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Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

Simple Summary Wilms’ tumour (WT) is one of the most common solid paediatric malignancies and arises in the developing kidney. Treatment of most WTs is relatively successful, with the notable exception of tumours with anaplastic histology. Anaplastic WT survival rates can be as low as 30–50%, emphasising the need for a better understanding of their molecular aetiology, in order to facilitate the development of novel therapeutics for poor-prognosis WT. Previous studies have implicated increases in MYCN at the DNA and RNA level in WTs with anaplasia, although the biological functions of MYCN protein have not been reported. In this study, we define the expression pattern of MYCN protein in WTs and also modulate MYCN protein levels in anaplastic WT cell-lines to define the functions of MYCN in WT. We show that MYCN depletion leads to decreased cell growth and identify MYCN-regulated genes and pathways that may represent therapeutic vulnerabilities in poor-prognosis WT. Abstract The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma, where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated overexpression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms’ tumours with poorer outcomes, including tumours with diffuse anaplasia. Surprisingly, however, the expression and functions of the MYCN protein in Wilms’ tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms’ tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms’ tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms’ tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes including the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms’ tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of RE1-Silencing Transcription factor (REST) target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor-prognosis Wilms’ tumour.

Wilms tumour (WT), a childhood kidney cancer with embryonal origins, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of fetal kidney with two WT cell lines, using methyl-CpG immunoprecipitation. We filtered our results to remove common cancer-associated epigenetic changes, and to enrich for genes involved in early kidney development. This identified four candidate genes that were hypermethylated in WT cell lines compared to fetal kidney, of whichESRP2(epithelial splicing regulatory protein 2), was the most promising gene for further study.ESRP2was commonly repressed by DNA methylation in WT, and this was shown to occur early in WT development (in nephrogenic rests).ESRP2expression could be reactivated by DNA methyltransferase inhibition in WT cell lines. WhenESRP2was overexpressed in WT cell lines, it acted as an inhibitor of cellular proliferationin vitro,andin vivoit suppressed tumour growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets, in addition to well-characterised targets of ESRP2. We propose a model in which the mesenchymal to epithelial transition that is essential for early kidney development, can be disrupted in to generate WT, either by genetic abnormalities such asWT1mutations, or by epigenetic defects, such asESRP2methylation.

International audience; To discover epigenetic changes that may underly neuroblastoma pathogenesis, we identified differentially methylated genes in neuroblastoma cells compared to neural crest cells, the presumptive precursors cells for neuroblastoma, by using genome-wide DNA methylation analysis. We previously described genes that were hypermethylated in neuroblastoma; in this paper we report on 67 hypomethylated genes, which were filtered to select genes that showed transcriptional over-expression and an association with poor prognosis in neuroblastoma, highlighting GATA3 for detailed studies. Specific methylation assays confirmed the hypomethylation of GATA3 in neuroblastoma, which correlated with high expression at both the RNA and protein level. Demethylation with azacytidine in cultured sympathetic ganglia cells led to increased GATA3 expression, suggesting a mechanistic link between GATA3 expression and DNA methylation. Neuroblastomas that had completely absent GATA3 methylation and/or very high levels of protein expression, were associated with poor prognosis. Knock-down of GATA3 in neuroblastoma cells lines inhibited cell proliferation and increased apoptosis but had no effect on cellular differentiation. These results identify GATA3 as an epigenetically regulated component of the neuroblastoma transcriptional control network, that is essential for neuroblastoma proliferation. This suggests that the GATA3 transcriptional network is a promising target for novel neuroblastoma therapies. Neuroblastoma (NB) is one of the commonest extra-cranial solid malignancies of childhood, which arises as a result of disordered development of the sympathetic nervous system from neural crest cells 1,2. Neuroblastoma is clinically heterogeneous, with younger patients (18months) mostly have disseminated tumours at diagnosis and poor outcomes 3. The clinical heterogeneity of neuroblastoma is reflected in its molecular pathogenesis, where no single pathway has been identified as being critical for tumour development. Oncogene activations were the first genetic alterations identified in neuroblastoma; initially MYCN amplification was identified in high-risk tumours 4 and later ALK mutations were discovered in inherited neuroblastoma and some sporadic high-risk tumours 5,6. Mutations in tumour suppressor genes such as PHOX2B 7 and NF1 8 have also been reported. Recent genome-wide analyses have identified genomic mutations and other alterations in chromatin remodelling genes such as ATRX, ARID1A and ARID1B, in components of the RAC-RHO pathway 9-11 and in TERT 12,13 , with relapsed tumours demonstrating an increased mutation rate 14,15. Like most childhood cancers, neuroblastomas contain fewer mutations than adult cancers 16,17 , with some tumours apparently containing no detectable driver mutations 9-11. In low-risk neuroblastomas, copy-number changes may drive tumorigenesis 18 , but the lack of driver mutations in many cases, emphasises the need to consider other mechanisms of pathogenesis, such as epigenetic alterations 19 .

The neural crest, which has been referred to as the fourth germ layer, comprises a multipotent cell population which will specify diverse cells and tissues, including craniofacial cartilage and bones, melanocytes, the adrenal medulla and the peripheral nervous system. These cell fates are known to be determined by gene regulatory networks (GRNs) acting at various stages of neural crest development, such as induction, specification, and migration. Although transcription factor hierarchies and some of their interplay with morphogenetic signalling pathways have been characterised, the full complexity of activities required for regulated development remains uncharted. Deregulation of these pathways may contribute to tumourigenesis, as in the case of neuroblastoma, a frequently lethal embryonic cancer thought to arise from the sympathoadrenal lineage of the neural crest.In this conceptual analysis, we utilise next generation sequencing data from neuroblastoma cells and tumours to evaluate the possible influences of Wnt signalling on neural crest GRNs and on neuroblastoma cell lineages. We provide evidence that Wnt signalling is a major determinant of regulatory networks that underlie mesenchymal/NCC-like cell identities through PRRX1 and YAP/TAZ transcription factors. Furthermore, Wnt may also co-operate with Hedgehog signalling in driving proneural differentiation programmes along the adrenergic lineage. We propose that elucidation of Signalling Regulatory Networks can augment and complement GRNs in characterising cell identities, which will in turn contribute to the design of improved therapeutics tailored to primary and relapsing neuroblastoma.

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers., Highlights • The small molecule inhibitor YK-4-279 can trigger p53-independent apoptosis in neuroblastoma cell lines. • YK-4-279 induces mitotic arrest with multipolar, fragmented and unseparated spindles. • YK-4-279 acts differently from paclitaxel and vincristine. • YK-4-279 can overcome resistance to vincristine and synergize with other inhibitors of mitosis.

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma–carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription–polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=−0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=−0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma–carcinoma transition. Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma-carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription-polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=-0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=-0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma-carcinoma transition.Oncogene advance online publication,16 January 2012; doi:10.1038/onc.2011.609.

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5′ and 3′ ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10 4 . A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

During our search for transcriptional regulators that control the developmentally regulated expression of the enkephalin (ENK) gene, we identified AUF1. ENK, a peptide neurotransmitter, displays precise cell-specific expression in the adult brain. AUF1 (also known as heterogeneous nuclear ribonucleoprotein D) has been known to regulate gene expression through altering the stability of AU-rich mRNAs. We show here that in the developing brain AUF1 proteins are expressed in a spatiotemporally defined manner, and p37 and p40/42 isoforms bind to an AT-rich double-stranded (ds) DNA element of the rat ENK (rENK) gene. This AT-rich dsDNA sequence acts as a cis-regulatory DNA element and is involved in regulating the cell-specific expression of the ENK gene in primary neuronal cultures. The AT-rich dsDNA elements are present at approximately 2.5 kb 5'upstream of the rat, human, and mouse ENK genes. AUF1 proteins are shown here to provide direct interaction between these upstream AT-rich DNA sequences and the TATA region of the rENK gene. Double immunohistochemistry demonstrated that in the developing brain AUF1 proteins are expressed by proliferating neural progenitors and by differentiating neurons populating brain regions, which will not express the ENK gene in the adult, suggesting a repressor role for AUF1 proteins during enkephalinergic differentiation. Their subnuclear distribution and interactions with AT-rich DNA suggest that in the developing brain they can be involved in complex nuclear regulatory mechanisms controlling the development- and cell-specific expression of the ENK gene.

A Correction on: \ud LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma\ud Gabriella Cunha Vieira, S. Chockalingam, Zsombor Melegh, Alexander Greenhough, Sally Malik, Marianna Szemes, Ji Hyun Park, Abderrahmane Kaidi, Li Zhou, Daniel Catchpoole, Rhys Morgan, David O. Bates, Peter J. Gabb and Karim Malik\ud Original article: Oncotarget. 2015; 6:40053-67. DOI: 10.18632/oncotarget.5548.\ud \ud The originally Figure 5 contains duplicate total-ERK panels.\ud \ud The proper Figure 5 is attached. The authors sincerely apologize for this error.

Although tumour suppressor gene hypermethylation is a universal feature of cancer cells, little is known about the necessary molecular triggers. Here, we show that Wilms’ tumour 1 (WT1), a developmental master regulator that can also act as a tumour suppressor or oncoprotein, transcriptionally regulates the de novo DNA methyltransferase 3A (DNMT3A) and that cellular WT1 levels can influence DNA methylation of gene promoters genome-wide. Specifically, we demonstrate that depletion of WT1 by short-interfering RNAs leads to reduced DNMT3A in Wilms’ tumour cells and human embryonal kidney-derived cell lines. Chromatin immunoprecipitation assays demonstrate WT1 recruitment to the DNMT3A promoter region and reporter assays confirm that WT1 directly transactivates DNMT3A expression. Consistent with this regulatory role, immunohistochemical analysis shows co-expression of WT1 and DNMT3A proteins in nuclei of blastemal cells in human fetal kidney and Wilms’ tumours. Using genome-wide promoter methylation arrays, we show that human embryonal kidney cells over-expressing WT1 acquire DNA methylation changes at specific gene promoters where DNMT3A recruitment is increased, with hypermethylation being associated with silencing of gene expression. Elevated DNMT3A is also demonstrated at hypermethylated genes in Wilms’ tumour cells, including a region of long-range epigenetic silencing. Finally, we show that depletion of WT1 in Wilms’ tumour cells can lead to reactivation of gene expression from methylated promoters, such as TGFB2, a key modulator of epithelial–mesenchymal transitions. Collectively, our work defines a new regulatory modality for WT1 involving elicitation of epigenetic alterations which is most likely crucial to its functions in development and disease.

Background Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system. Results The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays. Conclusion This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.