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Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB’s mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB’s cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

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.

The MYC family of transcription factors is a major driver of human cancer and potential therapeutic target. However, no clinically viable drugs have been yet developed that are able to directly tackle MYC oncoproteins. In our laboratory, we are exploring alternative approaches aiming to disturb signalling downstream of MYC. MYCN is frequently activated in neuroblastoma, a paediatric solid malignancy that, in its metastatic form, has a very poor prognosis. An important pathway regulated by MYC is the CKS1/SKP2/p27kip1 axis. In this study, we have repurposed the anti-psychotic drug Prozac to disrupt CKS1/SKP2/p27Kip1 signalling and assess its potential as an anti-neuroblastoma agent in vitro and in vivo. Using DNA editing technology, we show that stabilisation of p27Kip1 operated by Prozac in MYC-activated cells is essential for the anti-neuroblastoma activity of the drug. Furthermore, dosing mice with a concentration of Prozac equivalent to that used in long-term clinical trials in children with psychiatric disorders caused a significant reduction of metastatic disease in two models of high-risk neuroblastoma. The favourable toxicity profile of Prozac suggests that long-term treatments might be implemented in children with MYC/CKS1high neuroblastomas.

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 (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.

Background: Individuals with Down syndrome suffer from mental retardation due to severe neurogenesis impairment.

Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy, remarkably refractory to therapeutic treatments. One of the most powerful independent prognostic indicators for this disease is the amplification of the MYCN oncogene, which occurs at high levels in approximately 25% of neuroblastomas. Interestingly, amplification and not just expression of MYCN has a strong prognostic value, although this fact appears quite surprising as MYCN is a transcription factor that requires dimerising with its partner MAX, to exert its function. This observation greatly suggests that the role of MYCN in neuroblastoma should be examined in the context of MAX expression. In this report, we show that, in contrast to what is found in normal cells, MAX expression is significantly different among primary NBs, and that its level appears to correlate with the clinical outcome of the disease. Importantly, controlled modulation of MAX expression in neuroblastoma cells with different extents of MYCN amplification, demonstrates that MAX can instruct gene transcription programs that either reinforce or weaken the oncogenic process enacted by MYCN. In general, our work illustrates that it is the MAX to MYCN ratio that can account for tumour progression and clinical outcome in neuroblastoma and proposes that such a ratio should be considered as an important criterion to the design and development of anti-MYCN therapies.

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.

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.

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.

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.

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Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (1). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells.

Myc oncoproteins exert tumorigenic effects by regulating the 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. DOT1L is the only known histone methyltransferase that catalyses H3K79 methylation. Here, we showed that N-Myc up-regulated DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. Knocking down DOT1L reduced mRNA and protein expression of the N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knocking down 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 xenografted with neuroblastoma cells stably expressing doxycycline-inducible DOT1L small hair-pin RNA, 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 data identify DOT1L as a novel co-factor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis, and DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Citation Format: Matthew Wong, Andrew Tee, Giorgio Milazzo, Jessica Bell, Stefan Hüttelmaier, Patsie Polly, Giovanni Perini, Christopher J. Scarlett, Tao Liu. The histone methyltransferase DOT1L promotes neuroblastoma by regulating gene transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-080. doi:10.1158/1538-7445.AM2017-LB-080

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.

In this work we have investigated the molecular basis of the neuronal damage induced by the prion peptide by searching for a surface receptor whose activation could be the first step of a cascade of events responsible for cell death. By using a human neuroblastoma cell line lacking all the neurotrophin receptors and derived clones expressing the full-length or truncated forms of the low affinity neurotrophin receptor (p75(NTR)), we have been able to demonstrate that the neuronal death induced by the prion protein fragment PrP-(106-126) is an active process mediated by a) the binding of the peptide to the extracellular region of p75(NTR), b) the signaling function of the intracytoplasmic region of the receptor, and c) the activation of caspase-8 and the production of oxidant species.

The hepatitis B virus pX protein is a potent transcriptional activator of viral and cellular genes whose mechanism of action is poorly understood. Here we show that pX dramatically stimulates in vitro DNA binding of a variety of cellular proteins that contain basic region/leucine zipper (bZIP) DNA binding domains. The basis for increased DNA binding is a direct interaction between pX and the conserved bZIP basic region, which promotes bZIP dimerization and the increased concentration of the bZIP homodimer then drives the DNA binding reaction. Unexpectedly, we found that the DNA binding specificity of various pX-bZIP complexes differs from one another and from that of the bZIP itself. Thus, through recognition of the conserved basic region, pX promotes dimerization, increases DNA binding, and alters DNA recognition. These properties of pX are remarkably similar to those of the human T-cell lymphotrophic virus type I Tax protein. Although Tax and pX are not homologous, we show that the regions of the two proteins that stimulate bZIP binding contain apparent metal binding sites. Finally, consistent with thisin vitro activity, we provide evidence that both Tax and pX activate transcription in vivo, at least in part, by facilitating occupancy of bZIPs on target promoters.

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.

N-Myc gene amplification occurs in one quarter of human neuroblastoma tissues, and is a marker for poor patient prognosis. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1NB1, which were most considerably differentially expressed between N-Myc gene amplified and nonamplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPD was one of the few genes considerably downregulated in neuroblastoma cells after RP1NB1 depletion. Chromatin immunoprecipitation assays showed that knocking down RP1NB1 expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPD gene promoter. Luciferase assays demonstrated that knocking down RP1NB1 decreased DEPD gene promoter activity. Depletion of RP1NB1 or DEPD with two independent siRNAs or shRNAs significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking down RP1NB1 with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNAs. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNA led to tumor eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1NB1 gene expression correlated with DEPD gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1NB1 as an important regulator of N-Myc protein stability and neuroblastoma tumorigenesis. Citation Format: Andrew E. Tee, Pei Y. Liu, Giorgio Milazzo, Kate M. Hannan, Jesper Maag, Nenad Bartonicek, Renhua Song, Chen C. Jiang, Xu D. Zhang, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Jinyan Li, Jo Vandesompele, John S. Mattick, Pieter Mestdagh, Giovanni Perini, Ross D. Hannan, Marcel E. Dinger, Tao Liu. Eradication of neuroblastoma by suppressing the expression of a single noncoding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2453.

Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.

Background In X-linked dilated cardiomyopathy due to dystrophin mutations which abolish the expression of the M isoform (5'-XLDC), the skeletal muscle is spared through the up-regulation of the Brain (B) isoform, a compensatory mechanism that does not appear to occur in the heart of affected individuals. Methods We quantitatively studied the expression topography of both B and M isoforms in various human heart regions through in-situ RNA hybridization, Reverse-Transcriptase and Real-Time PCR experiments. We also investigated the methylation profile of the B promoter region in the heart and quantified the B isoform up regulation in the skeletal muscle of two 5'-XLDC patients. Results Unlike the M isoform, consistently detectable in all the heart regions, the B isoform was selectively expressed in atrial cardiomyocytes, but absent in ventricles and in conduction system structures. Although the level of B isoform messenger in the skeletal muscle of 5'-XLDC patients was lower that of the M messenger present in control muscle, it seems sufficient to avoid an overt muscle pathology. This result is consistent with the protein level in XLDC patients muscles we previously quantified. Methylation studies revealed that the B promoter shows an overall low level of methylation at the CG dinucleotides in both atria and ventricles, suggesting a methylation-independent regulation of the B promoter activity. Conclusions The ventricular dilatation seen in 5'-XLDC patients appears to be functionally related to loss of the M isoform, the only isoform transcribed in human ventricles; in contrast, the B isoform is well expressed in heart but confined to the atria. Since the B isoform can functionally replace the M isoform in the skeletal muscle, its expression in the heart could potentially exert the same rescue function. Methylation status does not seem to play a role in the differential B promoter activity in atria and ventricles, which may be governed by other regulatory mechanisms. If these mechanisms could be deduced, de-silencing of the B isoform may represent a therapeutic strategy in 5'-XLDC patients.

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.

Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. ©2011 AACR.

THC2, an autosomal-dominant thrombocytopenia described so far in only two families, has been ascribed to mutations in MASTL or ACBD5. Here, we show that ANKRD26, another gene within the THC2 locus, and neither MASTL nor ACBD5, is mutated in eight unrelated families. ANKRD26 was also found to be mutated in the family previously reported to have an ACBD5 mutation. We identified six different ANKRD26 mutations, which were clustered in a highly conserved 19 bp sequence located in the 5' untranslated region. Mutations were not detected in 500 controls and are absent from the 1000 Genomes database. Available data from an animal model and Dr. Watson's genome give evidence against haploinsufficiency as the pathogenetic mechanism for ANKRD26-mediated thrombocytopenia. The luciferase reporter assay suggests that these 5' UTR mutations might enhance ANKRD26 expression. ANKRD26 is the ancestor of a family of primate-specific genes termed POTE, which have been recently identified as a family of proapoptotic proteins. Dysregulation of apoptosis might therefore be the pathogenetic mechanism, as demonstrated for another thrombocytopenia, THC4. Further investigation is needed to provide evidence supporting this hypothesis.

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.

MYCN amplification occurs in around 25% of neuroblastomas, and is associated with rapid tumor progression and poor prognosis. MYCN plays a paradoxical role in driving cellular proliferation and inducing apoptosis. We previously observed nuclear p53 accumulation in neuroblastoma and hypothesize that MYCN regulates p53 in neuroblastoma. Immunohistochemical analysis of 82 neuroblastoma tumors demonstrated an association between high p53 expression and MYCN expression and MYCN amplification. In a panel of 5 MYCN amplified and 5 non-amplified neuroblastoma cell lines and also the Tet21N regulatable MYCN expression system there was a correlation between p53 expression and MYCN expression. Knockdown of MYCN in 2 MYCN amplified cell lines led to a decrease in p53 expression. Tet21N MYCN+ cells expressed higher p53 mRNA and protein, and had greater p53 transcriptional activity, in comparison with Tet21N MYCN− cells. Using chromatin immunoprecipitation and reporter gene assays, MYCN was found to bind directly to an E-Box motif located close to the transcriptional start site within the p53 promoter and initiate transcription. Mutation of the E-Box led to a decrease in MYCN driven transcriptional activity. Microarray analysis of Tet21N MYCN+/− cells showed that several p53 regulated genes were upregulated in the presence of MYCN, including MDM2 and PUMA. Knockdown of MYCN and p53 in a MYCN amplified cell line led to reduced PUMA levels and other markers of apoptosis. We conclude that MYCN transcriptionally upregulates p53 expression in neuroblastoma and may be an important mechanism by which MYCN induces apoptosis.

Increased expression of specific ATP-binding cassette (ABC) transporters is known to mediate the efflux of chemotherapeutic agents from cancer cells. Therefore, establishing how ABC transporter genes are controlled at their transcription level may help provide insight into the role of these multifaceted transporters in the malignant phenotype. We have investigated ABC transporter gene expression in a large neuroblastoma data set of 251 tumor samples. Clustering analysis demonstrated a strong association between differential ABC gene expression patterns in tumor samples and amplification of the MYCN oncogene, suggesting a correlation with MYCN function. Using expression profiling and chromatin immunoprecipitation studies, we show that MYCN oncoprotein coordinately regulates transcription of specific ABC transporter genes, by acting as either an activator or a repressor. Finally, we extend these notions to c-MYC showing that it can also regulate the same set of ABC transporter genes in other tumor cells through similar dynamics. Overall our findings provide insight into MYC-driven molecular mechanisms that contribute to coordinate transcriptional regulation of a large set of ABC transporter genes, thus affecting global drug efflux.

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.

In childhood neuroblastoma, amplification of the transcription factor MycN is associated with poor outcome and a multidrug resistant phenotype. We have previously shown that MycN regulates the expression of several ATP-binding cassette (ABC) transporter genes, and that these genes represent powerful prognostic markers in neuroblastoma (1,2,3). Although these transporters are able to efflux a wide range of cytotoxic drugs, they do not confer resistance to several important cytotoxic agents used to treat neuroblastoma, including cisplatin and cyclophosphamide, hence 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, and 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. Multivariate analyses, including age at diagnosis, tumor stage and MYCN amplification status, were conducted on the two larger cohorts and demonstrated independent prognostic significance of GSTP1 expression levels. We also explored the regulation of GSTP1 by MycN using chromatin immunoprecipitation (ChIP) and luciferase reporter assays in neuroblastoma cell lines and found that GSTP1 is a direct transcriptional target of MycN. We conclude that N-Myc regulates multiple components of drug metabolism and efflux pathways, including GSTP1 and transporters of the ABC superfamily. The regulation of these key multidrug resistance mechanisms may have particular significance for malignancies with amplification of Myc family genes. 1) J Biol Chem, 285:19532-19543, 2010 2) J Natl Cancer Institute, 103:1236-51, 2011 4) Nat Rev Cancer, 10:147-156, 2010 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 789. doi:1538-7445.AM2012-789

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.

Myc oncoproteins induce cancer cell proliferation by transcriptional modulation. Histone deacetylases (HDACs) enhance cancer cell proliferation and survival, in part, by effects on tumour suppressor gene transcription, and thus HDAC inhibitors are among the most promising new classes of anticancer drugs. Here we show that N-Myc and c-Myc up-regulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Affymetrix microarray revealed that a subset of genes, including cyclin G2 (CCNG2), was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells. PCR analysis confirmed that CCNG2 gene transcription 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. BrdU incorporation assays demonstrated that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc and HDAC2-induced cell proliferation. Dual cross-linking chromatin immunoprecipitation and protein co-immunoprecipitation assays showed that N-Myc acted as a transrepressor, by recruiting the HDAC2 protein to an Sp1-binding site at the CCNG2 core promoter, in a manner distinct from its action as a transactivator. Moreover, HDAC2 was up-regulated, and CCNG2 down-regulated, in pre-cancerous and neuroblastoma tissues from MYCN (N-Myc) transgenic mice, and c-Myc over-expression correlated with up-regulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of up-regulation 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4869.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated “CpG island” at the 5′-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.

Abstract 3252 Poster Board III-1 A better understanding of the mechanisms which regulate drug resistance is critical for improving therapy for patients at risk of poor response. Even for CML patients the long-term benefits of the treatment are limited by the emergence of resistance, although the therapy has been dramatically improved by Imatinib Mesylate, which inhibits Bcr-Abl activity. One of the most important mechanisms of resistance is the disregulation of various members of the highly conserved family of transmembrane proteins characterized by an ATP-binding cassette domain, called ABC superfamily of transporters (Dean et al., Genome Res 2001). In CML cells an aberrant expression of ABC transporter genes has been described to be mediated by specific transcription factors, which are in turn affected by an aberrant activity of Bcr-Abl. It has been shown that Bcr-Abl can indirectly activate c-Myc via the JAK2 pathway, which increases a translation of c-Myc mRNA. In addition, in a subgroup of CML patients a chromosome 8 trysomy, has been observed, which can be associated with c-Myc amplification. In this study we have investigated whether c-Myc can regulate transcription of ABC tranporter genes in CML. Initially, ABC transporter gene expression has been monitored in HL60, a human promyelocytic cell line in which c-MYC is overexpressed. We have examined the expression level of all 48 human ABCs transporters as a function of c-MYC silencing. Our results have demonstrated that c-Myc regulates the transcription of several ABC genes, such as ABCA2, ABCB9, ABCB10, ABCC1, ABCC4, ABCE1, ABCF1, ABCF2, a majority of which has been found implicated in drug resistance. Furthermore, by performing chromatin immunoprecipitation (ChIP) we have shown a direct binding of c-Myc to the promoters of those ABC transporter genes in HL60 cell line. In addition, by ChIP we have demonstrated that c-MYC is physically bound to the promoter of tested ABC genes in CML cell lines KG-1a and K562. Based on those findings we have investigated the expression level of c-Myc in CD34+ progenitor cells derived from CML patients. Also, we have evaluated the effects of highly expressed c-Myc on ABC transporters. Our results have shown in a group of 20 newly diagnosed chronic phase (CP)-CML patients an increased expression of c-Myc in CD34+ cell fraction, when compared to the expression level of c-Myc in the entire population of mononuclear cells from which CD34+ cell fraction has been purified. In those cells we have identified in association with an increased level of c-Myc an increased expression of the same subset of ABC genes, which we have observed in cell lines. Furthermore, we have evaluated whether a differential expression of c-Myc and ABC transporter genes is associated with low Sokal risk and high Sokal risk patients. This analysis has been performed on CD34+ cells purified from 19 CML patients of whom 12 were scored as low Sokal risk and 7 as high Sokal risk. Our results have shown that expression of c-Myc and ABCC4 is different in those two patients population (p Disclosures: No relevant conflicts of interest to declare.

Abstract 854 It has been proposed that most protein-encoding genes may be regulated by small multifunctional RNAs which can control transcript turnover and/or protein translation (siRNA and miRNA). Specifically, miRNAs have been shown to act predominantly at the level of translation by blocking the access or sliding of ribosomes to mRNAs (3'UTR). Several studies have shown that multiple miRNAs can be disregulated in tumours as compared to normal tissues. For example, the miR-17-92 cluster is up regulated in CML, suggesting their involvement in leukemiagenesis (Venturini et al., Blood 2007). However, a role of miRNA in preventing tumor development and progression has also been suggested. Bueno et al have shown that hsa-miR-203 can specifically target BCR-ABL and reduce its expression in CML derived cell lines (Bueno et al., Cancer Cell 2008). Since multiple miRNAs seem to act in a combinatorial fashion to regulate mRNA translation, we hypothesised that other miRNAs in addition to hsa-miR-203 might be involved in BCR-ABL expression control. To test this hypothesis we conducted a search for miRNAs specifically targeting Bcr-Abl, using the miRBASE program to scan human genome (http://microrna.sanger.ac.uk/). This search identified 15 miRNAs potentially able to target the BCR-ABL 3' UTR. Further investigation showed that only hsa-miR-451, hsa-miR-515-3p and hsa-miR-760 had a sufficiently high score to predict genuine interactions with the 3'UTR of BCR-ABL and therefore only these miRNAs were utilized for further analyses. Initially, the three miRNAs were transfected in K562 Ph+ cells, together with hsa-miR-203 as a positive control and a scrambled miRNA used as a negative control. BCR-ABL expression was monitored at both mRNA ( qRT-PCR) and protein level (Western Blotting). Our results demonstrated that BCR-ABL mRNA expression was unaffected by miRNAs, whereas protein expression was significantly reduced. Considering the combinatorial function of miRNAs on their target mRNAs, we also tested whether a pool of the four identified miRNAs could be effective in suppressing BCR-ABL expression. In those experiments, the molar concentration of each miRNA was a quarter of that used for a single miRNA transfection. The results have shown that the pool of miRNAs worked efficiently in suppressing Bcr-Abl expression, which suggested a cooperative function of the four miRNAs in controlling both the expression and translation of Bcr-Abl. To further confirm that miRNAs directly targeted BCR-ABL, the 3'UTR of the gene was cloned downstream of a reporter renilla luciferase gene. The reporter was co-transfected in HEK293 cells together with single miRNAs or a pool of them, and luciferase activity was quantified. Those results show that the presence of each miRNAs significantly reduced the luciferase activity as compared to that obtained by transfecting cells with a scrambled miRNA. These experiments therefore confirmed a direct effect of the four miRNAs on the 3'UTR of BCR-ABL. Again the “pool” of miRNAs showed the strongest effect on the luc reporter expression. Through an additional deletion analysis we mapped the regions of 3'UTR of BCR-ABL targeted by the four miRNAs, in order to confirm that the predicted binding regions of miRNAs are critical to mediate repression of BCR-ABL. In conclusion, our study identified three new human miRNAs having a potential to specifically target BCR-ABL and suppress its translation and expression in CML cells. BCR-ABL, which plays a critical role in CML, is effectively suppressed by TK inhibitors, exemplified by Gleevec and our findings provide a rationale to exploit miRNA as an alternative therapeutic approache which could further improve CML treatment, or to complement TK inhibitors in an effort to eradicate minimal residual disease. Supported by: Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures: No relevant conflicts of interest to declare.

We describe the purification and cloning of human DNA replicated at the onset of S phase in HL60 cells synchronized with aphidicolin. A survey of the overall structural properties of these sequences did not show any distinctive features except for an enrichment in Cot0 DNA. The two longer fragments were completely sequenced and studied in more detail. Both were shown to contain transcriptional signals associated with promoters and/or enhancers, such as the binding sites of Sp1, T antigen and nuclear factor III. In one instance, a binding site for a known cellular transcription factor (USF/MLTF) was located inside the sequence by footprinting. Accordingly, by CAT assay and Northern blot, the same sequence was shown to contain an active promoter. The significance of these findings with respect to the role of transcription in initiation of DNA replication at the origin is discussed. None of the tested fragments exhibited autonomously replicating sequence (ARS) activity in transfected cells. The problems connected with the detection of ARS activity in human cells are critically examined.