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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.

CENP-C is a fundamental component of the inner kinetochore plate and contributes to the formation of functional centromeres in eukaryotic organisms. Recruitment of CENP-C to kinetochore requires other centromere proteins, particularly CENP-A, CENP-H, and CENP-I. However, how CENP-C is correctly localized at the kinetochore is not clearly determined, mainly due to the functional variety of its domains, which hints at a complex recruitment mechanism. Here, by both immunofluorescent labeling and chromatin/immunoprecipitation we could show that human CENP-C contains two distinct domains, one in the central region, between amino acids 426 and 537, and the second one in the carboxyl terminal region, between amino acids 638 and 943, which are both capable of localizing at centromeres and binding α-satellite DNA. The presence of two domains that iterate the same function despite being significantly different in their amino acid sequence and structure suggests that CENP-C may target the centromere by establishing multiple contacts with both the DNA and protein constituents of the kinetochore. © 2002 Elsevier Science (USA). All rights reserved.

CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro,the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo;and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.

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.

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph(+) acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph(+) ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph(+) ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph(+) ALL patients. (Blood. 2008; 112:3847-3855)

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.

Background Imatinib has so far been the first-choice treatment in chronic myeloid leukemia with excellent results. However, only a proportion of patients achieve major molecular response – hence the need to find biological predictors of outcome to select the optimal therapeutic strategy now that more potent inhibitors are available. Design and methods We investigated a panel of 20 polymorphisms in 7 genes, potentially associated with the pharmacogenetics of imatinib, in a subset of 189 newly diagnosed chronic myeloid leukemia patients enrolled in the TOPS trial. Included in this analysis were polymorphisms in the transporters hOCT1, MDR1, ABCG2, OCTN1, and OATP1A2, and in the metabolizing genes CYP3A4 and CYP3A5. Results In the overall population, the OCTN1 C allele (rs1050152), a simple combination of polymorphisms in the hOCT1 gene and in the genes involved in imatinib uptake were significantly associated with major molecular response. The combination of polymorphisms in imatinib uptake was also significantly associated with complete molecular response. Analyses restricted to Caucasians highlighted the significant association of MDR1 CC (rs60023214) genotype with complete molecular response. Conclusions We demonstrate the usefulness of a pharmacogenetic approach for stratification of chronic myeloid leukemia patients in terms of likelihood to achieve major or complete molecular response on imatinib. This represents an attractive opportunity for therapy optimization, worth testing in clinical trials.

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4), which plays important roles in cancer progression, has been reported to be a direct transcriptional target of MYC. In this study, we have shown that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome and furthermore that siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells impaired migration and colony formation, and led to an increased proportion of cells in G1/S phase of the cell cycle. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that TFAP4 expression is positively regulated by MYCN through direct promoter binding. In addition, when MYCN was overexpressed in neuroblastoma cells, TFAP4 was required for the observed increase in cell migration. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study unveils a complex regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying relevant therapeutic targets for aggressive forms of this disease. Citation Format: Chengyuan Xue, Denise M. Yu, Samuele Gherardi, Jessica Koach, Giorgio Milazzo, Laura Gamble, Bing Liu, Amanda Russell, Tao Liu, Belamy B. Cheung, Glenn M. Marshall, Giovanni Perini, Michelle Haber, Murray D. Norris. MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2450.

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.

Research over the past decade has confirmed that epigenetic alterations act in concert with genetic lesions to deregulate gene expression in acute myeloid leukemia and myelodysplastic syndromes. Epigenetic alterations may serve as markers of disease, and may potentially be used for classification, prognostication and to monitor minimal residual disease. In addition, we now have the capability to pharmaceutically target epigenetic modifications, and there is an urgent need for early validation of the efficacy of the drugs. Also, an improved understanding of the functionality of epigenetic modifications may further pave the road towards individualized therapy. The recent advances in biotechnology and bioinformatics provide a plethora of novel tools for characterizing the epigenome in clinical samples, but at this point the practical, clinical utility of these methodologies needs further exploration. Here, we provide the pros and cons of the currently most feasible methods used for characterizing the methylome in clinical samples, and give a brief introduction to novel approaches to sequencing that may revolutionize our abilities to characterize the genomes and epigenomes in acute myeloid leukemia and myelodysplastic syndrome patients.

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.

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

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.

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.

Abstract 2574 Recently, using genome-wide single nucleotide polymorphism arrays and gene candidate deep exon sequencing, we identified lesions in CDKN2A gene, encoding p16/INK4A and p14/ARF tumor suppressors, in 27% (32/117) adult newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients and in 47% (14/30) relapsed cases. Clinically, in our cohort CDKN2A deletions were associated by univariate analysis to a worse outcome in terms of overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) (OS: 27.7 vs 38.2 months, p = 0.0206; DFS: 10.1 vs. 56.1 months, p = 0.0010; CIR: 73.3 vs 38.1, p = 0.0014). Noteworthy, the negative prognostic impact of CDKN2A deletion on DFS was also confirmed by the multivariate analysis (p = 0.0051). These results showed that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation and tumor cell growth by the action of MDM2, a negative regulator of p53, we investigated the preclinical activity of the MDM2 antagonist RG7112 in primary B-ALL patient samples and leukemic cell line models. BV-173, SUPB-15 and K562 Ph+ cell lines were incubated with increasing concentration of RG7112 (0.5–10 μM) and its inactive enantiomer for 24, 48 and 72 hours (hrs). MDM2 inhibition by RG7112 resulted in a dose and time-dependent cytotoxicity with IC50 (24 hrs) of 2 μM for BV-173 and SUPB-15 which harbor homozygous deletion of CDKN2A but wild-type p53. No significant changes in cell viability were observed in K562 p53-null cell line after incubation with RG7112. 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/Propidium Iodide analysis showed a significant increase in apoptosis after 24 hrs in BV-173, SUPB-15 and in primary leukemia blasts, whereas no apoptosis was observed in K562 cells. To examine the possible mechanisms underlying RG7112-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 upon treatment with RG7112 after 24 hrs of incubation with concentrations equal to the IC50. These data demonstrate the ability of RG7112 to activate the intrinsic apoptotic pathway by a p53-dependent mechanism. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was next performed, comparing sensitive cell lines (BV-173 and SUPB-15) after 24 hrs exposure to 2 μM RG7112 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). They include genes involved in cell cycle and apoptosis control (e.g. Histone H1, TOP2, GAS41, H2AFZ) and in the down-regulation of the Hedgehog signaling (e.g. BMI1, BMP7, CDKN1C, POU3F1, CTNNB1, PTCH2) with a strong repression of stemness genes and re-activation of INK4/ARF as illustrated in Figure 1. Actually, both GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. In our data they were strongly down-regulated (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, suggesting that these genes have a potential as new biomarkers of activity. In conclusion, inhibition of the p53–MDM2 interaction by RG7112 can activate the p53 pathway, resulting in apoptosis and inhibition of stemness genes in B-ALL with wild-type p53. Our findings provide a strong rational for further clinical investigation of RG7112 in Ph+ ALL. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione–Università 2007–2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

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.

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.

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.

Nitric oxide (NO) exerts its function in several cell and organ compartments. Recently, several lines of evidence have been accrued showing that NO can play a critical role in oncogenesis. Here we summarize some of these findings and highlight the role of NO as a possible target for antineoplastic drugs. Specifically, NO appears to affect some aspects of neuronal tumour progression, particularly the chemoresistance phenotype, through inhibition of MYC activity and expression of a large set of ATP binding cassette transporters. Here we provide lines of evidence supporting the view that MYCN can alter expression of several members of the ABC transporter family thus influencing the chemoresistance phenotype of neuroblastoma cells. Furthermore, we show that increased intracellular NO concentration either through addition of NO donors to culture medium or through forced expression of nNOS in neuroblastoma cells leads to decreased expression of MYCN and ABC drug transporter genes. Overall, data reviewed here and novel results presented, unveil a NO-MYCN-ABC transporters axis with important implication on development and control of the chemoresistance phenotype in neuronal tumours.

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.

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.

Dyskerin is a nucleolar RNA-binding protein that functions as a core component of the telomerase holoenzyme, as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. This study investigates the clinical and functional relevance of dyskerin expression in the paediatric cancer neuroblastoma. Here it is shown, in two independent neuroblastoma patient cohorts, that high expression of the gene encoding dyskerin (DKC1) very strongly correlates with poor event-free and overall survival. The prognostic power of DKC1 expression was independent of the established indicators used in the management of this disease, namely diagnosis age > 1.5 yr, stage of disease and amplification of the MYCN oncogene (P Citation Format: Rosemary O'Brien, Michelle F. Maritz, Cheng Fei Kong, Stefania Purgato, Bing Liu, Chen Yang, Amanda Russell, Jayne Murray, Claudia Flemming, Michelle Haber, Giovanni Perini, Murray D. Norris, Jamie I. Fletcher, Karen L. Mackenzie. High DKC1 expression supports neuroblastoma tumor cell proliferation and is strongly associated with poor patient outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2015-488

N-Myc is a transcription factor that forms heterodimers with the protein Max and binds gene promoters by recognizing a DNA sequence, CACGTG, called E-box. The identification of N-myc target genes is an important step for understanding N-Myc biological functions in both physiological and pathological contexts. In this study, we describe the identification of N-Myc-responsive genes through chromatin immunoprecipitation and methylation-sensitive restriction analysis. Results show that N-Myc is a direct regulator of several identified genes, and that methylation of the CpG dinucleotide within the E-box prevents the access of N-Myc to gene promoters in vivo . Furthermore, methylation profile of the E-box within the promoters of EGFR and CASP8 , two genes directly controlled by Myc, is cell type-specific, suggesting that differential E-box methylation may contribute to generating unique patterns of Myc-dependent transcription. This study illuminates a central role of DNA methylation in controlling N-Myc occupancy at gene promoters and modulating its transcriptional activity in cancer cells.

The neurodegenerative changes in Alzheimer's disease (AD) are elicited by the accumulation of beta-amyloid peptides (Abeta), which damage neurons either directly by interacting with components of the cell surface to trigger cell death signaling or indirectly by activating astrocytes and microglia to produce inflammatory mediators. It has been recently proposed that the p75 neurotrophin receptor (p75(NTR)) is responsible for neuronal damage by interacting with Abeta. By using neuroblastoma cell clones lacking the expression of all neurotrophin receptors or engineered to express full-length or various truncated forms of p75(NTR), we could show that p75(NTR) is involved in the direct signaling of cell death by Abeta via the function of its death domain. This signaling leads to the activation of caspases-8 and -3, the production of reactive oxygen intermediates and the induction of an oxidative stress. We also found that the direct and indirect (inflammatory) mechanisms of neuronal damage by Abeta could act synergistically. In fact, TNF-alpha and IL-1beta, cytokines produced by Abeta-activated microglia, could potentiate the neurotoxic action of Abeta mediated by p75(NTR) signaling. Together, our results indicate that neurons expressing p75(NTR), mostly if expressing also proinflammatory cytokine receptors, might be preferential targets of the cytotoxic action of Abeta in AD.

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

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.

Background: Polyamines are highly regulated essential cations that are elevated in rapidly proliferating tissues. We have previously shown that the MYC/MYCN target ornithine decarboxylase (ODC1), rate-limiting for polyamine biosynthesis, is a therapeutic target for neuroblastoma (Hogarty et al, Cancer Res 2008; 68: 9735). We have also shown that targeting multiple steps in the polyamine pathway enhances therapeutic efficacy in complementary pre-clinical models (Haber et al, Adv Neurobl Res, 2012, OR 88). An international Phase I trial for refractory neuroblastoma, coordinated by the New Approaches to Neuroblastoma Treatment (NANT) consortium USA, will open shortly using ODC1 inhibition by high-dose difluoromethylornithine and SAT1 induction by celecoxib. We have now examined the prognostic impact of all polyamine pathway genes in a large cohort of neuroblastoma tumors. Methods: Gene-expression profiles of 650 primary untreated neuroblastomas were analyzed for all polyamine pathway genes, and related to clinical outcome. Gene expression was evaluated by Real-Time PCR (RT-PCR) and correlated with MYCN levels in neuroblastoma cell lines. Activation and repression of polyamine genes by MYCN were analyzed by chromatin immunoprecipitation (ChIP). Polyamine gene promoters were cloned into luciferase reporter vectors and tested as a function of MYCN expression. Results: Expression of all polyamine pathway genes was highly prognostic of neuroblastoma outcome. High levels of each synthetic gene and low levels of each catabolic gene predicted poor outcome. Multivariate analysis showed 6/11 genes retained independent prognostic significance following adjustment for MYCN, age and stage. In neuroblastoma cell lines, in the presence or absence of MYCN expression, RT-PCR analysis demonstrated a correlation of high MYCN with high biosynthetic polyamine gene expression, but an inverse correlation of high MYCN with low catabolic polyamine gene expression. ChIP assays confirmed that MYCN associates with biosynthetic gene promoters by binding to E-box sites in order to activate transcription. In contrast, MYCN binds the promoters of the catabolic genes in proximity to the transcription start sites by interacting with the Sp1 protein in order to repress transcription of these genes. Direct activation by MYCN of biosynthetic genes and down-regulation by MYCN of catabolic genes was confirmed by luciferase activity assays. Conclusions: These results provide a remarkable demonstration of an oncogene coordinately regulating the expression of every gene in a metabolic pathway in order to drive cell proliferation. The findings highlight the importance of polyamines in neuroblastoma and identify target enzymes in addition to ODC1 for potential therapeutic intervention. Citation Format: Murray D. Norris, Laura D. Gamble, Jayne Murray, Stefania Purgato, Giorgio Milazzo, Lesley J. Ashton, Andrei Oberthuer, Wendy B. London, Mike D. Hogarty, Mattias Fischer, David Ziegler, Glenn M. Marshall, Giovanni Perini, Michelle Haber. MYCN coordinately regulates the entire polyamine gene pathway to drive polyamine biosynthesis in neuroblastoma, with expression of every synthetic and catabolic polyamine pathway gene being highly prognostic of clinical outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3808. doi:10.1158/1538-7445.AM2013-3808

HUMAN T-cell leukaemia virus type I (HTLV-I) Tax protein increases the DNA binding of many cellular transcription factors that contain a basic regiona¤-leucine zipper (bZIP) DNA-binding domain1a¤-3. bZIP domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact. How Tax recognizes diverse bZIPs is not understood. Here we show that no specific sequence of the leucine zipper is required for a Tax response. In contrast, the basic region is essential for the Tax-mediated DNA-binding increase, which can be eliminated by single substitutions of several conserved amino acids. Surprisingly, Tax alters the relative affinity of a bZIP for different DNA binding sites. Thus, through recognition of the conserved basic region, Tax increases DNA binding and modifies DNA site selection. Tax provides a model for how a single auxiliary factor can regulate multiple sequence-specific DNA-binding proteins.

Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. Principal Findings: Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. Conclusions: Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.

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.

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting low-level to 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 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2011-3027

We have previously shown that high levels of the multidrug transporters ABCC1/MRP1 (1,2) and ABCC4/MRP4 (3) are strongly predictive of poor outcome in the childhood cancer, neuroblastoma. Although the prognostic significance of ABCC1 may be explained in terms of cytotoxic drug resistance, none of the drugs used to treat children in these studies were ABCC4 substrates. This suggests that multidrug transporters can contribute to the malignant phenotype, independent of cytotoxic drug efflux, as we have recently outlined (4). To address this hypothesis, a MYCN-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain or alternatively, treated with an ABCC1 inhibitor. Pharmacological inhibition or genetic depletion of ABCC1 significantly inhibited neuroblastoma development in MYCN transgenic mice, while knockdown of ABCC1 using siRNA reduced cell motility and clonogenicity in cultured neuroblastoma cells, and induced morphological differentiation. Analysis of a large neuroblastoma cohort of 209 primary untreated tumors revealed that amongst the 12 members of the ABCC gene family, expression of only ABCC1, ABCC4 and also ABCC3 were predictive of neuroblastoma outcome. We confirmed a highly significant association between high levels of either ABCC1 or ABCC4 and poor outcome (p 1) New Engl J Med 334:231-238, 1996 2) J Clin Oncol 24:1546-53, 2006 3) Mol Cancer Ther 4:547-53, 2005 4) Nat Rev Cancer, 10:147-156, 2010 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2011-954

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.

Abstract 670 The availability of multiple options for chronic myeloid leukemia (CML) treatment is not paralleled by the availability of biological predictors of outcome allowing to identify patients (pts) who are more likely to benefit from dasatinib or nilotinib rather than imatinib (IM). Pharmacogenetics has proven a potential source of biomarkers given the known influence of polymorphisms in key genes encoding drug transporters and metabolizing enzymes on drug delivery – hence effectiveness. In CML, only two studies had so far explored this field, but both were conducted in heterogeneous populations including pts at different stages of disease, not all receiving IM first-line. We thus aimed to investigate a panel of 20 single nucleotide polymorphisms (SNPs) in ABCB1, ABCG2, SLC22A1, OATP1A2, OCTN1, CYP3A4 and CYP3A5 genes that can be hypothesized to influence IM transport and metabolism in 189 newly diagnosed CML pts enrolled in the TOPS phase III trial (Cortes et al, J Clin Oncol 2010). Pts selection was exclusively based on availability of written informed consent and sufficient amount of archived material. Median age was 46 years; male to female ratio was 103 to 86; 156 (83%) pts were Caucasian and 23 (12%) were Asian; low, intermediate and high Sokal risk pts were 84 (44.4%), 65 (34.4%) and 40 (21.2%), respectively. Baseline demographic/clinical features did not differ significantly from those of the overall population. Treatment outcomes (complete cytogenetic response [CCyR]; major molecular response [MMR] and complete molecular response [CMR]) were compared according to i) each candidate genotype ii) summary measures based on combinations of SNPs in the same gene and iii) summary measures based on combinations of SNPs in functionally related genes (uptake; efflux). CC genotype in OCTN1 had a favorable impact on the achievement of MMR at 12 months (MMR@12m; P = 0.03). With respect to the summary measures, combination of SNPs in the SLC22A1 gene was significantly correlated with MMR@12m (P = 0.03). When considering summary measures of uptake and efflux, the former was found to be associated with both MMR@12m and CMR@12m (P = 0.003 and P = 0.01, respectively). A separate analysis limited to Caucasian pts (n=156) yielded similar results (Table 1). In addition, the analysis in the Caucasian subgroup evidenced a significant association between the CC genotype in ABCB1 rs60023214 and MMR@12m (P = 0.005) (Table 1). Cumulative incidence plots based on the Kaplan-Meier method were also analyzed in the overall population and in Caucasians, with comparable results. Representative plots are shown in Figure 1. There was evidence for difference among MMR cumulative incidence curves for 2 single SNPs and 2 score measures. Presence of the major allele in OCTN1 (CC) and of the minor allele in CYP3A4 rs2740574 (GG) were associated with increased MMR rate (P = 0.028 and P = 0.042, respectively, in the overall population and P = 0.027 and P = 0.038, respectively, in Caucasians). Similarly, an increase in the number of favorable alleles in the SLC22A1 gene was associated with increased MMR rate (P = 0.030 and P = 0.043 in the overall population and in Caucasians, respectively). In addition, the combination of favorable alleles in the genes involved in IM uptake was associated with increased rates of both MMR and CMR (P = 0.004 and P = 0.015, respectively, in the overall population and P = 0.005 and P = 0.009, respectively, in Caucasians). Our results suggest that SNP genotyping might be helpful in selecting pts who are more likely to benefit from first-line use of more potent inhibitors. Further assessment of the SNPs here identified in larger series of pts is warranted. Table 1 . Summary of significant associations Overall population (n=189) Caucasians (n=156) Gene, rs Outcome P Hazard ratio[95% CI] P Hazard ratio[95% CI] OCTN1 - rs1050152 MMR@12m 0.03 0.78 (0.63, 0.98) 0.03 0.77 (0.60, 0.97) ABCB1 (MDR1) - rs60023214 CMR@12m 0.06 0.71 (0.49, 1.02) 0.005 0.56 (0.37, 0.84) SLC22A1 (hOCT1) - SNP combination MMR@12m 0.03 1.24 (1.02, 1.50) 0.04 1.24 (1.00, 1.54) Uptake - SNP combination MMR@12m 0.003 1.21 (1.06, 1.37) 0.005 1.24 (1.06, 1.44) Uptake - SNP combination CMR@12m 0.01 1.30 (1.05, 1.61) 0.01 1.37 (1.08, 1.74) Download : Download high-res image (197KB) Download : Download full-size image Figure 1: . Cumulative incidence of pts achieving MMR and CMR according to the # of major alleles in uptake-related gene (SLC22A1, OATP1A2, OCTN1) SNPs Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures: Hughes: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria. White: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Saglio: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Hatfield: Novartis: Employment. Martinelli: Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

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 3283 Poster Board III-1 IM is the first-choice treatment in CML and allows to obtain excellent response rates. Some pts, however, experience suboptimal response or resistance, which highlights the need to find biological predictors of outcome in order to guide therapy optimization. Since blood and tissue concentrations of drugs may be influenced by variations between individuals (single nucleotide polymorphisms, SNPs) in genes encoding drug metabolizing enzymes and drug transporters, we have investigated if SNPs influencing the delivery if IM to target cells may account for differences in response. To test that hypothesis, we have genotyped a panel of SNPs known to affect the activity of the cytochrome p450 family isoforms and of the transporters that have been implicated in IM metabolism and transport in a subset of pts enrolled in the TOPS trial -; a randomized phase III study of IM 400 mg/d vs IM 800 mg/d in newly diagnosed CML (Cortes et al, ASH 2008). Included in this analysis were 18 SNPs in 7 genes: hOCT1 (ins/del, rs12208357, rs683369, rs4646277, rs4646278, rs2282143); ABCB1 (rs60023214, rs1128503, rs10245483, rs3213619, rs1128501), ABCG2 (rs2231137, rs2231142); OCTN1 (rs1050152); OATP1A2 (rs11568563); CYP3A5 (rs28365083, rs776746); CYP3A4 (rs28371759). Genotype distributions were tested for Hardy-Weinberg equilibrium (HWE). Additive models were used to assess association between individual SNPs and three response outcomes: major molecular response (MMR) at 12 months, optimal CgR and optimal response as defined by European LeukemiaNet (ELN) recommendations (Baccarani, Blood 2006). Summary measures based on SNPs from the same gene and those related functionally (uptake and efflux) were defined as the number of alleles, across the genes of interest, hypothesized to be associated with favorable response. Association of individual SNPs and summary variables with response was assessed with logistic regression models and likelihood ratio and exact tests. A total of 166 pts were genotyped, out of which 133 whites from Italy (N=21), the U.S.A. (N=66) and Australia (N=46); 22 Asian from Korea (N=17), Italy (N=1), and Australia (N=4); and 11 non-white and non-Asian from the U.S.A (N=6) and Australia (N=5). Selection of pts was based exclusively on availability of a written informed consent for correlative substudies and of a sufficient amount of archived DNA material. The response rates by 12 months among the subset of pts analyzed were 51% for MMR, 87% for optimal cytogenetic response according to ELN, and 85% for optimal response according to ELN. Our analyses have identified four monomorphic, non-informative SNP ( hOCT1: rs4646278, rs2282143; MDR1: rs1128501; and CYP3A4: rs28371759). The results have shown, when analyzing the patient population as a whole, that no association could be found between individual SNPs and any of the response variables considered. However, among whites, but not other pts, we have observed a trend of association between achievement of MMR and two SNPs in the hOCT1 and CYP3A5 genes. Namely, the wild type allele for hOCT1 rs683369 was associated with a higher response rate compared to heterozygote and homozygote mutants (60% vs 36%, p=0.057). Also, the mutant allele for CYP3A5 rs28365083 was associated with a higher response rate (p=0.058). In the subset of pts from the U.S.A, there was a evidence of association between achievement of MMR and three SNPs in the hOCT1 and MDR1 genes. Those with the wild type allele for hOCT1 ins/del had a lower MMR rate compared to those carrying at least one minor allele (48% vs 81%, p=0.08). The wild type for hOCT1 rs683369 was associated with a higher response rate compared to heterozygote and homozygote mutants (65% vs 36%, p=0.05). MDR1 rs60023214 (p=0.08) and the MDR1 gene summary measure of total number of favorable alleles among all MDR1 SNPs (p=0.08) were correlated with MMR, with improved response in the wild type group. Our study suggests for the first time a role of ethnicity in determining correlations between SNP-based molecular signature and response to treatment. If confirmed by larger studies, this suggests that ethnicity should be taken in consideration to better interpret pharmacogenetic analyses in CML and could explain the differences in results of the studies published so far. Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Baccarani:Novartis: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau.

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.