EED deletion from neural stem cells (NSCs) or cerebellar granule cell progenitors (GCPs) leads to reduced GCPs proliferation, cell death, cerebellar hypoplasia, and motor deficits in mice. Joint profiling of transcripts and ChIP-seq analysis in cerebellar granule cells reveals that EED regulates bunches of genes involved in cerebellar development. EED ablation exhibits overactivation of a developmental repressor long non-coding RNA H19. Importantly, an obvious H3K27ac enrichment is found at Ctcf, a trans-activator of H19, and H3K27me3 enrichment at the H19 imprinting control region (ICR), suggesting that EED regulates H19 in an H3K27me3-dependent manner. Intriguingly, H19 deletion reduces EED expression and the reprogramming of EED-mediated H3K27me3 profiles, resulting in increased proliferation, differentiation, and decreased apoptosis of GCPs. Finally, molecular and genetic evidence provides that increased H19 expression is responsible for cerebellar hypoplasia and motor defects in EED mutant mice. Thus, this study demonstrates that EED, H19 forms a negative feedback loop, which plays a crucial role in cerebellar morphogenesis and controls cerebellar development.

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

An Individualistic Homoeopathic Approach in a Case of Infected Epidermal Naevus—A Case Report

AbstractNaevi are circumscribed developmental defects or the lack of one or more components of the skin that manifest as overgrowths or hamartomas derived from embryonic ectoderm. The majority of naevi are present at birth or appear soon after birth. Naevi can be infected by various organisms, particularly by Staphylococcus, without other systemic involvement, similar to the skin. A 30-year-old male patient presented with an infected, painful congenital epidermal naevus on the anterior side of his right thigh. He experienced severe itching, burning, and sudden blister formation. After treatment, the infection completely resolved, leaving the naevus without any complications. Possible causal attribution of these changes was determined using the Modified Naranjo Criteria (score: 8 out of 13). No recurrence of further infection was reported in the next year, demonstrating the positive curative role of individualized homeopathic treatment in a case of infected epidermal naevus.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

Genomic context- and H2AK119 ubiquitination-dependent inheritance of human Polycomb silencing.

Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to epigenetic inheritance of mammalian Polycomb repression are not fully understood. Here, we show that, in human cells, ectopically induced Polycomb silencing at initially active developmental genes, but not near ubiquitously expressed housekeeping genes, is inherited for many cell divisions. Unexpectedly, silencing is heritable in cells with mutations in the H3K27me3 binding pocket of the Embryonic Ectoderm Development (EED) subunit of PRC2, which are known to disrupt H3K27me3 recognition and lead to loss of H3K27me3. This mode of inheritance is less stable and requires intact PRC2 and recognition of H2AK119ub1 by PRC1. Our findings suggest that maintenance of Polycomb silencing is sensitive to local genomic context and can be mediated by PRC1-dependent H2AK119ub1 and PRC2 independently of H3K27me3 recognition.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Abstract ND04: Discovery of ORIC-944, a novel inhibitor of PRC2 with best-in-class properties for the treatment of prostate cancer

Abstract First generation inhibitors of PRC2 (Polycomb Repressive Complex 2) targeting EZH2 (Enhancer of Zeste Homolog 2) achieved clinical proof-of-concept in follicular lymphoma and epithelioid sarcoma, and emerging clinical data recently revealed promising activity in prostate cancer in combination with AR pathway inhibitors (ARPIs). Here we describe the discovery and development of a second generation PRC2 inhibitor, ORIC-944, a potent, selective, allosteric inhibitor of PRC2 that binds the EED (Embryonic Ectoderm Development) subunit within the complex. Co-crystallization of previously disclosed triazolopyrimidine-based compounds demonstrated binding to EED in the histone H3K27me3 binding pocket, adopting a Lys211-in conformation. We evaluated the apo structure of EED and hypothesized that the Lys211-in conformation may be energetically unfavorable. Using structure-based drug design we pursued a strategy for differentiation of SAR and physicochemical properties by progressing from an early 2-substituted imidazopyrimidine that targets the cryptic pocket with a Lys211-out conformation. Key optimization criteria focused on metabolic stability and solubility, and resulted in the discovery of ORIC-944, an orally bioavailable inhibitor of PRC2 with best-in-class properties. Single agent tumor growth inhibition was observed for ORIC-944 in prostate cancer xenograft models. ORIC-944 also improved ARPI antitumor activity in preclinical combination studies and demonstrated in vitro synergy. Characterization of ORIC-944 revealed superiority to first generation clinical PRC2 inhibitors across a variety of preclinical assessments including potency, solubility, CYP inhibition/induction, PK and oral bioavailability. Together, the preclinical results indicate the desired properties were achieved in ORIC-944 to meet a superior target candidate profile, and the ongoing phase 1b trial (NCT05413421) demonstrates a clinical half-life of approximately 20 hours consistent with once daily dosing and a potential best-in-class profile. Citation Format: Matthew A. Marx, Aaron C. Burns, Nidhi Arora, Thomas P. Bobinski, David M. Briere, Andrew Calinisan, Robin J. Gunn, John M. Ketcham, David Lawson, Matthew Lee, Peter Olson, Christopher R. Smith, Niranjan Sudhakar, James G. Christensen, Chelsea X. Chen, Natalie Yuen, Livia Ulicna, Frank Duong, Christophe Colas, Anjana Ramnath, Ron Xu, Edna Chow Maneval, Pratik S. Multani, Rupal Patel, Archana Kumar, Anneleen Daemen, Anthony Romero, Melissa R. Junttila, Lori S. Friedman. Discovery of ORIC-944, a novel inhibitor of PRC2 with best-in-class properties for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND04.

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.

Abstract 6586: ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models

Abstract Polycomb repressive complex 2 (PRC2) dysregulation occurs in multiple tumor types and is associated with poor prognosis in patients with prostate cancer. In patients treated with androgen receptor pathway inhibitors (ARPI), emergence of tumor cell plasticity is associated with resistance to therapy. Dysregulation of epigenetic reprogramming factors, including PRC2, creates an environment that is permissive for such lineage plasticity. Inhibition of the PRC2 complex therefore may provide an opportunity to overcome or prevent the emergence of plasticity in prostate cancer. Notably, emerging clinical trial data has demonstrated the potential of ARPI and PRC2 inhibitor combination therapy to improve outcomes in patients with metastatic prostate cancer. PRC2 tri-methylates histone H3 at lysine 27 (H3K27me3), leading to long-term transcriptional silencing, a key mechanism regulating cellular functions such as cell growth and differentiation. Three core subunits comprise PRC2: the catalytic subunit enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). EED is essential for the histone methyltransferase activity of PRC2. First-generation PRC2 inhibitors, which target EZH2, exhibit poor drug properties, drug-drug interaction liabilities and short half-life requiring twice daily dosing at high doses in patients. We developed a second generation PRC2 inhibitor, ORIC-944, an allosteric inhibitor of PRC2 that binds the EED subunit. ORIC-944 is a potent, highly selective, orally bioavailable inhibitor with best-in-class properties. ORIC-944 demonstrated single agent tumor growth inhibition in a spectrum of in vivo prostate cancer models, including AR-positive, AR-mutant, ARv7, ARPI-responsive and ARPI-resistant models. In vitro analysis of the combination of ARPIs and ORIC-944 showed synergy in multiple prostate cancer cells, utilizing BLISS and other quantitative methods. In vivo studies revealed that ORIC-944 demonstrated increased efficacy in combination with ARPI, consistent with the in vitro synergy. RNA-seq analysis of transcriptional changes induced by ORIC-944 provided mechanistic insight into the role of PRC2 in prostate cancer lineage plasticity and combination response. These results position ORIC-944 as a best-in-class PRC2 inhibitor for evaluation in combination with ARPI in patients with metastatic prostate cancer. A phase 1b trial of ORIC-944 is ongoing (NCT05413421). Citation Format: Anneleen Daemen, Natalie Yuen, Amber Wang, Aleksandr Pankov, Livia Ulicna, Chelsea Chen, Frank L. Duong, Jason Long, Matthew A. Marx, James G. Christensen, Lori S. Friedman, Melissa R. Junttila. ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models [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 6586.

Isothiocyanates Potentiate Tazemetostat-Induced Apoptosis by Modulating the Expression of Apoptotic Genes, Members of Polycomb Repressive Complex 2, and Levels of Tri-Methylating Lysine 27 at Histone 3 in Human Malignant Melanoma Cells.

In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.

Mechanical plasticity of cell membranes enhances epithelial wound closure

During epithelial wound healing, cell morphology near the healed wound and the healing rate vary strongly among different developmental stages even for a single species like . We develop deformable particle (DP) model simulations to understand how variations in cell mechanics give rise to distinct wound closure phenotypes in the embryonic ectoderm and larval wing disc epithelium. We find that plastic deformation of the cell membrane can generate large changes in cell shape consistent with wound closure in the embryonic ectoderm. Our results show that the embryonic ectoderm is best described by cell membranes with an elasto-plastic response, whereas the larval wing disc is best described by cell membranes with an exclusively elastic response. By varying the mechanical response of cell membranes in DP simulations, we recapitulate the wound closure behavior of both the embryonic ectoderm and the larval wing disc. Published by the American Physical Society 2024

Insight into the Inhibitory Mechanism of Embryonic Ectoderm Development Subunit by Triazolopyrimidine Derivatives as Inhibitors through Molecular Dynamics Simulation.

Inhibition of the Embryonic Ectoderm Development (EED) subunit in Polycomb Repressive Complex 2 (PRC2) can inhibit tumor growth. In this paper, we selected six experimentally designed EED competitive Inhibitors of the triazolopyrimidine derivatives class. We investigated the difference in the binding mode of the natural substrate to the Inhibitors and the effects of differences in the parent nuclei, heads, and tails of the Inhibitors on the inhibitory capacity. The results showed that the binding free energy of this class of Inhibitors was close to or lower compared to the natural substrate, providing an energetic basis for competitive inhibition. For the Inhibitors, the presence of a strong negatively charged group at the 6-position of the parent nucleus or the 8'-position of the head would make the hydrogen atom on the head imino group prone to flip, resulting in the vertical movement of the parent nucleus, which significantly decreased the inhibitory ability. When the 6-position of the parent nucleus was a nonpolar group, the parent nucleus would move horizontally, slightly decreasing the inhibitory ability. When the 8'-position of the head was methylene, it formed an intramolecular hydrophobic interaction with the benzene ring on the tail, resulting in a significant increase in inhibition ability.

Epidermal Barrier Development via Corneoptosis: A Unique Form of Cell Death in Stratum Granulosum Cells.

Epidermal development is responsible for the formation of the outermost layer of the skin, the epidermis. The establishment of the epidermal barrier is a critical aspect of mammalian development. Proper formation of the epidermis, which is composed of stratified squamous epithelial cells, is essential for the survival of terrestrial vertebrates because it acts as a crucial protective barrier against external threats such as pathogens, toxins, and physical trauma. In mammals, epidermal development begins from the embryonic surface ectoderm, which gives rise to the basal layer of the epidermis. This layer undergoes a series of complex processes that lead to the formation of subsequent layers, including the stratum intermedium, stratum spinosum, stratum granulosum, and stratum corneum. The stratum corneum, which is the topmost layer of the epidermis, is formed by corneoptosis, a specialized form of cell death. This process involves the transformation of epidermal keratinocytes in the granular layer into flattened dead cells, which constitute the protective barrier. In this review, we focus on the intricate mechanisms that drive the development and establishment of the mammalian epidermis to gain insight into the complex processes that govern this vital biological system.

Title: Single-Cell Transcriptome Analyses Further Elucidate the Mechanism of Action of Eed Inhibition on HbF Induction

Mutations in the gene encoding the hemoglobin β (HBβ) subunit lead to red blood cell disorders such as Sickle Cell Disease (SCD) and β-thalassemia. Hemoglobin γ subunit, a fetal counterpart of HBβ, can relieve the disease symptoms by forming a functional complex with hemoglobin a subunit, thereby compensating for the mutated HBβ. The expression of β-like hemoglobins is tightly regulated during development, with a switch from the fetal ortholog (γ) to the adult ortholog (β) shortly after birth. Maintenance of HBγ is therapeutically beneficial for SCD patients and is observed clinically in individuals with mutations for both SCD and hereditary persistent fetal hemoglobin (HPFH) who exhibit less severe or even no SCD-related symptoms. Polycomb repressive complex (PRC) proteins have been shown to regulate the expression of β-like hemoglobins. Our previous research has demonstrated inhibitors targeting the PRC2 protein embryonic ectoderm development (EED) upregulate fetal hemoglobin (HbF) expression, leading to the formation of HbF complexes in both human CD34 + HSC-derived erythroid cells and the Townes SCD mouse model. However, the functional mechanisms of PRC complexes in this regulatory process and their target genes remain to be clarified. In our study, we conducted single-cell transcriptome analyses on erythroid cells derived from CD34 + HSCs collected at various stages of differentiation. We identified several erythroid subpopulations, from early progenitors to maturing cells, within the CD34 + derived erythroid cell cultures. HbF + and HbF - cell populations were also identified using known markers. To explore the mechanisms underlying HbF upregulation by EED inhibition, we compared single-cell transcriptome profiles of samples treated with and without an EED inhibitor. Our findings reveal that EED inhibitor treatment consistently leads to increased HbF + populations across multiple donors without impacting erythroid cell differentiation. Differential gene expression analysis revealed sequential changes in the expression of previously identified HbF regulators during differentiation in the presence of EED inhibitor treatment. Through pseudotime analyses, we identified erythroid progenitor cells undergoing fate bifurcation, leading to an increase in the lineage of mature cells that express HBγ. Finally, by comparing the pseudotime trajectories of different samples, we identified genes whose expression changed along the differentiation trajectories in response to EED inhibitor treatment which influence cell fate. Overall, this study provides a deeper understanding of the relevant biological pathways that respond to EED inhibition and potential for the identification of novel molecular targets for HbF induction.

Embryonic Ectoderm Development (EED) Inhibitor APG5918 Improves Chronic Kidney Disease- (CKD)-Induced Hemoglobin Insufficiency in Preclinical Models of Anemia

Introduction Anemia can be caused by infections, malignancies, CKD, or chemotherapy. Increasing Hb or fetal hemoglobin (HbF) can prevent or reduce anemia-related pathophysiology. Polycomb inhibitory complex 2 (PRC2), consisting of EZH2, EED, and SUZ12, is a subunit of the major transcriptional corepressor complex of BCL11A, and targeting EED may be one novel approach for treatment of patients with anemia. We report the effects of APG-5918, a small-molecule selective EED inhibitor, on Hb and HbF induction in human CD34⁺ hematopoietic stem cells (HSCs) and the efficacy of APG-5918 in a CKD-induced anemia model in vivo. Methods The pharmacodynamic (PD) effects of APG-5918 were evaluated in human cord blood CD34⁺ HSCs by flow cytometry, RT-qPCR, and ELISA. In vivo, the antianemia effect of APG-5918 was evaluated in adenine-induced renal anemic Sprague-Dawley rats. Results APG-5918 inhibited H3K27me3 inCD34⁺ HSCs, the critical epigenetic mark catalyzed by PRC2. APG-5918 also increased γ-globin coding gene ( HBG) and β-globin coding gene ( HBB) mRNA levels in a dose-dependent manner. After 7 days of treatment with APG-5918 at 1 μM, HBG and HBB mRNA increased to 12.36-fold and 4.76-fold of the control group, respectively, which was pharmacologically superior to effects observed with hydroxyurea at 33 µM (~6.53-fold and 2.27-fold increases in HBG and HBB mRNA, respectively). Hb and HbF protein were also upregulated in a dose-dependent manner. A total of 1.69-fold and 1.9-fold increases in HbF and Hb protein expression were observed after 7 days of treatment with APG-5918 at 1 μM.HbF and Hb protein showed no significant difference after hydroxyurea treatment. In an adenine-induced renal anemic rat model, APG-5918 was administered orally once daily at 3 dose levels (5, 15, and 45 mg/kg). Both body weight and the blood parameters erythrocytes, Hb, hematocrit (HCT), and reticulocytes (RET) improved significantly in a dose-dependent manner compared to the model (adenine) group ( p < 0.05, 0.01, or 0.001) (Figure 1). APG-5918 showed similar in vivo efficacy with erythropoietin (EPO): body weight and blood parameters in the group treated with APG-5918 45 mg/kg were comparable to those in the EPO group. In addition, blood parameters in the APG-5918 (15 mg/kg) plus EPO group recovered to the level of the control group, which was superior to the APG-5918 and EPO monotherapy groups. Conclusions APG-5918 demonstrated its effects as a single agent on inhibition of H3K27me3 inCD34⁺ HSCs, HbF and Hb induction, and normalization of hematologic parameters in a CKD-induced anemia model. APG-5918 also showed synergistic antianemia effects when combined with EPO. These findings support APG-5918 as a novel treatment option for CKD-induced anemia.

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.

Retinal Tumors Associated with Phacomatoses and Isolated Retinal Hamartoma

The phacomatoses or neurocutaneous syndromes are a heterogeneous group of genetic disorders arising from the embryonic ectoderm, characterized by multiple congenital anomalies affecting the eye, skin, and central nervous system. Two of the most common phacomatoses are neurofibromatosis and tuberous sclerosis, and retinal manifestations may be seen in both these entities. Retinal astrocytic hamartomas are benign glial tumors that originate from astrocytes in the retinal nerve fiber layer. While they are classically associated with systemic phacomatoses such as tuberous sclerosis complex and neurofibromatosis, these lesions can also present as incidental findings in otherwise healthy individuals. It should be kept in mind that retinal involvement may accompany phacomatoses and detailed ophthalmological evaluation should be performed in these patients.

Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.

The sulfotransferase XB5850668.L is required to apportion embryonic ectodermal domains.

Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up-regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase. Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss- and gain-of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild-type protein. Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity.