Histone Methyltransferase Activity Research Articles

Structure-based design of conformationally constrained cyclic peptidomimetics to target the MLL1-WDR5 protein–protein interaction as inhibitors of the MLL1 methyltransferase activity

Abstract We described herein structure-based design, synthesis and evaluation of conformationally constrained, cyclic peptidomimetics to block the MLL1-WDR5 protein–protein interaction as inhibitors of the MLL1 histone methyltransferase activity. Our study has yielded cyclic peptidomimetics with very high binding affinities to WDR5 (Ki values

Ribavirin as a tri-targeted antitumor repositioned drug.

The aim of the present study was to demonstrate that ribavirin, a known inhibitor of eIF4E and inosine 5'-phosphate dehydrogenase (IMPDH), also inhibits histone methyltransferase zeste homolog 2 (EZH2). A computational searching revealed that ribavirin has a high structural similarity to 3-deazaneplanocin A (DZNep). The growth inhibitory effects of ribavirin as well as its effects upon epigenetic enzymes were evaluated in various cancer cell lines. siRNA assays were used to downregulate eIF4E, EZH2 and IMPDH to determine the contribution of these targets to the growth inhibitory effects of ribavirin. Ribavirin decreased EZH2 expression, inhibited histone methyltransferase activity and decreased H3K27 trimethylation. Ribavirin induced variable growth inhibition in a number of cell lines and downregulation of the targets, EZH2, eIF4E and IMPDH1 and 2 by siRNA led to comparable growth inhibition while no significant further reduction in viability was observed when siRNA transfected cells were treated with ribavirin. The results showed that ribavirin inhibits these cancer targets and should thus be studied for cancer therapy.

ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development.

ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.

Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming.

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic “memory” and to establish the pluripotent state. Accordingly, alterations of chromatin regulators including Ezh2 influence iPSC generation. While the role of individual transcription factors in resetting the chromatin landscape during iPSC generation is increasingly evident, their engagement with chromatin modulators remains to be elucidated. In the current study, we demonstrate that histone methyl transferase activity of Ezh2 is required for mesenchymal to epithelial transition (MET) during human iPSC generation. We show that the H3K27me3 activity favors induction of pluripotency by transcriptionally targeting the TGF-β signaling pathway. We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET. Unique association of Ezh2 with c-Myc was required to silence the aforementioned circuitry. Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation.

Histone methylation patterns in astrocytes are influenced by age following ischemia

In animal models, middle-aged females sustain greater ischemia-induced infarction as compared to adult females. This age difference in infarct severity is associated with reduced functional capacity of astrocytes, a critical neural support cell. The impaired response of astrocytes following stroke in middle-aged females may be related to epigenetic alterations, including histone acetylation or methylation. The present study measured the activity of enzymes that regulate histone acetylation and methylation in cerebral cortical astrocytes of adult (6 month) and middle-aged (11+ month) female rats 48 h following middle cerebral artery occlusion. H3K4 histone methyltransferase activity was decreased in astrocytes from middle-aged females. The next experiment therefore examined H3K4me3 (transcriptional enhancer) and H3K9me3 (transcriptional repressor) in astrocytes from adult and middle-aged females using ChIP-seq analysis. Adult females had more enriched H3K4me3 peaks (304 vs. 26) at transcriptional start sites and fewer H3K9me3 enriched peaks than middle-aged females (4 vs. 22), indicating a pattern of less active chromatin in astrocytes in the older group following ischemia. DAVID clustering analysis of H3K4me3 enriched genes found several functional categories, including cell motility, regulation of apoptosis and the vascular endothelial growth factor (VEGF) pathway. H3K4me3 was enriched at the miR-17-20 cluster and VEGFa, and analysis of a separate set of astrocytes confirmed that VEGF protein expression and miR-20 mRNA expression were significantly greater following ischemia in adult females compared to middle-aged females. These data indicate that astrocytes display less active chromatin with aging and provide new insight into possible mechanisms for differences in stroke severity observed during aging.

Recognition of H3K9 methylation by GLP is required for efficient establishment of H3K9 methylation, rapid target gene repression, and mouse viability.

GLP and G9a are major H3K9 dimethylases and are essential for mouse early embryonic development. GLP and G9a both harbor ankyrin repeat domains that are capable of binding H3K9 methylation. However, the functional significance of their recognition of H3K9 methylation is unknown. Here, we report that the histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes that are premethylated at H3K9. These stimulation events function in cis and are dependent on the H3K9 methylation binding activities of ankyrin repeat domains of GLP and G9a. Disruption of the H3K9 methylation-binding activity of GLP in mice causes growth retardation of embryos, ossification defects of calvaria, and postnatal lethality due to starvation of the pups. In mouse embryonic stem cells (ESCs) harboring a mutant GLP that lacks H3K9me1-binding activity, critical pluripotent genes, including Oct4 and Nanog, display inefficient establishment of H3K9me2 and delayed gene silencing during differentiation. Collectively, our study reveals a new activation mechanism for GLP and G9a that plays an important role in ESC differentiation and mouse viability.

CB1-receptor knockout neonatal mice are protected against ethanol-induced impairments of DNMT1, DNMT3A, and DNA methylation.

The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type-1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol-induced activation of caspase 3. Together, these data demonstrate that ethanol-induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis. Schematic mechanism of action by which ethanol impairs DNA methylation. Studies have demonstrated that ethanol has the capacity to bring epigenetic changes to contribute to the development of fetal alcohol spectrum disorder (FASD). However, the mechanisms are not well studied. P7 ethanol induces the activation of caspase 3 and impairs DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) proteins (→). The inhibition or genetic ablation of cannabinoid receptor type-1 or inhibition of histone methyltransferase (G9a) by Bix (-----) or inhibition of caspase 3 activation by Q- quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) () rescue loss of DNMT1, DNMT3A as well as DNA methylation. Hence, the putative DNMT1/DNMT3A/DNA methylation mechanism may have a potential regulatory role in FASD.

PRDM9 drives evolutionary erosion of hotspots in Mus musculus through haplotype-specific initiation of meiotic recombination.

Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent's Prdm9 allele and the opposite parent's chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.

Gain-of-function mutation of chromatin regulators as a tumorigenic mechanism and an opportunity for therapeutic intervention.

Somatic gain-of-function mutations that drive cancer pathogenesis are well established opportunities for therapeutic intervention, as demonstrated by the clinical efficacy of kinase inhibitors in kinase-mutant malignancies. Here, we discuss the recently discovered gain-of-function mutations in chromatin-regulatory machineries that promote the pathogenesis of cancer. The current understanding of the underlying molecular mechanisms and the therapeutic potential for direct chemical inhibition will be reviewed. Point mutations that increase the catalytic activity of EZH2 and NSD2 histone methyltransferases are found in distinct subsets of B-cell neoplasms, which promote cell transformation by elevating the global level of H3K27 tri-methylation or H3K36 di-methylation, respectively. In addition, mutations in histone H3 have been identified in certain pediatric cancers which cause reprogramming of H3K27 and H3K36 methylation by interfering with the histone methyltransferase activity. Finally, chromosomal translocations involving chromatin regulator genes can lead to the formation of fusion oncoproteins that directly modify chromatin as their mechanism of action. Although relatively rare in aggregate, gain-of-function mutations in chromatin regulators represent compelling therapeutic targets in genetically defined subsets of cancer patients. However, a broader clinical impact for epigenetic therapies in oncology will require an increased understanding of how nonmutated chromatin regulators function as cancer-specific dependencies.

Deconvoluting MLL1-dependent pathways in hematopoiesis and leukemogenesis

The Mixed Lineage Leukemia gene (MLL1, previously termed HRX, ALL-1, MLL) was identified in humans as a gene at the site of common chromosomal translocations in infant leukemia.1 It is now clear that MLL1 represents a family of histone methyltransferases (HMTs) including its closest relative MLL2 and MLL3, MLL4, SETD1A, SETD1B and possibly MLL5.2 Most of these proteins possess histone H3, lysine 4 (H3K4) HMT activity encoded by the SET domain (for Suvar 3–9, Enhancer of Zeste, Trithorax, epigenetic regulators). Studies of the Drosophila homeotic regulator and MLL1 ortholog, Trithorax, established the paradigm for mechanisms by which these proteins function in vivo.3 For example, the fact that Trithorax regulates clustered homeodomain (Hox) genes prompted this line of investigation in the first loss-of-function mouse Mll1 mutants, which were demonstrated to mis-express multiple Hox genes.4, 5, 6 Since the discovery that the SET domain encodes HMT activity,7, 8 great attention has been paid to this particular enzymatic activity in regulating downstream target genes. To determine the function of MLL1 in hematopoietic stem/progenitor cells (HSPCs), we previously analyzed conditional knockout animals and determined that deletion of Mll1 rapidly reduced HSPCs through proliferation and cell fate defects.9 Using this model system, we identified genes that were deregulated upon Mll1 deletion in the hematopoietic stem cell (HSC)-enriched LSK/CD48neg population. Small-scale chromatin immunoprecipitation experiments identified a series of transcriptional regulators that were direct MLL1 target genes including Mecom, Pbx1 and Prdm16, which are known to control HSC self-renewal and quiescence.10 To determine whether maintenance of these target genes required the HMT activity of MLL1, a mouse strain in which homozygous germline deletion of the SET domain of MLL1 (ΔSET) was used for the analysis of the hematopoietic system.6 Surprisingly, we found that H3K4 methylation at target genes such as Hoxa9, Mecom and Prdm16 was absolutely normal in hematopoietic cells of ΔSET-mutant animals, as was the expression level of these genes. We also found that retroviral MLL-AF9 expression generated leukemia in ΔSET HSPCs that was indistinguishable in latency or phenotype from leukemia generated in wild-type HSPCs. Collectively, none of the known biological functions of MLL1 in the hematopoietic system required the HMT activity. To understand how MLL1 maintains the expression of its target genes, a survey of histone modifications that undergo changes upon deletion of Mll1 was performed. We identified only histone H4 lysine 16 acetylation (H4K16Ac) as immediately corresponding to the decrease in transcriptional initiation upon MLL1 removal from the target gene promoter regions. This modification is performed by the histone acetyl transferase MOF (also called Myst1 or KAT8), previously discovered as part of the MLL1 protein complex.11 Furthermore, inhibition of SIRT1, a deacetylase that removes H4K16Ac, was sufficient to prevent the loss of transcription at MLL1 targets. These observations together with our data from the MLL1▵SET knockout mouse provide a clear but unexpected mechanism for the maintenance of target gene expression by MLL1 in the hematopoietic cells.12 These studies highlight differences between endogenous MLL1 and MLL-fusion oncoprotein gene regulation mechanisms, suggesting that differential targeting in leukemia may be feasible.

SUVR2 is involved in transcriptional gene silencing by associating with SNF2-related chromatin-remodeling proteins in Arabidopsis.

The SU(VAR)3-9-like histone methyltransferases usually catalyze repressive histone H3K9 methylation and are involved in transcriptional gene silencing in eukaryotic organisms. We identified a putative SU(VAR)3-9-like histone methyltransferase SUVR2 by a forward genetic screen and demonstrated that it is involved in transcriptional gene silencing at genomic loci targeted by RNA-directed DNA methylation (RdDM). We found that SUVR2 has no histone methyltransferase activity and the conserved catalytic sites of SUVR2 are dispensable for the function of SUVR2 in transcriptional silencing. SUVR2 forms a complex with its close homolog SUVR1 and associate with three previously uncharacterized SNF2-related chromatin-remodeling proteins CHR19, CHR27, and CHR28. SUVR2 was previously thought to be a component in the RdDM pathway. We demonstrated that SUVR2 contributes to transcriptional gene silencing not only at a subset of RdDM target loci but also at many RdDM-independent target loci. Our study suggests that the involvement of SUVR2 in transcriptional gene silencing is related to nucleosome positioning mediated by its associated chromatin-remodeling proteins.

Epigenetic approaches in glioblastoma multiforme and their implication in screening and diagnosis.

Epigenetic modifications have been reported in a number of non-germ-line tumor types. Epigenetic modifications to the genome, especially DNA methylation and histone modifications, affect gene expression causing increased risk for cancers and other diseases. We have summarized information about DNA methylation percentages in Glioblastoma multiforme (GBM) line HTB-12, alveolar cell carcinoma, and acute lymphocytic leukemia samples and determined H3 (K27) methyltransferase activity in GBM and leukemia cells and made comparisons to H3 (K27) methyltransferase activity in normal astrocyte, lung, and lymphocyte cells. GBM and alveolar cell carcinoma gDNA possessed lower gDNA methylation percentages compared to normal cells. Methyl-sensitive cut counting analysis (MSCC) showed fold decreases in GBM CpG methylation sites for genes PBK, KIF23, COL6A3, and LOX. There was no significant difference in CpG DNA methylation, but less histone methyltransferase activity in acute lymphocytic leukemia compared to normal cells. GBM possessed increased histone methyltransferase activity compared to normal samples. Challenges in the field in diagnosis and prognosis for cancer risk especially with regard to the results of this work are discussed.

Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor buttonhead.

The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.

Abstract 541: The role of long noncoding RNA SChLAP1 in prostate cancer

Abstract Prostate cancer is the second most common epithelial cancer and second leading cause of cancer death in men. Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority of patients. The molecular basis for this clinical heterogeneity remains incompletely understood. Long noncoding RNAs (lncRNAs) have emerged as a prominent layer of transcriptional regulation implicated in various biological and disease processes, including several types of cancer. Therefore, we hypothesized that lncRNAs may play a role in mediating aggressive prostate cancer. Previously, our lab utilized RNA sequencing across a cohort of prostate tissues to discover differentially expressed lncRNAs in cancer versus benign samples. We identifed a novel transcript termed SChLAP1 (Second Chromosome Locus Associated with Prostate-1) overexpressed in a subset of localized and metastatic cancers. Clinical analyses showed that SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo experiments indicated that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, we found that SChLAP1 binds to and antagonizes the genome-wide localization of the tumor-suppressive SWI/SNF nucleosome-remodeling complex. Given the scaffolding capabilities of lncRNAs, previous studies describing an inhibitory role of SWI/SNF on PRC2 function, and the known oncogenic role of PRC2 in prostate cancer, we hypothesized that SChLAP1 may mediate PRC2 activity in conjunction with SWI/SNF to promote aggressive disease. Here, we show that a SChLAP1-associated gene signature nominates PRC2-related concepts, and in vitro studies indicate an interaction between SChLAP1 and PRC2 that enhances PRC2 genome-wide localization as well as its histone methyltransferase activity. These results suggest a mechanistic model in which SChLAP1 simultaneously engages multiple epigenetic complexes, inhibiting tumor-suppressive functions of SWI/SNF while enhancing oncogenic activity of PRC2, which leads to aggressive prostate cancer. Citation Format: Anirban Sahu, Matthew K. Iyer, John R. Prensner, Arul M. Chinnaiyan. The role of long noncoding RNA SChLAP1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2014-541

Abstract 5162: Effects of combined epigenetic therapy with histone methyltransferase EZH2 inhibitor 3-deazaneplanocin and histone deacetylase inhibitor SAHA on non-small cell lung cancer cells

Abstract [Background] EZH2, a polycomb group protein, has histone methyltransferase activity, and is involved in malignant transformation of several cancers. We have previously shown that an EZH2 inhibitor, DZNep, inhibits growth of non-small cell lung cancer(NSCLC) cell lines via G1 arrest and apoptosis. Recently, cotreatment with DZNep and an histone deacetylase (HDAC) inhibitor has been shown to induce apoptosis synergistically in AML, ovarian cancers and renal cancers. Therefore, we determined the combined effect of DZNep and an HDAC inhibitor, SAHA, on NSCLC cells. [Methods] We evaluated the effect of combined therapy with DZNep and SAHA against four human NSCLC cell lines_H1299, H1975, A549 and PC-3. Cell proliferation was measured by MTT assay. Percentage of apoptotic cells was measured using Annexin V-FITC with a flow cytometer. Western blot analysis was performed on total cell lysates. For in vivo assay, H1975 cells were subcutaneously implanted into the flank of BALB/cAJcl nu/nu nude mice. When the average tumor volume reached approximately 50∼100mm3, the following treatments were intraperitoneally administered twice per week for 6 weeks; vehicle alone (5% DM SO), DZNep (4mg/kg), SAHA (40mg/kg), and DZNep (4mg/kg) plus SAHA (40mg/kg). [Results] Co-treatment with DZNep and SAHA inhibited cell proliferation synergistically, and reduced EZH2 expression and histone H3 lysine 27 trimethylation more effectively compared with each agent alone. The co-treatment greatly induced accumulation of p27 Kip1 and decrease in cyclin A expression. Flow cytometry analysis demonstrated that the apoptotic fraction was increased in an additive or synergistic manner by the combination therapy. These effects were more evident in H1975 and PC-3 cells with EGFR mutation, in which the co-treatment markedly reduced phosphorylation of EGFR, AKT and ERK1/2. The combined therapy reduced the levels of β-catenin, and its downstream pro-proliferative targets cyclin D1 and EGFR. The co-treatment with DZNep and SAHA also caused significantly greater inhibition of tumor growth of H1975 xenografts than each agent alone in nude mice without visible toxicity. [Conclusion] Combined epigenetic therapy with an EZH2 inhibitor and an HDAC inhibitor may represent an effective approach for NSCLCs. Citation Format: Taichi Takashina, Ichiro Kinoshita, Junko Kikuchi, Yasushi Shimizu, Jun Sakakibara Konishi, Satoshi Oizumi, Masaharu Nishimura, Hirotoshi Dosaka Akita. Effects of combined epigenetic therapy with histone methyltransferase EZH2 inhibitor 3-deazaneplanocin and histone deacetylase inhibitor SAHA on non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5162. doi:10.1158/1538-7445.AM2014-5162

Abstract 1375: Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models

Abstract Background: Sunitinib is first-line therapy for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite the clinical efficacy of sunitinib, in the majority of patients the disease eventually develops resistance and progresses. Epigenetic modifications of histone protein in chromatins have been shown to play a role in the regulation of gene transcription patterns in cells primarily by the catalytic activity of histone methyltransferase. EZH2 has been shown to contribute to tumor angiogenesis by inactivating anti-angiogenic factors via methylation at their promoter region. In this study, we examined epigenetic changes that may be associated with response and the resistant phenotype. Methods: Human ccRCC xenograft models RP-01 (isolated from a skin metastasis in a patient with sporadic ccRCC) and RP-02 (isolated from a skin metastasis in a patient with hereditary ccRCC) were implanted (∼1mm pieces) subcutaneously into SCID mice and were randomly assigned into two groups (sunitinib and vehicle). Mice were treated 5 days/week with sunitinib at 40mg/kg. Tumor volumes and body weights were assessed weekly by caliper measurements and weigh scale, respectively. Tumor tissues and blood were collected prior treatments and when tumors became resistant to treatment. Tissues collected were used for immunohistochemistry studies and RNA analysis. Results: Our results showed that RP-01 and RP-02 tumors, although initially responsive to treatment at 40 mg/kg, eventually became resistant to treatment. Immunohistochemistry analysis of RP-01 tumor samples indicated tumors to be hypo-vasculature when responding to sunitinib, but then became hyper-vascularized at point of resistance by CD31 staining. Analysis also revealed an associated increase in the expression of the methyltransferase EZH2 when tumors became resistant to sunitinib. However, there was no change in the expression levels of its EZH2 associated histone make H3K27me3. In addition, we noticed an increase in the levels of H3k9me2 and H3k4me2 in tumors resistant to sunitinib as compared to controls. Conclusion: Overall, our results suggest the potential role of epigenetic changes that that may be associated with sunitinib-induced resistance in ccRCC Citation Format: REMI ADELAIYE, Kiersten Marie Miles, Eric Ciamporcero, Dylan Conroy, Swathi Ramakrishnan, Ashley Orillion, Sheng Yu Ku, May Elbanna, Li shen, Sreenivasulu Chintala, Roberto Pili. Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2014-1375

Abstract 5160: Development of a non-radioactive method to determine Protein-Methyl-Transferase activity in-vitro

Abstract The epigenetic regulation of gene expression has been of increasing interest in the last couple of years. It has been shown that methylation of DNA may pass information epigenetically even from one cellular generation to the next without changing the DNA sequence. Epigenetic information may also be transferred by acetylation, methylation and phosphorylation of DNA binding proteins, e.g. transcription factors and histones. The latter constitute the core of the nucleosomal particles, which are essential for structuring the DNA into chromatin. Histones are methylated on specific lysine or arginine residues by a family of enzymes called HMTs (Histone Methyl Transferases). It leads to either compacting or loosening of the chromatin structure which influences the transcriptional activity of the DNA bound to these histones. Histones in each nucleosomal particle may be differentially methylated and the exact information coded into such methylation patterns is not yet well understood. Overexpression of several HMTs, however, has been found in a number of pathological conditions including cancer. Therefore HMTs are a promising target class for pharmacological intervention in such diseases. To start a compound development process targeting specific HMTs, but also to enable profiling against many different HMTs, robust in-vitro assays are required that allow the generation of scientifically valid results, are preferably automatable, economically feasible and safe, and are readily transferable to all HMTs. Current methods to detect HMT activity monitor the transfer of the methyl-group from the co-substrate SAM (S-adenosyl-methionine) onto the protein substrate, either directly by tracing the 3H-labelled methyl-group from SAM to a substrate, or indirectly by using methylation site specific antibodies. Alternatively the reaction co-product SAH (S-adenosyl-homocysteine) may be detected directly by antibodies or indirectly via conversion of the SAH into other compounds which may be traced by various methods. While the use of 3H is often limited due to safety reasons, antibody based detection is frequently limited by antibody specificity, is costly and, in the case of site specific antibodies, results may only to a limited degree be compared when different sites are methylated by different HMTs. We have therefore developed an universal, non radioactive, non-antibody based in-vitro assay which detects HMT activity by its co-product SAH that is converted by a two-step, enzymatically catalysed reaction to AMP which in turn is quantified using Promegas AMP-Glo technology. Currently we are in the process of recombinantly expressing and purifying at least 35 members of the HMT family. Data is presented on the production of the HMTs, assay coupling enzymes and on the development of validated in-vitro activity assays for the human HMT proteins EHMT1 and EHMT2. Citation Format: Daniel Mueller, Christian Beisenherz-Huss, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Michael H.G. Kubbutat. Development of a non-radioactive method to determine Protein-Methyl-Transferase activity in-vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5160. doi:10.1158/1538-7445.AM2014-5160

Abstract 5527: Determining the apoptotic pathways required for the EZH2 inhibitor, GSK-126, in a mouse model of B cell lymphoma

Abstract Background: Approximately 40% of B-cell lymphomas are characterized by the presence of recurrent chromosomal translocation affecting the Myc/8q24 locus. Further, similar breakpoints may also occur as secondary events giving rise to “Double Hit” (DH) lymphomas. DH lymphomas frequently involve Myc with concurrent, Bcl-2 or Bcl6 translocations. These Myc associated B cell lymphomas are linked with poor survival, with a median overall survival of only 0.2-1.5 years, and novel therapeutic strategies are urgently required. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is shown to correlate with disease progression in many cancer types. EZH2 is involved in gene repression via tri-methylation of histone H3 lysine 27 (H3K27me3) and its expression is demonstrated to be increased by Myc, highlighting a potential therapeutic target towards Myc driven B cell lymphomas. In this study we determined the antitumor activity of a specific EZH2 inhibitor, GSK-126. Taking advantage of the well characterized Eμ-myc B cell lymphoma mouse model, we screened tumors with defined genetic alterations in apoptotic pathways to determine GSK-126 antitumor activity. Methods: The anti-tumor effects of GSK126 or ectopic expression of an EZH2 mutant deficient for histone methyltransferase activity (ΔSET-EZH2) was assessed towards Eµ-myc B cell lymphomas, and compound mutant B cell lymphomas (Eµ-myc/Bcl2, Eµ-myc/p53-/-, Eµ-myc/p19-/-, and Eµ-myc/apaf1-/-) by uptake of propidium iodide. Further apoptotic assessment was performed using annexin, cell cycle analysis, mitochondria damage and caspase activity by flow cytometry. GSK-126 inhibition of EZH2 activity was determined by H3K27me3 expression by immunoblot. In vivo anti-tumor activity was determined by transplanting Eµ-myc lymphomas and Eµ-myc compound mutants expressing control of ΔSET-EZH2 plasmids to wild type mice. Overall survival was determined by Kaplan-Meier analysis. Results: Preliminary data demonstrates that GSK-126 treatment of Eµ-myc lymphomas results in rapid apoptotic cell death in a dose dependent manner. GSK-126 mediated apoptosis is independent of p53, though requires mitochondria damage. Further experiments described above are currently being performed. Conclusion: Our current data demonstrate that EZH2 inhibition induces apoptosis in Eµ-myc B cell lymphomas independent of p53, but requires mitochondrial damage. Overall, this study will lead to a more comprehension of the apoptotic mechanisms underlying GSK-126 therapeutic effect and will provide translational tools to predict the therapeutic response in DH lymphomas overexpressing Bcl2 or Bcl6. Citation Format: Elena Lasorsa, Kevin Schaarschuch, Zafardjan Dalimov, Leigh Ellis. Determining the apoptotic pathways required for the EZH2 inhibitor, GSK-126, in a mouse model of B cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5527. doi:10.1158/1538-7445.AM2014-5527

FOXA1 antagonizes EZH2-mediated CDKN2A repression in carcinogenesis

CDKN2A (p16INK4a) is a crucial tumor suppressor involved in many cancers. Our recent investigations revealed that FOXA1 as a forkhead transcription factor mediates CDKN2A activation in cellular senescence. However, the contribution of this axis in carcinogenesis remains unclear. Here, using a comprehensive collection of cancer microarray data, we found FOXA1 is down-regulated in many cancers compared to their normal counterparts and the positive correlation between FOXA1 and CDKN2A could be observed in prostate and breast cancers with lower EZH2 (epigenetic repressor for CDKN2A) expression. Experimentally, epistasis analysis in prostate and breast cancer cells indicated that higher expression of FOXA1 opposes EZH2-mediated CDKN2A repression, as further depletion of FOXA1 reverts the de-silencing of CDKN2A caused by EZH2 inhibition. Concomitantly, EZH2-depletion suppresses cancer cell cycle progression and this regulation is optimized in the presence of FOXA1 and CDKN2A. A further oncogenic transformation assay suggested that overexpression of EZH2 is insufficient to block RAS-induced CDKN2A activation and loss of FOXA1 is mandatory to potentiate EZH2-mediated CDKN2A silencing and to bypass the senescence barrier. Importantly, using an in vitro histone methyltransferase (HMTase) system, we found FOXA1 directly inhibits EZH2’s histone methyltransferase activity through its C-terminal histone binding motif. These data support that positive regulation of CDKN2A by FOXA1 counteracts its tumorigenic repression of by EZH2 in cancers.

A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element.

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.