REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma.

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)-a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein - a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

The NFIB/CARM1 partnership is a driver in preclinical models of small cell lung cancer.

The coactivator associated arginine methyltransferase (CARM1) promotes transcription, as its name implies. It does so by modifying histones and chromatin bound proteins. We identified nuclear factor I B (NFIB) as a CARM1 substrate and show that this transcription factor utilizes CARM1 as a coactivator. Biochemical studies reveal that tripartite motif 29 (TRIM29) is an effector molecule for methylated NFIB. Importantly, NFIB harbors both oncogenic and metastatic activities, and is often overexpressed in small cell lung cancer (SCLC). Here, we explore the possibility that CARM1 methylation of NFIB is important for its transforming activity. Using a SCLC mouse model, we show that both CARM1 and the CARM1 methylation site on NFIB are critical for the rapid onset of SCLC. Furthermore, CARM1 and methylated NFIB are responsible for maintaining similar open chromatin states in tumors. Together, these findings suggest that CARM1 might be a therapeutic target for SCLC.

Licochalcone A is a Natural Selective Inhibitor of Arginine Methyltransferase 6.

Arginine methylation is a post-translational modification that is implicated in multiple biological functions including transcriptional regulation. The expression of protein arginine methyltransferases (PRMT) has been shown to be upregulated in various cancers. PRMTs have emerged as attractive targets for the development of new cancer therapies. Here, we describe the identification of a natural compound, licochalcone A, as a novel, reversible and selective inhibitor of PRMT6. Since expression of PRMT6 is upregulated in human breast cancers and is associated with oncogenesis, we used the human breast cancer cell line system to study the effect of licochalcone A treatment on PRMT6 activity, cell viability, cell cycle, and apoptosis. We demonstrated that licochalcone A is a non-S-adenosyl L-methionine (SAM) binding site competitive inhibitor of PRMT6. In MCF-7 cells, it inhibited PRMT6-dependent methylation of histone H3 at arginine 2 (H3R2), which resulted in a significant repression of estrogen receptor activity. Licochalcone A exhibited cytotoxicity towards human MCF-7 breast cancer cells, but not MCF-10A human breast epithelial cells, by upregulating p53 expression and blocking cell cycle progression at G2/M, followed by apoptosis. Thus, licochalcone A has potential for further development as a therapeutic agent against breast cancer.

Medulloblastoma epigenetics and the path to clinical innovation.

INTRODUCTION: In the last decade, a number of genomic and pharmacological studies have demonstrated the importance of epigenetic dysregulation in medulloblastoma initiation and progression. High throughput approaches including gene expression array, next-generation sequencing (NGS), and methylation profiling have now clearly identified at least four molecular subgroups within medulloblastoma, each with distinct clinical and prognostic characteristics. These studies have clearly shown that despite the overall paucity of mutations, clinically relevant events do occur within the cellular epigenetic machinery. Thus, this review aims to provide an overview of our current understanding of the spectrum of epi-oncogenetic perturbations in medulloblastoma. METHODS: Comprehensive review of epigenetic profiles of different subgroups of medulloblastoma in the context of molecular features. Epigenetic regulation is mediated mainly by DNA methylation, histone modifications and microRNAs (miRNA). Importantly, epigenetic mis-events are reversible and have immense therapeutic potential. CONCLUSION: The widespread epigenetic alterations present in these tumors has generated intense interest in their use as therapeutic targets. We provide an assessment of the progress that has been made towards the development of molecular subtypes-targeted therapies and the current status of clinical trials that have leveraged these recent advances.

CARM1 inhibition reduces histone acetyltransferase activity causing synthetic lethality in CREBBP/EP300-mutated lymphomas.

Somatic mutations affecting CREBBP and EP300 are a hallmark of diffuse large B-cell lymphoma (DLBCL). These mutations are frequently monoallelic, within the histone acetyltransferase (HAT) domain and usually mutually exclusive, suggesting that they might affect a common pathway, and their residual WT expression is required for cell survival. Using in vitro and in vivo models, we found that inhibition of CARM1 activity (CARM1i) slows DLBCL growth, and that the levels of sensitivity are positively correlated with the CREBBP/EP300 mutation load. Conversely, treatment of DLBCLs that do not have CREBBP/EP300 mutations with CARM1i and a CBP/p300 inhibitor revealed a strong synergistic effect. Our mechanistic data show that CARM1i further reduces the HAT activity of CBP genome wide and downregulates CBP-target genes in DLBCL cells, resulting in a synthetic lethality that leverages the mutational status of CREBBP/EP300 as a biomarker for the use of small-molecule inhibitors of CARM1 in DLBCL and other cancers.

Novel 3,5-bis(bromohydroxybenzylidene)piperidin-4-ones as coactivator-associated arginine methyltransferase 1 inhibitors: enzyme selectivity and cellular activity.

Coactivator-associated arginine methyltransferase 1 (CARM1) represents a valuable target for hormone-dependent tumors such as prostate and breast cancers. Here we report the enzyme and cellular characterization of the 1-benzyl-3,5-bis(3-bromo-4-hydroxybenzylidene)piperidin-4-one (7g) and its analogues 8a-l. Among them, 7g, 8e, and 8l displayed high and selective CARM1 inhibition, with lower or no activity against a panel of different PRMTs or HKMTs. In human LNCaP cells, 7g showed a significant dose-dependent reduction of the PSA promoter activity.

Xenoestrogens regulate the activity of arginine methyltransferases.

Arginine methylation is a common post-translational modification that has been strongly implicated in transcriptional regulation. The arginine methyltransferases (PRMTs) were first reported as transcriptional coactivators for the estrogen and androgen receptors. Compounds that inhibit these enzymes will provide us with valuable tools for dissecting the roles of these enzymes in cells, and will possibly also have therapeutic applications. In order to identify such inhibitors of the PRMTs, we have previously performed a high-throughput screen using a small molecule library. These compounds were named arginine methyltransferase inhibitors (AMIs). The majority of these inhibitors were polyphenols, and one in particular (AMI-18) shared additional features with a group of known xenoestrogens. We, thus, tested a panel of xenoestrogens and found that a number of them possess the ability to inhibit PRMT activity, in vitro. These inhibitors primarily target CARM1, and include licochalcone A, kepone, benzyl 4-hydroxybenzoate, and tamoxifen. We developed a cell-based reporter system for CARM1 activity, and showed that tamoxifen (IC(50) =30 µM) inhibits this PRMT. The ability of these compounds to regulate the activity of transcriptional coactivators may be an unappreciated mechanism of action for xenoestrogens, and might also explain the efficacy of high-dose tamoxifen treatment on estrogen receptor negative cancers.

Enzymatic activity is required for the in vivo functions of CARM1.

CARM1 is one of nine protein arginine methyltransferases that methylate arginine residues in proteins. CARM1 is recruited by many different transcription factors as a positive regulator. Gene targeting of CARM1 in mice has been performed, and knock-out mice, which are smaller than their wild-type littermates, die just after birth. It has been proposed that CARM1 has functions that are independent of its enzymatic activity. Indeed, CARM1 is found to interact with a number of proteins and may have a scaffolding function in this context. However, CARM1 methylates histone H3, PABP1, AIB1, and a number of splicing factors, which strongly suggests that its impact on transcription and splicing is primarily through its ability to modify these substrates. To unequivocally establish the importance of CARM1 enzymatic activity in vivo, we generated an enzyme-dead knock-in of this protein arginine methyltransferase. We determined that knock-in cells and mice have defects similar to those seen in their knock-out counterparts with respect to the time of embryo lethality, T cell development, adipocyte differentiation, and transcriptional coactivator activity. CARM1 requires its enzymatic activity for all of its known cellular functions. Thus, small molecule inhibitors of CARM1 will incapacitate all of the enzyme's cellular functions.

epigenetic multiple ligands: mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (sirtuin) inhibitors.

A number of new compounds bearing two ortho-bromo- and ortho, ortho-dibromophenol moieties linked through a saturated/unsaturated, linear/(poly)cyclic spacer (compounds 1- 9) were prepared as simplified analogues of AMI-5 (eosin), a recently reported inhibitor of both protein arginine and histone lysine methyltransferases (PRMTs and HKMTs). Such compounds were tested against a panel of PRMTs (RmtA, PRMT1, and CARM1) and against human SET7 (a HKMT), using histone and nonhistone proteins as a substrate. They were also screened against HAT and SIRTs, because they are structurally related to some HAT and/or SIRT modulators. From the inhibitory data, some of tested compounds ( 1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f) were able to inhibit PRMTs, HKMT, HAT, and SIRTs with similar potency, thus behaving as multiple ligands for these epigenetic targets (epi-MLs). When tested on the human leukemia U937 cell line, the epi-MLs induced high apoptosis levels [i.e., 40.7% ( 4l) and 42.6% ( 7b)] and/or massive, dose-dependent cytodifferentiation [i.e., 95.2% ( 1c) and 96.1% ( 4j)], whereas the single-target inhibitors eosin, curcumin, and sirtinol were ineffective or showed a weak effect.

CARM1 promotes adipocyte differentiation by coactivating PPARgamma.

The coactivator-associated arginine methyltransferase 1 (CARM1) is recruited to gene promoters by many transcription factors. To identify new pathways that use CARM1, we carried out a comprehensive transcriptome analysis of CARM1-knockout embryos. By using complementary DNA microarrays and serial analysis of gene expression, we identified various genes involved in lipid metabolism that were underrepresented in CARM1-knockout embryos, indicating an important role for this coactivator in adipose tissue biology. We also observed that the amount of brown fat in CARM1-knockout embryos is reduced. Furthermore, cells lacking CARM1 have a severely curtailed potential to differentiate into mature adipocytes. Reporter experiments and chromatin immunoprecipitation analysis show that CARM1 regulates these processes by acting as a coactivator for peroxisome proliferator-activated receptor gamma (PPARgamma). Together, these results show that CARM1 promotes adipocyte differentiation by coactivating PPARgamma-mediated transcription and thus might be important in energy balance.

Arginine methylation of the histone H3 tail impedes effector binding.

Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding.

Ribosomal protein rpS2 is hypomethylated in PRMT3-deficient mice.

PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.

The activity and stability of the transcriptional coactivator p/CIP/SRC-3 are regulated by CARM1-dependent methylation.

The transcriptional coactivator p/CIP(SRC-3/AIB1/ACTR/RAC3) binds liganded nuclear hormone receptors and facilitates transcription by directly recruiting accessory factors such as acetyltransferase CBP/p300 and the coactivator arginine methyltransferase CARM1. In the present study, we have established that recombinant p/CIP (p300/CBP interacting protein) is robustly methylated by CARM1 in vitro but not by other protein arginine methyltransferase family members. Metabolic labeling of MCF-7 breast cancer cells with S-adenosyl-L-[methyl-(3)H]methionine and immunoblotting using dimethyl arginine-specific antibodies demonstrated that p/CIP is specifically methylated in intact cells. In addition, methylation of full-length p/CIP is not supported by extracts derived from CARM1(-/-) mouse embryo fibroblasts, indicating that CARM1 is required for p/CIP methylation. Using mass spectrometry, we have identified three CARM1-dependent methylation sites located in a glutamine-rich region within the carboxy terminus of p/CIP which are conserved among all steroid receptor coactivator proteins. These results were confirmed by in vitro methylation of p/CIP using carboxy-terminal truncation mutants and synthetic peptides as substrates for CARM1. Analysis of methylation site mutants revealed that arginine methylation causes an increase in full-length p/CIP turnover as a result of enhanced degradation. Additionally, methylation negatively impacts transcription via a second mechanism by impairing the ability of p/CIP to associate with CBP. Collectively, our data highlight coactivator methylation as an important regulatory mechanism in hormonal signaling.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

Techniques in protein methylation.

Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated.

Small molecule regulators of protein arginine methyltransferases.

Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.

Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening.

Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis. In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert. GaCDfn and GaCD were purified to homogeneity and refolded in vitro. GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions. GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions. GaCD is unstable in the presence of calcium and zinc ions. The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities. The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5. Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn. EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities. A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL. Further analysis by using MMP inhibitors indicated there is a correlation between IC(50) values on GaCDfn and GaCD. A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification.

Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening.

AIM: To obtain a catalytically active human macrophage elastase catalytic domain (hMECD) and to establish an efficient high-throughput method for screening macrophage elastase inhibitors. METHODS: Catalytic domain of human macrophage elastase was expressed in E coli and characterized to establish a high-throughput screening assay using a colorimetric method. A set of 8560 pure compounds and mixtures were screened. RESULTS: We have constructed an efficient E coli system for this human protein expression, and the recombinant hMECD protein was purified to homogeneity using anion-exchange chromatography after in vitro refolding from inclusion bodies. The yield of active hMECD protein was 23 mg from one liter of E coli culture after purification. Calcium and zinc ions were required both in refolding and enzymatic activity, but high concentration of zinc inhibited the refolding and activity. The hMECD cleaved several synthetic substrates including a chromogenic thiopeptolide and fluorogenic peptides with optimal activity around pH 8.0. Screening of 8560 compounds and mixtures led to identify 27 pure compounds and 14 natural products with inhibitory activity higher than 80 % at 20 mg/L. CONCLUSION: An efficient expression and purification method for hMECD protein has been established, and the assay is effective, reliable, and fast in identifying the recombinant protein inhibitors.