Histone Methyltransferase Inhibitor Research Articles

Two‐Dimensional Transition Metal Dichalcogenides Trigger Trained Immunity in Human Macrophages through Epigenetic and Metabolic Pathways

Trained immunity is a recently described phenomenon whereby cells of the innate immune system undergo long-term epigenetic and/or metabolic reprogramming following a short-term interaction with microbes or microbial products. Here, it is shown that 2D transition metal dichalcogenides (TMDs) trigger trained immunity in primary human monocyte-derived macrophages. First, aqueous dispersions of 2D crystal formulations of MoS2 and WS2 are tested, and no cytotoxicity is found despite avid uptake of these materials by macrophages. However, when macrophages are pre-exposed to TMDs, followed by a resting period, this causes a marked modulation of immune-specific gene expression upon subsequent challenge with a microbial agent (i.e., bacterial lipopolysaccharides). Specifically, MoS2 triggers trained immunity through an epigenetic pathway insofar as the histone methyltransferase inhibitor methylthioadenosine reverses these effects. Furthermore, MoS2 triggers an elevation of cyclic adenosine monophosphate (cAMP) levels in macrophages and increased glycolysis is also evidenced in cells subjected to MoS2 training, pointing toward a metabolic rewiring of the cells. Importantly, it is observed that MoS2 triggers the upregulation of Mo-dependent enzymes in macrophages, thus confirming that Mo is bioavailable in these cells. In conclusion, MoS2 is identified as a novel inducer of trained immunity. Thus, TMDs could potentially be harnessed as immunomodulatory agents.

Selective histone methyltransferase G9a inhibition reduces metastatic development of Ewing sarcoma through the epigenetic regulation of NEU1

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.

Enhanced Cytotoxic Effects in Human Oral Squamous Cell Carcinoma Cells Treated with Combined Methyltransferase Inhibitors and Histone Deacetylase Inhibitors.

Combined treatment of human oral squamous cell carcinoma (OSCCs) with DNA methyltransferase inhibitors (DNMTis), histone methyltransferase inhibitors (HMTis), and histone deacetylase inhibitors (HDACis), and the molecular mechanisms underlying their anticancer effects, have not been fully elucidated. Herein, we investigated the cytotoxic effects of combined DNMTis (5-Aza-deoxycytidine: 5-Aza-dC, RG108), HMTis (3-deazaneplanocin A: DZNep), and HDACis (trichostatin A: TSA) treatment on human OSCC cells and explored their molecular mechanisms. Combined 5-Aza-dC, or RG108, and TSA treatment significantly decreased HSC-2 and Ca9-22 cell viability. Combinatorial DZNep and TSA treatment also decreased Ca9-22 cell viability. Although caspase 3/7 activation was not observed in HSC-2 cells following combined treatment, caspase activity was significantly increased in Ca9-22 cells treated with DZNep and TSA. Moreover, combined treatment with 5-Aza-dC, RG108, and TSA increased the proportion of HSC-2 and Ca9-22 cells in the S and G2/M phases. Meanwhile, increased phosphorylation of the histone variant H2A.X, a marker of double-stranded DNA breaks, was observed in both cells after combination treatment. Hence, the decreased viability induced by combined treatment with epigenomic inhibitors results from apoptosis and cell cycle arrest in S and G2/M phases. Thus, epigenomic therapy comprising combined low concentrations of DNMTi, HMTi, and HDACi is effective against OSCC.

TSLP regulates mitochondrial ROS-induced mitophagy via histone modification in human monocytes

BackgroundThymic stromal lymphopoietin (TSLP) is a Th2-like cytokine involved in asthma pathogenesis. Excessive reactive oxygen species (ROS) production can lead to airway inflammation, hyperresponsiveness and remodeling. Mitophagy, followed by ROS production, is the selective degradation of mitochondria by autophagy and often occurs in defective mitochondria. In the present study, we aimed to examine the effects of TSLP on ROS production and mitophagy in human monocytes and to investigate the underlying mechanisms, including epigenetic regulation.ResultsTSLP induced ROS generation, and the effects were reversed by the antioxidant N-acetylcysteine (NAC) in THP-1 cells. Transmission electron microscopy images showed donut-shaped mitochondria that lost the cristae ultrastructure after TSLP stimulation. A decrease in mitochondrial membrane potential, decreased MTCO2 expression, and increased mitochondrial DNA release after TSLP stimulation were found. TSLP enhanced mitochondrial complex I and complex II/III activity and increased mitochondrial copy numbers and the expression of the complex II SHDA gene. TSLP-induced SHDA expression was inhibited by the histone acetyltransferase inhibitor anacardic acid (AA) and the histone methyltransferase inhibitor methylthioadenosine (MTA), and chromatin immunoprecipitation assays revealed that TSLP enhanced H3 acetylation, H4 acetylation, and H3K4 and H3K36 trimethylation in the SHDA promoter. Confocal laser microscopy showed that TSLP treatment increased the signals of the mitophagy-related proteins PINK1, LC3, phospho-parkin and phospho-ubiquitin, and pretreatment with AA and MTA reduced TSLP-induced PINK1 and LC3 accumulation in mitochondria. Western blot analysis showed that TSLP significantly increased phosphor-AMPK signal intensity, and the effects were inhibited by the antioxidant NAC. The increased signal intensities of the mitophagy-related proteins PINK1, Parkin and LC3 I/II were decreased by dorsomorphin, an AMPK inhibitor. TSLP decreased M1-related cytokine CXCL-10 production and increased M2-related cytokine CCL-1 and CCL-22 production, which was suppressed by the mitophagy inhibitor Mdivi-1 and PINK1 gene knockdown.ConclusionsEpithelial-derived TSLP regulates ROS production and mitophagy through AMPK activation and histone modification and alters M1/M2 chemokine expression in human monocytes.Graphical

BRD4770 functions as a novel ferroptosis inhibitor to protect against aortic dissection

Smooth muscle cell (SMC) loss is the characteristic feature in the pathogenesis of aortic dissection (AD), and ferroptosis is a novel iron-dependent regulated cell death driven by the excessive lipid peroxidation accumulation. However, whether targeting ferroptosis is an effective approach for SMC loss and AD treatment remains unclear. Here, we found that the iron level, ferroptosis-related molecules TFR, HOMX1, ferritin and the lipid peroxidation product 4-hydroxynonenal were increased in the aorta of AD. Then, we screened several inhibitors of histone methyltransferases and found that BRD4770 had a protective effect on cystine deprivation-, imidazole ketone erastin- or RSL3-induced ferroptosis of SMCs. The classic ferroptosis pathways, System Xc--GPX4, FSP1-CoQ10 and GCH1-BH4 pathways which were inhibited by ferroptosis inducers, were re-activated by BRD4770 via inhibiting mono-, di- and tri- methylated histone H3 at lysine 9 (H3K9me1/2/3). RNA-sequencing analysis revealed that there was a positive feedback regulation between ferroptosis and inflammatory response, and BRD4770 can reverse the effects of inflammation activation on ferroptosis. More importantly, treatment with BRD4770 attenuated aortic dilation and decreased morbidity and mortality in a β-Aminopropionitrile monofumarate-induced mouse AD model via inhibiting the inflammatory response, lipid peroxidation and ferroptosis. Taken together, our findings demonstrate that ferroptosis is a novel and critical pathological mechanism that is involved in SMC loss and AD development. BRD4770 is a novel ferroptosis inhibitor and has equivalent protective effect to Ferrostatin-1 at the optimal concentration. Translating insights into the anti-ferroptosis effects of BRD4770 may reveal a potential therapeutic approach for targeting SMC ferroptosis in AD.

Vitamin C promotes anti-leukemia of DZNep in acute myeloid leukemia

The epigenetic treatment by 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, shows great potential against acute myeloid leukemia (AML). However, the variant sensitivity and incomplete response to DZNep are commonly observed. Here, we reveal that vitamin C (Vc) dramatically promotes DZNep response against leukemic cells in different cell lines and primary AML samples. Vc enhances apoptosis and differentiation induced by DZNep in different AML cell lines in vitro and reduces leukemia progression in vivo. At the molecular level, Vc downregulates an enzyme of serine synthesis named D-3-phosphoglycerate dehydrogenase (PHGDH), as well as BCL2, an anti-apoptotic gene. Over-expression of PHGDH reverses the Vc-enhanced anti-leukemic effect of DZNep in AML cells. Therefore, our findings provide an effective approach to reduce the resistance against epigenetic treatment by Vc, which shows a potential improvement of their combination in AML patients.

The Role of Oxidative Stress in Epigenetic Changes Underlying Autoimmunity.

Significance: Epigenetic dysregulation plays an important role in the pathogenesis and development of autoimmune diseases. Oxidative stress is associated with autoimmunity and is also known to alter epigenetic mechanisms. Understanding the interplay between oxidative stress and epigenetics will provide insights into the role of environmental triggers in the development of autoimmunity in genetically susceptible individuals. Recent Advances: Abnormal DNA and histone methylation patterns in genes and pathways involved in interferon and tumor necrosis factor signaling, cellular survival, proliferation, metabolism, organ development, and autoantibody production have been described in autoimmunity. Inhibitors of DNA and histone methyltransferases showed potential therapeutic effects in animal models of autoimmune diseases. Oxidative stress can regulate epigenetic mechanisms via effects on DNA damage repair mechanisms, cellular metabolism and the local redox environment, and redox-sensitive transcription factors and pathways. Critical Issues: Studies looking into oxidative stress and epigenetics in autoimmunity are relatively limited. The number of available longitudinal studies to explore the role of DNA methylation in the development of autoimmune diseases is small. Future Directions: Exploring the relationship between oxidative stress and epigenetics in autoimmunity will provide clues for potential preventative measures and treatment strategies. Inception cohorts with longitudinal follow-up would help to evaluate epigenetic marks as potential biomarkers for disease development, progression, and treatment response in autoimmunity. Antioxid. Redox Signal. 36, 423-440.

Eradication of leukemia stem cells by inhibitors of DNA methyltransferase, EZH2 and G9a histone methyltransferases

Complete eradication of leukemic stem cells (LSCs) in patients with acute myeloid leukemia (AML) is required for curative therapy. Epigenetic alterations that involve gene-silencing by DNA methylation by DNMT1, methylation of H3K27 by EZH2 histone methyltransferase (HMT) and methylation of H3K9 by G9a HMT may play a major role in the development of AML. The major action of these epigenetic alterations is the silencing of the genes that program differentiation of AML cells. Inhibitors of DNA and histone methylation have the potential to reverse this block in differentiation. If tumor suppressor genes (TSGs) contain two gene-silencing markers, such as DNA methylation and H3K27me3, they may not be fully reactivated with only an inhibitor of DNA methylation, such as 5-aza-2’-deoxycytidine (5-AZA-CdR), but may also require an inhibitor of EZH2. In support of this model is the synergistic antileukemic action as shown by a colony assay on AML cells using 5-AZA-CdR in combination with 3-deazaneplanocin A (DZNep), a potent inhibitor of EZH2. A similar type of interaction can occur when TSGs are silenced by DNA methylation and the G9a methylation of H3K9me2, a second gene-silencing marker. Treatment of these AML cells with 5-AZA-CdR and BIX01294, an inhibitor of G9a, also results in a synergistic antileukemic action. Leukemic cells that contain 3 different gene-silencing markers: DNA methylation, H3K27me3 and H3K9me2 may require 3 different inhibitors for maximal antineoplastic activity. This result was observed when the AML cells were treated in with 5-AZA-CdR, DZNep and BIX01294. The aim of this study was to demonstrate that epigenetic agents that target DNA and histone methylation have remarkable antineoplastic activity against myeloid leukemia cells. The second aim was to propose a dose-schedule for these epigenetic agents that can be evaluated in a clinical trial in patients with advanced AML for its potential to eradicate LSCs. One of the most sensitive targets for chemotherapeutic intervention in LSCs is the block in differentiation due to gene-silencing by DNA and histone methylation. Epigenetic agents that have the potential to reverse this block merit clinical investigation with high priority.

Screening for Small-Molecule Inhibitors of Histone Methyltransferases.

Potent and highly selective small-molecule inhibitors are needed to unravel the biological complexities of histone methyltransferases and to reveal their therapeutic potential. A prerequisite to developing these inhibitors is the identification of validated chemical matter for initiating a medicinal chemistry campaign. For the most part, finding these initial starting points occurs through screening of large, unbiased compound libraries. The size and nature of these libraries, coupled with the complexities of the bisubstrate utilizing histone methyltransferases, necessitates that the primary screen and subsequent hit triage be carefully considered.In this chapter, using EZH2 as a representative example, we describe a screening and hit triage campaign that identified validated chemical matter allowing initiation of medicinal chemistry studies. Moreover, we discuss a cell-based assay to support lead identification and optimization. The approach described here entailing a mixture of biochemical, biophysical and cell-based assays should be applicable to identifying validated starting points for other histone methyltransferases.

A novel screening strategy to identify histone methyltransferase inhibitors reveals a crosstalk between DOT1L and CARM1.

Epigenetic regulation is a dynamic and reversible process that controls gene expression. Abnormal function results in human diseases such as cancer, thus the enzymes that establish epigenetic marks, such as histone methyltransferases (HMTs), are potentially therapeutic targets. Noteworthily, HMTs form multiprotein complexes that in concert regulate gene expression. To probe epigenetic protein complexes regulation in cells, we developed a reliable chemical biology high-content imaging strategy to screen compound libraries simultaneously on multiple histone marks inside cells. By this approach, we identified that compound 4, a published CARM1 inhibitor, inhibits both histone mark H3R2me2a, regulated also by CARM1, and H3K79me2, regulated only by DOT1L, pointing out a crosstalk between CARM1 and DOT1L. Based on this interaction, we combined compound 4 and DOT1L inhibitor EPZ-5676 resulting in a stronger inhibition of cell proliferation and increase in apoptosis, indicating that our approach identifies possible effective synergistic drug combinations.

Small molecule targeting of chromatin writers in cancer.

More than a decade after the launch of DNA methyltransferase and histone deacetylase inhibitors for the treatment of cancer, 2020 heralded the approval of the first histone methyltransferase inhibitor, revitalizing the concept that targeted manipulation of the chromatin regulatory landscape can have profound therapeutic impact. Three chromatin regulatory pathways-DNA methylation, histone acetylation and methylation-are frequently implicated in human cancer but hundreds of potentially druggable mechanisms complicate identification of key targets for therapeutic intervention. In addition to human genetics and functional screening, chemical biology approaches have proven critical for the discovery of key nodes in these pathways and in an ever-increasing complexity of molecularly defined human cancer contexts. This review introduces small molecule targeting approaches, showcases chemical probes and drug candidates for epigenetic writer enzymes, illustrates molecular features that may represent epigenetic dependencies and suggests translational strategies to maximize their impact in cancer therapy.

BIX01294 inhibits EGFR signaling in EGFR-mutant lung adenocarcinoma cells through a BCKDHA-mediated reduction in the EGFR level

BIX01294 (BIX), an inhibitor of the G9a histone methyltransferase, has been reported to have antitumor activity against a variety of cancers. However, the molecular mechanisms underlying its anticancer effects, particularly those against lung cancer, remain unclear. Here, we report that BIX induces apoptotic cell death in EGFR-mutant non-small cell lung cancer (NSCLC) cells but not in their wild-type counterparts. Treatment with BIX resulted in a significant reduction in the EGFR level and inhibition of EGFR signaling only in EGFR-mutant NSCLC cells, leading to apoptosis. BIX also inhibited mitochondrial metabolic function and decreased the cellular energy levels that are critical for maintaining the EGFR level. Furthermore, BIX transcriptionally downregulated the transcription of branched-chain α-keto acid dehydrogenase (BCKDHA), which is essential for fueling the tricarboxylic acid (TCA) cycle. Interestingly, this BCKDHA downregulation was due to inhibition of Jumanji-domain histone demethylases but not the G9a histone methyltransferase. We observed that KDM3A, a Jumonji histone demethylase, epigenetically regulates BCKDHA expression by binding to the BCKDHA gene promoter. BIX exposure also led to a significant decrease in the EGFR level, causing apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Taken together, our current data suggest that BIX triggers apoptosis only in EGFR-mutant NSCLC cells via inhibition of BCKDHA-mediated mitochondrial metabolic function.

Venetoclax and Epigenetic Modifiers: Promising Novel Combinations for the Treatment of Multiple Myeloma

Multiple Myeloma (MM) is a haematological malignancy characterised by clonal proliferation of plasma cells within the bone marrow (Landgren and Weiss, 2009). Unfortunately, despite the major improvements to the treatment of MM within the last decade, it remains an incurable disease. Therefore, novel innovative combinations of less toxic therapies are warranted, especially for elderly patients with relapsed/refractory disease.The anti-apoptotic BCL-2 family of proteins (BCL-2, BCL-XL and MCL-1) are critical regulators of the intrinsic apoptotic pathway and determine the survival of human MM cells (Letai et al., 2004, Del Gaizo Moore et al., 2008). Recently, Venetoclax, a selective BCL-2 inhibitor, was FDA approved for the treatment of chronic lymphocytic leukaemia (CLL) and acute mylogenous leukemia (AML), in combination with demethylating agents (Roberts et al., 2016, DiNardo et al., 2018, DiNardo et al., 2019) It is known that some MM patients with t(11;14) have a good response to combination treatment with venetoclax, however certain patients who do not have t(11;14) also respond to venetoclax. Therefore, a biomarker for response is urgently required in MM, as it has heterogeneous anti-apoptotic dependencies. In AML, venetoclax is combined with the epigenetic modifier 5-azacytidine. Highlighting, that screening for epigenetic modifier's, maybe a useful approach to identify synergistic combination of treatments with venetoclax in MM.Methods:To assess anti-apoptotic protein dependence in MM cell lines (JJN3, RPMI-8226, KMS18, MM1S, U266) and primary patient samples, BH3 profiling was used. Briefly, cells are exposed to a series of BH3-only peptides (20-23 mer in length) following gentle permeabilisation of cell membrane with low concentrations of digitonin. The loss of mitochondrial potential (JC-1) or the release of cytochrome c (cytochrome-c-FITC antibody) was assessed by plate reader or by flow cytometry. Cell death was assessed by Annexin V/ propidium iodide staining by flow cytometry. Using primary patient samples, CD138 + cells were isolated using the Miltenyi MAC sorter. For the epigenetic screen cell viability was assessed by CellTiter-Glo® and death was then confirmed by Annexin V/Pi staining.ResultsBH3 profiling was used to assess anti-apoptotic dependence in a panel of five MM cell lines. It is a functional assay that interrogates BCL-2 protein interactions using synthetic BH3 peptides to measure the loss of mitochondrial membrane potential. The BH3 profiling was correlated to the response of the cell lines to a series of BH3 mimetics : venetoclax (selective BCL-2 inhibitor), ABT-263 (BCL-2, BCL-XL and BCL-W inhibitor), WEHI-539 (BCL-XL inhibitor) and AMG-176 (MCL-1 inhibitor). This data highlighted that BH3 profiling is a powerful tool for identifying anti-apoptotic dependnece in MM. It also showed and that there is a diverse anti-apoptotic dependence in MM cell lines and primary patient samples. Remarkably, one patient with plasma cell leukemia showed BCL-2 dependence by BH3 profiling, a t(11:14) translocation and a sustained in-vivo single agent response to venetoclax (Glavey et al., 2020)..Next, we performed an unbiased epigenetic modifier screen in two MM cell lines JJN3 (MCL-1 dependent cell lines) and KMS-18 (mixed anti-apoptotic dependence) to induce BCL-2 dependence and sensitivity to venetoclax. The screen included the following classes of epigenetic modifiers: histone deacetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors and histone acetylase inhibitors. Interestingly, two classes of epigenetic drugs were synergistic with venetoclax in three different MM cell lines (CI <0.8). The combination was also validated by dynamic BH3 profiling in cell lines MM patient samples ex-vivo. Further work is required, in the form of a clinical trial, to validate our hypothesis that this provides a highly effective novel combination therapy option for myeloma patients.Conclusion.BH3 profiling is a powerful tool to identify the anti-apoptotic dependence in MM cell lines and MM patient samples, which we can exploit pharmacologically to kill MM cells in a personalised medicine approach. Combining epigenetic modifiers with venetoclax induces BCL-2 dependence in MM and enhances response to treatment. [Display omitted] DisclosuresFlanagan: AbbVie: Research Funding. O'Dwyer: Bristol Myers Squibb: Research Funding; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Quinn: Takeda: Honoraria. Glavey: Janssen: Honoraria, Research Funding; Celgene and BMS company: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Ni Chonghaile: AbbVie: Research Funding.

Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos.

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

P-094: ABT-199 and epigenetic modifiers: promising novel combinations for the treatment of Multiple Myeloma

Multiple Myeloma (MM) is a malignancy of the antibody-producing plasma cells. Despite improvements to treatment throughout the years, it remains an incurable and fatal disease [1]. Therefore, novel innovative therapies are needed for relapsed/refractory MM. The anti-apoptotic BCL-2 family of proteins (BCL-2, BCL-XL and MCL-1) are critical regulators of the intrinsic apoptotic pathway and determine the survival of human MM cells [2,3]. The anti-apoptotic BCL-2 proteins represent attractive therapeutic targets in MM. Recently, ABT-199, a selective BCL-2 inhibitor, was FDA approved for the treatment of CLL and AML. The aim of the study is to develop a biomarker to identify MM patients that are reliant on BCL-2 and could be treated with ABT-199. To assess anti-apoptotic dependence, we used BH3 profiling, a functional assay that interrogates BCL-2 protein interactions using synthetic BH3 peptides to measure the loss of mitochondrial membrane potential. We screened a panel of BH3 mimetics in MM cells and patient bone marrow samples; ABT-199 (selective BCL-2 inhibitor), ABT-263 (BCL-2, BCL-xL and BCL-W inhibitor), WEHI-539 (BCL-XL inhibitor) and AMG-176 (selective MCL-1 inhibitor). The BH3 profile data and BH3 mimetics sensitivity data revealed that there is a diverse anti-apoptotic dependence in MM cell lines and primary patient samples. In addition, we aim to identify novel combination treatments that will induce BCL-2 dependence in MM cells, enhancing sensitivity to ABT-199 treatment. We performed a small molecule screen to identify epigenetic modifiers that could induce BCL-2 dependence in two MM cell lines. The screen included the following classes of epigenetic drugs: histone deacetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors and BET inhibitors. Interestingly, two classes of the epigenetic drugs, were synergistic with ABT-199 in three MM cell lines, (CI <0.8). Furthermore, we confirmed enhanced cell death by Annexin V/PI staining following both ABT-199 and the two classes of epigenetic drugs in two patient MM samples ex-vivo. Future work will focus on determining the mechanism of enhanced cell death induced by the epigenetic drugs to improve the response to ABT-199 treatment.

Chaetocin enhances callus induction by decreasing the expression of major leaf polarity genes in Nicotiana tabacum

Using chemicals known as inhibitors of chromatin remodeling enzymes is a useful approach in understanding the regulation machinery that contributes to the maintenance of or conversion to a pluri- and totipotent states of the cells in multicellular organisms. They can serve as tools to reveal the molecular and cellular mechanisms required for reprogramming. A deeper understanding of reprogramming pathways that drive the cell into a dedifferentiated state might help us enhance plant micropropagation and green biotechnology applications. Chaetocin is one of the histone methyltransferase inhibitors known as key drivers in several cellular processes. It was shown that chaetocin enhanced the reprogramming of human fibroblasts. Here, by using an analogy approach, we tested the effects of chaetocin on the postgermination growth and callus induction in Nicotiana tabacum for the first time. We found that chaetocin retarded the leaf development in N. tabacum seedlings but increased the callus formation when applied in the post-germination growth phase (1.4 fold). The expression of AS1, KAN1, YAB3, FIL, major leaf polarity genes, significantly decreased when seeds were treated with Ch during the post-germination growth phase by 69, 45, 46, 68%, respectively. We also observed that chaetocin could not replace 2,4-Dichlorophenoxyacetic acid as a callus inducing modulator. The present study is a pioneer, exhibiting valuable data to understand the molecular mechanisms underlying the effects of chaetocin and the unknown properties of histone-modifying enzymes. Hence, novel approaches can be developed by using high throughput methods to elucidate epigenetic mechanisms of cellular differentiation/dedifferentiation processes in plant systems.

The progress of research on histone methylation in ischemic stroke pathogenesis.

Stroke, also known as cerebral stroke or cerebrovascular accident, refers to acute ischemic or hemorrhagic encephalopathy caused by a disturbance to cerebral blood flow. Ischemic stroke is the most common type of cerebral stroke, accounting for approximately 80% of the total incidence of clinical stroke. High morbidity, disability, and mortality rates place heavy burdens on the families of patients and society. An increasing number of studies have shown that histone modification plays an important role in the pathogenesis of ischemic stroke, but most studies on histone modification focus on acetylation, and studies on the role of histone methylation in the pathogenesis of ischemic stroke are limited. Here, we review the role of histone methylation and related histone methyltransferase (HMT) inhibitors in the pathogenesis of ischemic stroke and related HMT inhibitors in the treatment of ischemic stroke, which may open up a new avenue to the study of ischemic stroke.

H3K9me2 regulation of BDNF expression in the hippocampus and medial prefrontal cortex is involved in the depressive-like phenotype induced by maternal separation in male rats.

Early life stress (ELS) induces a depressive-like phenotype and increases the risk of depression. Brain-derived neurotrophic factor (BDNF) has been confirmed to be involved in the pathophysiology of depression. However, the mechanism by which ELS alters the epigenetic regulation of BDNF and changes susceptibility to depression has not been fully clarified. The present study used maternal separation (MS) and chronic unpredicted mild stress (CUMS) to establish an MS animal model and a depressive animal model. We assessed depressive-like behaviours, including anhedonia, locomotor activity, anxiety-like behaviour, and spatial memory, using the sucrose preference test, the open field test, the elevated plus maze test, and the Morris water maze test. We also investigated BDNF and H3K9me2 expression in the hippocampus and medial prefrontal cortex (mPFC) by immunohistochemistry, western blotting, and qPCR analysis. Additionally, we used Unc0642, a small molecule inhibitor of histone methyltransferase (G9a), as an intervention. The results showed that CUMS induced depressive-like behaviours in rats and resulted in increased H3K9me2 expression and decreased BDNF expression in the hippocampus and mPFC. More importantly, adult MS rats experiencing CUMS had more severe depressive behaviours, had higher expression of H3K9me2 in the hippocampus and mPFC, and had lower expression of BDNF in the hippocampus and mPFC. In addition, administration of the G9a inhibitor reversed most of the changes. Our study suggests that ELS changed BDNF and H3K9me2 expression in the rat brain, resulting in a depressive-like phenotype.

Abstract 2066: Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer

Abstract Defective DNA damage response (DDR) is a hallmark of high grade serous ovarian cancer (HGSOC). Genetic and epigenomic aberrations in DDR pathways including homologous recombination (BRCA1/BRCA2) and non-homologous end joining (PARP-1) make some HGSOC vulnerable to cisplatin and Poly (ADP-ribose) polymerase inhibitors (PARPi). However, mechanisms behind primary and acquired resistance to these agents remain unknown. We hypothesise aberrant epigenomic changes at DDR-associated genes contribute to PARPi resistance and can be reversed using a novel histone methyltransferase inhibitor (HKMT-1-005) which targets two histone methyltransferases (EZH2 and EHMT2) frequently upregulated in HGSOC. MTS assays were used to calculate the IC50 values for HKMT-1-005 and the PARPi Rucaparib in a panel of human HGSOC cell lines (n=8). Isobologram analysis using Combenefit software was used to determine synergy. RNA-seq and ATAC-seq was performed on cell lines derived from the same patient before (PEO1) and after acquired resistance to chemotherapy (PEO4) pre/post treatment with HKMT-1-005. Pathway analysis was used to identify pathways enriched in genes with altered expression and/or chromatin conformation after treatment with HKMT-1-005 and validated with qRT-PCR. Synergy between HKMT-1-005 and Rucaparib and changes in chromatin conformation with HKMT-1-005 treatment were further examined in CRIPSR modified ID8 murine ovarian cell lines with the genotypes: WT, P53-/-, P53-/-BRCA1-/- and P53-/-BRCA2-/-. Isobologram analysis revealed HKMT-1-005 in combination with Rucaparib was synergistic in multiple cell lines. Synergy was greater in PEO1 compared to PEO4. RNA-seq and ATAC-seq identified a significant (FDR &amp;lt;0.05) enrichment of genes associated with DDR pathways which had altered expression and/or chromatin accessibility in PEO1 compared to PEO4, including BRCA1 and BRCA2. qRT-PCR analysis confirmed treatment with HKMT-1-005 reduced expression of BRCA1 and BRCA2 in PEO1 but not PEO4. Synergy between HKMT-1-005 and Rucaparib was greater in P53-/-BRCA1-/- and P53-/-BRCA2-/- compared to WT or P53-/- ID8 mouse cell lines. ATAC-seq identified multiple DDR-associated genes had significantly (FDR &amp;lt;0.05) altered chromatin accessibility in P53-/-BRCA2-/- compared with P53-/- alone. HKMT-1-005 treatment in ID8 cell lines revealed changes in chromatin at multiple DDR-associated genes including BRCA2. HKMT-1-005 is synergistic with the PARP inhibitor Rucaparib and can modulate the expression and chromatin conformation of DDR-associated genes in multiple human and mouse ovarian cell lines. Depletion of BRCA2 may additionally regulate chromatin conformation of genes including those associated with DDR pathways and explain why greater synergy between HKMT-1-005 and Rucaparib was observed in BRCA deficient lines. Citation Format: Ian M. Garner, Zhuyan Su, Sawyer Hu, Yue Wu, Iain A. McNeish, Matthew J. Fuchter, Robert Brown. Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer [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 2066.

Abnormal H3K27 histone methylation of RASA1 gene leads to unexplained recurrent spontaneous abortion by regulating Ras-MAPK pathway in trophoblast cells.

Some studies suggest that the inactivation of the Ras-MAPK pathway in trophoblast cells can lead to recurrent abortion, but the molecular mechanism underlying the inactivation of this pathway in trophoblast cells is still unclear. This study aimed to explore the relationship between the mechanism of abnormal activation of RASA1, a regulatory protein of the Ras-MAPK pathway, and unexplained recurrent spontaneous abortion. RT-qPCR was used to detect the transcription levels of RASA1 gene. Immunohistochemistry and Western blot were used to detect the expression levels of the RASA1, Raf and MEK proteins. CCK-8, TUNEL and Transwell assays were used to detect the proliferative, apoptotic, and invasive capacities of HTR-8/SVneo cells. ChIP assays were used to detect the enrichment of H3K27me3 in RASA1 gene promoter. Abortion villi experiments showed that the enrichment of H3K27me3 in the RASA1 gene promoter was reduced, and that both RASA1 gene transcription and RASA1 protein expression were increased. Cell experiments confirmed that RASA1 could decrease the phosphorylated Raf and MEK proteins, inhibit the proliferation and invasion ability, and promote the apoptosis ability of HTR-8/SVneo cells. It was also found that the proliferation and invasion ability as well as the Ras-MAPK pathway activity of HTR-8/SVneo cells were inhibited when treated with histone methyltransferase inhibitor DZNep. RASA1 gene was abnormally activated in unexplained recurrent spontaneous abortion villi due to the decreased enrichment of H3K27me3 in the gene promoter. High expression of RASA1 could inhibit the activity of the Ras-MAPK pathway, and thus inhibit the proliferation and invasion ability of trophoblast cells.