Major Epigenetic Regulator Research Articles

Epigenetic Cross-Talk Between Sirt1 and Dnmt1 Promotes Axonal Regeneration After Spinal Cord Injury in Zebrafish.

Though spinal cord injury (SCI) causes irreversible sensory and motor impairments in human, adult zebrafish retain the potent regenerative capacity by injury-induced proliferation of central nervous system (CNS)-resident progenitor cells to develop new functional neurons at the lesion site. The hallmark of SCI in zebrafish lies in a series of changes in the epigenetic landscape, specifically DNA methylation and histone modifications. Decoding the post-SCI epigenetic modifications is therefore critical for the development of therapeutic remedies that boost SCI recovery process. Here, we have studied on Sirtuin1 (Sirt1), a non-classical histone deacetylase that potentially plays a critical role in neural progenitor cells (NPC) proliferation and axonal regrowth following SCI in zebrafish. We investigated the role of Sirt1 in NPC proliferation and axonal regrowth in response to injury in the regenerating spinal cord and found that Sirt1 is involved in the induction of NPC proliferation along with glial bridging during spinal cord regeneration. We also demonstrate that Sirt1 plays a pivotal role in regulating the HIPPO pathway through deacetylation-mediated inactivation of Dnmt1 and subsequent hypomethylation of yap1 promoter, leading to the induction of ctgfa expression, which drives the NPC proliferation and axonal regrowth to complete the regenerative process. In conclusion, our study reveals a novel cross-talk between two important epigenetic effectors, Sirt1 and Dnmt1, in the context of spinal cord regeneration, establishing a previously undisclosed relation between Sirt1 and Yap1 which provides a deeper understanding of the underlying mechanisms governing injury-induced NPC proliferation and axonal regrowth. Therefore, we have identified Sirt1 as a novel, major epigenetic regulator of spinal cord regeneration by modulating the HIPPO pathway in zebrafish.

Abstract 2636: Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma

Abstract Background Head and neck cancer is the 8th most common cancer worldwide. Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) overall survival rates remain low (25-40%) despite advances in treatments. CD8+ T cell differentiation into activated/cytotoxic (CTL), exhausted (TEX) and tissue-resident memory (TRM) phenotypes is a major determinant of successful cancer immunotherapy, which relies on the ability of CD8+ T cells to detect and eliminate cancer cells. Recent evidence suggests the importance of epigenetic regulation in the development of CD8+ T cell exhaustion and tissue residency; yet the epigenetic mechanisms that govern the transition between CTL, TEX, and TRM CD8+ T cell states remain unclear. This study investigates the epigenetic modifications associated with CTL, TEX, and TRM states using CD8+ T cells from healthy donors and HPV-negative HNSCC tumors. Methods An in vitro human CD8+ T cell stimulation assay using healthy donor cells was utilized to generate CTL, TEX and TRM phenotypes. Naïve T cells underwent CD3/28, TCR-independent stimulation for 9 days to promote CTL (3 days) and TEX (9 days) phenotypes, and TGF-β exposure (days 3-9) occurred to induce a TRM phenotype. Flow cytometry to confirm expression of cell-surface and intracellular markers for each phenotype is ongoing. Genome-wide mapping for activating (H3K4me1, H3K4me3) and repressive (H3K9me3, H3K27me3, H4K20me3) histone marks with CUT&Tag assays will be pursued for each of the in vitro CTL, TEX and TRM states, along with RNA-seq. CUT&Tag and RNA-seq will also characterize the epigenetic profiles of CD8+ T cells from single-cell suspensions generated from HPV-negative HNSCC tumors (n=5). Findings will be correlated with results from our in vitro T cell states. Results Preliminary flow cytometry confirmed increased expression of CD25, PD-1, Tim3 after 9 days of in vitro stimulation, supporting the induction of a CD8+ TEX phenotype. CD103 was also highly expressed in CD8+ T cells exposed to TGF-β, supporting the induction of the CD8+ TRM phenotype. Validation of the CTL state is ongoing. Phenotypic validation of cell states with flow cytometry (18 cell surface and intracellular markers) is ongoing. Feasibility of genome-wide mapping for activating and repressive histone marks with CUT&Tag on naïve CD8+ T cells has been confirmed. Conclusion This study investigates epigenetic changes that characterize the transition between CTL, TEX, and TRM states of CD8+ T cells using healthy donor cells through an in vitro assay and CD8+ T cells isolated from HPV-negative HNSCC tumors. Understanding the epigenetic profile of T cell states can help determine the major epigenetic regulators that lead to exhaustion and tissue residency, paving the way for improved T cell-based immunotherapies. Future experiments aim to validate our findings using TCR-specific in vitro and in vivo models. Citation Format: Marie K. Luff, Marco Craviero, Mohd Saleem Dar, Katherine McKinnon, Amir Kaskas, Clint T. Allen, Vassiliki Saloura. Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma [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 2636.

Combating castration-resistant prostate cancer by co-targeting the epigenetic regulators EZH2 and HDAC.

While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.

HELLS Is Negatively Regulated by Wild-Type P53 in Liver Cancer by a Mechanism Involving P21 and FOXM1.

Simple SummaryThe tumor suppressor protein P53 is a major player in preventing liver cancer development and progression. In this study we could show that P53 negatively regulates the expression of Helicase, lymphoid specific (HELLS), previously described as an important pro-tumorigenic epigenetic regulator in hepatocarcinogenesis. The regulatory mechanism included induction of the P53 target gene P21 (CDKN1A) resulting in repression of HELLS via downregulation of the transcription factor Forkhead Box Protein M1 (FOXM1). Our in vitro and in vivo findings indicate an important additional aspect of the tumor suppressive function of P53 in liver cancer linked to epigenetic regulation.The major tumor suppressor P53 (TP53) acts primarily as a transcription factor by activating or repressing subsets of its numerous target genes, resulting in different cellular outcomes (e.g., cell cycle arrest, apoptosis and senescence). P53-dependent gene regulation is linked to several aspects of chromatin remodeling; however, regulation of chromatin-modifying enzymes by P53 is poorly understood in hepatocarcinogenesis. Herein, we identified Helicase, lymphoid specific (HELLS), a major epigenetic regulator in liver cancer, as a strong and selective P53 repression target within the SNF2-like helicase family. The underlying regulatory mechanism involved P53-dependent induction of P21 (CDKN1A), leading to repression of Forkhead Box Protein M1 (FOXM1) that in turn resulted in downregulation of HELLS expression. Supporting our in vitro data, we found higher expression of HELLS in murine HCCs arising in a Trp53−/− background compared to Trp53+/+ HCCs as well as a strong and highly significant correlation between HELLS and FOXM1 expression in different HCC patient cohorts. Our data suggest that functional or mutational inactivation of P53 substantially contributes to overexpression of HELLS in HCC patients and indicates a previously unstudied aspect of P53′s ability to suppress liver cancer formation.

DNMT3A Regulates Hematopoietic Stem Cell Function Via DNA Methylation-Independent Functions

The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators that is essential for efficient HSC differentiation. DNMT3A mutations are prevalent in myeloid diseases that include acute myeloid leukemia (AML; ~22%) and myelodysplastic syndrome (MDS; ~10%) where they act as initiating events However, the precise molecular mechanisms of how DNMT3A regulates normal hematopoiesis and its mutations prime HSCs for leukemic formation are unclear. Although DNMT3A is described as a DNA methyltransferase enzyme, the lack of consistent correlation between changes in DNA methylation and differential gene expression in Dnmt3a-null HSCs in mouse models, and AML patients with DNMT3A mutations undermine the conventional understanding of DNMT3A's canonical role in hematopoietic cells. Hence, we hypothesized that DNMT3A may have novel functions outside of DNA methylation that regulate HSC fate decisions.To answer this question, we first ectopically expressed GFP-labeled Dnmt3a constructs (wild-type Dnmt3a, Dnmt3aE752A; complete DNA methylation dead, and Dnmt3aR832A; reduced DNA methylation target recognition) and empty vector (negative control) in Dnmt3a-null (Vav-Cre: Dnmt3afl/fl = Dnmt3a-/- in hematopoiesis) bone marrow (BM) cells. The result showed that similar to restoring wild-type Dnmt3a, ectopic expression of Dnmt3aE752A as well as Dnmt3aR832A showed a rescue effect of decreased engraftment of transduced cells in the peripheral blood as well as reduced HSC numbers in the BM. Analysis of DNA methylation by whole-genome bisulfite sequencing (WGBS) in transduced cells showed this phenotypic and functional rescue of the Dnmt3a-/- phenotype occurred in the absence of restored DNA methylation patterns. To study the importance of Dnmt3a-mediated DNA methyltransferase activity in a more physiological system, we generated knock-in mice that have one copy of either wild-type Dnmt3a, Dnmt3aE752A, or Dnmt3aR832A (CAGG-Cre-ER T2 = ER T2-Cre: Dnmt3afl/+, Dnmt3afl/E752A, and Dnmt3afl/R832A) to be compared to the Dnmt3a-null group (ER T2-Cre: Dnmt3afl/-). These mice contain one allele with loxP-flanked Dnmt3a that is deleted by tamoxifen-inducible Cre-mediated recombination and one allele of either wild-type Dnmt3a, Dnmt3aE752A, Dnmt3aR832A, or germline knockout Dnmt3anull. 5-weeks post-tamoxifen (~93% floxed allele recombination), competitive transplantation of 250 phenotypically defined test HSCs against with 2.5x10 5 congenic competitor BM cells was performed.Dnmt3afl/R832A recipients had higher engraftment (35.6 % +/- 6.1) than Dnmt3afl/+ (28.5% +/- 7.2) and Dnmt3afl/- (10.7% +/- 2.79), while Dnmt3afl/E752A had slightly higherengraftment (12.5% +/- 3) than Dnmt3afl/-. Analysis of the BM 18 weeks post-transplant showed that Dnmt3afl/E752A and Dnmt3afl/R832A HSCs phenocopied the HSC self-renewal potential phenotype of heterozygous Dnmt3a fl/+ HSCs (Fig. 1). The absolute count of donor-derived HSCs per mouse after the transplant were: ER T2-Cre control (675.7 +/- 299.3), Dnmt3afl/+ (1870 +/- 961.4), Dnmt3afl/- (3546 +/- 1019), Dnmt3afl/E752A (1130 +/- 362.7), and Dnmt3afl/R832A (1184 +/- 344.5) (mean +/- S.E.M.). While the described clonal expansion of Dnmt3a-null HSCs was observed, HSCs with one copy of full-length Dnmt3a but devoid of its methyltransferase capacity mimicked the heterozygous state rather than the homozygous loss-of-function. This is the first evidence to suggest that DNMT3A potentially regulates HSCs by non-canonical (DNA methylation independent) mechanisms. DNA methylation analysis by WGBS is ongoing to determine if Dnmt3afl/E752A and Dnmt3afl/R832A HSCs show a methylome comparable to Dnmt3a-null HSCs whilst having the functional potential of Dnmt3a-heterozygous HSCs, which will be complemented with other molecular analyses including gene expression. Our study opens new avenues for investigations into the molecular mechanisms of DNMT3A function in HSC biology, which could ultimately benefit clinical practice by identifying new therapeutic approaches for the patients with DNMT3A mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting INMT and interrupting its methylation pathway for the treatment of castration resistant prostate cancer

BackgroundCastration-resistant prostate cancer (CRPC) is associated with a very poor prognosis, and the treatment of which remains a serious clinical challenge.MethodsRNA-seq, qPCR, western blot and immunohistochemistry were employed to identify and confirm the high expression of indolethylamine N-methyltransferase (INMT) in CRPC and the clinical relevance. Chip assay was used to identify Histone-Lysine N-Methyltransferase (SMYD3) as a major epigenetic regulator of INMT. LC-MS/MS were used to identify new substrates of INMT methylation in CRPC tissues. Gene knockdown/overexpression, MTT and mouse cancer models were used to examine the role of INMT as well as the anticancer efficacy of INMT inhibitor N,N-dimethyltryptamine (DMT), the SMYD3 inhibitor BCl-12, the selenium compounds methaneseleninic acid (MSA) and Se-(Methyl)selenocysteine hydrochloride (MSC), and the newly identified endogenous INMT substrate Bis(7)-tacrine.ResultsWe found that the expression of INMT was highly increased in CRPC and was correlated with poor prognosis of clinical prostate cancer (PCa). INMT promoted PCa castration resistance via detoxification of anticancer metabolites. Knockdown of INMT or treatment with INMT inhibitor N,N-dimethyltryptamine (DMT) significantly suppressed CRPC development. Histone-Lysine N-Methyltransferase SMYD3 was a major epigenetic regulator of INMT expression, treatment with SMYD3 inhibitor BCl-121 suppressed INMT expression and inhibits CRPC development. Importantly, INMT knockdown significantly increased the anticancer effect of the exogenous selenium compounds methaneseleninic acid (MSA) and Se-(Methyl)selenocysteine hydrochloride (MSC) as well as the endogenous metabolite Bis(7)-tacrine.ConclusionsOur study suggests that INMT drives PCa castration resistance through detoxification of anticancer metabolites, targeting INMT or its regulator SMYD3 or/and its methylation metabolites represents an effective therapeutic avenue for CRPC treatment.

Abstract 2117: Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) causes approximately 10,000 deaths annually in the United States, with human-papilloma-virus (HPV)-negative patients having a particularly poor prognosis. Pembrolizumab, a programmed-death-1 (PD-1) antibody, was recently approved as a first-line therapy for patients with relapsed disease, however response rates are as low as 19%, underscoring the need for an improved understanding of immunotherapy resistance mechanisms. We have previously shown that HPV-negative HNSCC tumors with higher mRNA levels of SET and MYND Domain Containing 3 (SMYD3), a chromatin modifier known to trimethylated H3K4 (H3K4me3), have significantly lower mRNA levels of CD8 and CD8+ T-cell attracting chemokines, and that SMYD3 depletion in HNSCC cells in vitro leads to upregulation of CD8+ T-cell attracting chemokines. The goal of this work was to elucidate epigenetic mechanisms of immune evasion mediated by SMYD3 in HPV-negative HNSCC. Methods: siRNA- mediated SMYD3 knockdown was performed for 3 days to investigate immuno-modulatory epigenetic mechanisms mediated by SMYD3. RT-qPCR for immune-related genes, Western blotting for histone marks, ChIP-sequencing for H3K4me3, ChIP-qPCRs to determine genomic interactions of SMYD3 and ATAC-sequencing were performed. SMYD3 and CD8 immunohistochemical staining (IHC) of normal squamous, dysplastic epithelium and HPV-negative HNSCC tumors was also performed. Results: siRNA mediated knockdown of SMYD3 resulted in enrichment of inflammatory pathways and upregulation of type 1 interferon response and antigen presentation machinery genes in HNSCC cell lines. Immunoblotting for histone marks showed no reduction in H3K4me3 following SMYD3 depletion for 3 days. ChIP-sequencing for H3K4me3 showed differential distribution pattern with an increase of H3K4me3 peaks on immune-related genes, and a decrease of H3K4me3 on UHRF1, a reader of the repressive histone mark H3K9me3. RNA-seq, RT-PCR and ChIP-qPCR showed that SMYD3 may regulate the transcription of UHRF1 by binding to its promoter. SMYD3 depletion also led to decreased protein levels of UHRF1. ChIP for UHRF1 and H3K9me3 showed that UHRF1 binds directly on the promoters of CXCL9 and CXCL10 which are enriched for H3K9me3. IHC on Normal Squamous Epithelium and Squamous Cell Carcinoma tissues of increasing cancer stages showed increase in SMYD3 levels with the higher stage of cancer, and an inverse correlation with CD8+ T cell infiltration within the tumors. Conclusion: This is the first study reporting that the chromatin modifier SMYD3 may attenuate type I IFN responses in HPV-negative HNSCC by regulating the expression of a major epigenetic regulator, UHRF1, which may silence the expression of immune-related genes through H3K9me3. Citation Format: Nupur Nigam, Benjamin Jonah Bernard, Kyunghee Burkitt, Sohyoung Kim, Yvette Robbins, Richard L. Bennett, Jonathan D. Licht, Vassiliki Saloura. Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2117.

Cytokines and pregnancy: Potential regulation by histonedeacetylases.

Cytokines are important regulators of pregnancy and parturition. Aberrant expression of proinflammatory cytokines during pregnancy contributes towards preterm labor, pre-eclampsia, and gestational diabetes mellitus. The regulation of cytokine expression in human cells is highly complex, involving interactions between environment, transcription factors, and feedback mechanisms. Recent developments in epigenetic research have made tremendous advancements in exploring histone modifications as a key epigenetic regulator of cytokine expression and the effect of their signaling molecules on various organ systems in the human body. Histone acetylation and subsequent deacetylation by histone deacetylases (HDACs) are major epigenetic regulators of protein expression in the human body. The expression of various proinflammatory cytokines, their role in normal and abnormal pregnancy, and their epigenetic regulation via HDACs will be discussed in this review.

A natural DNMT1 mutation elevates the fetal hemoglobin level via epigenetic derepression of the γ-globin gene in β-thalassemia

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with β-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 β-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for β-hemoglobinopathies.

Prokaryotic DNA methylation and its functional roles.

DNA methylation is known as a universal mechanism of epigenetic regulation in all kingdoms of life. Particularly, given that prokaryotes lack key elements such as histones and nucleosomes that can structurally modify DNA, DNA methylation is considered a major epigenetic regulator in these organisms. However, because DNA methylation studies have focused primarily on eukaryotes, the mechanism of prokaryotic DNA methylation has been less studied than in eukaryotes. DNA methylation in prokaryotes plays an important role in regulating not only the host defense system, but also the cell cycle, gene expression, and virulence that can respond directly to the environment. Recent advances in sequencing techniques capable of detecting methylation signals have allowed for the characterization of prokaryotic genome-wide epigenetic regulation. In this review, we describe representative examples of cellular events regulated by DNA methylation in prokaryotes, from early studies to current applications.

P300 in Cardiac Development and Accelerated Cardiac Aging.

The heart is the first functional organ that develops during embryonic development. While a heartbeat indicates life, cessation of a heartbeat signals the end of life. Heart disease, due either to congenital defects or to acquired dysfunctions in adulthood, remains the leading cause of death worldwide. Epigenetics plays a key role in both embryonic heart development and heart disease in adults. Stress-induced vascular injury activates pathways involved in pathogenesis of accelerated cardiac aging that includes cellular dysfunction, pathological cardiac hypertrophy, diabetic cardiomyopathy, cardiac matrix remodeling, cardiac dysfunction and heart failure. Acetyltransferase p300 (p300), a major epigenetic regulator, plays a pivotal role in heart development during embryogenesis, as deficiency or abnormal expression of p300 leads to embryonic death at early gestation periods due to deformation of the heart and neural tube. Acetyltransferase p300 controls heart development through histone acetylation-mediated chromatin remodeling and transcriptional regulation of genes required for cardiac development. In adult hearts, p300 is differentially expressed in different chambers and epigenetically controls cardiac gene expression. Deregulation of p300, in response to prohypertrophic and profibrogenic stress signals, is associated with increased recruitment of p300 to several genes including transcription factors, increased acetylation of specific lysines in histones and transcription factors, altered chromatin organization, and increased hypertrophic and fibrogenic gene expression. Cardiac hypertrophy and myocardial fibrogenesis are common pathological manifestations of several stress-induced accelerated cardiac aging-related pathologies, including high blood pressure-induced or environmentally induced cardiac hypertrophy, myocardial infarction, diabetes-induced cardiomyopathy, and heart failure. Numerous studies using cellular and animal models clearly indicate that pharmacologic or genetic normalization of p300 activity has the potential to prevent or halt the progression of cardiac aging pathologies. Based on these preclinical studies, development of safe, non-toxic, small molecule inhibitors/epidrugs targeting p300 is an ideal approach to control accelerated cardiac aging-related deaths worldwide.

Role of chromatin modification and remodeling in stem cell regulation and meristem maintenance in Arabidopsis

In higher plants, pluripotent stem cells reside in the specialized microenvironment called stem cell niches (SCNs) harbored at the shoot apical meristem (SAM) and root apical meristem (RAM), which give rise to the aerial and underground parts of a plant, respectively. The model plant Arabidopsis thaliana (Arabidopsis) has been extensively studied to decipher the intricate regulatory mechanisms involving some key transcriptions factors and phytohormones that play pivotal roles in stem cell homeostasis, meristem maintenance, and organ formation. However, there is increasing evidence to show the epigenetic regulation of the chromatin architecture, gene expression exerting an influence on an innate balance between the self-renewal of stem cells, and differentiation of the progeny cells to a specific tissue type or organ. Post-translational histone modifications, ATP-dependent chromatin remodeling, and chromatin assembly/disassembly are some of the key features involved in the modulation of chromatin architecture. Here, we discuss the major epigenetic regulators and illustrate their roles in the regulation of stem cell activity, meristem maintenance, and related organ patterning in Arabidopsis.

HDAC4 Levels Control Sensibility toward Cisplatin in Gastric Cancer via the p53-p73/BIK Pathway.

Gastric cancer (GC) remains a health issue due to the low efficiency of therapies, such as cisplatin. This unsatisfactory situation highlights the necessity of finding factors impacting GC sensibility to therapies. We analyzed the cisplatin pangenomic response in cancer cells and found HDAC4 as a major epigenetic regulator being inhibited. HDAC4 mRNA repression was partly mediated by the cisplatin-induced expression of miR-140. At a functional level, HDAC4 inhibition favored cisplatin cytotoxicity and reduced tumor growth. Inversely, overexpression of HDAC4 inhibits cisplatin cytotoxicity. Importantly, HDAC4 expression was found to be elevated in gastric tumors compared to healthy tissues, and in particular in specific molecular subgroups. Furthermore, mutations in HDAC4 correlate with good prognosis. Pathway analysis of genes whose expression in patients correlated strongly with HDAC4 highlighted DNA damage, p53 stabilization, and apoptosis as processes downregulated by HDAC4. This was further confirmed by silencing of HDAC4, which favored cisplatin-induced apoptosis characterized by cleavage of caspase 3 and induction of proapoptotic genes, such as BIK, in part via a p53-dependent mechanism. Altogether, these results reveal HDAC4 as a resistance factor for cisplatin in GC cells that impacts on patients’ survival.

DNA methylation and mRNA expression of imprinted genes in blastocysts derived from an improved in vitro maturation method for oocytes from small antral follicles in polycystic ovary syndrome patients.

Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients? No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts. Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity. For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts. COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3-5days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24h containing C-type natriuretic peptide), followed by an IVM step (30h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets. CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups. The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known. A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.'s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest.

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.

Acetyltransferase p300 inhibitor reverses hypertension-induced cardiac fibrosis.

Epigenetic dysregulation plays a crucial role in cardiovascular diseases. Previously, we reported that acetyltransferase p300 (ATp300) inhibitor L002 prevents hypertension‐induced cardiac hypertrophy and fibrosis in a murine model. In this short communication, we show that treatment of hypertensive mice with ATp300‐specific small molecule inhibitor L002 or C646 reverses hypertension‐induced left ventricular hypertrophy, cardiac fibrosis and diastolic dysfunction, without reducing elevated blood pressures. Biochemically, treatment with L002 and C646 also reverse hypertension‐induced histone acetylation and myofibroblast differentiation in murine ventricles. Our results confirm and extend the role of ATp300, a major epigenetic regulator, in the pathobiology of cardiac hypertrophy and fibrosis. Most importantly, we identify the efficacies of ATp300 inhibitors C646 and L002 in reversing hypertension‐induced cardiac hypertrophy and fibrosis, and discover new anti‐hypertrophic and anti‐fibrotic candidates.

Histone methyltransferase NSD2 mediates the survival and invasion of triple-negative breast cancer cells via stimulating ADAM9-EGFR-AKT signaling.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with a poor prognosis due to the lack of an effective targeted therapy. Histone lysine methyltransferases (KMTs) have emerged as attractive drug targets for cancer therapy. However, the function of the majority of KMTs in TNBC has remained largely unknown. In the current study, we found that KMT nuclear receptor binding SET domain protein 2 (NSD2) is overexpressed in TNBC tumors and that its overexpression is associated with poor survival of TNBC patients. NSD2 regulates TNBC cell survival and invasion and is required for tumorigenesis and tumor growth. Mechanistically, NSD2 directly controls the expression of EGFR and ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family that mediates the release of growth factors, such as HB-EGF. Through its methylase activity, NSD2 overexpression stimulates EGFR-AKT signaling and promotes TNBC cell resistance to the EGFR inhibitor gefitinib. Together, our results identify NSD2 as a major epigenetic regulator in TNBC and provide a rationale for targeting NSD2 alone or in combination with EGFR inhibitors as a targeted therapy for TNBC.

Abstract 3180: Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma

Abstract Disease-free survival in relapsed Ewing's Sarcoma Family of Tumors (ESFT) has not improved over the past 25 years. Current standard-of-care (SOC) agents result in 70% survival in patients with localized ESFT; however, for relapsed patients the survival rates remain between 15-20%. Approximately 85% of ESFTs have the chromosomal translocation t(11;22)(q24;q12) which encodes for the oncogenic EWS/FL1 fusion protein. The EWS/FL1 functions as a potent transcription factor leading to the dysregulated expression of genes that promote and maintain tumorigenesis. A major epigenetic regulator that is a downstream target of EWS/FL1 is the enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Notably, it is overexpressed in ESFT and maintains tumor oncogenicity by tri-methylating histone 3 lysine 27 (H3K27me3) to modulate gene expression. Genome and transcriptome data obtained by the Pediatric Precision Genomics Program at Riley Hospital for Children at Indiana University Health (IUH) indicate that EZH2 is expressed at high levels in ESFT biopsies. Additionally, other groups have reported that high levels of EZH2 protein in ESFT and other cancers correlate with increased chemoresistance to SOC therapy. We are testing tested the hypothesis that EZH2 contributes to chemoresistance in ESFT by regulating critical growth and survival genes. In addition, we are investigating if pharmacological inhibition of EZH2 will enhance sensitivity to the cytotoxic effects of SOC agents. Pediatric primary and relapsed ESFT cell lines and ESFT xenografts were validated for the EWS/FLI, EZH2, and H3K27me3 signatures. In vitro- and in vivo-pharmacodynamic studies of EZH2 inhibition via tazemetostat were conducted to optimize dosing effect. In ESFT cell lines (TC71, A673, CHLA-9, and CHLA-10), tazemetostat dose-response experiments indicated a significant reduction of H3K27me3 by one day post-treatment which was either sustained or completely blocked by 7-days post-treatment compared to vehicle treated (p<0.001). EZH2-mediated gene regulation was confirmed via qPCR. Inhibition via siRNA or tazemetostat resulted in transcriptional up-regulation of p21 (p<0.05) and down-regulation of C-MYC (p<0.01) in all ESFT cell lines. In TC71 sarcoma xenografts, 200-400 mg/kg BID was well tolerated, and decreased H3K27me3 was evident by 5 and 10 days post-treatment (n=4-5 per cohort, vehicle vs treated, p<0.001). While decreased tumor growth was evident at day 5 post-treatment, growth had recovered by day 10. In vitro assays indicate that tazemetostat potentiates SOC agents, etoposide and irinotecan (SN-38), in mediating ESFT growth inhibition (p<0.001). These data provide rationale for systematic investigation of EZH2 inhibition in combination with SOC. New information will be obtained on how to reprogram the epigenome to mitigate therapeutic resistance in ESFT that overexpress EZH2. Citation Format: Pankita H. Pandya, Barbara Bailey, Adily E. Elmi, Heather R. Bates, Courtney N. Hemenway, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, M. Reza Saadatzadeh, Harlan E. Shannon, Jixin Ding, Mark S. Marshall, Michael J. Ferguson, Lijun Cheng, Lang Li, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3180.

Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.

Abstract 482: A Novel Small Molecule Inhibitor of Acetyltransferase p300 Ameliorates Hypertension-Induced Cardiac and Renal Fibrosis

Rationale: Fibrosis is a common pathophysiological manifestation of chronically injured or stress-affected organs. Additionally, cardiac and renal fibrosis remain leading causes of global morbidity and mortality. Meanwhile, no effective therapy is available to halt the onset and progression of cardiac and renal fibrogenesis. We have previously demonstrated that p300 with intrinsic factor acetyltransferase (FATp300) activity is an essential epigenetic regulator of profibrogenic signal-induced matrix protein synthesis. Objective: Here, we attempted to delineate the therapeutic efficacy of a small molecule inhibitor of FATp300, L002, in blocking i) TGF-b-induced profibrogenic processes in cardiac and renal cells in vitro, and ii) Angiotensin II-induced cardiac and renal fibrogenesis in mice. Methods and Results: Analysis of cardiac and renal cells demonstrates that L002 inhibits specific histone acetylation and blunts TGF-b induced profibrogenic processes, including cellular proliferation, migration, myofibroblast differentiation and extracellular matrix protein synthesis. The mRNA analysis further indicates that matrix protein, collagen, and myofibroblast marker, a-SMA are inhibited at the transcriptional level. L002 also prevents TGF-b-induced FATp300 upregulation suggesting that TGF-b-induced FATp300 expression is partly self-regulated. However, TGF-b-induced Smad activation is unaltered in the presence of L002, demonstrating its target specificity. Further, mice infused with Angiotensin II show increased blood pressure, cardiac hypertrophy and increased heart weight. Conversely, co-administration of L002 reduces Angiotensin II-induced cardiac hypertrophy and left ventricular wall thickness. Most importantly, L002 administration ameliorates Angiotensin II-induced cardiac and renal fibrosis in mice. Conclusions: Taken together this study demonstrates that specific pharmacological inhibition of elevated factor acetyltransferase activity of p300, a major epigenetic regulator, is an ideal approach to control hypertension- or other stress-induced cardiac and renal fibrogenesis as seen in a wide variety of cardiovascular and renal diseases.