H3K9 Dimethylation Research Articles

Sirtuin 7 Promotes Mesenchymal to Epithelial Transition by β-Catenin Redistribution and Stabilization.

SIRT7 belongs to the family of “NAD+ dependent deacetylases” called Sirtuins. In the present work we report a novel role of SIRT7 in regulating cellular polarity. SIRT7 overexpression in immortalized mouse fibroblasts (NIH3T3) induced epithelial transition. This transition was accompanied by typical N- to E- cadherin transition, stabilization of β-catenin, and the downregulation of transcription factors responsible for maintenance of mesenchymal phenotype (Snail, Slug, and Zeb1). Interestingly, a subpopulation of cells overexpressing SIRT7 exhibited an intermediate stage between mesenchymal and epithelial characters. Transformed epithelial cells showed a loss of heterochromatisation as evidenced by a loss of HP1α and H3K9 dimethylation staining. In conclusion, we report a role of SIRT7 in mesenchymal cells, which may have implications for health and disease.

The lncRNA LAMP5-AS1 drives leukemia cell stemness by directly modulating DOT1L methyltransferase activity in MLL leukemia

BackgroundMixed-lineage leukemia (MLL) gene rearrangements trigger aberrant epigenetic modification and gene expression in hematopoietic stem and progenitor cells, which generates one of the most aggressive subtypes of leukemia with an apex self-renewal. It remains a challenge to directly inhibit rearranged MLL itself because of its multiple fusion partners and the poorly annotated downstream genes of MLL fusion proteins; therefore, novel therapeutic targets are urgently needed.MethodsqRT-PCR, receiver operating characteristic (ROC), and leukemia-free survival analysis were used to validate LAMP5-AS1 (LAMP5 antisense 1) expression and evaluate its clinical value. We performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5-AS1 in MLL leukemia progression and leukemia cell stemness. RNA electrophoretic mobility shift assays (EMSA), histone methyltransferase assay, RNA pull-down assay, and RNA fluorescence in situ hybridization (FISH) were used to validate the relationship between LAMP5-AS1 and the methyltransferase activity of DOT1L. The downstream ectopic target genes of LAMP5-AS1/DOT1L were validated by the chromatin immunoprecipitation (ChIP) and western blot.ResultsWe discovered that a long noncoding RNA (lncRNA) LAMP5-AS1 can promote higher degrees of H3K79 methylation, followed by upregulated expression of the self-renewal genes in the HOXA cluster, which are responsible for leukemia stemness in context of MLL rearrangements. We found that LAMP5-AS1 is specifically overexpressed in MLL leukemia patients (n = 58) than that in the MLL-wt leukemia (n = 163) (p < 0.001), and the patients with a higher expression level of LAMP5-AS1 exhibited a reduced 5-year leukemia-free survival (p < 0.01). LAMP5-AS1 suppression significantly reduced colony formation and increased differentiation of primary MLL leukemia CD34+ cells. Mechanistically, LAMP5-AS1 facilitated the methyltransferase activity of DOT1L by directly binding its Lys-rich region of catalytic domain, thus promoting the global patterns of H3K79 dimethylation and trimethylation in cells. These observations supported that LAMP5-AS1 upregulated H3K79me2/me3 and the transcription of DOT1L ectopic target genes.ConclusionsThis is the first study that a lncRNA regulates the self-renewal program and differentiation block in MLL leukemia cells by facilitating the methyltransferase activity of DOT1L and global H3K79 methylation, showing its potential as a therapeutic target for MLL leukemia.

H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres

The epigenetic environment plays an important role in DNA damage recognition and repair, both at DNA double-strand breaks and at deprotected telomeres. To increase understanding on how DNA damage responses (DDR) at deprotected telomeres are regulated by modification and remodeling of telomeric chromatin we screened 38 methyltransferases for their ability to promote telomere dysfunction-induced genomic instability. As top hit we identified MMSET, a histone methyltransferase (HMT) causally linked to multiple myeloma and Wolf-Hirschhorn syndrome. We show that MMSET promotes non-homologous end-joining (NHEJ) at deprotected telomeres through Ligase4-dependent classical NHEJ, and does not contribute to Ligase3-dependent alternative NHEJ. Moreover, we show that this is dependent on the catalytic activity of MMSET, enabled by its SET-domain. Indeed, in absence of MMSET H3K36-dimethylation (H3K36me2) decreases, both globally and at subtelomeric regions. Interestingly, the level of MMSET-dependent H3K36me2 directly correlates with NHEJ-efficiency. We show that MMSET depletion does not impact on recognition of deprotected telomeres by the DDR-machinery or on subsequent recruitment of DDR-factors acting upstream or at the level of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair.

Polycomb-like 2 regulates PRC2 components to affect proliferation in glioma cells

IntroductionThe Polycomb group (PcG) is an important family of transcriptional regulators that controls growth and tumorigenesis. The PcG mainly consists of two complexes, PRC1 and Polycomb Repressive Complex 2 (PRC2). Polycomb-like 2 (PCL2) is known to interact with the PRC2 protein. The role of PCL2 in the development and progression of glioma is unclear.MethodsWe use The Cancer Genome Atlas (TCGA) database to detect the expression of PCL2 in various tumors. 117 cases of clinical glioma (WHOI–IV) were collected, and PCL2 expression and localization were detected by immunohistochemical staining. Glioma cells U87/U251 were infected with overexpressed and interfered PCL2. CCK8 assay, colony formation assay, EdU method, cell cycle and apoptosis were used to detect cell proliferation and apoptosis. Western blot was used to detect the expression of PRC2-related core proteins. After DZNeP intervention, PRC2 protein expression was again measured to discuss the mechanism of PCL2 action.ResultsTCGA database results and immunohistochemical staining results suggest that PCL2 is highly expressed in gliomas. We found that the PCL2 gene promoted tumor cell proliferation, enhanced the colony formation ability, and increased S phase in the cell cycle. The overexpression of PCL2 upregulated the expression levels of EZH2 and EED (two core members of PRC2), decreased the expression of SUZ12, increased the level of H3K27 trimethylation (H3K27me3), H3K4 dimethylation (H3K4me2), and decreased H3K9 dimethylation (H3K9me2). The result after interfering with PCL2 was the opposite.ConclusionsAs an important accessory protein of PRC2, PCL2 can not only change the expression of PRC2 components, but also affect the expression level of Histone methylation. Therefore, PCL2 may be an important hub for regulating the synergy among PRC2 members. This study revealed PCL2 as a new target for tumor research and open up a new avenue for future research in glioma.

WHSC1 monomethylates histone H1 and induces stem-cell like features in squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck (SCCHN) is a malignancy with poor outcomes, thus novel therapies are urgently needed. We recently showed that WHSC1 is necessary for the viability of SCCHN cells through H3K36 di-methylation. Here, we report the identification of its novel substrate, histone H1, and that WHSC1-mediated H1.4K85 mono-methylation may enhance stemness features in SCCHN cells. To identify proteins interacting with WHSC1 in SCCHN cells, WHSC1 immunoprecipitation and mass spectrometry identified H1 as a WHSC1-interacting candidate. In vitro methyltransferase assays showed that WHSC1 mono-methylates H1 at K85. We generated an H1K85 mono-methylation-specific antibody and confirmed that this methylation occurs in vivo. Sphere formation assays using SCC-35 cells stably expressing either wild-type (FLAG-H1.4-WT) or mutated (FLAG-H1.4K85A) vector with lysine 85 to alanine substitution which is not methylated, indicated a higher number of spheres in SCC-35 cells expressing the wild type than those with the mutant vector. SCC-35 cells expressing the wild type H1.4 proliferated faster than those expressing the mutated vector. RNA sequencing, RT-PCR and Western blotting of the FLAG-H1.4-WT or FLAG-H1.4K85A SCC-35 cells revealed that OCT4 levels were higher in wild type compared to mutant cells. These results were reproduced in SCC-35 cells genetically modified with CRISPR to express H1.4K85R. Chromatin immunoprecipitation showed that FLAG-H1.4K85A had decreased occupancy in the OCT4 gene compared to FLAG-H1.4-WT. This study supports that WHSC1 mono-methylates H1.4 at K85, it induces transcriptional activation of OCT4 and stemness features in SCCHN cells, providing rationale to target H1.4K85 mono-methylation through WHSC1 in SCCHN.

Leishmania donovani Subverts Host Immune Response by Epigenetic Reprogramming of Macrophage M(Lipopolysaccharides + IFN-γ)/M(IL-10) Polarization.

Reciprocal changes in histone lysine methylation/demethylation of M(LPS + IFN-γ)/M(IL-10) genes is one of the factors that direct macrophage polarization and contribute to host defense/susceptibility toward infection. Although, histone lysine methyltransferases and lysine demethylases orchestrate these events, their role remains elusive in visceral leishmaniasis, a disease associated with macrophage M(IL-10) polarization. In this study, we observed that L. donovani induced the expression of histone lysine methyltransferases Ash1l, Smyd2, and Ezh2 and histone lysine demethylases Kdm5b and Kdm6b in J774 macrophages and BALB/c mice. Chromatin immunoprecipitation analysis revealed that L. donovani facilitated H3K36 dimethylation at TNF-α promoter by Smyd2 and H3K27 trimethylation at inducible NO synthase promoter by Ezh2 to suppress their expression in macrophages. Furthermore, infection-induced Kdm5b and Kdm6b modulated H3K4 and H3K27 trimethylation at IL-12, TNF-α, and arginase-1 promoters, respectively, whereas H3K4 trimethylation by Ash1l at IL-10 promoter induced its expression. Analysis of transductional events revealed that HIF-1α upregulated Kdm5b and Kdm6b expression, whereas Ash1l and Ezh2 expression were induced by transcription factor MeCP2. Additionally, Smyd2 was induced by c-Myc in infected macrophages. Knockdown of Ash1l, Ezh2, Kdm5b, and Kdm6b by specific small interfering RNA and Vivo-Morpholino, as well as inhibition of Smyd2 by its specific inhibitor, AZ505, led to increased protective proinflammatory response and inhibited amastigote multiplication in infected J774 macrophages and BALB/c mice, respectively. Collectively, our findings demonstrate that L. donovani exploits specific histone lysine methyltransferases/demethylases to redirect epigenetic programming of M(LPS + IFN-γ)/M(IL-10) genes for its successful establishment within the host.

Early-Life m6A RNA Demethylation by Fat Mass and Obesity-Associated Protein (FTO) Influences Resilience or Vulnerability to Heat Stress Later in Life.

Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the preoptic anterior hypothalamus (PO/AH) thermal response center and is regulated by epigenetic mechanisms. Here, we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N6-methyladenosine (m6A) RNA methylation in long-term response to heat stress. Heat conditioning of 3-d-old chicks diminished m6A RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the m6A demethylase, fat mass and obesity-associated protein (FTO). Moreover, a week later, methylation of two heat stress-related transcripts, histone 3 lysine 27 (H3K27) methyltransferase, enhancer of zeste homolog 2 (EZH2) and brain-derived neurotrophic factor (BDNF), were downregulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of m6A levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in m6A modification was negatively correlated with the expression levels of both BDNF and EZH2 Antisense "knock-down" of FTO caused an elevation of global m6A RNA methylation, reduction of EZH2 and BDNF mRNA levels, and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along BDNF coding region, and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress.

Investigation into the Role of Histone H3 Lysine 4 in Developmental Gene Regulation

Histone H3 lysine 4 (H3K4) methylation is an evolutionarily‐conserved modification of chromatin closely linked to gene activation. H3K4 monomethylation (me1) is associated with active enhancers and flanks promoters. H3K4 dimethylation (me2) is enriched at tissue‐specific transcription factor binding sites, enhancers, and promoter edges. H3K4 trimethylation (me3) defines active transcriptional initiation at promoters. H3K4me1, me2, and me3 in Drosophila melanogaster is mediated by the methyltransferases Trr, dSet1, and Trx. The human homologs of these enzymes (MLL3/4, SET1A/B, and MLL1/2, respectively) perform conserved roles in human gene regulation and are commonly mutated in cancers. While these histone‐modifying enzymes have been found to be major regulators of development, differentiation, and disease, their enzymatic activity is not limited to histones and they also have non‐enzymatic functions. For these reasons, the specific role of H3K4 modification in transcriptional regulation is not understood. To directly test the function of H3K4 in animal development, we mutated H3K4 in Drosophila melanogaster. In this approach, the endogenous wild‐type histone genes are replaced with transgenic versions that have been mutated to encode a non‐modifiable residue (H3K4R). Notably, Drosophila is the only animal experimental system in which histone gene replacement can be performed because histone genes exist in high copy numbers and at multiple locations throughout the genome of most animals. By contrast, the canonical histone genes in Drosophila are clustered in a single chromosomal locus, allowing for its precise deletion (1). Previously, we showed that wild‐type replacement transgene supports the propagation of a stable stock lacking all endogenous histone genes (1). By contrast, here we find that H3K4R replacement transgenes result in embryonic lethality. Thus, H3K4 modification is essential for animal development. In future work, we will determine the effects of H3K4 mutation on gene expression through RNA‐seq analysis, as well as its impact on the histone post‐translational modification landscape through mass spectrometry. Together, this work will extend our understanding of the mechanistic role that histone modifications contribute to developmental gene regulation.Support or Funding InformationNIH to DJM R35‐GM128851

ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet

Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclicAMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7+/- mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7+/- mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.

H3K9 Demethylation-Induced R-Loop Accumulation Is Linked to Disorganized Nucleoli

The nucleolar structure and integrity are important for a range of cellular functions of the nucleoli. It has been shown that cells lacking histone H3 Lysine 9 (H3K9) methylation form fragmented nucleoli. However, the molecular mechanism involved remains poorly understood. Here, we present evidence suggesting that loss of H3K9 dimethylation (H3K9me2) triggers R-loop accumulation at the rDNA locus, which further leads to the multilobed nucleoli. We reveal that suppression of H3K9 methyltransferase G9a by the inhibitor BIX 01294 causes R-loop accumulation at the rDNA region as well as inducing formation of multiple nucleoli. SiRNA-mediated knockdown of RNase H1 which can hydrolyze the RNA chain in R-loops causes an increase in R-loop formation, which in turn results in multiple nucleoli in one nucleus, whereas H3K9me2 levels are not affected by R-loop accumulation. Inhibition of RNA polymerase I transcription elongation by small molecule inhibitors induces a substantial decrease in H3K9me2 levels, accumulation of R-loops at rDNA sites, and nucleolus fragmentation. These results provide a mechanistic insight into the role of H3K9me2 in the structural integrity and organization of nucleoli via regulating R-loop accumulation.

Maternal obesogenic diet combined with postnatal exposure to high-fat diet induces metabolic alterations in offspring.

Maternal obesity has been shown to impact the offspring health during childhood and adult life. This study aimed to evaluate whether maternal obesity combined with postnatal exposure to an obesogenic diet could induce metabolic alterations in offspring. Female CD1 mice were fed a control diet (CD, 11.1% of energy from fat) or with a high-fat diet (HFD, 44.3% of energy from fat) for 3 months. After weaning, pups born from control and obese mothers were fed with CD or HFD for 3 months. Both mothers and offspring were weighted weekly and several blood metabolic parameters levels were evaluated. Here, we present evidence that the offspring from mothers exposed to a HFD showed increased acetylation levels of histone 3 on lysine 9 (H3K9) in the liver at postnatal Day 1, whereas the levels of acetylation of H4K16, dimethylation of H3K27, and trimethylation of H3K9 showed no change. We also observed a higher perinatal weight and increased blood cholesterol levels when compared to the offspring on postnatal Day 1 born from CD-fed mothers. When mice born from obese mothers were fed with HFD, we observed that they gained more weight, presented higher blood cholesterol levels, and abdominal adipose tissue than mice born to the same mothers but fed with CD. Collectively, our results point toward maternal obesity and HFD consumption as a risk factor for epigenetic changes in the liver of the offspring, higher perinatal weight, increased weight gain, and altered blood cholesterol levels.

Protein kinase A accelerates the rate of early stage differentiation of pluripotent stem cells

In normal development, the rate of cell differentiation is tightly controlled and critical for normal development and stem cell differentiation. However, the underlying mechanisms regulating the rate of the differentiation are unknown, and manipulation of the rate of the stem cell differentiation is currently difficult. Here we show that activation of protein kinase A (PKA) accelerates the rate of mouse embryonic stem cell (ESC) differentiation through an early loss of ESC pluripotency markers and early appearance of mesodermal and other germ layer cells. The activation of PKA hastened differentiation by increasing the expression of a histone H3 lysine 9 (H3K9) dimethyltransferase, G9a protein, and the level of a negative epigenetic histone mark, H3K9 dimethylation (H3K9me2), in the promoter regions of the pluripotency markers Nanog and Oct4. These results elucidate a novel role of PKA on ESC differentiation and offer an experimental model for controlling the rate of ESC differentiation.

Epigenetic regulation affects gene amplification in Drosophila development.

In Drosophila melanogaster, in response to developmental transcription factors, and by repeated initiation of DNA replication of four chorion genes, ovarian follicle cells, form an onion skin-type structure at the replication origins. The DNA replication machinery is conserved from yeast to humans. Subunits of the origin recognition complex (ORC) is comprised of Orc1, Orc2, and Cdc6 genes. While mutations of Orc1 and Orc2 and not Cdc6can be lethal, overexpression of these genes lead to female sterility. Ecdysone, is a steroidal prohormone of the major insect molting hormone 20-hydroxyecdysone that in Drosophila, triggers molting, metamorphosis, and oogenesis. To this end, we identified several ecdysone receptor (EcR) binding sites around gene amplification loci. We also found that H3K4 was trimethylated at chorion gene amplification origins, but not at the act1 locus. Female mutants overexpressing Lsd1 (a dimethyl histone H3K4 demethylase) or Lid (a trimethyl histone H3K4 demethylase), but not a Lid mutant, were sterile. The data suggest that ecdysone signaling determines which origin initiates DNA replication and contributes to the development. Screening strategies using Drosophila offer the opportunity for development of drugs that reduce gene amplification and alter histone modification associated with epigenetic effects.

The histone methyltransferase Suv39h regulates 3T3-L1 adipogenesis

ABSTRACT We discovered a unique expression pattern of two histone methyltransferases Suv39h1 and Suv39h2 during 3T3-L1 adipogenesis, both of which preferentially catalyse the formation of H3K9 dimethylation (H3K9me2) and further H3K9 trimethylation (H3K9me3), a transcriptional repressive mark. The expression of Suv39h1 and Suv39h2 displayed a sharp increase at the early stage of 3T3-L1 differentiation, which peaked after differentiation induction, and then declined towards later stage of differentiation, suggesting a key role for these two histone methyltransferases in adipogenesis. Indeed, inactivating Suv39h1 or Suv39h2 via lentiviral shRNA knockdown inhibited adipogenesis, while overexpressing Suv39h1 promoted adipogenesis. Notably, overexpressing or knocking down Suv39h1 in 3T3-L1 cells was associated with reciprocal changes in the expression of Wnt10a, an anti-adipogenic regulator. Further, Wnt10a knockdown largely prevented the inhibitory effect of Suv39h1 on adipogenesis, indicating Wnt10a as a downstream target mediating Suv39h1’s action in adipogenesis. Mechanistically, our comprehensive approaches involving ChIP, co-immunoprecipitation and pyrosequencing analysis demonstrated that Suv39h1 may regulate Wnt10a expression via H3K9 methylation and interaction with DNA methyltransferase 1 (DNMT1) at the Wnt10a promoter, resulting in altered DNA methylation at the promoter. We conclude that Suv39h promotes adipogenesis by epigenetically down-regulating Wnt10a expression via H3K9me3 and DNA methylation at the Wnt10a promoter.Abbreviated title: Suv39h and 3T3-L1 Adipogenesis

Loss of Histone H3 K79 Methyltransferase Dot1l Facilitates Kidney Fibrosis by Upregulating Endothelin 1 through Histone Deacetylase 2.

The progression rate of CKD varies substantially among patients. The genetic and epigenetic contributions that modify how individual patients respond to kidney injury are largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l has an antifibrotic effect by repressing Edn1, which encodes endothelin 1 in the connecting tubule/collecting duct. To determine if deletion of the Dot1l gene is a genetic and epigenetic risk factor through regulating Edn1, we studied four groups of mice: wild-type mice, connecting tubule/collecting duct-specific Dot1l conditional knockout mice (Dot1lAC ), Dot1l and Edn1 double-knockout mice (DEAC ), and Edn1 connecting tubule/collecting duct-specific conditional knockout mice (Edn1AC ), under three experimental conditions (streptozotocin-induced diabetes, during normal aging, and after unilateral ureteral obstruction). We used several approaches (colocalization, glutathione S-transferase pulldown, coimmunoprecipitation, yeast two-hybrid, gel shift, and chromatin immunoprecipitation assays) to identify and confirm interaction of Dot1a (the major Dot1l splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating Edn1 transcription. In each case, Dot1lAC mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These Dot1lAC phenotypes were ameliorated in the double-knockout DEAC mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing Edn1 transcription by modulating H3 K79 dimethylation and H3 acetylation at the Edn1 promoter. Our study confirms Dot1l to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating Edn1 transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.

Mediator MED23 regulates inflammatory responses and liver fibrosis.

Liver fibrosis, often associated with cirrhosis and hepatocellular carcinomas, is characterized by hepatic damage, an inflammatory response, and hepatic stellate cell (HSC) activation, although the underlying mechanisms are largely unknown. Here, we show that the transcriptional Mediator complex subunit 23 (MED23) participates in the development of experimental liver fibrosis. Compared with their control littermates, mice with hepatic Med23 deletion exhibited aggravated carbon tetrachloride (CCl4)-induced liver fibrosis, with enhanced chemokine production and inflammatory infiltration as well as increased hepatocyte regeneration. Mechanistically, the orphan nuclear receptor RAR-related orphan receptor alpha (RORα) activates the expression of the liver fibrosis-related chemokines C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10), which is suppressed by the Mediator subunit MED23. We further found that the inhibition of Ccl5 and Cxcl10 expression by MED23 likely occurs because of G9a (also known as euchromatic histone-lysine N-methyltransferase 2 [EHMT2])-mediated H3K9 dimethylation of the target promoters. Collectively, these findings reveal hepatic MED23 as a key modulator of chemokine production and inflammatory responses and define the MED23-CCL5/CXCL10 axis as a potential target for clinical intervention in liver fibrosis.

Abstract LB-C10: Preclinical synergistic anti-tumor effect against multiple cancer types by EZH2 inhibitor EPZ-6438 plus imipridone ONC201 to mimic H3K27M mutation observed in DIPG tumors that respond to ONC201 in the clinic

Abstract ONC201, the founding member of the imipridone class of small molecules, has broad spectrum anti-tumor activity and achieved tumor regressions and prolonged disease stability in clinical trials, especially in patients with histone H3 K27M mutated glioma. Levels of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3) are reduced globally in H3 K27M patient samples. EZH2 is the catalytic component of the Polycomb Repressor Complex 2 (PRC2), a conserved multi-subunit that represses gene transcription by methylating lysine 27 on Histone H3 (H3K27). EZH2 is frequently overexpressed or mutated (activating) in numerous human malignancies and EZH2 inhibitors represent a promising therapeutic strategy. We hypothesized that cancer cells could be sensitized to ONC201 by EZH2 inhibition through suppression of H3K27 methylation that may mimic tumors such as DIPG with H3K27M mutations which are known to respond to ONC201 in the clinic and are defective in H3K27 methylation. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response and apoptosis in combination with EZH2 inhibitor EPZ-6438. There was greater induction of ATF4 in combination therapy-treated tumor cells versus monotherapy. Intriguingly, U-251 GBM cells that were resistant to ONC201 under hypoxia could be sensitized to ONC201 by EZH2 inhibitor EPZ-6438. Our findings provide a preclinical rationale for developing ONC201 and EZH2 inhibitors as a novel synergistic combinatorial regimen with the potential for clinical translation in the treatment of several human malignancies including GBM, pancreatic cancer, colorectal cancer, and breast cancer. Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Robyn Borsuk, Howard P. Safran, Attila A. Seyhan, Wafik S. El-Deiry. Preclinical synergistic anti-tumor effect against multiple cancer types by EZH2 inhibitor EPZ-6438 plus imipridone ONC201 to mimic H3K27M mutation observed in DIPG tumors that respond to ONC201 in the clinic [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C10. doi:10.1158/1535-7163.TARG-19-LB-C10

Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry.

Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modifications (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification of histone PTMs are still challenging. Although classic chemical derivatization in conjunction with trypsin digestion is widely used for histone PTMs analysis in a bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T (OmpT) was utilized as a protease for direct histone proteolysis and generated appropriate lengths of histone peptides for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysines and arginines was demonstrated and can be quantitatively described for the first time, making it useful for detecting natural modifications. Using the optimized digestion conditions, we succeeded in identifying 121 histone marks from HEK293T cells, 42 of which were previously unreported. Additionally, histone H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma cells and NSD2 selective knockout KMS11cells, revealing that NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.

Plasticity of histone modifications around Cidea and Cidec genes with secondary bile in the amelioration of developmentally-programmed hepatic steatosis

We recently reported that a treatment with tauroursodeoxycholic acid (TUDCA), a secondary bile acid, improved developmentally-deteriorated hepatic steatosis in an undernourishment (UN, 40% caloric restriction) in utero mouse model after a postnatal high-fat diet (HFD). We performed a microarray analysis and focused on two genes (Cidea and Cidec) because they are enhancers of lipid droplet (LD) sizes in hepatocytes and showed the greatest up-regulation in expression by UN that were completely recovered by TUDCA, concomitant with parallel changes in LD sizes. TUDCA remodeled developmentally-induced histone modifications (dimethylation of H3K4, H3K27, or H3K36), but not DNA methylation, around the Cidea and Cidec genes in UN pups only. Changes in these histone modifications may contribute to the markedly down-regulated expression of Cidea and Cidec genes in UN pups, which was observed in the alleviation of hepatic fat deposition, even under HFD. These results provide an insight into the future of precision medicine for developmentally-programmed hepatic steatosis by targeting histone modifications.

Inhibition of LSD1 in Myelodysplastic Syndrome Progenitors Restores Differentiation of CD141Hi Conventional Dendritic Cells

Background: Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hi conventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141Hi cDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141Hi cDCs due to impaired myeloid differentiation. Methods: Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results: We found fewer CD141Hi cDCs (p&lt;0.0001), CD1c+ cDCs (p&lt;0.005) and plasmacytoid DCs (CD123+ pDCs; p&lt;0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141Hi cDCs had superior survival (p &lt; 0.05). No differences in survival were seen in patients stratified by the CD1c+ cDCs (p = 0.96) and CD123+ pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141Hi cDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p&lt;0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141Hi cDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p&lt;0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141Hi cDCs compared to those with higher levels of IRF8 (n=10; p&lt;0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141Hi cDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8 expression in myeloid leukemia cells, would induce CD141Hi cDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+ cells, and in HD and MDS CD34+ progenitors (p&lt;0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p&lt;0.05) at a region -70 kb to the IRF8 transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8 locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+ cells increased the number of mature CD141Hi cDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+ cells increased the number of CD141Hi cDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+ cells from Irf8 knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+ cells resulted in increased numbers of CD141Hi cDCs in vitro (p&lt;0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+ cells did not result in differentiation of CD141Hi cDCs. These data suggest that LSD1 inhibition drives CD141Hi cDCs differentiation through IRF8. Conclusion: These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141Hi cDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients. Disclosures Sait: Celgene: Consultancy. Griffiths:Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Abbvie, Inc.: Consultancy; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial.