Dimethylated Histone H3 Research Articles

Abstract 1382: Novel dual inhibitors of LSD1-HDAC6/8 for treatment of cancer

Abstract LSD1 is an FAD-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4 (H3-K4) as a part of CoREST repressor complex. Recent studies have shown that there is crosstalk between two components of CoREST complex, LSD1 and HDAC, which provides an advantage to cancer cell proliferation and survival by regulating key signaling pathways. Accordingly, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting the growth of glioblastoma, AML and breast cancer. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating several cancers effectively without enhancing systemic toxicity mediated by administration of multiple drugs. To test the hypothesis, we have developed a set of molecules that either have LSD1 activity alone or dual activity on LSD1 and HDAC. Computational chemistry approaches were used to design LSD1-HDAC dual inhibitors. In vitro LSD1 potency was assessed by TR-FRET assay and in vitro HDAC activity by fluorimetric HDAC activity assay. Western blotting and RT qPCR were used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Multiple compounds from this series show an in vitro potency of less than 0.05 μM against LSD1 with more than 100-fold selectivity against MAOs. The dual inhibitors showed stronger activity on HDAC6/8. JBI-097, one such dual molecule with LSD1/HDAC/6/8 selectivity, showed an IC50 of 0.007uM on LSD1 and an IC50 of 0.06 and 0.1uM on HDAC6 and HDAC8, respectively, with about 100-fold selectivity against other HDAC isoforms. JBI-097 showed strong antiproliferative activity on leukemia and multiple myeloma cell lines. In MM.1S cell line, JBI-097 showed stronger potency when compared to LSD1 inhibitor GSK2879552 and HDAC6 inhibitor ACY1215. In cell-based and in vivo target engagement studies there was a concomitant increase in CD11b, CD86 and GFI1b and tubulin acetylation levels. JBI-097 was more efficacious in inhibiting the growth of HEL92.1.7 xenograft by oral administration when compared to IP administration of an HDAC inhibitor. In MM.1S tumor model, JBI-097 showed stronger effect in inhibiting tumor growth when compared to LSD1 inhibitor and HDAC6 inhibitor used as single agents. Further, combination with topotecan resulted in complete tumor growth inhibition of MM.1S xenograft. The data obtained demonstrate that it is feasible to design dual LSD1-HDAC6/8 inhibitors that retain individual activity and potently inhibit cell proliferation, and such inhibitors could serve as powerful therapeutic agents for cancer. Further understanding of mechanisms to identify the right tool compound is in progress. Citation Format: Sivanandhan Dhanalakshmi, Sridharan Rajagopal, Sreekala Nair, Chandru Gajendran, Dimpy Ghosh, Subramanyam Janardhan Tantry, Purushottam Dewang, Mahanandeesha Hallur, M Kannan, Srinatha KC, Damodara Kuntrapaku, M Dilipkumar, Radha Sharma, S Meghashree, D P. Kumar, Mohd Zainuddin, A B. Vinod, Sriram Rajagopal. Novel dual inhibitors of LSD1-HDAC6/8 for treatment of cancer [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 1382.

Abstract 5860: Novel, first-in-class dual inhibitors of lysine specific demethylase 1 (LSD1) and histone deacetylatse 1 (HDAC) for treatment of cancer

Abstract Introduction: LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4. Studies have shown that LSD1 contributes to acute myelogenous leukaemia pathogenesis and patients that are non responders to ATRA respond to LSD1-ATRA combination treatment in clinic. Recent studies have shown that there is cross-talk between two components of CoREST complex, LSD1 and HDAC1/2 and combined inhibition of these targets has been shown to be more efficacious in multiple cancers. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating cancers. To test the hypothesis, we have developed a set of molecules that have dual activity on LSD1 and HDACs. Methods: To assess in vitro LSD1potency, TR-FRET assay was used. For assessing HDAC activity fluorescence based HDAC activity assay was performed. Western blotting and RT-qPCR were used to assess biomarkers of LSD1 and HDAC1/2 inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Xenograft models were used to assess in vivo efficacy. Results: Dual inhibitors show strong in vitro potency comparable to advanced clinical candidates, against LSD1. On HDAC1/2 these molecules showed comparable IC50 as marketed HDAC1/2 specific drugs. Interestingly, these dual inhibitors lead to stronger and complete inhibition of cell proliferation in broader types of cancer cell lines, including, acute myeloid leukemia, multiple myeloma, hepatocellular carinoma and small cell lung cancer when compared to LSD1 specific inhibitor GSK2879552. Target engagement studies confirm the concomitant increase in mRNA levels of GFI1b, CD11b, CD86 and H3K9 acetylation levels. In vitro activity also shows a clear translation in efficacy studies. JBI128, one of the lead molecules from this series was tested in multiple in vivo studies, including, non-small cell and small cell lung cancer, where it not only shows single agent activity, but can also be combined with other targeted agents or SoC safely. In addition, combination of JBI128 with immune checkpoint inhibitor anti-PDL1 antibody in the CT26 syngeneic model showed much stronger efficacy as compared to either of the single agents. This clearly suggests that these dual inhibitors in addition to being epigenetic modulators in the cancer cells also have a role in immune modulation thereby contributing to stronger efficacy. Further studies in terms of gene expression analysis are underway to understand the molecular mechanism behind the additional activity/efficacy of these dual inhibitors. Conclusion: These LSD1-HDAC dual inhibitors have the potential to be developed as first-in-class therapies that could offer stronger clinical efficacy compared to LSD1 or HDAC inhibitor alone. Further mechanistic and combination studies with standard therapies are in progress. Citation Format: Dhanalakshmi Sivanandhan, Sreekala Nair, Subramaniyam Tantry, Sridharan Rajagopal, Mahanandeesha Hallur, Kannan M, Srinatha KC, Damodara K, Dilipkumar M, Chandru G, Dimpy Ghosh, Pradeep N, Anuj Kumar Singh, Mohd Zainuddin, Rudresh G, Radha Sharma, Meghashree S, Durga P. Kumar, Purushottam Dewang, Sriram Rajagopal. Novel, first-in-class dual inhibitors of lysine specific demethylase 1 (LSD1) and histone deacetylatse 1 (HDAC) for treatment of cancer [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 5860.

RNAi-dependent heterochromatin assembly in fission yeast Schizosaccharomyces pombe requires heat-shock molecular chaperones Hsp90 and Mas5

BackgroundHeat-shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA (siRNA) onto RNA interference (RNAi) effector complexes. While the RNAi process is coupled with heterochromatin assembly in several model organisms, it remains unclear whether the Hsps contribute to epigenetic gene regulation. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism and investigated the roles of Hsp90 and Mas5 (a nucleocytoplasmic type-I Hsp40 protein) in RNAi-dependent heterochromatin assembly.ResultsUsing a genetic screen and biochemical analyses, we identified Hsp90 and Mas5 as novel silencing factors. Mutations in the genes encoding these factors caused derepression of silencing at the pericentromere, where heterochromatin is assembled in an RNAi-dependent manner, but not at the subtelomere, where RNAi is dispensable. The mutations also caused a substantial reduction in the level of dimethylation of histone H3 at Lys9 at the pericentromere, where association of the Argonaute protein Ago1 was also abrogated. Consistently, siRNA corresponding to the pericentromeric repeats was undetectable in these mutant cells. In addition, levels of Tas3, which is a protein in the RNA-induced transcriptional silencing complex along with Ago1, were reduced in the absence of Mas5.ConclusionsOur results suggest that the Hsps Hsp90 and Mas5 contribute to RNAi-dependent heterochromatin assembly. In particular, Mas5 appears to be required to stabilize Tas3 in vivo. We infer that impairment of Hsp90 and Hsp40 also may affect the integrity of the epigenome in other organisms.

Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication

We report an optimised centrifugal counter-flow elutriation protocol for the rapid and direct isolation of G1 cell cycle synchronised populations of both the procyclic and bloodstream form stages of Trypanosoma brucei that yields viable and proliferative cells. The high quality of the synchronisation achieved can be judged by the uniform DNA content, narrow size distribution, synchronous division, and the maintenance of synchronicity into subsequent cell cycles. We show that early-eluting fractions represent different G1 subpopulations that progress through the cell cycle with distinct temporal profiles post-elutriation, as exemplified by the observation of the maturation of a second flagellar basal body in late G1 phase, DNA replication in S phase, and dimethylation of histone H3 in mitosis/cytokinesis. We use our temporal observations to construct a revised model of the relative timing and duration of the nuclear and kinetoplast cell cycle that differs from the current model.

Effects of adolescent social stress and antidepressant treatment on cognitive inflexibility and Bdnf epigenetic modifications in the mPFC of adult mice

Adolescent social stress (ASS) can increase susceptibility to depression in adulthood. However, the underlying psychological and neural mechanisms remain unclear. Cortically mediated cognitive dysfunctions are increasingly recognized as an independent and important risk factor of depression. Using social defeat stress, a classical animal model of depression, our previous studies found that mice subjected to this form of stress during early adolescence displayed cognitive inflexibility (CI) in adulthood. This change was accompanied by a down-regulation of Bdnf gene expression in the medial prefrontal cortex (mPFC); this gene encodes a key molecule involved in depression and antidepressant action. In the present paper, we identified epigenetic modification of Bdnf as a possible mechanism underlying the behavioral and molecular changes. ASS induced a set of depressive phenotypes, including increased social avoidance and CI, as well as reduced levels of total Bdnf and isoform IV but not isoform I or VI transcripts in the mPFC. In parallel with changes in Bdnf gene expression, previously stressed adult mice showed increased levels of dimethylation of histone H3 at lysine K9 (H3K9me2) immediately downstream of the Bdnf IV promoter. On the other hand, no differences were found in trimethylation of histone H3 at lysine K4 (H3K4me3) or in acetylation of histone H3 at lysine K9 (H3K9ac) or at K4 (H3K4ac) in the Bdnf IV promoter. Likewise, no alterations were found in DNA methylation of the Bdnf IV promoter. Additionally, treatment with the chronic antidepressant tranylcypromine reversed Bdnf epigenetic changes and related gene transcription while also reversing CI, but not social avoidance, in previously stressed adult mice. These results suggest that epigenetic changes to the Bdnf gene in the mPFC after adolescent social adversity may be involved in the regulation of cognitive dysfunction in depression and antidepressant action in adulthood.

Epigenetic Transcriptional Memory of GAL Genes Depends on Growth in Glucose and the Tup1 Transcription Factor in Saccharomyces cerevisiae.

Previously expressed inducible genes can remain poised for faster reactivation for multiple cell divisions, a conserved phenomenon called epigenetic transcriptional memory. The GAL genes in Saccharomyces cerevisiae show faster reactivation for up to seven generations after being repressed. During memory, previously produced Gal1 protein enhances the rate of reactivation of GAL1, GAL10, GAL2, and GAL7 These genes also interact with the nuclear pore complex (NPC) and localize to the nuclear periphery both when active and during memory. Peripheral localization of GAL1 during memory requires the Gal1 protein, a memory-specific cis-acting element in the promoter, and the NPC protein Nup100 However, unlike other examples of transcriptional memory, the interaction with NPC is not required for faster GAL gene reactivation. Rather, downstream of Gal1, the Tup1 transcription factor and growth in glucose promote GAL transcriptional memory. Cells only show signs of memory and only benefit from memory when growing in glucose. Tup1 promotes memory-specific chromatin changes at the GAL1 promoter: incorporation of histone variant H2A.Z and dimethylation of histone H3, lysine 4. Tup1 and H2A.Z function downstream of Gal1 to promote binding of a preinitiation form of RNA Polymerase II at the GAL1 promoter, poising the gene for faster reactivation. This mechanism allows cells to integrate a previous experience (growth in galactose, reflected by Gal1 levels) with current conditions (growth in glucose, potentially through Tup1 function) to overcome repression and to poise critical GAL genes for future reactivation.

Inhibition of LSD1 epigenetically attenuates oral cancer growth and metastasis.

Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

Role of the long non-coding RNA HOTAIR in hepatocellular carcinoma.

A number of recent studies have focused on the association between long non-coding RNAs (lncRNAs) and cancer. HOX transcript antisense RNA (HOTAIR), an lncRNA that functions as a transcriptional modulator, has been implicated in various fundamental biological activities. HOTAIR mediates the trimethylation of histone H3 at lysine 27 and the demethylation of histone H3 dimethyl Lys4 by recruiting the polycomb repressive complex 2 and the lysine-specific demethylase 1/co-repressor of RE1-silencing transcription factor (coREST)/REST complex to the target gene promoters, which leads to gene silencing. Overexpression of HOTAIR in hepatocellular carcinoma (HCC) is strongly associated with an unfavorable prognosis for patients with HCC. HOTAIR promotes the carcinogenic activity of HCC cells through the suppression of RNA binding motif protein 38, triggering the epithelial-mesenchymal transition, and by interacting with microRNAs that act as tumor suppressors. In the present review, the role of the lncRNA HOTAIR in HCC is examined. The potential use of HOTAIR as a biomarker to achieve more accurate prognostic predictions and as an effective therapeutic target for HCC is then discussed.

Histone H3K9 Demethylase JMJD2B Activates Adipogenesis by Regulating H3K9 Methylation on PPARγ and C/EBPα during Adipogenesis.

Previous studies have shown that tri- or di-methylation of histone H3 at lysine 9 (H3K9me3/me2) on the promoter of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) contribute to the repression of PPARγ and C/EBPα and inhibition of adipogenesis in 3T3-L1 preadipocytes. The balance of histone methylation is regulated by histone methyltransferases and demethylases. However, it is poorly understood which demethylases are responsible for removing H3K9me3/me2 on the promoter of PPARγ and C/EBPα. JMJD2B is a H3K9me3/me2 demethylase that was previously shown to activate adipogenesis by promoting mitotic clonal expansion. Nevertheless, it remains unclear whether JMJD2B plays a role in the regulation of adipogenesis by removing H3K9me3/me2 on the promoter of PPARγ and C/EBPα and subsequently activating PPARγ and C/EBPα expression. Here, we showed that JMJD2B decreased H3K9me3/me2 on the promoter of PPARγ and C/EBPα, which in turn stimulated the expression of PPARγ and C/EBPα. JMJD2B knockdown using siRNA in 3T3-L1 preadipocytes repressed the expression of PPARγ and C/EBPα, resulting in inhibition of adipogenesis. This was accompanied by increased enrichment of H3K9me3/me2 on the promoter of PPARγ and C/EBPα. In contrast, overexpression of JMJD2B increased the expression of PPARγ and C/EBPα, which was accompanied by decreased enrichment of H3K9me3/me2 on the promoter and activated adipogenesis. Together, these results indicate that JMJD2B regulates PPARγ and C/EBPα during adipogenesis.

Intracytoplasmic oxidative stress reverses epigenetic modifications in polycystic ovary syndrome.

In polycystic ovary syndrome (PCOS), substantial genetic and environmental alterations, along with hyperandrogenism, affect the quality of oocytes and decrease ovulation rates. To determine the mechanisms underlying these alterations caused specifically by an increase in plasma androgens, the present study was performed in experimentally-induced PCOS mice. As the study model, female B6D2F1 mice were treated with dehydroepiandrosterone (DHEA, 6mg per 100g bodyweight). After 20 days, oocytes at the germinal vesicle and metaphase II stages were retrieved from isolated ovaries and subsequent analyses of oocyte quality were performed for each mouse. DHEA treatment resulted in excessive abnormal morphology and decreased polar body extrusion rates in oocytes, and was associated with an increase in oxidative stress. Analysis of fluorescence intensity revealed a significant reduction of DNA methylation and dimethylation of histone H3 at lysine 9 (H3K9) in DHEA-treated oocytes, which was associated with increased acetylation of H4K12. Similarly, mRNA expression of DNA methyltransferase-1 and histone deacetylase-1 was significantly decreased in DHEA-treated mice. There was a significant correlation between excessive reactive oxygen species (ROS) production and increased histone acetylation, which is a novel finding and may provide new insights into the mechanism causing PCOS. The results of the present study indicate that epigenetic modifications of oocytes possibly affect the quality of maturation and ovulation rates in PCOS, and that the likely mechanism may be augmentation of intracytoplasmic ROS.

The Histone Methyltransferase Mixed Lineage Leukemia (MLL) 3 May Play a Potential Role in Clinical Dilated Cardiomyopathy

Histone modifications play a critical role In the pathological processes of dilated cardiomyopathy (DCM), while the role and expression pattern of histone methyltransferases (HMTs), especially mixed lineage leukemia (MLL) families, in DCM are unclear. To this end, 12 normal and 15 DCM heart samples were included in the present study. A murine cardiac remodeling model was induced by transverse aortic constriction (TAC). Real-time polymerase chain reaction was performed to detect the expression levels of MLL families in the mouse and human left ventricles. The mRNA level of MLL3 was significantly increased in the mouse hearts treated with TAC surgery. Compared with normal hearts, higher mRNA and protein level of MLL3 was detected in the DCM hearts, and its expression level was closely associated with left ventricular end diastolic diameter and left ventricular ejection fraction. However, there was no obvious change in the expression levels of other MLL families (MLL, MLL2, MLL4, MLL5, SETD1A and SETD1B) between control and DCM hearts or remodeled mouse hearts. Furthermore, the dimethylated histone H3 lysine 4 (H3K4me2) but not H3K4me3 was significantly increased in the DCM hearts. The protein levels of Smad3, GATA4 and EGR1, which might be regulated by MLL3, were remarkably elevated in the DCM hearts. Our hitherto unrecognized findings indicate that MLL3 has a potential role in the pathological processes of DCM by regulating H3K4me2 and the expression of Smad3, GATA4 and EGR1.

Effects of lipopolysaccharide on maturation of bovine oocyte in vitro and its possible mechanisms.

Lipopolysaccharide disturbs the secretion of gonadotropin, endometrial function and implantation efficiency. However, there is little information regarding the effects of lipopolysaccharide on cyclic ovary activity, especially oocyte maturation. Therefore, we aimed to investigate the effects of lipopolysaccharide on the maturation potential of bovine oocytes. We found that lipopolysaccharide exposure significantly decreased the first polar body extrusion rate and delayed the cell cycle progression. The abnormal spindle rate was significantly increased in lipopolysaccharide treatment group, accompanied by disrupted localization and level of phosphorylated mitogen-activated protein kinase (p-MAPK). Moreover, lipopolysaccharide treatment significantly increased intracellular reactive oxygen species (ROS) levels and the early apoptotic rate in oocytes. The pro-apoptotic caspase-3 and Bax mRNA levels and caspase-3 protein level were significantly increased, whereas the anti-apoptotic Bcl-2 and XIAP transcript abundance were significantly decreased in lipopolysaccharide exposure group. Furthermore, the dimethyl-histone H3 lysine 4 (H3K4me2) level was significantly increased, while the DNA methylation (5-mC) and dimethyl-histone H3 lysine 9 (H3K9me2) levels were markedly decreased in oocytes treated with lipopolysaccharide. In conclusion, lipopolysaccharide exposure inhibits the maturation potential of bovine oocytes by affecting cell cycle, cytoskeletal dynamics, oxidative stress, and epigenetic modifications.

A Genetically Encoded Allysine for the Synthesis of Proteins with Site-Specific Lysine Dimethylation.

Using the amber suppression approach, Nϵ -(4-azidobenzoxycarbonyl)-δ,ϵ-dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site-specific lysine dimethylation. Using this approach, dimethyl-histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.

Cooperative Gene Repression By DNA Methylation and LSD1-Mediated Enhancer Inactivation in Acute Myeloid Leukemia

Epigenetic silencing of tumor suppressor genes, mediated by aberrant DNA hypermethylation and repressive histone modifications, is a hallmark of acute myeloid leukemia (AML). Novel epigenetic therapies are emerging, but single-agent approaches targeting only one category of epigenetic marks have limited therapeutic activity. We show that combinatorial treatment strategy targeting epigenetic modifications of both promoter and enhancer elements can improve therapeutic intervention.As current cell lines do not accurately represent the heterogeneous nature of AML, we established an ex vivo culturing system enabling us to propagate primary AML specimens long enough to test epigenetic therapeutics (48 specimens expanded 4-6 weeks; 6 specimens expanded >12 months; 39 specimen fail to expand). We tested 52 expandable primary AMLs with a combination therapy utilizing the DNA demethylating agent 5-Azacytidine (5-Aza) and a novel small molecule inhibitor against LSD1, a histone demethylase that removes enhancer-associated marks (mono-/di-methylated histone H3 lysine 4; H3K4me1/2). In order to avoid DNA damage-induced cytotoxicity, we applied a non-DNA damaging dose of 5-Aza (≤ 200 nM). 5-Aza treatment had modest inhibition of cell growth for the majority of cases, but did not appreciably alter cell viability. Treatment with the LSD1 inhibitor (LSD1i) substantially impaired cell growth and survival of ~80% (p<0.001) of cultured primary AMLs. Notably, we found that dual combination therapy using LSD1i and 5-Aza demonstrated significantly improved inhibition of cell viability and growth in most LSD1i-resistant AML cases. Targeted resequencing analysis of 200 genes recurrently mutated in myeloid malignancies revealed an association of mutations with therapeutic activity; TET2mut AML cases without concurrent DNMT3A mutations were most substantially impaired by combinatorial treatment (p<0.001). Also, leukemia burden was significantly reduced after combination therapy in NSG mice transplanted with luciferase-labelled TET2mut patient-derived AML cells (p<0.0002).To gain insight into mechanism, we performed a genome-wide integrative analysis of the DNA methylome (Enhanced Reduced Representation Bisulfite Sequencing), LSD1 occupancy sites (ChIP-sequencing) and gene expression profiling (RNA-sequencing) in a TET2mut patient-derived AML sample. Single-agent LSD1i treatment led to induction of genes associated with myeloid differentiation. Even though 5-Aza induced substantial hypomethylation, there was only a minor effect on gene expression. However, combination therapy did induce greater de-repression of genes, in particular those where 5-Aza induced hypomethylation at promoters and at LSD1-occupied enhancers. Combination treatment especially potentiated induction of differentiation genes, suggesting potential anti-leukemia stem cell effects. Accordingly, combination therapy resulted in impaired colony formation potential (p=0.007). Moreover, limiting dilution analysis in NSG mice demonstrated significant impairment of leukemia-initiating cells (p=0.0025) after 5-Aza+LSD1i combination therapy when compared to either drug alone.To determine what genes mediate the effect of combination therapy, we studied genes preferentially induced by 5-Aza+LSD1i. GATA2, a transcription factor involved in hematopoietic differentiation, was more potently induced by combination therapy in primary AML samples vs. either drug alone. We found LSD1 occupied at the GATA2 distal hematopoietic enhancer and verified by using quantitative ChIP analysis that inhibition of LSD1 induced an increase of H3K4me2 and H3K27ac, a mark that reflects enhancer activation. Overexpression of GATA2 using viral transduction in two patient-derived AML cases caused partial differentiation and attenuated leukemia growth. These results indicated that cellular responses to combination therapy are in part mediated by induction of GATA2.In summary, our results provide a novel link for therapeutic approaches targeting redundant epigenetic silencing of tumor suppressors via cooperative enhancer and enhancer-promoter activation. DisclosuresDuy:GlaxoSmithKline: Research Funding. Mohammad:GlaxoSmithKline: Employment. Smitheman:GlaxoSmithKline: Employment. Guzman:Cellectis: Research Funding. Kruger:GlaxoSmithKline: Employment. Creasy:GlaxoSmithKline: Employment. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Melnick:Janssen: Research Funding.

TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation.

BackgroundTPA Induced Sequence 7 acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. We and others have shown that TIS7 regulates adult myogenesis through MyoD, one of the essential myogenic regulatory factors.ResultsHere, we present data identifying ICln as the specific, novel protein downstream of TIS7 controlling myogenesis. We show that TIS7/ICln epigenetically regulate myoD expression controlling protein methyl transferase activity. In particular, ICln regulates MyoD expression via its interaction with PRMT5 by an epigenetic modification that utilizes symmetrical di-methylation of histone H3 on arginine 8. We provide multiple evidences that TIS7 directly binds DNA, which is a functional feature necessary for its role in transcriptional regulation.ConclusionWe present here a molecular insight into TIS7-specific control of MyoD gene expression and thereby skeletal muscle differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0318-6) contains supplementary material, which is available to authorized users.

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.

Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation.

Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs.

Depletion of G9a gene induces cell apoptosis in human gastric carcinoma.

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Evidence suggests that G9a is required to maintain the malignant phenotype, but little documentation show the role of G9a function in mediating tumor growth. We retrospectively analyzed the protein of G9a and monomethylated histone H3 lysine 9 (H3K9 me1), and dimethylated histone H3 lysine 9 (H3K9 me2) in 175 cases of gastric carcinoma by immunohistochemistry. RNAi-based inhibition of G9a in MGC803 cancer cell line was studied. G9a depletion was done by transient transfection using Lipofectamine 2000. Depletion efficiency of G9a was tested using real-time PCR and western blot analysis. Cell apoptosis and proliferation were detected by TUNEL assay and MTT, respectively. The proteins of H3K9 me1, me2, trimethylation of H3K9 (H3K9 me3), monomethylated histone H3 lysine 27 (H3K27 me1), dimethylated histone H3 lysine 27 (H3K27 me2) and histone acetylated H3, apoptotic proteins were studied by western blot analysis. G9a and H3K9 me2 expression was higher in gastric cancer cells compared to the control (p<0.05). Both G9a and H3K9 me2 were positively correlated with the degree of differentiation, depth of infiltration, lymphatic invasions and tumor-node-metastasis stage in gastric carcinoma, (p<0.05). RNAi-mediated knockdown of G9a induced cell apoptosis and inhibited cell proliferation. Depletion of G9a reduced the levels of H3K9 me1 and me2, H3K27 me1 and me2. Nonetheless, it did not activate acetylation of H3 and H3K9 me3. These data suggest that G9a is required in tumorigenesis, and correlated with prognosis. Furthermore, G9a plays a critical role in regulating epigenetics. Depletion of G9a inhibits cell growth and induces cells apoptosis in gastric cancer. It might be of therapeutic benefit in gastric cancers.

The arginine methyltransferase PRMT5 regulates CIITA-dependent MHC II transcription

Class II major histocompatibility complex (MHC II) dependent antigen presentation serves as a key step in mammalian adaptive immunity and host defense. In antigen presenting cells (e.g., macrophages), MHC II transcription can be activated by interferon gamma (IFN-γ) and mediated by class II transactivator (CIITA). The underlying epigenetic mechanism, however, is not completely understood. Here we report that following IFN-γ stimulation, symmetrically dimethylated histone H3 arginine 2 (H3R2Me2s) accumulated on the MHC II promoter along with CIITA. IFN-γ augmented expression, nuclear translocation, and promoter binding of the protein arginine methyltransferase PRMT5 in macrophages. Over-expression of PRMT5 potentiated IFN-γ induced activation of MHC II transcription in an enzyme activity-dependent manner. In contrast, PRMT5 silencing or inhibition of PRMT5 activity by methylthioadenosine (MTA) suppressed MHC II transactivation by IFN-γ. CIITA interacted with and recruited PRMT5 to the MHC II promoter and mediated the synergy between PRMT5 and ASH2/WDR5 to activate MHC II transcription. PRMT5 expression was down-regulated in senescent and H2O2-treated macrophages rendering ineffectual induction of MHC II transcription by IFN-γ. Taken together, our data reveal a pathophysiologically relevant role for PRMT5 in MHC II transactivation in macrophages.