Aberrant Histone Methylation Research Articles

Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives.

Post-translational methylation of histone lysine or arginine residues plays important roles in gene regulation and other physiological processes. Aberrant histone methylation caused by a gene mutation, translocation, or overexpression can often lead to initiation of a disease such as cancer. Small molecule inhibitors of such histone modifying enzymes that correct the abnormal methylation could be used as novel therapeutics for these diseases, or as chemical probes for investigation of epigenetics. Discovery and development of histone methylation modulators are in an early stage and undergo a rapid expansion in the past few years. A number of highly potent and selective compounds have been reported, together with extensive preclinical studies of their biological activity. Several compounds have been in clinical trials for safety, pharmacokinetics, and efficacy, targeting several types of cancer. This review summarizes the biochemistry, structures, and biology of cancer-relevant histone methylation modifying enzymes, small molecule inhibitors and their preclinical and clinical antitumor activities. Perspectives for targeting histone methylation for cancer therapy are also discussed.

Role of several histone lysine methyltransferases in tumor development.

The field of cancer epigenetics has been evolving rapidly in recent decades. Epigenetic mechanisms include DNA methylation, histone modifications and microRNAs. Histone modifications are important markers of function and chromatin state. Aberrant histone methylation frequently occurs in tumor development and progression. Multiple studies have identified that histone lysine methyltransferases regulate gene transcription through the methylation of histone, which affects cell proliferation and differentiation, cell migration and invasion, and other biological characteristics. Histones have variant lysine sites for different levels of methylation, catalyzed by different lysine methyltransferases, which have numerous effects on human cancers. The present review focused on the most recent advances, described the key function sites of histone lysine methyltransferases, integrated significant quantities of data to introduce several compelling histone lysine methyltransferases in various types of human cancers, summarized their role in tumor development and discussed their potential mechanisms of action.

H3K27 Demethylase JMJD3 Employs the NF-κB and BMP Signaling Pathways to Modulate the Tumor Microenvironment and Promote Melanoma Progression and Metastasis.

Histone methylation is a key epigenetic mark that regulates gene expression. Recently, aberrant histone methylation patterns caused by deregulated histone demethylases have been associated with carcinogenesis. However, the role of histone demethylases, particularly the histone H3 lysine 27 (H3K27) demethylase JMJD3, remains largely uncharacterized in melanoma. Here, we used human melanoma cell lines and a mouse xenograft model to demonstrate a requirement for JMJD3 in melanoma growth and metastasis. Notably, in contrast with previous reports examining T-cell acute lymphoblastic leukemia and hepatoma cells, JMJD3 did not alter the general proliferation rate of melanoma cells in vitro. However, JMJD3 conferred melanoma cells with several malignant features such as enhanced clonogenicity, self-renewal, and transendothelial migration. In addition, JMJD3 enabled melanoma cells not only to create a favorable tumor microenvironment by promoting angiogenesis and macrophage recruitment, but also to activate protumorigenic PI3K signaling upon interaction with stromal components. Mechanistic investigations demonstrated that JMJD3 transcriptionally upregulated several targets of NF-κB and BMP signaling, including stanniocalcin 1 (STC1) and chemokine (C-C motif) ligand 2 (CCL2), which functioned as downstream effectors of JMJD3 in self-renewal and macrophage recruitment, respectively. Furthermore, JMJD3 expression was elevated and positively correlated with that of STC1 and CCL2 in human malignant melanoma. Moreover, we found that BMP4, another JMJD3 target gene, regulated JMJD3 expression via a positive feedback mechanism. Our findings reveal a novel epigenetic mechanism by which JMJD3 promotes melanoma progression and metastasis, and suggest JMJD3 as a potential target for melanoma treatment.

Epigenetics Directing Therapy in Acute Lymphocytic Leukemia: Emerging Roles and New Therapies

In the last 15 years, our understanding of the contribution of epigenetic mechanisms to ALL leukemogenesis has increased exponentially. Epigenetic alterations are particularly interesting from a therapeutic standpoint, as they are potentially reversible and amenable to pharmacologic modulation. Indeed, inhibitors of DNA methylation, histone deactylation and histone methylation all have entered clinical trials, and early results for several of these agents are encouraging. Incorporating epigenetic modulation into ALL therapy may therefore hold great promise in improving tolerability and outcomes. Specifically, this session will address: - Patterns of aberrant DNA methylation and clinical trials combining "demethylating" agents with standard chemotherapy for relapsed ALL.DNA methylation patterns have been shown to correlate with outcome. Basic studies failed to conclusively link the regulation of specific genes or pathways to aberrant DNA methylation, and upfront methylation patterns poorly predict response to demethylating agents. Nevertheless, several clinical trials combining demethylating agents and chemotherapy in relapsed ALL are ongoing, with encouraging early results. These included responses of patients treated with decitabine and HyperCVAD who had previously failed to respond HyperCVAD alone. - Patterns of aberrant histone acetylation, the biology of histone acetyl transferases (HATs) and histone deacetylases (HDACS) in ALL, and clinical trials modulating histone acetylation. Histone acetylation patterns and a high frequency of deletions of the HAT CBP at relapse suggest that aberrant histone acetylation contributes to relapse and resistance. HDAC inhibitors show efficacy on ALL cells in preclinical studies, and clinical trials are ongoing. - The role of the histone acetylation "reader" protein Brd4 in ALL. In addition to "writers" (HAT) and "erasers" (HDACs), readers of histone acetylation may play a role in leukemia. Inhibition of Brd4 showed activity in several lymphoblastic leukemia models, and two Brd4 inhibitors are currently in clinical trials for relapsed/refractory hematologic malignancies including ALL. - Patterns of aberrant histone methylation, biology and therapeutic possibilities. Aberrant histone methylation is also emerging as a contributor to leukemogenesis, and may offer opportunities for therapeutic intervention. Loss of function mutations of the H3K27 histone methyltransferase EZH2 or other members of its complex (SUZ12, EED) are often found in early T-cell precursor ALL (ETP), and loss of EED or EZH2 contributes to aberrant T-cell development and leukemic transformation in mouse models. Gain of function of the H3K36 histone methyltransferase NSD2 have recently been described in pre-B ALL, raising the obvious question whether these leukemias depend on H3K36 hypermethylation, and NSD2 inhibition would have a therapeutic effect. Finally, aberrant recruitment of the histone methyltransferase DOT1L is observed in MLL-rearranged ALL, and an inhibitor of DOT1L is in clinical trials for MLL-rearranged malignancies, including ALL. Basic and translational studies support a critical role for epigenetic mechanisms in ALL leukemogenesis, drug resistance and relapse. Early results from clinical trials demonstrate that pharmacologic modulation of epigenetic modifiers can produce clinically meaningful responses. The next few years will likely see an increased number of compounds that modulate epigenetics enter clinical trials. Current assignment of patients to studies/compounds is crude, based on tumor type and status (i.e. "relapsed hematologic malignancy") or cytogenetics (i.e. MLL-rearrangement), rather than epigenetic profiling or an understanding of the biology that drives an individual patient's leukemia. As our diagnostic and therapeutic tools improve, epigenetic modulation may become an important component of ALL therapy. Disclosures Off Label Use: This presentation will include discussion of the use of decitabine, azacitidine, vorinostat, rhomidepsin and EPZ-5676 for ALL..

Abstract IA26: Aberrant histone methylation in multiple myeloma.

Abstract MMSET (Multiple Myeloma SET domain) is a histone methyltransferase (HMT) overexpressed as a result of the translocation t(4;14). MMSET contains a SET domain, which encodes histone methyltransferase activity, and protein and DNA interaction domains, including PHD and PWWP domains. Overexpression of MMSET induces a global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. The HMT activity of MMSET is essential for growth stimulation by MMSET as re-expression of MMSET in a t(4;14) myeloma cell line which the rearranged MMSET allele was disrupted (KMS11-TKO), rescued growth only when the HMT activity of the protein was intact. The complete H3K36/H3K27 switch mediated by MMSET requires all PHD finger domains of the protein, the second PWWP domain and the functional SET domain. Despite the global change in histone methylation in response to MMSET, microarray and RNA-Seq analysis showed that only ~1000 genes are appreciably changed in response to MMSET. Many genes activated by MMSET display a peak of H3K27me3 near the transcription start site in MMSET low cells, which is absent in MMSET overexpressing cells, displaced by a broad pattern of H3K36me2 modification. We also found a subset of genes repressed in response to MMSET overexpression. While on a genome-wide basis, H3K27 methylation is decreased in MMSET overexpressing cells, at repressed genes, H3K27me3 levels were increased in association with increased occupancy by EZH2. These regions did not show an increase in H3K36 methylation and are enriched for GC-rich elements, representing putative polycomb complex recruitment sites. The role of MMSET in malignancies extends beyond MM as a recurrent heterozygous point of the gene is found in the Broad Cell Line Encyclopedia, as well as in cases of CLL, ALL and MM. Cell lines harboring such mutations had elevated H3K36 and depressed H3K27 methylation indicating that the mutation leads to a gain of function. MMSET overexpression/increased function is also linked to advanced, metastatic solid tumors and is associated with epithelial-mesenchymal transition and up-regulation of TWIST1. Given that MMSET gain of function is associated with malignancy- it is an attractive therapeutic target. In mouse xenografts the efficacy of chemotherapy against a t(4;14) cell line was enhanced by knockdown of MMSET. Using the previously identified structure of the SET domain of NSD1, an MMSET homologue, we performed an in silico screen against a library of compounds to identify putative MMSET inhibitors. We tested the in silico hits in a high throughput screen (HTS) that combines Self-Assembled Monolayer targets and matrix-assisted laser Desorption Ionization time-of-flight (SAMDI). From this screen several potential hits have been identified. These compounds have IC50 values in the micromolar range and Differential Scanning Fluorimetry (DSF) confirmed the binding of the small molecule to MMSET. Other mutations in MM lead to altered histone methylation. H3K27 methylation a repressive mark that is “erased” by the UTX/KDM6A H3K27 demethylase. The UTX/KDM6A gene is mutated/deleted in > 30% of multiple myeloma. shRNA mediated depletion of UTX in cells wild-type for the protein slowed growth and changed cell adhesion while re-expression of UTX slowed myeloma cell growth. Examination of the Broad Cell line encyclopedia dataset showed evidence of disruption of the MLL3 and MLL2 proteins in MM. Examination of gene expression array datasets showed that patients with very low expression of UTX and presumably mutation/deletion of the protein had a distinct pattern of gene expression. Remarkably when patients were analyzed based upon very low expression of MLL2 or MLL3 they showed a gene expression pattern that highly coincided with that of UTX low patients. This suggests that UTX, MLL2 and MLL3 work in concert and deletion of UTX, MLL2 or MLL3 may drive aberrant expression of a common set of genes. Pathways affected included p53, Wnt and JAK/STAT signaling. Collectively, these data indicate that aberrant histone methylation, generally affecting H3K27 is a common theme in multiple myeloma and other lymphoid malignancies. Citation Format: Jonathan D. Licht. Aberrant histone methylation in multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA26.

Abstract 100: PTEN regulates polycomb repressive EZH2 expression and its deficiency correlates with active transcription mark H3K4Me3

Abstract Phosphatase and tensin homolog (PTEN) is one of the most commonly lost tumor suppressors in human cancers. Frequent genetic inactivation of PTEN occurs in glioblastoma, endometrial cancer, and prostate cancer; and reduced expression is found in many other tumor types such as lung and breast cancer. PTEN is mostly found in the cytosol and it transiently associates with the inner surface of the plasma membrane and removes a phosphate group from phosphatidylinositol 3,4,5-trisphosphate (PIP3) and by this, it regulates various cellular functions. However in the nucleus, PTEN mediates phosphatase independent functions and is believed to be important for its tumor suppressive roles. Functional studies further confirm the transcriptional regulatory role of the PTEN; however, the process by which PTEN acts as a potential modulator of chromatin structure and histone modification is unexplored. We observed that the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a histone methyltransferase is upregulated in PTEN null mouse embryonic fibroblasts (MEFs) as compared to wild type counterparts, which suggested an inverse relation between EZH2 expression and PTEN expression. We further observed that phospho EZH2 levels (pEZH2ser21) were specifically upregulated in the PTEN null cells and coincided with the high pEZH2:EZH2 ratio in these settings, indicating that the majority of the upregulated EZH2 exists in the phosphorylated form. We also found that the pEZH2ser21 is mainly localized in the nucleus and only a small fraction exists in the cytoplasm, suggesting additional roles for pEZH2ser21 after partnering with cytoplasmic proteins. However, the non-phosphorylated form of the EZH2 is totally a nuclear protein under these conditions. Interestingly in PTEN null MEFs, transcriptional repression mark such as H3K27Me3 expression was downregulated with the simultaneous upregulation of H3K4Me3, an active transcription mark. Also, the increased expression of EZH2 primarily linked with the pause of active transcription, was associated with corresponding decrease in the H3K27Me3 repression mark which subsequently influenced the expression of various genes including senescence associated genes such as p21. Since, these aberrant histone methylation marks are hallmarks of various cancer pathologies, targeting EZH2 or its phosphorylation component could be a potential therapeutic strategy in cancers involving loss of PTEN. Taken together, these results demonstrate that in PTEN null processes, the pEZH2ser21 is capable of translocation, altering the histone marks and influencing genes associated with transcriptional repression and senescence. We also suggest that EZH2 could serve as important target for novel chemotherapeutics against cancers that are associated with loss of PTEN. Citation Format: Abid Hamid, Mohammad Imran Khan, Vaqar Mustafa Adhami, Hasan Mukhtar. PTEN regulates polycomb repressive EZH2 expression and its deficiency correlates with active transcription mark H3K4Me3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 100. doi:10.1158/1538-7445.AM2015-100

IL-22 induces colon cancer proliferation through STAT3 and PRC2 signaling (TUM10P.1036)

Abstract Background: IL-22(interleukin-22) is a newly identified cytokine which has been recently highlighted owing to its biological significance in carcinogenesis. However, the role of IL-22 in colon cancer proliferation has remained unclear. Since both JAK-STAT signaling and Polycomb Repression complex (PRC) 2 play crucial roles in epithelial proliferation. We investigated the mechanism by which IL-22 induces colon cancer proliferation. Methods: Colon cancer cells proliferation was detected by FACS and H3 Thymidine Incorporation. Cell cycle related genes were tested by Real-Time PCR. Lentiviral vector encoding gene specific shRNAs (STAT3, EZH2, SUZ12) and scramble particles were used to transfect colon cancer cells. ChIP assays were performed to detect the binding of different proteins and DNA. Results: IL-22 significantly induces colon cancer cells proliferation through STAT3 signaling. This increased proliferation involved the inhibition of p16 and p18 through aberrant promoter histone methylation H3K27me3. PRC2 catalyzes promoter H3K27me3 of p16 and p18, which can be up-regulated by IL-22. Furthermore, STAT3 can bind to PRC2 members (SUZ12 and EED), which also can be up-regulated by IL-22 stimulation. Conclusion: IL-22 can induce human colon cancer cells proliferation by activation of STAT3, which was phosphorylated and directed bind to the PRC2 complex, especially SUZ12 and EED, and consequently catalyzed the H3K27m3 of cell cycle check-point genes p16 and p18.

Cadmium induces histone H3 lysine methylation by inhibiting histone demethylase activity.

Cadmium is an established human lung carcinogen with weak mutagenicity. However, the mechanisms underlying cadmium-induced carcinogenesis remain obscure. It has been suggested that epigenetic mechanisms may play a role in cadmium-induced carcinogenesis. In this study, we investigated the effects of cadmium on histone methylation and histone demethylases, and the role of histone methylation in transformation of immortalized normal human bronchial epithelial (BEAS-2B) cells. Exposure to 0.625, 1.25, 2.5, and 5.0 μM of cadmium for 6, 24, and 48 h increased global trimethylated histone H3 on lysine 4 (H3K4me3) and dimethylated histone H3 on lysine 9 (H3K9me2) in BEAS-2B cells compared with untreated cells, and most of these changes remained after the removal of cadmium (P < .05 or P < .01 for most modifications). Meanwhile, cadmium inhibited the activities of histone H3 on lysine 4 (H3K4) and histone H3 on lysine 9 (H3K9) demethylases which were detected by histone demethylation assay. However, there was no significant change in the protein levels of the H3K4 demethylase lysine-specific demethylase 5A (KDM5A) and the H3K9 demethylase lysine-specific demethylase 3A (KDM3A). Interestingly, during transformation of BEAS-2B cells by 20 weeks of exposure to 2.0 μM cadmium as assessed by anchorage-independent growth in soft agar, global H3K4me3, and H3K9me2 were significantly increased at 4 weeks (P < .05 or P < .01), whereas no significant change was observed at 8, 12, 16, and 20 weeks compared with control. Our study suggests that cadmium increases global H3K4me3 and H3K9me2 by inhibiting the activities of histone demethylases, and aberrant histone methylation that occurs early (48 h) and at 4 weeks is associated with cadmium-induced transformation of BEAS-2B cells at the early stage.

MDR1 Mediated Drug Resistance to a Histone Methyltansferase Inhibitor (KMT)

Aberrant histone methylation is emerging as a major contributor to oncogenesis, and inhibitors of histone lysine methyltransferases (KMTs) are starting to enter early clinical studies. Currently, inhibitors for two KMTs are in phase I/II clinical trials, an inhibitor of the histone 3 lysine 79 (H3K79) methyltransferase DOT1L for MLL-rearrange leukemias (NCT02141828), and inhibitors for the H3K27 methyltransferase EZH2 for lymphomas or solid tumors with activating EZH2 mutations (NCT01897571 and NCT02082977). The next few months and years are likely to see more KMT inhibitors entering clinical development, such as compounds directed against the H3K36 methyltransferase MMSET/NSD2/WHSC1, which is commonly translocated in multiple myeloma and carries activating mutations in a subset of acute lymphoblastic leukemia. Typically, KMT inhibitors are structurally similar and are derived from the methyl donor S-adenosyl-methionine (SAM). Very little is known about possible mechanisms of resistance to these compounds.We exposed a panel of leukemia cell lines to varying concentrations of the DOT1L inhibitor EPZ4777 and assessed the decrease of H3K79 di-methylation by Western Blotting. Our analysis revealed a dose-dependent decrease of H3K79 dimethylation in most cell lines. Interestingly, we identified a pair of cell lines, KG-1 and KG-1a, which were near completely resistant to EPZ4777 mediated DOT1L inhibition, with no effect of 10 μM EPZ4777 (4 day exposure). Even after exposure to EPZ4777 for 15 days, KG-1 showed only a slight decrease in H3K79me2 marking and no decrease in marking was seen for KG-1a up to a concentration of 3 μM. We searched the encode database for expression levels of DOT1L, as well as higher expression of proteins previously described as mediating drug resistance in AML cell lines. We found no significantly higher DOT1L expression level, suggesting that overexpression of the target is not the predominant mechanisms of resistance in KG-1/KG-1a. We confirmed similar baseline H3K79 methylation levels in KG1/KG1a and the other cell lines in our panel by Western Blotting. However, we noted a higher expression of multidrug resistance protein 1 (MDR1/p-glycoprotein/ABCB1) in the KG-1 and KG-1a cell lines compared to the other cell lines in our panel. To explore whether DOT1L inhibition by EPZ4777 could be restored in KG1 and KG1a cells when MDR1 was inhibited, cells were exposed to 0.3 μM EPZ4777 and varying concentrations of Cyclosporine A (CSA), a known inhibitor of MDR1. Increasing concentration of CSA with a fixed dose of 0.3 μM EPZ4777 led to a dose dependent decrease in H3K79me2 in KG-1/KG-1a, suggesting that inhibition of MDR1 increased the intracellularly effective dose of EPZ4777. CSA at a concentration of 0.3 μM and 0.3 μM EPZ4777 near completely abolished H3K79 methylation. In summary, our results suggest that KMT inhibitors can be subject to classic drug resistance mechanisms such as the MDR1/p-glycoprotein/ABCB1 efflux pump. These data provide insight for the use and design of small-molecule inhibitors for KMTs. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5213: microRNA-mediated loss of KDM5B expression leads to suppression of the malignant bladder cancer phenotype

Abstract We recently identified KDM5B as a putative oncogene. The expression of KDM5B correlated with increased cellular proliferative capacity and was found to be overexpressed in many cancers including bladder cancer (BC). KDM5B is a histone demethylase enzyme that modifies chromatin structure to specify cellular transcriptional states. It is a cancer/testis antigen; whereby in normal conditions, expression is limited to early embryonic stages and the testis in adults. Thus, KDM5B is an excellent therapeutic target. Since the expression of KDM5B in embryonic stem cells is orchestrated by miRNAs and that the expression of miRNAs are altered in BC, we hypothesized that miRNAs may be the switch that can abate the expression of KDM5B and thus affect the malignant phenotype of BC cells. This study was aimed at identifying and modulating the expression of miRNAs that are able to regulate KDM5B expression and BC phenotypes. By coupling in silico screens with RT-QPCR analysis of in vitro expression of BC cells of different malignant potential, we predicted several miRNAs as potential regulators of KDM5B transcripts. The functional effects of specific miRNAs on KDM5B expression, cell cycle, proliferation and invasion of BC cells was determined by modulating the expression of these miRNAs using mimics (upregulation) and antagomirs (downregulation). Expression of miR-137 in BC cells was found to be inversely correlated with KDM5B expression. Ectopic overexpression of miR-137 led to the repression of KDM5B expression, reduced cell growth and invasion as well as increased apoptosis of BC cells. In contrast, the downregulation of miR-137 expression led to reverse effects. The expression modulation of miR-137 also affected the expression of other BC-associated genes such as JDP2 and EGR2. While our results suggest that miR-137 can mitigate the KDM5B-associated BC phenotype, further studies on understanding the effect on aberrant histone methylation patterns are warranted. Citation Format: Abhimanyu Veerakumarasivam, Radha Kodiappan, Soon Choy Chan, Azad Hassan Abdul Razack, Teng Aik Ong. microRNA-mediated loss of KDM5B expression leads to suppression of the malignant bladder cancer phenotype. [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 5213. doi:10.1158/1538-7445.AM2014-5213

ICR Noncoding RNA Expression Controls Imprinting and DNA Replication at the Dlk1-Dio3 Domain

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.

MECHANISMS OF TOXICITY AND THERAPEUTIC APPROACHES FOR C9FTD/ALS

A significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two diseases commonly seen in comorbidity, carry an expanded non-coding hexanucleotide repeat in the C9orf72 gene, a condition commonly referred to as c9FTD/ALS. We and others have demonstrated that the RNA structure of GGGGCC repeats may cause neurodegeneration via their accumulation into discrete structures in the nucleus, termed RNA foci, and by serving as a template for the synthesis of aggregation-prone “c9RAN proteins” by repeat-associated non-ATG (RAN) translation. Expanded repeat carriers also exhibit reduced C9orf72 mRNA levels, suggesting that C9orf72 loss of function may contribute to neurodegeneration. Using a variety of approaches, including the generation of novel antibodies, in vitro and in vivo assays, as well as analyses of human tissue, we examine how bidirectional transcription of the expanded C9orf72 repeat and reduced C9orf72 mRNA expression lead to c9FTD/ALS. We discover antisense transcripts result from bidirectional transcription of the expanded repeat, and we find ectopic expression of CCCCGG repeats, much like GGGGCC repeats, leads to foci formation and RAN translation in cultured and transfected cells. We observe neuronal foci composed of antisense transcripts, as well as cytoplasmic inclusions of expanded (CCCCGG)RAN proteins [poly(PR), poly(GP) and poly(PA)] in various brain tissues in c9FTD/ALS; however, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell suggesting separate disease mechanisms. In other studies, we demonstrate aberrant methylation of histone tail lysine residues, which is detectable in brain tissue, fibroblasts and blood of C9orf72 mutation carriers, mediates C9orf72 mRNA reduced expression and potentially contributes to disease pathogenesis. We identify aberrant histone methylation specific to C9orf72 mutation carriers as a potential disease biomarker. In addition, our findings suggest therapeutic strategies designed to target the RNA structures necessary for RAN translation and foci formation and/or to modulate methylation of C9orf72 -bound histone residues in c9FTD/ALS hold promise.

Aberrant histone methylation and the effect of Suv39H1 siRNA on gastric carcinoma.

The present study aimed to investigate the involvement of the Suv39H1 histone methyltransferase in the epigenetic changes in the euchromatic promoter in gastric carcinoma. We retrospectively analyzed the protein of Suv39H1 and tri-methylated histone H3 lysine9 (H3K9) and histone H3 lysine4 (H3K4) in 175cases of gastric carcinoma by immunohistochemistry. Suv39H1 was depleted by siRNA, and cell apoptosis and cell proliferation were assessed by TUNEL and MTT assays, respectively. Histone methylated H3K9 and histone acetylated H3 and H4 were evaluated by western blotting. We found that the expression of Suv39H1 and tri-methylated H3K9 in gastric carcinoma was higher than that in benign gastric diseases (p<0.05). Tri-methylated H3K4 was similar in both tissue types (p>0.05). Both Suv39H1 and tri-methylated H3K9 were positively correlated with the degree of differentiation, depth of infiltration and lymphatic invasion (p<0.05) in gastric carcinoma. In addition, tri-methylated H3K9 was positively correlated with tumor stage, and node and metastatic statuses (p<0.05). Activation of Suv39H1 and overexpression of H3K9 tri-methylation may play an important role in tumorigenesis. They may be useful as a predictor for poor prognosis in gastric carcinoma. Silencing of the Suv39H1 gene decreased tri-methylated H3K9 and increased histone H3 acetylation, which caused activation of gene transcription, while there was no change in histone H4 acetylation. Depletion of Suv39H1 induced apoptosis and inhibited cell proliferation in the gastric cancer MGC803 cell line, while decreasing BCL-2, pro-caspase-9, pro-caspase-3 and C-myc. Suv39H1 may be a potential gene target for anti-gastric carcinoma therapy.

Characterization of DNA hypermethylation in the cerebellum of c9FTD/ALS patients

A significant number of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two diseases commonly seen in comorbidity, carry an expanded noncoding hexanucleotide repeat in the C9orf72 gene, a condition collectively referred to as c9FTD/ALS. Repeat expansions, also present in other neurodegenerative diseases, have been shown to alter epigenetic mechanisms and consequently lead to decreased gene expression, while also leading to toxic RNA gain-of-function. As expression of multiple C9orf72 transcript variants is known to be reduced in c9FTD/ALS cases, our group and others have sought to uncover the mechanisms causing this reduction. We recently demonstrated that histones H3 and H4 undergo trimethylation at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) in all pathogenic repeat carrier brain samples, confirming the role of altered histone methylation in disease. It was also reported that about 40% of c9ALS cases show hypermethylation of the CpG island located at the 5' end of the repeat expansion in blood, frontal cortex, and spinal cord. To determine whether the same CpG island is hypermethylated in the cerebella of cases in whom aberrant histone methylation has been identified, we bisulfite-modified the extracted DNA and PCR-amplified 26 CpG sites within the C9orf72 promoter region. Among the ten c9FTD/ALS (4 c9ALS, 6 c9FTD), nine FTD/ALS, and eight disease control samples evaluated, only one c9FTD sample was found to be hypermethylated within the C9orf72 promoter region. This study is the first to report cerebellar hypermethylation in c9FTD/ALS, and the first to identify a c9FTD patient with aberrant DNA methylation. Future studies will need to evaluate hypermethylation of the C9orf72 promoter in a larger cohort of c9FTD patients, and to assess whether DNA methylation variation across brain regions reflects disease phenotype.This article is part of a Special Issue entitled RNA Metabolism 2013.

Aberrant histone methylation in the patients with immune thrombocytopenia

The aim of the present study was to investigate alterations in histone methylation in patients with primary immune thrombocytopenia (ITP). Global histone H3K4/H3K9 methylation in CD4+ T cells from 35 ITP patients and 15 healthy controls were measured using the EpiQuikTM global histone H3K4/H3K9 methylation assay kits. The mRNA expression of SUV39H1, SUV39H2 and EZH2 were detected by real-time quantitative polymerase chain reaction (RT-PCR). The results showed that global histone H3K9 hypomethylation in CD4+ T cells of active ITP, compared with ITP in remission and controls, while the global histone H3K4 methylation were not significantly different between ITP patients and healthy controls. The expression of EZH2 and SUV39H2 were significantly down-regulated in active ITP patients, when compared with ITP in remission and controls. There were not different between ITP patients and controls in the expression SUV39H1. In conclusion, the aberrant histone methylation was involved in the pathogenesis of ITP.

Epigenetic Down-Regulation Of AML2 Expression By EZH2 Contributes To NF-E2 Overexpression In Myeloproliferative Neoplasms

The transcription factor NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPN). In Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) NF-E2 levels are elevated independent of the presence or absence of the JAK2V617Fmutation. We have recently shown that NF-E2 overexpression in a murine model leads to an MPN phenotype followed by spontaneous transformation to acute leukemia in a subset of mice.We have demonstrated that increased NF-E2 transcription is mediated by the transcription factor and proto-oncogene AML-1, which is overexpressed in MPN patients, again irrespective of the JAK2V617Fstatus. AML-1 binds to a conserved enhancer region located 3500 bp upstream of the NF-E2 transcriptional start site. Since AML-1 responsive genes are often also inversely regulated by the tumor suppressor AML-2 (RUNX3), we investigated whether NF-E2 expression is affected by AML-2 as well.Using chomatin immunoprecipitation assays (ChIP) we show that AML-2 binds the NF-E2 enhancer in vivo at a site distinct from but in proximity to the three AML-1 sites in the -3500bp region. AML-2 binding strongly represses transcription off the NF-E2 enhancer in reporter gene assays. This repression is abrogated by site directed mutagenesis of the AML-2 recognition sequence. Likewise, lentivirally induced AML-2 expression drastically reduces the amount of NF-E2 protein in erythroid and myeloid cells. These data clearly demonstrate that NF-E2 expression is directly regulated by AML-2. AML-2 thus serves as a repressor on the hematopoietic NF-E2 gene, a function previously noted mainly in solid tumors.In primary cells from patients with polycythemia vera (PV) AML-2 mRNA expression is significantly reduced. Moreover, ChIP assays demonstrate that in primary PV cells, significantly less AML-2 is bound to the NF-E2 enhancer than in healthy controls. Decreased repression by AML-2 thus cooperates with increased AML-1 induced transcription to elevate NF-E2 levels in PV patients.It has been demonstrated that AML-2 expression can be regulated by two distinct epigenetic mechanisms. For one, DNA methylation of the AML-2 promoter has been reported to silence AML-2 expression in gastric, colorectal and bladder cancers. On the other hand, aberrant histone methylation in the promoter region can also silence AML-2 expression. Here we show that DNA methylation of the AML-2 promoter is unaltered in PV patients. Rather, PV patients display aberrant histone methylation in the AML-2 promoter. Compared to healthy controls, H3K27me3 is significantly increased and H3K4me3 is significantly decreased in primary PV cells. This results in an inactive chromatin conformation on the AML-2 promoter in PV patients. Moreover, we show here that the histone-lysine-methyl-transferase “enhancer of zeste homologue 2” (EZH2), which confers the K3K27me3 mark, binds to the AML-2 promoter and decreases AML-2 expression. PV patients demonstrate significantly increased levels of EZH2 binding to the AML-2 promoterTreatment of primary PV cells and MPN cell lines with 2'-deoxy-5-azacytidine (DAC, Decitabine) ex vivo reverses the altered histone methylation, restoring the pattern found in healthy controls and decreases EZH2 binding to the AML-2 promoter. At the same time, Decitabine treatment induces AML-2 protein expression, decreases EZH2 expression and reduces the elevated NF-E2 levels. Moreover, a post-PV AML patient receiving Decitabine, displayed normalization of AML-2 promoter histone methylation and EZH2 binding on day 8 and day 15 of treatment.Taken together our data demonstrate that epigenetic silencing of AML-2 contributes to the elevated NF-E2 expression observed in MPN patients. Treatment with Decitabine restores physiological histone modifications to the AML-2 locus and decreases NF-E2 levels by reactivating AML-2 expression. These data provide a molecular rationale for extending the clinical investigation of epigenetic modifiers such as Decitabine or Azacitidine, currently used in MDS and AML, to patients with MPNs. A phase I study using Azacitidine in high risk PMF patients is currently being planned by the MPD-RC. Disclosures:No relevant conflicts of interest to declare.

Differential methylation of the arsenic (III) methyltransferase promoter according to arsenic exposure

Inorganic arsenic is methylated in the body by arsenic (III) methyltransferase (AS3MT). Arsenic methylation is thought to play a role in arsenic-related epigenetic phenomena, including aberrant DNA and histone methylation. However, it is unclear whether the promoter of the AS3MT gene, which codes for AS3MT, is differentially methylated as a function of arsenic exposure. In this study, we evaluated AS3MT promoter methylation according to exposure, assessed by urinary arsenic excretion in a stratified random sample of 48 participants from the Strong Heart Study who had urine arsenic measured at baseline and DNA available from 1989 to 1991 and 1998-1999. For this study, all data are from the 1989-1991 visit. We measured AS3MT promoter methylation at its 48 CpG loci by bisulphite sequencing. We compared mean % methylation at each CpG locus by arsenic exposure group using linear regression adjusted for study centre, age and sex. A hypomethylated region in the AS3MT promoter was associated with higher arsenic exposure. In vitro, arsenic induced AS3MT promoter hypomethylation, and it increased AS3MT expression in human peripheral blood mononuclear cells. These findings may suggest that arsenic exposure influences the epigenetic regulation of a major arsenic metabolism gene.

Induction of Chromosomal Instability via Telomere Dysfunction and Epigenetic Alterations in Myeloid Neoplasia

Chromosomal instability (CIN) is a characteristic feature of cancer. In this review, we concentrate on mechanisms leading to CIN in myeloid neoplasia, i.e., myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The pathogenesis of myeloid neoplasia is complex and involves genetic and epigenetic alterations. Chromosome aberrations define specific subgroups and guide clinical decisions. Genomic instability may play an essential role in leukemogenesis by promoting the accumulation of genetic lesions responsible for clonal evolution. Indeed, disease progression is often driven by clonal evolution into complex karyotypes. Earlier studies have shown an association between telomere shortening and advanced MDS and underlined the important role of dysfunctional telomeres in the development of genetic instability and cancer. Several studies link chromosome rearrangements and aberrant DNA and histone methylation. Genes implicated in epigenetic control, like DNMT3A, ASXL1, EZH2 and TET2, have been discovered to be mutated in MDS. Moreover, gene-specific hypermethylation correlates highly significantly with the risk score according to the International Prognostic Scoring System. In AML, methylation profiling also revealed clustering dependent on the genetic status. Clearly, genetic instability and clonal evolution are driving forces for leukemic transformation. Understanding the mechanisms inducing CIN will be important for prevention and for novel approaches towards therapeutic interventions.

Abstract 3: Targeted inhibition of LSD1 in castration-resistant prostate cancer.

Abstract Epigenetic dysfunction is recognized as a driver in the pathology of various cancers. Promoter DNA hypermethylation, genomic hypomethylation, and aberrant histone acetylation and methylation contribute to epigenetic lesions. Further, genetic mutations in important epigenetic regulators have been identified across various solid and hematological malignancies. These aberrant marks are reversible due to the dynamic nature of epigenetic regulation and present attractive therapeutic targets. While epigenetic therapies, like DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, have shown survival benefit in the clinic there are several potential epigenetic targets whose therapeutic potential remains undefined. The histone lysine specific demethylase 1 (LSD1) is such a target. LSD1 is a FAD-dependent amine oxidase which catalyzes the oxidative demethylation of mono- and dimethyl marks from both lysines 4 and 9 on histone H3 (H3K4 and H3K9). Overexpression of LSD1 correlates with aggressive tumor biology, dedifferentiation, and poor prognosis in various solid malignancies, including prostate, breast, bladder, lung, liver and colorectal tumors. In prostate cancer, upregulation of LSD1 is sufficient to promote ligand-independent androgen-receptor-dependent transcription through demethylation of the repressive histone 3 lysine 9 dimethyl mark (H3K9me2). Currently reported inhibitors of LSD1 exhibit poor potency and specificity, solubility issues, and irreversibility and preclinical studies using these compounds have offered little in the way of single agent efficacy in solid tumors. Furthermore, inducible LSD1 knockdown experiments in vivo raise significant concerns about the potential for LSD1 inhibitors to block terminal hematopoetic differentiation. More potent and reversible inhibitors are required to discern which solid malignancies present an opportunity for LSD1 enzymatic inhibition as a single-agent and if there is a therapeutic window between single-agent efficacy and hematopoetic toxicity. HCI-2509 was previously reported as a potent and specific inhibitor for LSD1 with in vitro efficacy in breast cancer and in vivo efficacy in acute myeloid leukemia (AML). Here we report single-agent efficacy of HCI-2509 dosed both orally and intraperitoneally over the course of 21 days using the castration-resistant prostate cancer (CRPC) PC-3 xenografts in vivo. We observed target modulation with increased levels of H3K9me2 in both treatment groups. Additionally, the literature-predicted hematopoetic toxicity was not observed. This suggests that reversible and potent LSD1 inhibition provides a viable therapeutic strategy for CRPC and that dosing strategies can be optimized to avoid serious hematological toxicity. Our efforts are now focused on building a pharmacokinetic/pharmacodynamic model to better understand the clinical potential of LSD1 inhibition in CRPC. Citation Format: Emily Theisen, Jared Bearss, Venkataswamy Sorna, David Bearss, Sunil Sharma. Targeted inhibition of LSD1 in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1003. doi:10.1158/1538-7445.AM2013-1003

DNA and histone methylation in gastric carcinogenesis

Epigenetic alterations contribute significantly to the development and progression of gastric cancer, one of the leading causes of cancer death worldwide. Epigenetics refers to the number of modifications of the chromatin structure that affect gene expression without altering the primary sequence of DNA, and these changes lead to transcriptional activation or silencing of the gene. Over the years, the study of epigenetic processes has increased, and novel therapeutic approaches that target DNA methylation and histone modifications have emerged. A greater understanding of epigenetics and the therapeutic potential of manipulating these processes is necessary for gastric cancer treatment. Here, we review recent research on the effects of aberrant DNA and histone methylation on the onset and progression of gastric tumors and the development of compounds that target enzymes that regulate the epigenome.