Histone Demethylase Inhibitor Research Articles

Recent Progress in Histone Demethylase Inhibitors.

There is increasing interest in targeting histone N-methyl-lysine demethylases (KDMs) with small molecules both for the generation of probes for target exploration and for therapeutic purposes. Here we update on previous reviews on the inhibition of the lysine-specific demethylases (LSDs or KDM1s) and JmjC families of N-methyl-lysine demethylases (JmjC KDMs, KDM2-7), focusing on the academic and patent literature from 2014 to date. We also highlight recent biochemical, biological, and structural studies which are relevant to KDM inhibitor development.

Docking and Linking of Fragments To Discover Jumonji Histone Demethylase Inhibitors.

Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600,000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼ 3 log-orders to yield compound 35 (Ki = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors.

HIV-Induced Epigenetic Alterations in Host Cells.

Human immunodeficiency virus (HIV), a member of the Retroviridae family, is a positive-sense, enveloped RNA virus. HIV, the causative agent of acquired immunodeficiency syndrome (AIDS) has two major types, HIV-1 and HIV-2 In HIV-infected cells the single stranded viral RNA genome is reverse transcribed and the double-stranded viral DNA integrates into the cellular DNA, forming a provirus. The proviral HIV genome is controlled by the host epigenetic regulatory machinery. Cellular epigenetic regulators control HIV latency and reactivation by affecting the chromatin state in the vicinity of the viral promoter located to the 5' long terminal repeat (LTR) sequence. In turn, distinct HIV proteins affect the epigenotype and gene expression pattern of the host cells. HIV-1 infection of CD4(+) T cells in vitro upregulated DNMT activity and induced hypermethylation of distinct cellular promoters. In contrast, in the colon mucosa and peripheral blood mononuclear cells from HIV-infected patients demethylation of the FOXP3 promoter was observed, possibly due to the downregulation of DNA methyltransferase 1. For a curative therapy of HIV infected individuals and AIDS patients, a combination of antiretroviral drugs with epigenetic modifying compounds have been suggested for the reactivation of latent HIV-1 genomes. These epigenetic drugs include histone deacetylase inhibitors (HDACI), histone methyltransferase inhibitors (HMTI), histone demethylase inhibitors, and DNA methyltransferase inhibitors (DNMTI).

Inhibition of Histone Demethylases Offers a Novel and Promising Approach for the Treatment of Cancer and Other Diseases.

Cancer and HDME-dependent proliferative diseases Biological Target: Histone demethylases (HDMEs) Summary: The invention in this patent application relates to heterocyclic aromatic compounds represented generally by formula (I), which arecapableofmodulatingtheactivitiesofhistonedemethylases(HDMEs)andmayprovideusefultherapytotreatcancerandother HDME-dependent proliferative diseases. Nuclear DNA is too large to exist as a linear structure; instead, it exists as a condensed, much smaller but more complex and highlyordered structurecalled chromatin. In additiontoDNA,chromatincontainsproteinandRNA. Chromatinis organizedinto smaller repeat units called nucleosomes composed of the DNA wrapped around a spool made of octamer of histones. Covalent chemical derivatizations of the chromatin components can cause changes in the ordered chromatin structure. Many reversible chemicalmodificationsoccuronthechromatincomponentsthatareessentialtothefunctionsofchromatintodetermineregionsof active and silenced transcription. They also have profound effects on the fundamental cellular processes such as differentiation, proliferation, and apoptosis. Chemical modifications such as methylations of DNA and/or histones are part of epigenetic regulations, which include any heritable changes in gene expression other than those mediated at the DNA sequencing level. Histones, the nucleosome core proteins, contain basic amino acid residues such as lysine and arginine. The amino groups on the lysine or arginine residues are subjected to several covalent modifications such as N-methylation, N-acetylation, and N-phosphorylation. These modifications can affect the histone functions based on their nature and location. Histone tails contain numerous lysine sites; a lysine (K) site is identified by its location on a specific histone (H) subunit, for example, H3K4 refers to a lysine residue in the fourth position on histone H3. The methylated sites are further identified by adding me1, me2, or me3 to denote mono-, di-, or trimethyl derivatives. N-Methylations of the histonelysine residues play critical roles in many epigenetic events. The methylation reaction is regulated by histone methyltransferases and functions as a mechanism to regulate DNA transcription. New evidence suggests that site-specific methylations are linked to specific biological processes ranging from transcriptional regulation to epigenetic silencing. Evidence also suggests that histone methylation may provide a stable genomic imprint that may serve to regulate gene expression as well as other epigenetic phenomena. The reverse process, i.e., the demethylation of methylated histones, is regulated by histone demethylases. There are two main classes of histone demethylases: the amine oxidases catalyzed by flavin adenine dinucleotide (FAD) and the dioxygenases catalyzed by Fe(II)/α-ketoglutarate. Studies have shown that methylation and demethylation of a specific H3 lysine residue can be associated with epigenetic marks that define transcriptionally active or inactive chromatin. For example, while methylation of H3K9 is usually associated with epigenetically silenced chromatin, the methylation of H3K4 is associated with transcriptionally active chromatin. Similarly, di- and trimethylation of H3K27 marks are repressive, while the methylation of H3K36 mark is associated with gene activation. Dysregulation of histone methyltransferases and/or demethylases may result in significant changes in histone lysine methylation, which, in turn, may be responsible for the pathogenesis of several diseases including cancer as well as metabolic, inflammatory, neurodegenerative, and cardiovascular diseases. Therefore, selective modulation of aberrant functions of these enzymes may provide an effective treatment for the above diseases.

Abstract LB-B06: Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer that has a limited response to treatment (median survival after diagnosis is only 9 months). Historically, DIPG research has been limited by a dearth of tumor tissue available for study and a lack of experimental model systems. Recently, both cell cultures derived from patients with DIPG and orthotopic xenograft models have been established. We performed a chemical screen in 16 patient-derived DIPG cultures using 83 drugs, selected by pediatric neurooncologists as either promising small-molecule compounds or traditional chemotherapeutic agents. Of these 83 drugs, 14 showed activities against three or more DIPG cultures. Notable ‘misses’ that highlight the resistance of this tumor to traditional chemotherapies included temozolomide, carboplatin, and vincristine, while DIPG cell cultures were reported to have substantial sensitivity to histone deacetylase inhibitors, consistent with the highly recurrent H3K27M driving mutations in ∼80% of DIPG. Panobinostat was one of the most effective drugs screened, with 12/16 DIPG cultures showing sensitivity to this drug. Compared to vorinostat, another HDAC inhibitor, panobinostat exhibited substantially greater potency against DIPG cells. Treatment of DIPG cultures with panobinostat yielded a time and dose-dependent decrease in viability (RNA-mediated knockdown of HDAC1 and HDAC2 confirmed the mechanism of action). Treatment of DIPG cultures with panobinostat also increased H3 acetylation and H3K27 trimethylation, and led to a partial rescue of the H3K27M induced global hypotrimethylation phenotype. Increased K27 trimethylation was an unexpected effect of the drug, but it is consistent with recent findings that acetylated H3K27 can ‘detoxify’ K27M-induced inhibition of PCR2. RNA-seq performed on panobinostat- or vehicle-treated DIPG cells revealed sweeping changes in gene expression, including normalization of the K27M gene expression signature and decrease of the oncogenic MYC target gene-expression signature. In addition, the multi-histone deacetylase inhibitor panobinostat demonstrated efficacy in DIPG orthotopic xenograft models administered using convection enhanced delivery. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4, recently shown to decrease viability of mutant DIPG cells, revealed that the two had synergistic effects. Together, these data suggest a promising FDA-approved therapeutic strategy for DIPG that could be rapidly translated for use in the clinic, further indicating that epigenetic modifying therapies are emerging as the most promising class of agents for the treatment of DIPG. Citation Format: Catherine S. Grasso. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B06.

KDM4/JMJD2 Histone Demethylase Inhibitors Block Prostate Tumor Growth by Suppressing the Expression of AR and BMYB-Regulated Genes

Histone lysine demethylase KDM4/JMJD2s are overexpressed in many human tumors including prostate cancer (PCa). KDM4s are co-activators of androgen receptor (AR) and are thus potential therapeutic targets. Yet to date few KDM4 inhibitors that have anti-prostate tumor activity in vivo have been developed. Here, we report the anti-tumor growth effect and molecular mechanisms of three novel KDM4 inhibitors (A1, I9, and B3). These inhibitors repressed the transcription of both AR and BMYB-regulated genes. Compound B3 is highly selective for a variety of cancer cell lines including PC3 cells that lack AR. B3 inhibited the in vivo growth of tumors derived from PC3 cells and ex vivo human PCa explants. We identified a novel mechanism by which KDM4B activates the transcription of Polo-like kinase 1 (PLK1). B3 blocked the binding of KDM4B to the PLK1 promoter. Our studies suggest a potential mechanism-based therapeutic strategy for PCa and tumors with elevated KDM4B/PLK1 expression.

Abstract 1719: Highly adaptable triple-negative breast cancer cells as a suitable model for testing epigenetic therapies

Abstract A major obstacle in developing effective therapies against solid tumors stems from an inability to adequately model the rare subpopulation of panresistant cancer cells that may often drive the disease. We propose a strategy for optimally modeling highly abnormal and highly adaptable human triple-negative breast cancer (TNBC) cells, and evaluating therapies for their ability to eradicate such cells. To overcome the shortcomings often associated with cell culture models, we have incorporated several features in our model including a selection of highly adaptable cancer cells based on their ability to survive a metabolic challenge. We have shown that metabolically adaptable (MA) TNBC cells efficiently metastasize to multiple organs in nude mice. Recently we have found that MA cells selected from SUM149 cell line feature an embryo-like gene expression, which may be important in generating tumor heterogeneity and therapy-resistance. Our results obtained thus far with a variety of anticancer agents support the validity of the model of realistic panresistance and suggest that it could be used for developing anticancer agents that would overcome panresistance. The cancer cells most responsible for panresistance are heterogeneous, and the likelihood that a particular test therapy will affect these cells is difficult to predict. Our testing strategy, which involves long-term evaluation of anticancer agents on MA cells in parallel with the parental cell line, is optimized to investigate the effects of potential therapeutic compounds on panresistance. This approach can not only help choose superior therapies for clinical trials, but also suggest strategies for overcoming therapeutic resistance in a timely manner. Considering an important role of chromatin in panresistance, we evaluated two promising chromatin modifiers- a bromodomain inhibitor JQ1 and a Jumonji histone demethylase inhibitor JIB-04 for their ability to overcome panresistance in our model. Both these compound appear promising in different cancer models. We are evaluating these compounds for efficacy in eradicating MA cells; we are testing these compounds to determine whether they are effective as single-agents and/or whether they can sensitize MA cells to chemotherapies. Our results indicate that a long treatment with either JQ1 or JIB-04 sensitizes MA cells to chemotherapeutic drugs doxorubicin and paclitaxel. Our results support the utility of our approach in discovering effective anticancer therapies against a heterogeneous and evolving disease like TNBC. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Balraj Singh, Ryan D. Milligan, Hannah E. Kinne, Anna Shamsnia, Anthony Lucci. Highly adaptable triple-negative breast cancer cells as a suitable model for testing epigenetic therapies. [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 1719. doi:10.1158/1538-7445.AM2015-1719

Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening

Jumonji C domain-containing histone demethylases (JHDMs) are epigenetic proteins capable of demethylating methylated lysine residues on histones proteins and for which high-quality chemical probes and eventual therapeutic leads are highly desirable. To expand the extent of known scaffolds targeting JHDMs, we initiated an unbiased high-throughput screening approach using a fluorescence polarization (FP)–based competitive binding assay we recently reported for JHDM1A (aka KDM2A). In total, 14,400 compounds in the HitFinder collection v.11 were screened, which represent all the distinct skeletons of the Maybridge Library. An eventual three compounds with two new scaffolds were discovered and further validated, which not only show in vitro binding for two different JHDMs, JHDM1A and JMJD2A (aka KDM4A), but also induce hypermethylation of their substrate in cells. These represent novel scaffolds as JHDM inhibitors and provide a basis for future optimization of affinity and selectivity.

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.

Diffuse Intrinsic Pontine Glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNAseq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated efficacy in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat with histone demethylase inhibitor GSKJ4 revealed synergy. Together, these data suggest a promising therapeutic strategy for DIPG.

The Role of Histone Demethylase KDM4B in Myc Signaling in Neuroblastoma

Epigenetic alterations, such as histone methylation, modulate Myc signaling, a pathway central to oncogenesis. We investigated the role of the histone demethylase KDM4B in N-Myc-mediated neuroblastoma pathogenesis. Spearman correlation was performed to correlate MYCN and KDM4B expression. RNA interference, microarray analysis, gene set enrichment analysis, and real-time polymerase chain reaction were used to define the functions of KDM4B. Immunoprecipitation and immunofluorescence were used to assess protein-protein interactions between N-Myc and KDM4B. Chromatin immunoprecipitation was used to assess the binding of Myc targets. Constitutive and inducible lentiviral-mediated KDM4B knockdown with shRNA was used to assess the effects on tumor growth. Kaplan-Meier survival analysis was used to assess the prognostic value of KDM4B expression. All statistical tests were two-sided. KDM4B and MYCN expression were found to be statistically significantly correlated in a variety of cancers, including neuroblastoma (R = 0.396, P < .001). Functional studies demonstrated that KDM4B regulates the Myc pathway. N-Myc was found to physically interact with and recruit KDM4B. KDM4B was found to regulate neuroblastoma cell proliferation and differentiation in vitro and xenograft growth in vivo (5 mice/group, two-tailed t test, P ≤ 0.001). Finally, together with MYCN amplification, KDM4B was found to stratify a subgroup of poor-prognosis patients (122 case patients, P < .001). Our findings provide insight into the epigenetic regulation of Myc via histone demethylation and proof-of-concept for inhibition of histone demethylases to target Myc signaling in cancers such as neuroblastoma.

Environmental enrichment reverses histone methylation changes in the aged hippocampus and restores age-related memory deficits.

A decline in long-term memory (LTM) formation is a common feature of the normal aging process, which corresponds with abnormal expression of memory-related genes in the aged hippocampus. Epigenetic modulation of chromatin structure is required for proper transcriptional control of genes, such as the brain-derived neurotrophic factor (Bdnf) and Zif268 in the hippocampus during the consolidation of new memories. Recently, the view has emerged that aberrant transcriptional regulation of memory-related genes may be reflective of an altered epigenetic landscape within the aged hippocampus, resulting in memory deficits with aging. Here, we found that baseline resting levels for tri-methylation of histone H3 at lysine 4 (H3K4me3) and acetylation of histone H3 at lysine 9 and 14 (H3K9,K14ac) were altered in the aged hippocampus as compared to levels in the hippocampus of young adult rats. Interestingly, object learning failed to increase activity-dependent H3K4me3 and di-methylation of histone H3 at lysine 9 (H3K9me2) levels in the hippocampus of aged adults as compared to young adults. Treatment with the LSD-1 histone demethylase inhibitor, t-PCP, increased baseline resting H3K4me3 and H3K9,K14ac levels in the young adult hippocampus, while young adult rats exhibited similar memory deficits as observed in aged rats. After environmental enrichment (EE), we found that object learning induced increases in H3K4me3 levels around the Bdnf, but not the Zif268, gene region in the aged hippocampus and rescued memory deficits in aged adults. Collectively, these results suggest that histone lysine methylation levels are abnormally regulated in the aged hippocampus and identify histone lysine methylation as a transcriptional mechanism by which EE may serve to restore memory formation with aging.

Histone Demethylase Inhibition As a Novel Therapeutic Strategy in Myeloma

Epigenetic dysregulation plays an important role in myeloma pathogenesis. Histone methyltransferases have been implicated in disease initiation and progression; for example MMSET, acting at H3K36 is the oncogenic driver in the 15% of patients with the initiating event t(4;14), whilst MLL, acting at H3K4, is recurrently mutated. It is logical to presume that enzymes with the opposite action at histone methyl marks, the demethylases, also play a role in myeloma and recurrent mutations and deletions are seen in KDM6A (UTX), a H3K27 demethylase. We further investigated the role of demethylases using linked gene expression and survival data from 3 large studies of newly diagnosed patients, a small molecule 2-oxoglutarate dependent histone demethylase inhibitor (GSK-J4, inhibitor of KDM5B+KDM6A/B) and knockdown with locked nucleic acids (LNAs), to define a potential new targeted therapeutic strategy for myeloma patients.Linked gene expression and survival data were available for the MRC Myeloma IX study (n=259), HOVON65/GMMG study (n=246) and Total Therapy 2+3 (TT2+3) studies (n=559). Patients with high expression of KDM5B, a H3K4 demethylase, had a significantly shorter overall survival (OS) than patients with lower expression. (Myeloma IX: high expression median OS 31.6 months vs 45.5 months, log rank p=0.020. HOVON65/GMMG-H4: high expression median OS 54.8 months vs median OS not reached, log rank p <0.001. TT2+3: medians not reached, same trend toward significance). No other demethylase gene had a consistent association between expression and survival.Inhibition of viability with GSK-J4 treatment occurred in a time and dose-dependent manner in a panel of 11 myeloma cell lines; GC50s ranging from 0.5 -3uM at 72 hours. Treatment induced apoptosis was demonstrated by increased annexin/PI staining, by flow cytometry, and increased caspase activity in the Caspase-Glo® 3/7 luminescent assay. Gene expression arrays (Illumina HumanHT12-v4), using RNA extracted from JJN-3 cells treated at 2uM for 6 and 24 hours, showed induction of expression of the metallothionein family of genes including MT1X, associated with intracellular metal-ion homeostasis, and genes associated with the cellular stress response including DDIT3 and INHBE, both ATF4 downstream target genes. These genes were validated by RT-PCR in 5 myeloma cell lines (e.g. RPMI-8226 24hrs 2.5uM GSK-J4 treatment mean RQ values: MT1X 176, DDIT 45, INHBE 70).Locked Nucleic Acid knockdown targeting KDM6A+6B reduced cell viability and induced the same transcriptional response seen with GSK-J4. Reduced expression of KDM5B was also detected following treatment with this LNA. Knockdown targeting KDM5B alone did not affect viability or induce the same transcriptional response, suggesting inhibition of all three demethylases is required for the response seen with GSK-J4. Chip-seq analysis of JJN-3 cells treated at 2uM GSK-J4 for 6 and 48 hours or LNA knockdown of KDM6A+B for 7 days, showed increased H3K4me3 marks globally, with specific peaks upstream of the metallothionein genes and ATF4 target genes, indicating that the effects seen with GSK-J4 treatment are mediated via an epigenetic mechanism.To evaluate the relevance of our findings to human disease, viability experiments using CD138+ plasma cells from myeloma patients were performed for cells alone and in co-culture with bone marrow stroma. GSK-J4 treatment reduced the viability of CD138 selected cells both alone and in co-culture compared to control. (Mean viability at 48hrs cells alone: 4uM GSK-J4 46%, 8uM 27%, 12uM 5%, co-cultured: 4uM 76%, 8uM 59%, 12uM 26%.) The transcriptional response seen in cell lines was conserved in primary patient cells following 24hr 2uM GSK-J4 treatment (Mean RQ values MT1X 105, INHBE 13, DDIT3 4). Treatment of bone marrow stromal cells and peripheral blood mononuclear cells with GSK-J4 demonstrated a therapeutic index between normal and malignant tissue.This work demonstrates a pro-apoptotic effect of demethylase inhibition in myeloma, a class of drug targets not previously investigated for this disease. A complex interplay between the demethylases affected by the GSK-J4 chemical probe is demonstrated and suggests that the modulation of KDM5B, KDM6A and KDM6B simultaneously delivers the apoptotic signal. The enrichment of H3K4me3 marks at genes involved in the transcriptional response along with survival data suggests KDM5B might be key to the response. DisclosuresLindow:Santaris: Employment. Obad:Santaris: Employment. Oerum:Santaris: Employment.

S129 Inflammatory Cytokines Influence Respiratory Epithelial Anti-viral Immune Responses Via Inducible Epigenetic Control Of Rig1 Expression: A Model Of Early Life Origins Of Asthma?

The development of asthma is linked to early life environmental exposures and the occurrence of severe viral infections. Rapid maturation of adaptive immunity from a tolerant (Th2) to an anti-infective (Th1) state occurs in the neonatal period. We hypothesised that the airway inflammatory milieu, driven by the maturing immune response to environmental exposures may have important effects on the development of anti-viral innate immunity at the level of the epithelium. We studied whether the inflammatory environment of the airway epithelium modulates gene expression via epigenetic regulation of anti-viral genes as a model of the development of a long term abnormality which is a hallmark of asthma. We optimised an in-vitro model using AALEB, human immortalised bronchial epithelial-derived cells which were pre-treated for 24 h with cytokines that mimic Th1 environment (IFNy, 10 ng/ml) and Th2 (IL-13, 10 ng/ml) before being infected with RSV A, MOI=2 for 48 h. Quantitative real-time PCR with Taqman primers was used to assess expression of innate genes. Cells were collected after 48 h and stored in Trizol. Chromatin Immuno Precipitation (ChIP) with antibodies against histone modifications was used to assess epigenetic controls. In order to confirm epigenetic regulation of innate genes we used a panel of HAT, HDAC and histone demethylase inhibitors. We initially studied the impact of cytokines on a range of innate anti-viral genes. RIG1 was differentially expressed and reductions in expression associated with higher viral titres. IFNy priming induced increases in RIG1 mRNA at 48 h that correlated at the promoter with enrichment of H3K9ac and RNApolII (active-promoters) and reduction of H3K9me3 (repressive-promoters). We observed a statistically significant increase of RIG1 expression by IFNγ when co-incubated with SAHA (HDAC I and II inhibitors) and JIB-04 (Pan-Jumanji histone demethylase inhibitor). No effects of Th2 priming were seen at the level of antiviral responses. This in-vitro study suggests the inflammatory environment of naive epithelial cells can induce epigenetic modulation of innate immune responses at the level of histone methylation and acetylation and hence potentially lead to long term impacts on anti-viral immunity. The presence of a Th1 milieu appears key to the development of effective anti-viral responses.

Epigenetic changes in bone marrow progenitor cells influence the inflammatory phenotype and alter wound healing in type 2 diabetes.

Classically activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in type 2 diabetes (T2D); however, the mechanism responsible for the dominant proinflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages toward a proinflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL-12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese). These epigenetically “preprogrammed” macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions the H3K27 demethylase Jmjd3 drives IL-12 production in macrophages and that IL-12 production can be modulated by inhibiting Jmjd3. Using human T2D tissue and murine models, we have identified a previously unrecognized mechanism by which macrophages are programmed toward a proinflammatory phenotype, establishing a pattern of unrestrained inflammation associated with nonhealing wounds. Hence, histone demethylase inhibitor–based therapy may represent a novel treatment option for diabetic wounds.

Abstract 5161: The development of therapeutic inhibitors of the KDM5 histone demethylases

Abstract Histone methyl marks contribute to the regulation of chromatin architecture and of DNA-based processes such as replication, repair and gene transcription. There is increasing evidence for alterations in histone methylation patterns in cancer and other human diseases. Importantly, aberrant methylation can contribute to deregulated gene expression and thereby to excessive proliferation, survival and metastasis of cancer cells. Histone methyl transferases and demethylases establish a dynamic equilibrium of histone modifications, and the expression and activity of several members of these enzyme families are deregulated in human cancer. The jumonji C-domain histone H3K4me3/2 demethylase KDM5B/PLU1/JARID1B as well as its close relative KDM5A/JARID1A are potential oncogenes. Both proteins are overexpressed in human tumors and the gene encoding KDM5B is e.g. amplified in invasive breast cancer cells. Results obtained with RNA interference and targeted deletions in mice have provided additional support for a critical role of KDM5 demethylases in cancer cells. EpiTherapeutics is developing novel cancer drugs targeting the enzymatic activity of histone demethylases. Here we present our recent progress in obtaining selective, potent, and cell-permeable inhibitors of the KDM5 demethylases. Biochemical and cellular activities, pharmacological data, and in vivo antitumor activity will be discussed. Citation Format: Heidi R. Hudlebusch, Bo Heinemann, Jesper M. Nielsen, Daniela Kleine-Kohlbrecher, Camilla Hauge, Thomas Boesen, Marc Labelle, Lars-Ole Gerlach, Peter Staller, Peter Birk. The development of therapeutic inhibitors of the KDM5 histone demethylases. [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 5161. doi:10.1158/1538-7445.AM2014-5161

Abstract 5143: KDM3A promotes cell growth and cisplatin-resistance in ovarian cancer cells

Abstract Background: Ovarian cancer is the most lethal gynecological cancer affecting the women worldwide. One of the main challenging hurdles in ovarian cancer treatment is the high recurrence rate due to therapeutic resistance. Recent evidences showed that JmjC-domain (JMJD) containing histone demethylases plays a critical role in controlling gene expression during normal development and cancer progression. Aims: To determine whether histone demethylase plays a role in ovarian cancer progression and therapeutic resistance. Methods: We developed cisplatin-resistance ovarian cancer cell lines SKOV-3 and OVCAR-5 and isolated total RNA to screen the expression pattern of JMJD family genes by real-time RT-PCR. We performed stable knock-down of KDM3A by lentiviral mediated ShRNA transfection and studied its effects on cell proliferation and apoptosis by MTT assay and western blot analysis (PARP, caspase-3), respectively. We also determined the effect of KDM3A knock-down on cell cycle and apoptosis regulators by western blot. Results: Our observations indicated the differential expression of various JMJD family proteins between cisplatin-resistance and sensitive cell lines. To further evaluate our results, we also tested the expression of those differentially expressed JMJD proteins in a widely used cisplatin-resistance and sensitive A2780 cell lines. We observed that KDM3A/JMJD1A is consistently overexpressed in all the cisplatin-resistance cell lines studied. Next, we stably knock-down KDM3A in cisplatin-resistance OVCAR-5 and A2780 cell lines. More interestingly, we found that KDM3A depletion inhibited cell proliferation, induced apoptosis and enhanced the sensitivity to cisplatin treatment. Further, we confirmed that the important regulators of cell cycle and apoptosis including p21 and Bcl-2 were modulated by KDM3A knock-down. Conclusions: Our results indicate that KDM3A is a critical epigenetic factor required for the ovarian cancer growth by promoting cell proliferation and cisplatin-resistance. Our findings might have important clinical implications in targeting ovarian cancer cells by using specific histone demethylase inhibitors. Grant support: This work was supported by Carl and Roberta Deutsch, and Kelly Day Foundations. Citation Format: Sivakumar Ramadoss, Suvajit Sen, Gautam Chaudhuri, Robin Farias-Eisner. KDM3A promotes cell growth and cisplatin-resistance in ovarian 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 5143. doi:10.1158/1538-7445.AM2014-5143

Epigenomic networking in drug development: from pathogenic mechanisms to pharmacogenomics.

Different epigenetic alterations (DNA methylation, histone modifications, chromatin remodeling, noncoding RNA dysregulation) are associated with the phenotypic expression of complex disorders in which genomic, epigenomic, proteomic, and metabolomic changes, in conjunction with environmental factors, are involved. As epigenetic modifications are reversible and can be potentially targeted by pharmacological and dietary interventions, a series of epigenetic drugs have been developed, including DNA methyltransferase inhibitors (nucleoside analogs, small molecules, bioproducts, antisense oligonucleotides, miRNAs), histone deacetylase inhibitors (short-chain fatty acids, hydroxamic acids, cyclic peptides, benzamides, ketones, sirtuin inhibitors, sirtuin activators), histone acetyltransferase modulators, histone methyltransferase inhibitors, histone demethylase inhibitors, and noncoding RNAs (miRNAs), with potential effects against myelodysplastic syndromes, different types of cancer, and neurodegenerative disorders. Pharmacogenetic and pharmacoepigenetic studies are required for the proper evaluation of efficacy and safety issues in clinical trials with epigenetic drugs.

Substrate- and cofactor-independent inhibition of histone demethylase KDM4C.

Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging, as the active sites of KDM1A-B and KDM4A-D histone demethylases are highly conserved. Most inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide sequence or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation, and chemical modifications. Hydrogen/deuterium exchange mass spectrometry revealed that the peptide-based inhibitors target KDM4C through substrate-independent interactions located on the surface remote from the active site within less conserved regions of KDM4C. The sites discovered in this study provide a new approach of targeting KDM4C through substrate- and cofactor-independent interactions and may be further explored to develop potent selective inhibitors and biological probes for the KDM4 family.

Inhibition of histone demethylase, LSD2 (KDM1B), attenuates DNA methylation and increases sensitivity to DNMT inhibitor-induced apoptosis in breast cancer cells

Increasing evidence suggests that dysfunction of histone lysine demethylase is associated with abnormal chromatin remodeling and gene silencing, contributing to breast tumorigenesis. In silico analysis shows that the newly identified histone demethylase lysine-specific demethylase 2 is highly expressed in breast cancer, especially in invasive tumors. However, it is currently unknown how LSD2 regulates chromatin remodeling and gene expression regulation in breast cancer. Using short hairpin RNA, we stably knocked down LSD2 (LSD2-KD) in MDA-MB-231 breast cancer cells. LSD2-KD led to accumulation of H3K4me1/2 without changing methylation levels of other key histone lysine residues, suggesting that LSD2 acts as a bona fide H3K4 demethylase in breast cancer cells. LSD2-KD resulted in decreased colony formation and attenuated global DNA methylation in MDA-MB-231 cells. Additionally, treatment with the DNMT inhibitor, 5-aza-deoxycytidine (DAC), synergistically increased mRNA expression of aberrantly silenced genes important in breast cancer development, including PR, RARβ, ERα, SFRP1, SFRP2, and E-cadherin in LSD2-KD cells. Furthermore, LSD2-KD cells are more susceptible to cell death than scramble controls, and combined treatment with tranylcypromine, an LSD2 inhibitor, and DAC resulted in synergistic growth inhibition of breast cancer cells. DNMT inhibition by DAC in LSD2-KD cells led to internucleosomal DNA fragmentation, enhanced PARP cleavage and increased sub-G1 apoptotic cell population. These results demonstrate an important role for LSD2 in regulation of DNA methylation and gene silencing in breast cancer, and suggest that inhibition of LSD2 in combination with DNA methyltransferase inhibition represents a novel approach for epigenetic therapy of breast cancer.

Sensitivity of MLL-rearranged AML cells to all-trans retinoic acid is associated with the level of H3K4me2 in the RARα promoter region.

All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML–RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPɛ and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML.