Modifications Of Histone Proteins Research Articles

Novel histone modifications in microglia derived from a mouse model of chronic pain

As the resident immune cells in the central nervous system, microglia play an important role in the maintenance of its homeostasis. Dysregulation of microglia has been associated with the development and maintenance of chronic pain. However, the relevant molecular pathways remain poorly defined. In this study, we used a mass spectrometry-based proteomic approach to screen potential changes of histone protein modifications in microglia isolated from the brain of control and cisplatin-induced neuropathic pain adult C57BL/6J male mice. We identified several novel microglial histone modifications associated with pain, including statistically significantly decreased histone H3.1 lysine 27 mono-methylation (H3.1K27me1, 54.8% of control) and H3 lysine 56 tri-methylation (7.5% of control), as well as a trend suggesting increased H3 tyrosine 41 nitration. We further investigated the functional role of H3.1K27me1 and found that treatment of cultured microglial cells for 4 consecutive days with 1-10μM of NCDM-64, a potent and selective inhibitor of lysine demethylase 7A, an enzyme responsible for the demethylation of H3K27me1, dose-dependently elevated its levels with a greater than a two-fold increase observed at 10μM compared to vehicle-treated control cells. Moreover, pretreatment of mice with NCDM-64 (10 or 25mg/kg/day, i.p.) prior to cisplatin treatment prevented the development of neuropathic pain in mice. The identification of specific chromatin marks in microglia associated with chronic pain may yield critical insight into the contribution of microglia to the development and maintenance of pain, and opens new avenues for the development of novel nonopioid therapeutics for the effective management of chronic pain.

POS-404 LINC00355 MEDIATES CTNNBIP1 PROMOTOR METHYLATION AND PROMOTES ER STRESS-INDUCED PODOCYTE INJURY IN DIABETIC NEPHROPATHY

Podocyte injury is a crucial feature of many glomerular disease,including diabetic nephropathy (DN) which is one of the most common microvascular complications of diabetes mellitus and the main cause of death and disability in diabetic patients. Recently,a series of studies have shed lighti on the role of epigenetic changes such as histone protein modifications played in podocyte injury of glomerular disease.Endoplasmic reticulum stress (ER stress) plays an important role in podocyte injury in diabetic nephropathy.

On-going consequences of in utero exposure of Pb: An epigenetic perspective.

Epigenetic modifications by toxic heavy metals are one of the intensively investigated fields of modern genomic research. Among a diverse group of heavy metals, lead (Pb) is an extensively distributed toxicant causing an immense number of abnormalities in the developing fetus via a wide variety of epigenetic changes. As a divalent cation, Pb can readily cross the placental membrane and the fetal blood brain barrier leading to far-reaching alterations in DNA methylation patterns, histone protein modifications, and micro-RNA expression. Over recent years, several human cohorts and animal model studies have documented hypermethylation and hypomethylation of developmental genes along with altered DNA methyl-transferase expression by in utero Pb exposure in a dose-, duration-, and sex-dependent manner. Modifications in the expression of specific histone acetyltransferase enzymes along with histone acetylation and methylation levels have been reported in rodent and murine models. Apart from these, down-regulation and up-regulation of certain microRNAs crucial for fetal development have been shown to be associated with in utero Pb exposure in human placenta samples. All these modifications in the developing fetus during the prenatal and perinatal stages reportedly caused severe abnormalities in early or adult age, such as impaired growth, obesity, autism, diabetes, cardiovascular diseases, risks of cancer development, and Alzheimer's disease. In this review, currently available information on Pb-mediated alterations in the fetal epigenome is summarized. Further research on Pb-induced epigenome modification will help to understand the mechanisms in detail and will enable us to formulate safety guidelines for pregnant women and developing children.

Epigenetic Modifications in Tumor-Associated Macrophages: A New Perspective for an Old Foe.

Tumorigenesis is frequently accompanied by chronic inflammation, and the tumor microenvironment (TME) can be considered an ecosystem that consists of tumor cells, endotheliocytes, fibroblasts, immune cells and acellular components such as extracellular matrix. For tumor cells, their survival advantages are dependent on both genetic and epigenetic alterations, while other cells mainly present epigenetic modifications. Macrophages are the most plastic type of immune cells and undergo diverse epigenetic alterations in the TME. Some of these epigenetic modifications mitigate against cancer progression, and others accelerate this process. Due to the complex roles of macrophages in the TME, it is urgent to understand their epigenetic modifications associated with the TME. Here, we mainly summarize recent findings on TME-associated epigenetic alterations of tumor-associated macrophages (TAMs), including DNA methylation, posttranslational modifications of histone proteins, chromatin remodeling, and noncoding RNA-mediated epigenetic regulation. At the end of this review, we also discuss the translational potential of these epigenetic modifications for developing novel cancer therapies targeting TAMs.

Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry.

Chemical modification of histone proteins by methylation plays a central role in chromatin regulation by recruiting epigenetic "readers" via specialized binding domains. Depending on the degree of methylation, the exact modified amino acid, and the associated reader proteins histone methylations are involved in the regulation of all DNA-based processes, such as transcription, DNA replication, and DNA repair. Here we present methods to identify histone methylation readers using a mass spectrometry-linked nucleosome affinity purification approach. We provide detailed protocols for the generation of semisynthetic methylated histones, their assembly into biotinylated nucleosomes, and the identification of methylation-specific nucleosome-interacting proteins from nuclear extracts via nucleosome pull-downs and label-free quantitative proteomics. Due to their versatility, these protocols allow the identification of readers of various histone methylations, and can also be adapted to different cell types and tissues, and other types of modifications.

A VIN3-like Protein OsVIL1 Is Involved in Grain Yield and Biomass in Rice.

In chromatin remodeling, the post-translational modification of histone proteins is mediated by multimeric protein complexes. VERNALIZATION INSENSITIVE3 (VIN3) forms a complex with Polycomb Repressive Complex 2 (PRC2), which mediates the trimethylation of H3K27 to repress target gene expression. In rice, four genes (OsVIL1-OsVIL4) encoding the VIN3-like proteins are expressed ubiquitously in various tissues. Null mutants of osvil2 display pleiotropic phenotypes such as altered flowering time, floral organ defects, and reduced tiller size. In contrast, osvil1 mutants did not show significant phenotypes except in fertilization compared with the wild type. However, transgenic plants overexpressing OsVIL1 showed phenotypes of increased biomass and grain yield. Cross-sections of the basal region of elongating stems revealed that the increased biomass was mediated by inducing cell proliferation in the meristem. Chromatin immunoprecipitation assay indicated that OsVIL1 repressed expression of cytokinin oxidase/dehydrogenase gene (OsCKX2) by binding to the promoter and genic regions of OsCKX2. We also observed that OsVIL1 modified the levels of H3K27me3 in the OsCKX2 chromatin. Because OsCKX2 encodes an enzyme that degrades active cytokinin, we conclude that OsVIL1 functions in the regulation of endogenous active cytokinin levels, thereby increasing plant height and productivity.

Improved Prediction Model of Protein Lysine Crotonylation Sites Using Bidirectional Recurrent Neural Networks

Histone lysine crotonylation (Kcr) is a post-translational modification of histone proteins that is involved in the regulation of gene transcription, acute and chronic kidney injury, spermatogenesis, depression, cancer, and so forth. The identification of Kcr sites in proteins is important for characterizing and regulating primary biological mechanisms. The use of computational approaches such as machine learning and deep learning algorithms have emerged in recent years as the traditional wet-lab experiments are time-consuming and costly. We propose as part of this study a deep learning model based on a recurrent neural network (RNN) termed as Sohoko-Kcr for the prediction of Kcr sites. Through the embedded encoding of the peptide sequences, we investigate the efficiency of RNN-based models such as long short-term memory (LSTM), bidirectional LSTM (BiLSTM), and bidirectional gated recurrent unit (BiGRU) networks using cross-validation and independent tests. We also established the comparison between Sohoko-Kcr and other published tools to verify the efficiency of our model based on 3-fold, 5-fold, and 10-fold cross-validations using independent set tests. The results then show that the BiGRU model has consistently displayed outstanding performance and computational efficiency. Based on the proposed model, a webserver called Sohoko-Kcr was deployed for free use and is accessible at https://sohoko-research-9uu23.ondigitalocean.app.

A Review on Epigenetic Inheritance of Experiences in Humans.

If genetics defines the inheritance of DNA, epigenetics aims to regulate and make it adaptable. Epigenetic alterations include DNA methylation, chromatin remodelling, post-translational modifications of histone proteins and activity of non-coding RNAs. Several studies, especially in animal models, have reported transgenerational inheritance of epigenetic marks. However, evidence of transgenerational inheritance in humans via germline in the absence of any direct exposure to the driving external stimulus remains controversial. Most of the epimutations exist in relation with genetic variants. The present review looks at intergenerational and transgenerational inheritance in humans, (both father and mother) in response to diet, exposure to chemicals, stress, exercise, and disease status. If not transgenerational, at least intergenerational human studies could help to understand early processes of inheritance. In humans, female and male germline development follow separate paths of epigenetic events and both oocyte and sperm possess their own unique epigenomes. While DNA methylation alterations are reset during epigenetic reprogramming, non-coding RNAs via human sperm provide evidence of being reliable carriers for transgenerational inheritance. Human studies reveal that one mechanism of epigenetic inheritance cannot be applied to the complete human genome. Multiple factors including time, type, and tissue of exposure determine if the modified epigenetic mark could be transmissible and till which generation. Population-specific differences should also be taken into consideration while associating inheritance to an environmental exposure. A longitudinal study targeting one environmental factor, but different population groups should be conducted at a specific geographical location to pinpoint heritable epigenetic changes.

Regulation of Epigenetic Modifications in the Placenta during Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells.

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

Trichostatin A Relieves Growth Suppression and Restores Histone Acetylation at Specific Sites in a FUS ALS/FTD Yeast Model.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that often occurs concurrently with frontotemporal dementia (FTD), another disorder involving progressive neuronal loss. ALS and FTD form a neurodegenerative continuum and share pathological and genetic features. Mutations in a multitude of genes have been linked to ALS/FTD, including FUS. The FUS protein aggregates and forms inclusions within affected neurons. However, the precise mechanisms connecting protein aggregation to neurotoxicity remain under intense investigation. Recent evidence points to the contribution of epigenetics to ALS/FTD. A main epigenetic mechanism involves the post-translational modification (PTM) of histone proteins. We have previously characterized the histone PTM landscape in a FUS ALS/FTD yeast model, finding a decreased level of acetylation on lysine residues 14 and 56 of histone H3. Here, we describe the first report of amelioration of disease phenotypes by controlling histone acetylation on specific modification sites. We show that inhibiting histone deacetylases, via treatment with trichostatin A, suppresses the toxicity associated with FUS overexpression in yeast by preserving the levels of H3K56ac and H3K14ac without affecting the expression or aggregation of FUS. Our data raise the novel hypothesis that the toxic effect of protein aggregation in neurodegeneration is related to its association with altered histone marks. Altogether, we demonstrate the ability to counter the repercussions of protein aggregation on cell survival by preventing specific histone modification changes. Our findings launch a novel mechanistic framework that will enable alternative therapeutic approaches for ALS/FTD and other neurodegenerative diseases.

Epigenetic Mechanisms in Memory and Cognitive Decline Associated with Aging and Alzheimer's Disease.

Epigenetic mechanisms, which include DNA methylation, a variety of post-translational modifications of histone proteins (acetylation, phosphorylation, methylation, ubiquitination, sumoylation, serotonylation, dopaminylation), chromatin remodeling enzymes, and long non-coding RNAs, are robust regulators of activity-dependent changes in gene transcription. In the brain, many of these epigenetic modifications have been widely implicated in synaptic plasticity and memory formation. Dysregulation of epigenetic mechanisms has been reported in the aged brain and is associated with or contributes to memory decline across the lifespan. Furthermore, alterations in the epigenome have been reported in neurodegenerative disorders, including Alzheimer’s disease. Here, we review the diverse types of epigenetic modifications and their role in activity- and learning-dependent synaptic plasticity. We then discuss how these mechanisms become dysregulated across the lifespan and contribute to memory loss with age and in Alzheimer’s disease. Collectively, the evidence reviewed here strongly supports a role for diverse epigenetic mechanisms in memory formation, aging, and neurodegeneration in the brain.

Dynamic alterations of H3K4me3 and H3K27me3 at ADAM17 and Jagged‐1 gene promoters cause an inflammatory switch of endothelial cells

Histone protein modifications control the inflammatory state of many immune cells. However, how dynamic alteration in histone methylation causes endothelial inflammation and apoptosis is not clearly understood. To examine this, we explored two contrasting histone methylations; an activating histone H3 lysine 4 trimethylation (H3K4me3) and a repressive histone H3 lysine 27 trimethylation (H3K27me3) in endothelial cells (EC) undergoing inflammation. Through computer-aided reconstruction and 3D printing of the human coronary artery, we developed a unique model where EC were exposed to a pattern of oscillatory/disturbed flow as similar to in vivo conditions. Upon induction of endothelial inflammation, we detected a significant rise in H3K4me3 caused by an increase in the expression of SET1/COMPASS family of H3K4 methyltransferases, including MLL1, MLL2, and SET1B. In contrast, EC undergoing inflammation exhibited truncated H3K27me3 level engendered by EZH2 cytosolic translocation through threonine 367 phosphorylation and an increase in the expression of histone demethylating enzyme JMJD3 and UTX. Additionally, many SET1/COMPASS family of proteins, including MLL1 (C), MLL2, and WDR5, were associated with either UTX or JMJD3 or both and such association was elevated in EC upon exposure to inflammatory stimuli. Dynamic enrichment of H3K4me3 and loss of H3K27me3 at Notch-associated gene promoters caused ADAM17 and Jagged-1 derepression and abrupt Notch activation. Conversely, either reducing H3K4me3 or increasing H3K27me3 in EC undergoing inflammation attenuated Notch activation, endothelial inflammation, and apoptosis. Together, these findings indicate that dynamic chromatin modifications may cause an inflammatory and apoptotic switch of EC and that epigenetic reprogramming can potentially improve outcomes in endothelial inflammation-associated cardiovascular diseases.

HAT/HDAC: The epigenetic regulators of inflammatory gene expression (Review)

Inflammation is a condition through which the body responds to infection or tissue injury. It is typically characterized by the expression of a plethora of genes involved in inflammation, that are regulated by transcription factors, transcriptional co‑regulators, and chromatin remodeling events. Differential mitotically heritable patterns of gene expression without changes in the DNA sequence are essentially controlled by epigenetic regulation. Epigenetic mechanisms, such as histone modifications and DNA methylation have a profound effect on inflammatory gene transcription. Histone protein modifications, which include acetylation and the ubiquitination of lysine residues, the methylation of lysine and arginine, and the phosphorylation of serine have been found to modulate chromatin dynamics, thus altering the levels of gene expression. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate the addition and removal of acetyl groups from lysine residues on histones respectively. Nuclear factor <em>(NF)‑κB</em>, tumor necrosis factor <em>(TNF)‑α</em> and interleukin <em>(IL)‑6</em> are the pro‑inflammatory genes known to promote inflammatory responses in cells. By contrast, 15‑lipoxygenase‑1 <em>(15‑LOX‑1)</em> and monoamine oxidase‑A <em>(MAO‑A)</em> are the genes that can act against inflammation in certain specific conditions. 15‑LOX‑1, a lipid peroxidative enzyme, is associated with the development of inflammatory disorders, such as atherosclerosis, rheumatoid arthritis, asthma and renal injury. MAO‑A catalyzes degradation of biogenic amines and has been reported to cause oxidative stress, atherosclerosis and neuroinflammation. 15‑LOX‑1 has been shown to be co‑expressed along with MAO‑A, in both primary human monocytes and A549 lung carcinoma cells upon treatment with Th2 cytokines, such as IL‑4 and IL‑13. The present review aimed to discuss the HAT‑ and HDAC‑mediated epigenetic machinery which governs the expression of pro‑inflammatory genes, such as <em>IL‑6</em>, <em>TNF‑α</em>, etc., as well as the expression of anti‑inflammatory genes, such as <em>15‑LOX‑1</em> and <em>MAO‑A</em>, responsible for modulating the process of inflammation. On the whole, the present review aims to provide deeper insight into the underlying molecular mechanisms involved in the epigenetic regulation of inflammation, which may have novel implications in designing small molecule inhibitors that target the epigenetic machinery for the effective treatment of a variety of inflammation‑related diseases.

Regulation of autophagy by protein methylation and acetylation in cancer.

Autophagy is a highly conserved mechanism responsible for cellular homeostasis and integrity in a variety of physiological conditions. Materials targeted for degradation are directed to autophagosomes and autolysosomes, where they are broken down into their base components. Aberrant regulation of autophagy is significantly associated with various cancers and neurodegenerative diseases. Recently, accumulating evidence has revealed that the coordinated regulation of histone and non-histone protein modification is associated with autophagy. In this review, we highlight the recent progress that has been made in elucidating the molecular basis of protein methylation and acetylation associated with autophagy at the transcriptional and posttranslational levels. Furthermore, we discuss the importance of describing causality between protein methylation/acetylation and autophagy regulation as compelling therapeutic opportunities in cancer pathogenesis and progression.

Applying Peptide and Protein Synthesis to Study Post-translational Modifications in Epigenetics and Beyond.

Epigenetics research focuses on the study of heritable gene regulatory mechanisms that do not involve changes of the DNA sequence. Such mechanisms include post-translational modifications of histone proteins that organize the genome in the nucleus into a nucleoprotein complex called chromatin, and which are of key importance in development and disease. Chemical biology tools as developed by my group, in particular synthetic peptide and protein chemistry, have been critical to elucidate epigenetic signaling mechanisms. As outlined below, they allow the reconstitution of chromatin carrying defined modifications and thus the elucidation of detailed molecular mechanisms.

Histone Modification Patterns Using RPPA-Based Profiling Predict Outcome in Acute Myeloid Leukemia Patients

Background: Prognostic factors based on cytogenetic and molecular abnormalities combined with clinical features like age, performance status, or history of an antecedent hematological disorder, are currently used to predict clinical outcome in acute myeloid leukemia (AML). (Döhner H. et al. 2010) Although, sequencing of AML has identified many driver mutations in genes associated with epigenetic regulation, e.g. post-translational histone tail modifications. Altered activity of post-translational histone tail modifications has been shown to contribute to AML leukemogenesis via regulation of gene transcription. (Fong C.Y. et al. 2014) The functions and effects of modulating histone-modifying proteins (HMP) are already typically studied by altering an individual protein of interest. However in AML, it is the net consequence of the combined influences of all the HMP, rather than a single protein, that determines the net effect on the cell. A global analysis of the level and patterns of expression of multiple HMP and the effect of different patterns of expression on outcome and prognosis has not been investigated in AML patients.Methods: We analyzed 20 HMP by reverse phase protein array (RPPA) in a cohort of 205 newly diagnosed AML patients evaluated at the University of Texas M.D. Anderson Cancer Center. (Kornblau S.M. et al. 2009) Protein levels were correlated with patient and disease characteristics, including survival and mutational state.Results: Different protein clusters were identified and characterized by higher (more on) or lower (more off) expression of HMP, relative to normal CD34+ cells (figure 1A). We found that the more on state of HMP was associated with poorer overall survival (P= 0.007, figure 1B) and more chemo resistance (P < 0.001) compared to normal -like and a more off state. Furthermore, FLT3 mutated AML patients were significantly overrepresented in the more on state (P= 0.025). Identification of the markedly upregulated proteins: KDM1A, hnRNPK, NCL, SIRT1, ASH2L, WTAP in all, and BRD4 and NPM1 in some on state patients suggest targets to develop drugs against that may be the most efficacious when used individually or in combination.Conclusions: These findings showed for the first time that multiple HMP form recurrent patterns of expression and that these significantly correlate with prognosis, chemo resistance, and FLT3 mutational state in newly diagnosed AML patients.F igure legend 1:(A) RPPA-based heatmap containing 20 histone modification related proteins showing only 205 fresh AML patient samples. Clusters were determined by a progeny clustering based algorithm coupled with k-means, which computationally calculated the optimal number of five clusters. The five cluster colors along the top bar delineate the five fresh clusters. Cluster 1 (red) was defined as a normal -like pattern by principal component analysis, whereas clusters 2, 3, 6 and 7 were only seen with leukemic samples. Expression is normalized for each protein to range from the lowest (blue) to the highest (red). Cluster 2 and 7 were defined as the more on clusters and cluster 3 and 6 as the more off clusters.(B) Overall Survival of patients from the normal -like histone-modifying proteins (HMP) cluster (n=61), more on HMP clusters (n=131) and the more off HMP clusters (n=13) as determined in our cohort of newly diagnosed AML fresh patient samples by RPPA-based profiling. The survival of patients with the more on state of HMP was significantly reduced compared to the more off state and the normal- like HMP signatures. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Phase 1/2 Study of Oral 5-Azacitidine and Romidepsin in Patients with Lymphoid Malignancies Reveals Promising Activity in Heavily Pretreated Peripheral T-Cell Lymphoma (PTCL)

Introduction: Patients with PTCL are characterized by frequent alterations of epigenetic operations, including recurrent mutations in important genes governing genome wide methylation (i.e., TET2, IDH1/2, and DNMT3). This feature creates the rationale for the use of hypomethylating agents (HMA) in these diseases. Indeed, a recent study showed impressive preliminary activity of 5-azacitidine (AZA) in patients with angioimmunoblastic T-cell lymphoma (AITL). Histone deacetylases (HDACs), transcriptional modulators that operate through modification of histone proteins, are also overexpressed in select types of PTCL. HDAC inhibitors (HDACi), such as romidepsin (ROMI), have shown consistent class activity in patients with R/R PTCL, and received regulatory approval for this indication. Our group has previously shown that the combination of the HDACi and the HMA can ‘reverse’ the malignant signature of T-cell lymphoma lines in vitro. In addition, the combination induced the expression of a unique set of genes not induced by either single agent, including a unique methylation pattern. We therefore launched a phase 1/2 trial of combined AZA and ROMI in patients with lymphoid neoplasms, including PTCL. Herein we present updated analysis of the results of the phase 1 and 2 portions of the trial combined.Methods: Patients with any from of lymphoma were eligible for the phase 1, while the phase 2 exclusively enrolled patients with PTCL. Patients with any number of prior therapies, including transplantation, were eligible for enrollment. In the phase 2 patients with newly diagnosed PTCL were also allowed to participate. Other inclusion criteria were ECOG PS <2 and adequate organ and marrow function. The phase 1 study followed a 3 + 3 dose-escalation design. The starting doses were AZA 100 mg/day and ROMI 10 mg/m2 with progressively increasing dose intensity in subsequent cohorts (7 cohorts). The primary objectives of the study were the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) of the combination. Secondary objectives included ORR (CR + PR), progression free survival (PFS), and duration of response (DOR).Results: 30 patients were enrolled in total, 27 in phase 1 (26 accrued, 1 screening failure) and 3 in phase 2, of whom one has been restaged. In the intention-to-treat population the median age was 49 years (23-79), male-to-female ratio 17:13, and median number of prior therapies 5 (0-18). Sixteen patients had had autologous stem cell transplant, and 1 prior allogeneic transplant. Lymphoma histology's included Hodgkin/other (12, 40%), B-cell (8, 27%), and T-cell (10, 33%). Twenty-seven patients are evaluable for toxicity. We recorded 5 DLTs across all cohorts, 3 of which occurred at the highest dose level (i.e., AZA 300 mg days 1-21 and ROMI 14 mg/m2 days 8, 15, and 22) and included neutropenia (N = 2) and thrombocytopenia (N = 1). Two additional DLTs were 1 pleural effusion and 1 missed dose. The MTD for the combination was AZA 300 mg days 1-14 and ROMI 14 mg/m2 days 8, 15, and 22. Other grade 3-4 toxicities included febrile neutropenia (N = 3), and thrombocytopenia (N = 5), only 2 being grade 4. Among 25 patients evaluable for efficacy (all NHL subtypes), the ORR was 28% (7/25) including a 16% (4/25) CR. Substantially higher response rates were seen in patients with PTCL, where the ORR and CR were 83% (5/6) and 50% (3/6). First dose PK revealed the AUC for R 10 and 14 mg/m2 were 2021.5 ± 1461.3 h*ng/mL, and 1765.9 ± 1002.0 h*ng/mL, respectively. The median t1/2 was 4.8 and 2.4 h, respectively. These data are comparable to historical single agent values.Conclusions: Albeit in a small population, these promising clinical results suggest that a doublet of epigenetic modifiers is well tolerated and may represent a viable backbone to build the next generation of active regimens in PTCL. The complete response seen in the AITL patient enrolled in the phase 2 portion of the study is encouraging and is being specifically expanded. The study is now multicenter, and results on additional patients enrolled in the phase 2 portion will be presented at the meeting. This study is registered on www.clinicaltrials.gov (NCT01998035). DisclosuresO'Connor:Trillium Therapeutics: Research Funding; Celgene: Honoraria, Research Funding.

Histone acylations and chromatin dynamics: concepts, challenges, and links to metabolism.

In eukaryotic cells, DNA is tightly packed with the help of histone proteins into chromatin. Chromatin architecture can be modified by various post‐translational modifications of histone proteins. For almost 60 years now, studies on histone lysine acetylation have unraveled the contribution of this acylation to an open chromatin state with increased DNA accessibility, permissive for gene expression. Additional complexity emerged from the discovery of other types of histone lysine acylations. The acyl group donors are products of cellular metabolism, and distinct histone acylations can link the metabolic state of a cell with chromatin architecture and contribute to cellular adaptation through changes in gene expression. Currently, various technical challenges limit our full understanding of the actual impact of most histone acylations on chromatin dynamics and of their biological relevance. In this review, we summarize the state of the art and provide an overview of approaches to overcome these challenges. We further discuss the concept of subnuclear metabolic niches that could regulate local CoA availability and thus couple cellular metabolisms with the epigenome.

Targeting super-enhancers reprograms glioblastoma central carbon metabolism.

The concept that tumor cells demand a distinct form of metabolism was appreciated almost a century ago when the German biochemist Otto Warburg realized that tumor cells heavily utilize glucose and produce lactic acid while relatively reducing oxidative metabolism. How this phenomenon is orchestrated and regulated is only partially understood and seems to involve certain transcription factors, including c-Myc, HIF1A and others. The epigenome eintails the posttranslational modification of histone proteins which in turn are involved in regulation of transcription. Recently, it was found that cis-regulatory elements appear to facilitate the Warburg effects since several genes encoding for glycolysis and associated pathways are surrounded by enhancer/super-enhancer regions. Disruption of these regions by FDA-approved HDAC inhibitors suppressed the transcription of these genes and elicited a reversal of the Warburg effect with activation of transcription factors facilitating oxidative energy metabolism with increases in transcription factors that are part of the PPARA family. Therefore, combined targeting of HDACs and oxidative metabolism suppressed tumor growth in patient-derived xenograft models of solid tumors, including glioblastoma.

Copper ions impair zebrafish skeletal myofibrillogenesis via epigenetic regulation.

Unbalanced copper (Cu2+ ) homeostasis is associated with the developmental defects of vertebrate myogenesis, but the underlying molecular mechanisms remain elusive. In this study, it was found that Cu2+ stressed zebrafish embryos and larvae showed reduced locomotor speed as well as loose and decreased myofibrils in skeletal muscle, coupled with the downregulated expression of muscle fiber markers mylpfa and smyhc1l and the irregular arrangement of myofibril and sarcomere. Meanwhile, the Cu2+ stressed zebrafish embryos and larvae also showed significant reduction in the expression of H3K4 methyltransferase smyd1b transcripts and H3K4me3 protein as well as in the binding enrichment of H3K4me3 on gene mylpfa promoter in skeletal muscle cells, suggesting that smyd1b-H3K4me3 axis mediates the Cu2+ -induced myofibrils specification defects. Additionally, whole genome DNA methylation sequencing unveiled that the gene smyd5 exhibited significant promoter hyper-methylation and increased expression in Cu2+ stressed embryos, and the ectopic expression of smyd5 in zebrafish embryos also induced the myofibrils specification defects as those observed in Cu2+ stressed embryos. Moreover, Cu2+ was shown to suppress myofibrils specification and smyd1b promoter transcriptional activity directly independent of the integral function of copper transporter cox17 and atp7b. All these data may shed light on the linkage of unbalanced copper homeostasis with specific gene promoter methylation and epigenetic histone protein modification as well as the resultant signaling transduction and the myofibrillogenesis defects.