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MYCN activation is a hallmark of advanced neuroblastoma (NB) and a known master regulator of metabolic reprogramming, favoring NB adaptation to its microenvironment. We found that the expression of the main regulators of the molecular clock loops is profoundly disrupted in MYCN-amplified NB patients, and this disruption independently predicts poor clinical outcome. MYCN induces the expression of clock repressors and downregulates the one of clock activators by directly binding to their promoters. Ultimately, MYCN attenuates the molecular clock by suppressing BMAL1 expression and oscillation, thereby promoting cell survival. Reestablishment of the activity of the clock activator RORα via its genetic overexpression and its stimulation through the agonist SR1078, restores BMAL1 expression and oscillation, effectively blocks MYCN-mediated tumor growth and de novo lipogenesis, and sensitizes NB tumors to conventional chemotherapy. In conclusion, reactivation of RORα could serve as a therapeutic strategy for MYCN-amplified NBs by blocking the dysregulation of molecular clock and cell metabolism mediated by MYCN., MYCN is frequently amplified in neuroblastomas. Here, the authors show that MYCN disrupts the molecular clock by downregulating clock activator RORα and that the reactivation of RORα restores BMAL1 activity, and inhibits lipid metabolism and neuroblastoma growth

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology., The transcriptional regulator PRDM15 is expressed at low levels in normal tissues but overexpressed in B-cell lymphomas. Here, the authors show that PRDM15 depletion does not affect adult somatic cell homeostasis but leads to a metabolic crisis which impairs B-cell lymphomagenesis.

Half of high-risk neuroblastoma patients have MYCN amplification. Here, the authors show that MYCN induces fatty acid uptake and synthesis to support neuroblastoma and inhibition of a fatty acid transporter impairs tumor progression in preclinical models.Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy., Although the gain in chromosome 17q21-ter is commonly associated with neuroblastoma, it is not clear which gene of this region mediates tumorigenesis. Here, the authors are showing that JMJD6, which locates in that region, is a neuroblastoma tumorigenic factor.

PRDM12 is a member of the PRDI-BF1 (positive regulatory domain I-binding factor 1) homologous domain (PRDM)-containing protein family, a subfamily of Kruppel-like zinc finger proteins, controlling key processes in the development of cancer. PRDM12 is expressed in a spatio-temporal manner in neuronal systems where it exerts multiple functions. PRDM12 is essential for the neurogenesis initiation and activation of a cascade of downstream pro-neuronal transcription factors in the nociceptive lineage. PRDM12 inactivation, indeed, results in a complete absence of the nociceptive lineage, which is essential for pain perception. Additionally, PRDM12 contributes to the early establishment of anorexigenic neuron identity and the maintenance of high expression levels of pro-opiomelanocortin, which impacts on the program bodyweight homeostasis. PRDMs are commonly involved in cancer, where they act as oncogenes/tumor suppressors in a “Yin and Yang” manner. PRDM12 is not usually expressed in adult normal tissues but its expression is re-activated in several cancer types. However, little information is currently available on PRDM12 expression in cancers and its mechanism of action has not been thoroughly described. In this review, we summarize the recent findings regarding PRDM12 by focusing on four main biological processes: neurogenesis, pain perception, oncogenesis and cell metabolism. Moreover, we wish to highlight the importance of future studies focusing on the PRDM12 signaling pathway(s) and its role in cancer onset and progression.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

Neuroblastoma (NB) is one of the most frequently occurring neurogenic extracranial solid cancers in childhood and infancy. Over the years, many pieces of evidence suggested that NB development is controlled by gene expression dysregulation. These unleashed programs that outline NB cancer cells make them highly dependent on specific tuning of gene expression, which can act co-operatively to define the differentiation state, cell identity, and specialized functions. The peculiar regulation is mainly caused by genetic and epigenetic alterations, resulting in the dependency on a small set of key master transcriptional regulators as the convergence point of multiple signalling pathways. In this review, we provide a comprehensive blueprint of transcriptional regulation bearing NB initiation and progression, unveiling the complexity of novel oncogenic and tumour suppressive regulatory networks of this pathology. Furthermore, we underline the significance of multi-target therapies against these hallmarks, showing how novel approaches, together with chemotherapy, surgery, or radiotherapy, can have substantial antineoplastic effects, disrupting a wide variety of tumorigenic pathways through combinations of different treatments.

Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain‐of‐function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti‐tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients., This manuscript utilizes three different models to demonstrate that the chromatin assembly factor 1 subunit p150 (CHAF1A) blocks neuronal differentiation and promotes neuroblastoma oncogenesis. CHAF1A functions in part by reprogramming cell metabolism and activating polyamine synthesis. Blocking polyamine synthesis is a clinically translatable approach to improve retinoic acid‐based differentiation therapy.

The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets., MYCN amplification is common in neuroblastomas. Here, the authors identify a long noncoding RNA, lncNB1 in these cancers and show that it promotes tumorigenesis by binding to ribosomal protein, RPL35 to enhance E2F1 and DEPDC1B protein synthesis, which phosphorylates ERK to stabilise N-Myc.

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN–PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN–PA2G4 protein–protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. Significance: Competitive chemical inhibition of the PA2G4–MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

// Giorgia Giordani 1, 5, * , Marilena Barraco 1, 6, * , Angela Giangrande 2 , Giovanni Martinelli 3 , Viviana Guadagnuolo 3 , Giorgia Simonetti 3 , Giovanni Perini 1, 4 , Roberto Bernardoni 1, 4 1 Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy 2 Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP 67404 Illkirch, France 3 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Institute of Hematology "L. e A. Seragnoli", University of Bologna, Bologna, Italy 4 Health Sciences and Technology - Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Ozzano Emilia, Italy 5 Present address: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, UK 6 Present address: Institute of Hematology, “L e A Seragnoli”, S. Orsola-Malpighi Hospital, Bologna, Italy * These authors contributed equally to this work Correspondence to: Roberto Bernardoni, email: roberto.bernardoni@unibo.it Giovanni Perini, email: giovanni.perini@unibo.it Keywords: PF-04449913, Smoothened inhibitor, leukemia, Drosophila, prohemocytes Received: December 09, 2015 Accepted: June 26, 2016 Published: July 28, 2016 ABSTRACT The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.

The oncoprotein BCR-ABL1 triggers chronic myeloid leukemia. It is clear that the disease relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. We describe and validate a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. We show that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate our Drosophila model, which promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators in order to better elucidate the mechanism of leukemia onset and progression.

Background Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. Methods A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. Results Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the "poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). Conclusion ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention

Most evolutionary new centromeres (ENC) are composed of large arrays of satellite DNA and surrounded by segmental duplications. However, the hypothesis is that ENCs are seeded in an anonymous sequence and only over time have acquired the complexity of “normal” centromeres. Up to now evidence to test this hypothesis was lacking. We recently discovered that the well-known polymorphism of orangutan chromosome 12 was due to the presence of an ENC. We sequenced the genome of an orangutan homozygous for the ENC, and we focused our analysis on the comparison of the ENC domain with respect to its wild type counterpart. No significant variations were found. This finding is the first clear evidence that ENC seedings are epigenetic in nature. The compaction of the ENC domain was found significantly higher than the corresponding WT region and, interestingly, the expression of the only gene embedded in the region was significantly repressed.

Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene–amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc–mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522–33. ©2017 AACR.

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2 Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct ‘positional alleles’, each one extending for about 80–160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles. Electronic supplementary material The online version of this article (doi:10.1007/s00412-014-0493-6) contains supplementary material, which is available to authorized users.

The biological complexity of NGF action is achieved by binding two distinct neurotrophin receptors, TrkA and p75(NTR). While several reports have provided lines of evidence on the interaction between TrkA and p75(NTR) at the plasma membrane, much fewer data are available on the consequence of such an interaction in terms of intracellular signaling. In this study, we have focused on how p75(NTR) may affect TrkA downstream signaling with respect to neuronal differentiation. Here, we have shown that cooperation between p75(NTR) and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite outgrowth is concomitant with a selective enhancement of the AP-1 activity and the transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be involved in the differentiation process. Collectively, our results unveil a functional link between the specific expression profile of neurotrophin receptors in neuronal cells and the NGF-mediated regulation of the differentiation process possibly through a persistent ERKs activation and the selective control of the AP-1 activity. The biological complexity of NGF action is achieved by binding two distinct neurotrophin receptors, TrkA and p75(NTR). While several reports have provided lines of evidence on the interaction between TrkA and p75(NTR) at the plasma membrane, much fewer data are available on the consequence of such an interaction in terms of intracellular signaling. In this study, we have focused on how p75(NTR) may affect TrkA downstream signaling with respect to neuronal differentiation. Here, we have shown that cooperation between p75(NTR) and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite outgrowth is concomitant with a selective enhancement of the AP-1 activity and the transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be involved in the differentiation process. Collectively, our results unveil a functional link between the specific expression profile of neurotrophin receptors in neuronal cells and the NGF-mediated regulation of the differentiation process possibly through a persistent ERKs activation and the selective control of the AP-1 activity.

ncRNAs are the most recently identified class of regulatory RNAs with vital functions in gene expression regulation and cell development. Among the variety of roles they play, their involvement in human diseases has opened new avenues of research towards the discovery and development of novel therapeutic approaches. Important data come from the field of hereditary muscle dystrophies, like Duchenne muscle dystrophy and Myotonic dystrophies, rare diseases affecting 1 in 7000–15,000 newborns and is characterized by severe to mild muscle weakness associated with cardiac involvement. Novel therapeutic approaches are now ongoing for these diseases, also based on splicing modulation. In this review we provide an overview about ncRNAs and their behavior in muscular dystrophy and explore their links with diagnosis, prognosis and treatments, highlighting the role of regulatory RNAs in these pathologies.

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G0/G1 phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5., Highlights ► CDKL5 enhances neuronal differentiation. ► CDKL5 arrests cell cycle of neuronal precursor cells. ► MYCN directly represses transcription of CDKL5. ► These results may help explain the neurological symptoms of RTT patients.

MYCN gene amplification in neuroblastoma drives a gene expression program that correlates strongly with aggressive disease. Mechanistically, trimethylation of histone H3 lysine 4 (H3K4) at target gene promoters is a strict prerequisite for this transcriptional program to be enacted. WDR5 is a histone H3K4 presenter that has been found to have an essential role in H3K4 trimethylation. For this reason, in this study, we investigated the relationship between WDR5-mediated H3K4 trimethylation and N-Myc transcriptional programs in neuroblastoma cells. N-Myc upregulated WDR5 expression in neuroblastoma cells. Gene expression analysis revealed that WDR5 target genes included those with MYC-binding elements at promoters such as MDM2. We showed that WDR5 could form a protein complex at the MDM2 promoter with N-Myc, but not p53, leading to histone H3K4 trimethylation and activation of MDM2 transcription. RNAi-mediated attenuation of WDR5 upregulated expression of wild-type but not mutant p53, an effect associated with growth inhibition and apoptosis. Similarly, a small-molecule antagonist of WDR5 reduced N-Myc/WDR5 complex formation, N-Myc target gene expression, and cell growth in neuroblastoma cells. In MYCN-transgenic mice, WDR5 was overexpressed in precancerous ganglion and neuroblastoma cells compared with normal ganglion cells. Clinically, elevated levels of WDR5 in neuroblastoma specimens were an independent predictor of poor overall survival. Overall, our results identify WDR5 as a key cofactor for N-Myc–regulated transcriptional activation and tumorigenesis and as a novel therapeutic target for MYCN-amplified neuroblastomas. Cancer Res; 75(23); 5143–54. ©2015 AACR.

BACKGROUND Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN oncogene have poor prognoses. METHODS Bioinformatics data were used to discover a novel long noncoding RNA, lncUSMycN, at the 130-kb amplicon. RNA-protein pull-down assays were used to identify proteins bound to lncUSMycN RNA. Kaplan-Meier survival analysis, multivariable Cox regression, and two-sided log-rank test were used to examine the prognostic value of lncUSMycN and NonO expression in three cohorts of neuroblastoma patients (n = 47, 88, and 476, respectively). Neuroblastoma-bearing mice were treated with antisense oligonucleotides targeting lncUSMycN (n = 12) or mismatch sequence (n = 13), and results were analyzed by multiple comparison two-way analysis of variance. All statistical tests were two-sided. RESULTS Bioinformatics data predicted lncUSMycN gene and RNA, and reverse-transcription polymerase chain reaction confirmed its three exons and two introns. The lncUSMycN gene was coamplified with MYCN in 88 of 341 human neuroblastoma tissues. lncUSMycN RNA bound to the RNA-binding protein NonO, leading to N-Myc RNA upregulation and neuroblastoma cell proliferation. High levels of lncUSMycN and NonO expression in human neuroblastoma tissues independently predicted poor patient prognoses (lncUSMycN: hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.06 to 3.28, P = .03; NonO: HR = 2.48, 95% CI = 1.34 to 4.57, P = .004). Treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice statistically significantly hindered tumor progression (P < .001). CONCLUSIONS Our data demonstrate the important roles of lncUSMycN and NonO in regulating N-Myc expression and neuroblastoma oncogenesis and provide the first evidence that amplification of long noncoding RNA genes can contribute to tumorigenesis.

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Amplification of the transcription factor MYCN is associated with poor outcome and a multidrug-resistant phenotype in neuroblastoma. N-Myc regulates the expression of several ATP-binding cassette (ABC) transporter genes, thus affecting global drug efflux. Because these transporters do not confer resistance to several important cytotoxic agents used to treat neuroblastoma, we explored the prognostic significance and transcriptional regulation of the phase II detoxifying enzyme, glutathione S-transferase P1 (GSTP1). Using quantitative real-time PCR, GSTP1 gene expression was assessed in a retrospective cohort of 51 patients and subsequently in a cohort of 207 prospectively accrued primary neuroblastomas. These data along with GSTP1 expression data from an independent microarray study of 251 neuroblastoma samples were correlated with established prognostic indicators and disease outcome. High levels of GSTP1 were associated with decreased event-free and overall survival in all three cohorts. Multivariable analyses, including age at diagnosis, tumor stage, and MYCN amplification status, were conducted on the two larger cohorts, independently showing the prognostic significance of GSTP1 expression levels in this setting. Mechanistic investigations revealed that GSTP1 is a direct transcriptional target of N-Myc in neuroblastoma cells. Together, our findings reveal that N-Myc regulates GSTP1 along with ABC transporters that act to control drug metabolism and efflux. Furthermore, they imply that strategies to jointly alter these key multidrug resistance mechanisms may have therapeutic implications to manage neuroblastomas and other malignancies driven by amplified Myc family genes. Cancer Res; 72(4); 845–53. ©2011 AACR.

Mental retardation in Down syndrome (DS) appears to be related to severe neurogenesis impairment during critical phases of brain development. Recent lines of evidence in the cerebellum of a mouse model for DS (the Ts65Dn mouse) have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. By using an in vitro model of NPCs obtained from the subventricular zone and hippocampus, we found that trisomic NPCs had an increased expression of the Shh receptor Patched1 (Ptch1), a membrane protein that suppresses the action of a second receptor, Smoothened (Smo), thereby maintaining the pathway in a repressed state. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. The overexpression of Ptch1 in trisomic NPCs resulted from increased levels of AICD [a transcription-promoting fragment of amyloid precursor protein (APP)] and increased AICD binding to the Ptch1 promoter. Our data provide novel evidence that Ptch1 overexpression underlies derangement of the Shh pathway in trisomic NPCs with consequent proliferation impairment. The demonstration that Ptch1 overexpression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.

The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.

Myc oncoproteins and histone deacetylases (HDACs) modulate gene transcription and enhance cancer cell proliferation, and HDAC inhibitors are among the most promising new classes of anticancer drugs. Here, we show that N-Myc and c-Myc upregulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells, respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Cyclin G2 (CCNG2) was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. 5-bromo-2'-deoxyuridine incorporation assays showed that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc- and HDAC2-induced cell proliferation. Dual crosslinking chromatin immunoprecipitation assay demonstrated that N-Myc acted as a transrepressor by recruiting the HDAC2 protein to Sp1-binding sites at the CCNG2 gene core promoter. Moreover, HDAC2 was upregulated, and CCNG2 downregulated, in pre-cancerous and neuroblastoma tissues from N-Myc transgenic mice, and c-Myc overexpression correlated with upregulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of upregulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers.

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repressionin vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activityin vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.

Nuclear lamins are intermediate filament-type proteins forming a fibrillar meshwork that underlies the inner nuclear membrane. We have previously reported the identification of the human lamin B2 gene that maps to the subtelomeric band p13.3 of chromosome 19 in close proximity of a human DNA replication origin. Here we report the identification within the human lamin B2 gene of a novel repeated element ( v ariable n umber of t andem r epeats: VNTR) that appears to have a very recent origin, being absent in the genome of mouse and primates such as cercopitheques, lemurs and macaques. The VNTR is adjacent to exon 8 of the lamin B2 gene which, albeit encoding the nuclear localization signal of the protein, is highly divergent both at amino acid and nucleotide level among species. Moreover the VNTR, characterized by a repeated unit of about 100 bp, covers most of intron 8 of the gene and reiterates both the last 7 bp of the upstream exon and the exon/intron junction. RT–PCR experiments carried out on HeLa cell RNA suggest that none of the downstream junctions is used during the processing of the lamin B2 pre-mRNA.

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc–induced neuroblastoma., Author Summary The class III histone deacetylase SIRT1 is repressed by tumor suppressor genes and exerts divergent effects on tumorigenesis depending on its down-stream targets. Small molecule SIRT1 inhibitors have shown promising anti-cancer effects both in vitro and in vivo. Here we identified SIRT1 as a gene directly up-regulated by N-Myc and identified SIRT1-mediated transcriptional repression as a novel mechanism responsible for maintaining N-Myc oncoprotein stability. Moreover, SIRT1 contributed to N-Myc–induced cell proliferation, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in N-Myc transgenic mice. Our data identify SIRT1 as an important co-factor for N-Myc oncogenesis and provide important evidence for the potential application of SIRT1 inhibitors in the prevention and therapy of N-Myc–induced neuroblastoma.

We have searched the human genome for genes encoding new proteins that may be involved in three nuclear gene expression processes: transcription, pre-messenger RNA splicing and polyadenylation. A plethora of potential new factors are implicated by sequence in nuclear gene expression, revealing a substantial but selective increase in complexity compared with Drosophila melanogaster and Caenorhabditis elegans. Although the raw genomic information has limitations, its availability offers new experimental approaches for studying gene expression.