MYCN Levels Research Articles

Reciprocal antagonistic regulation of N-myc mRNA by miR‑17 and the neuronal-specific RNA-binding protein HuD.

Neuroblastoma is a childhood cancer originating from embryonic neural crest cells. Amplification of the proto-oncogene N-myc, seen in ~30% of neuroblastoma tumors, is a marker for poor prognosis. Recently discovered small regulatory RNAs, microRNAs (miRNAs), are implicated in cancers, including neuroblastoma. miRNAs downregulate the expression of genes by binding to the 3-untranslated regions (3′-UTRs), thereby inhibiting translation or inducing degradation of cognate mRNAs. Our study sought to identify miRNAs that regulate N-myc expression and thereby malignancy in neuroblastoma. miRNAs whose expression negatively correlates with N-myc expression were identified from a miRNA microarray of 4 N-myc-amplified neuroblastoma cell lines. Three of these miRNAs (miR-17, miR-20a and miR-18a) belong to the miR-17-92 cluster, previously shown to be upregulated by N-myc. qPCR validation of these miRNAs in a larger panel of cell lines revealed that levels of miR-17 were inversely proportional to N-myc mRNA amounts in the N-myc-amplified cell lines. Notably, miR-17 also downregulated N-myc protein synthesis in the N-myc-amplified cells, thereby generating a negative feedback regulatory loop between the proto-oncogene and this miRNA. Moreover, the neuronal-specific RNA-binding protein HuD (ELAVL4), which regulates the processing/stability of N-myc mRNA, competes with miR-17 for a binding site in the 3′-UTR of N-myc. Thus, N-myc levels appear to be modulated by the antagonistic interactions of both miR-17, as a negative regulator, and HuD, as a positive regulator, providing further evidence of the complex cellular control mechanisms of this oncogene in N-myc-amplified neuroblastoma cells.

Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation.

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

MYCL is a target of a BET bromodomain inhibitor, JQ1, on growth suppression efficacy in small cell lung cancer cells

We aimed to elucidate the effect of JQ1, a BET inhibitor, on small cell lung cancers (SCLCs) with MYCL amplification and/or expression. Fourteen SCLC cell lines, including four with MYCL amplification, were examined for the effects of JQ1 on protein and gene expression by Western blot and mRNA microarray analyses. The sensitivity of SCLC cells to JQ1 was assessed by cell growth and apoptosis assays. MYCL was expressed in all the 14 cell lines, whereas MYC/MYCN expression was restricted mostly to cell lines with gene amplification. ASCL1, a transcription factor shown to play a role in SCLC, was also expressed in 11/14 cell lines. All SCLC cell lines were sensitive to JQ1 with GI50 values ≤1.23 μM, with six of them showing GI50 values <0.1 μM. Expression of MYCL as well as MYCN, ASCL1 and other driver oncogenes including CDK6 was reduced by JQ1 treatment, in particular in the cell lines with high expression of the respective genes; however, no association was observed between the sensitivity to JQ1 and the levels of MYCL, MYCN and ASCL1 expression. In contrast, levels of CDK6 expression and its reduction rates by JQ1 were associated with JQ1 sensitivity. Therefore, we concluded that CDK6 is a novel target of JQ1 and predictive marker for JQ1 sensitivity in SCLC cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

NCYM is upregulated by lncUSMycN and modulates N-Myc expression.

Neuroblastoma is the most common solid tumor in early childhood. Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN, MYCN antisense NCYM and lncUSMycN genes show poor prognosis. BET bromodomain inhibitors show anticancer efficacy against neuroblastoma partly by reducing MYCN gene transcription and N-Myc mRNA and protein expression. We have previously shown that the long nocoding RNA lncUSMycN upregulates N-Myc mRNA expression by binding to the RNA-binding protein NonO. In this study, we found that lncUSMycN upregulated NCYM expression, and knocking-down lncUSMycN reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the NCYM gene promoter. NCYM upregulated N-Myc mRNA expression, NCYM RNA formed a complex with NonO protein, and knocking down NCYM expression reduced neuroblastoma cell proliferation. Importantly, treatment with BET bromodomain inhibitors reduced NCYM expression. In human neuroblastoma patients, high levels of NCYM expression in tumor tissues correlated with high levels of N-Myc, NonO and lncUSMycN expression as well as poor patient prognosis. Taken together, our findings suggest that lncUSMycN upregulates NCYM expression by activating its gene transcription, and that NCYM RNA upregulates N-Myc mRNA expression by binding to NonO. Our findings also provide further evidence for the application of BET bromodomain inhibitors for the therapy of neuroblastoma characterized by MYCN/NCYM gene locus amplification.

Glycolytic Metabolism Plays a Functional Role in Regulating Human Pluripotent Stem Cell State

The rate of glycolytic metabolism changes during differentiation of human embryonic stem cells (hESCs) and reprogramming of somatic cells to pluripotency. However, the functional contribution of glycolytic metabolism to the pluripotent state is unclear. Here we show that naive hESCs exhibit increased glycolytic flux, MYC transcriptional activity, and nuclear N-MYC localization relative to primed hESCs. This status is consistent with the inner cell mass of human blastocysts, where MYC transcriptional activity is higher than in primed hESCs and nuclear N-MYC levels are elevated. Reduction ofglycolysis decreases self-renewal of naive hESCs and feeder-free primed hESCs, but not primed hESCs grown in feeder-supported conditions. Reduction of glycolysis in feeder-free primed hESCs also enhances neural specification. These findings reveal associations between glycolytic metabolism and human naive pluripotency and differences inthe metabolism of feeder-/feeder-free cultured hESCs. They may also suggest methods for regulating self-renewal and initial cell fate specification of hESCs.

Abstract 2474: DFMO targets cancer stem cells in neuroblastoma

Abstract Background: Difluoromethylornithine (DFMO) has shown promise in recent clinical trials as a therapeutic agent in treating neuroblastoma (NB). High-risk NB patients have an approximately 50% relapse rate, after which survival rate drops to lower than 10%. By targeting the cancer stem cells (CSC) we could decrease relapse rate in high-risk patients and improve long-term survival. DFMO is known to inhibit ornithine decarboxylase (ODC) impeding key polyamine biosynthesis as well as the LIN28/Let7 pathway and disrupting the glycolytic metabolism pathways both of which have been shown to be important in CSC models. We propose that DFMO may be an effective treatment against neuroblastoma CSCs. Methods: BE(2)C and SMS-KCNR NB cell lines were studied. Limiting dilution assays of xenograft studies were performed. Untreated, 10-day 5mM DFMO-pretreated, or 20-day 5mM DFMO-pretreated cells were injected into mice between 10-5000 cells/mouse and followed for tumor formation. Xenograft Be2C mice were treated with either vehicle or 2%DFMO in drinking water and harvested at 7-14 days for tumor analysis. Tumors and cell cultures of untreated and DFMO-treated cells were analyzed by RT-qPCR, Western Blot analysis, and GeneChip gene expression analysis measuring levels of ODC1, LIN28B, mirLet7, MYCN, SLC2A4 (a glucose transporter), and CSC markers such as CXCR4, POU5F1, NANOG, SOX2, and KLF4. Riboflavin induced autofluorescence (AF) was used to identify CSC. This method detected cells after a 24-hour bath in 30μM riboflavin and excitement at 488nm in a MoFlo cell sorter at the University of Michigan Flow Core, after which cells are sorted into AF- and AF+ sub-populations for imaging, neurosphere assays with or without DFMO, RT-qPCR and Western Blot analysis. Knock-down of ODC1 is achieved by siRNA transfection via Lipofectamine in cells and analyzed by RT-qPCR, Western Blot, and neurosphere assays with or without DFMO. Results: Pretreatment with DFMO in BE(2)C and SMS-KCNR cells in vitro and in vivo inhibits tumor formation in neurosphere assays and in limiting dilution xenograft models respectively. Tumors harvested from xenografts show a decrease in expression of CSC biomarkers and neurosphere formation capability in those treated with DFMO. AF+ cells showed an increase in stem cell marker expression and increase neurosphere formation relative to AF- cells, identifying them as cancer stem cells. In addition, AF+ cells are more sensitive to DFMO treatment with a greater decrease in neurosphere formation. Cells transfected with ODC siRNA or subjected to DFMO show a reduction in CSC biomarker RNA and protein and a decrease in neurospheres formation. Conclusion: These results suggest that DFMO targets the CSC subpopulation in neuroblastoma, disrupting cell growth pathways and inhibiting tumor formation. This concept is under further investigation in a Phase II clinical trial to prevent relapse in high risk neuroblastoma patients. Citation Format: Tracey Avequin, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Ebrahim Azizi, Max Wicha, Giselle L. Saulnier Sholler. DFMO targets cancer stem cells in neuroblastoma. [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 2474.

Abstract 2480: Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells

Abstract Neuroblastoma is the most common extra-cranial solid tumor in children. Despite aggressive therapy prognosis of high risk neuroblastoma remains guarded and also unpredictable because of heterogeneity of tumor response to treatment. Radio-resistance has been recognized as a significant contributor to aggressive clinical behavior of neuroblastoma. Characterization of genetic alterations have been used for predicting clinical outcome in neuroblastomas and one of the most studied genetic markers in aggressive neuroblastoma is nMyc/MYCN. The MYCN gene belongs to the MYC family of transcription factors and nMyc expression is to a large extent inversely correlated with cMyc expression and nMyc expression is also associated poor clinical outcomes. In this study, we have attempted to correlate the expression of nMyc in neuroblastoma cells NB1691 and SKNBE2 to radiation resistance. We exposed neuroblastoma cells NB1691 and SKNBE2 to chronic radiation doses with a cumulative high energy X-ray radiation dose of 25Gy achieved by dosing at 5Gy increments over a period of 15 days. Cells surviving after the 25Gy cumulative radiation dose were selected and designated as NB1691R and SKNBE2R. Expression levels of nMyc, cMyc and uPA were determined. We observed that the radiation tolerant cells showed at least 164 fold increase in the expression of uPA in NB1691R cells and at least 97 fold increase in SKNBE2R cells. We also observed that the expression levels of nMyc decreased in both NB1691R and SKNBE2R cells to almost undetectable levels. Further, the expression levels of cMyc revealed that NB1691R cells showed a 208 fold increase and SKNBE2R a 71 fold increase when compared to parental cells. We further determined whether the expression of uPA can modulate the expression of nMyc. We silenced uPA expression in NB1691 cells and observed that downregulation of uPA moderately rescued nMyc expression. Cell cycle analysis revealed that acute radiation of NB1691 cells induced accumulation of cells in the G2/M phase and un-radiated NB1691R cells continued to show cells accumulated in the G2/M phase similar to acute radiated NB1691 cells. Our study shows that: 1. Chronic radiation exposure suppresses the expression of nMyc and induces the expression of cMyc. 2. cMyc expression was accompanied with the expression of uPA and suppression of uPA expression moderately rescued the expression of nMyc. Our preliminary findings suggest that chronic exposure to radiation alters the nMyc/cMyc ratio in neuroblastoma cells and induces the expression of the pro-invasive molecule uPA. Citation Format: Manu Gnanamony, David Pinson, Reuben Antony, Karen S. Fernández, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells. [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 2480.

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

EYA1 expression and subcellular localization in neuroblastoma and its association with prognostic markers.

Neuroblastoma, the most frequently occurring extracranial solid tumor of childhood, arises from neural crest-derived cells that are arrested at an early stage of differentiation in the developing sympathetic nervous system. There is an urgent need to identify clinically relevant biomarkers for better prognosis and treatment of this aggressive malignancy. Eyes Absent 1 (EYA1) is an essential transcriptional coactivator for neuronal developmental programs during organogenesis. Whether or not EYA1 is implicated in neuroblastoma and subcellular localization of EYA1 is relevant to clinical behaviour of neuroblastoma is not known. We studied EYA1 expression and subcellular localization by immunohistochemistry in tissue microarrays containing tumor specimens from 98 patients, 66 of which were characterized by known clinical prognostic markers of neuroblastoma. Immunostaining results were evaluated and statistically correlated with the degree of histologic differentiation and with neuroblastoma risk stratification group characteristics, including stage of disease, patient age, tumor histology and mitosis–karyorrhexis index (MKI), respectively. We found that EYA1 levels were significantly higher in neuroblastomas than in ganglioneuromas and ganglioneuroblastomas. EYA1 was more highly expressed in stage 1,2,3 or 4S tumors as compared to stage 4 tumors (P<0.01). Tumors with high levels of nuclear EYA1 were more frequently associated with high nuclear MYCN levels. These results suggest that modulation of expression and intracellular localization of EYA1 in neural crest cells may provide a novel direction for therapeutic strategies.

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition

Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR.

Abstract A59: CHAF1A blocks neuroblastoma differentiation and promotes tumor growth via metabolic reprogramming

Abstract Neuroblastoma (NB), an embryonal tumor arising in tissues of the sympathetic nervous system, accounts for 13% of all deaths due to childhood malignancies. NB arises from embryonal neural crest secondary to a block in differentiation and long-term survival inversely correlates with the degree of neuronal differentiation. Importantly, treatment with differentiation agents, such as retinoic acid (RA), has modestly improved survival. We have recently demonstrated a novel function for the histone chaperone CHAF1A in promoting tumor progression by blocking NB differentiation. CHAF1A is a MYCN target gene and a subunit of the Chromatin Assembly Factor-1 (CAF1) which regulates H3K9-trimethylation and DNA methylation. Here, in order to define the mechanism for CHAF1A-mediated inhibition of differentiation, we performed transcriptome analyses (Affymetrix U133+2.0 arrays) in NB cells upon CHAF1A silencing. Notably, gene set enrichment analysis (GSEA) revealed repression of oncogenic pathways and enrichment of cell metabolism pathways (valine, leucine, and isoleucine degradation, glutamate metabolism and insulin pathways). Furthermore, Q-PCR confirmed that CHAF1A significantly affects the expression of critical genes regulating glucose uptake. Based on these findings and the fact that cancer cells have defective mitochondria, which forces them to depend on aerobic glycolysis, we then examined the sensitivity of NB cells to different glycolytic inhibitors. We hypothesized that targeting glucose metabolism will decrease NB cell growth and restore sensitivity to RA. The effect of the glycolytic inhibitors metformin, dichloroacetate (DCA), and 2-Deoxy-D- glucose (2-DG) on cell proliferation was determined by MTT-Assay in several NB lines under normoxic and hypoxic conditions (1% O2). 2-DG treatment significantly blocked cell proliferation in all NB lines tested, with enhanced effect under hypoxic conditions. However, metformin and DCA's anti-proliferative effects were dependent on MYCN status and levels of O2 deprivation. Notably, MYCN non-amplified lines showed greater sensitivity to metformin and DCA under normoxia than under hypoxic conditions (IC50 normoxia: 6.25 mM vs. IC50 hypoxia: &amp;gt; 50 mM). In contrast, MYCN-amplified cell lines maintained high proliferation under hypoxia and revealed high sensitivity to metformin and DCA both under normoxic and hypoxic conditions (IC50 normoxia: 25 mM vs. IC50 hypoxia: 22.5 mM). In conclusion, we have shown that: 1) the differentiation of NB cells upon CHAF1A silencing induces metabolic reprogramming by affecting genes controlling glucose uptake, and 2) NB cells are extremely sensitive to glycolytic inhibition, dependently on MYCN status and O2 levels. A complete characterization of the metabolic changes occurring in NB cells with high and low CHAF1A expression, as well as a definition of MYCN regulation of mitochondrial functions under hypoxic conditions is currently undergoing. This will help identify vulnerable metabolic points to block NB cell growth and guide the development of novel differentiating therapies. Citation Format: Myrthala Moreno-Smith, Zaowen Chen, Eugene S. Kim, Jason M. Shohet, Eveline Barbieri. CHAF1A blocks neuroblastoma differentiation and promotes tumor growth via metabolic reprogramming. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A59.

Abstract B165: Optimizing selective CDK7 inhibition in MYCN-driven neuroblastoma

Abstract Amplification of the MYCN gene defines approximately 50% of high-risk neuroblastomas (NB), and is associated with aggressive disease and a poor clinical outcome. We recently demonstrated that MYCN-amplified NBs are dependent on MYCN-driven global transcriptional amplification, and that they can be selectively targeted using a first-in-class covalent cyclin-dependent kinase (CDK) 7/12 inhibitor, THZ1. CDK7, CDK9 and CDK12 directly phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAPII) to regulate gene transcription. NB cells expressing high levels of MYCN were 10 times more sensitive to THZ1 than normal cells or NB cells not driven by amplified MYCN. THZ1 caused tumor growth inhibition in murine models of human NB, without general toxicity indicating the clinical potential of this therapeutic strategy. To identify mechanisms of resistance to THZ1, we generated THZ1-resistant NB cells (THZ1-R) by exposing sensitive cells to escalating sub-lethal doses of THZ1. THZ1-R cells exhibited no changes MYCN levels or RNAPII activity, eliminating alterations in CDK7 itself and/or emergence of compensatory pathways to account for the resistance. A survey of common multidrug resistance mechanisms revealed that extended THZ1 exposure exclusively enhanced the transcript levels of ATP-Binding Cassette sub-family member B1 (ABCB1, also MDR1) by 500-fold, which was translated to the protein level as well. The importance of ABCB1 in THZ1-resistance was demonstrated by rescue of THZ1 activity using the selective ABCB1 inhibitor tariquidar and shRNA-mediated gene knockdown. Subsequent screening of CDK inhibitors of the SBI-E class in these ABCB1-overexpressing NB cells afforded the identification of a covalent CDK9/12 inhibitor (SBI-E-9) devoid of efflux-substrate characteristics. Regardless of the expression levels of ABCB1, SBI-E-9 selectively suppressed MYCN expression and MYCN-associated transcriptional activity. In addition, we evaluated the in vitro efficacy of a series of covalent CDK7 inhibitors with distinctive scaffolds from THZ1. Although these compounds do not escape ABCB1-mediated drug efflux, their specific targeting of CDK7 over CDK12 correlated with greater differential selectivity in MYCN-amplified vs. MYCN non-amplified NB cells (30-fold versus 10-fold with THZ1). In conclusion, we report, for the first time, ABCB1-regulated drug efflux as a major mechanism of resistance to the first-generation covalent CDK7/12 inhibitor, THZ1, which can be overcome by a newly identified, covalent CDK9/12 inhibitor, SBI-E-9. Additionally, we have identified second generation covalent CDK7 inhibitors that display a higher selectivity against MYCN-driven NB through augmented CDK7 activity. Hence, these covalent CDK inhibitors represent attractive therapeutic options for MYCN-driven NB, and perhaps other MYC-dependent cancers. Citation Format: Yang Gao, Yanke Liang, Tinghu Zhang, Nathanael S. Gray, Rani E. George. Optimizing selective CDK7 inhibition in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B165.

CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

Previous studies have indicated the important roles of MYCN in tumorigenesis and progression of neuroblastoma (NB), the most common extracranial solid tumor derived from neural crest in childhood. However, the regulatory mechanisms of MYCN expression in NB still remain largely unknown. In this study, through mining public microarray databases and analyzing the cis-regulatory elements and chromatin immunoprecipitation data sets, we identified CCCTC-binding factor (CTCF) as a crucial transcription factor facilitating the MYCN expression in NB. RNA immunoprecipitation, RNA electrophoretic mobility shift assay, RNA pull down and in vitro binding assay indicated the physical interaction between CTCF and MYCN opposite strand (MYCNOS), a natural noncoding RNA surrounding the MYNC promoter. Gain- and loss-of-function studies revealed that MYCNOS facilitated the recruitment of CTCF to its binding sites within the MYCN promoter to induce chromatin remodeling, resulting in enhanced MYCN levels and altered downstream gene expression, in cultured NB cell lines. CTCF cooperated with MYCNOS to suppress the differentiation and promote the growth, invasion and metastasis of NB cells in vitro and in vivo. In clinical NB tissues and cell lines, CTCF and MYCNOS were upregulated and positively correlated with MYCN expression. CTCF was an independent prognostic factor for unfavorable outcome of NB, and patients with high MYCNOS expression had lower survival probability. Taken together, these results demonstrate that CTCF cooperates with noncoding RNA MYCNOS to exhibit oncogenic activity that affects the aggressiveness and progression of NB through transcriptional upregulation of MYCN.

Abstract B37: Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma

Abstract Oncogenic Myc is known to alter cellular metabolism resulting in glutamine addiction, and the MYCN-amplified subclass of neuroblastoma is associated with extremely poor prognosis. Successful identification of therapeutic agents that are effective in neuroblastoma, particularly in tumors with MYCN amplification, could significantly increase patient survival. To explore the effect of inhibiting glutamine metabolism in neuroblastoma, we used the glutamine antagonist DON (6-diazo-5-oxo-norleucine). DON is an irreversible competitive inhibitor which alkylates enzymes that utilize glutamine. Although first studied in the 1950s as a possible cancer chemotherapeutic, DON was associated with severe nausea which limited its use in patients. DON remains a potent inhibitor, and a useful tool to study the effect of modulating glutamine metabolism in vivo. We tested the DON sensitivity of six human neuroblastoma cell lines (Kelly, IMR-32, SK-N-BE2, SK-N-AS, SK-N-FI, and SH-SY5Y) compared to immortalized BJ fibroblast control cells. DON effectively inhibited all neuroblastoma cell lines tested with minimal effect on BJ cells. Furthermore, MycN overexpression in SK-N-AS cells, a cell line which is not MYCN-amplified and expresses low levels of cMyc, was sufficient to make cells significantly more sensitive to DON. Next, we tested the effect of DON treatment on subcutaneous xenografts. For these experiments we used the cell lines SK-N-AS (not MYCN-amplified), SK-N-BE2 (MYCN-amplified with moderate MycN levels), and IMR-32 (MYCN-amplified with high MycN levels). Mice were treated twice per week by intraperitoneal injection with DON (50 or 100mg/kg) or water control. Treatment with 50mg/kg DON significantly inhibited tumor growth of all three cell lines, suggesting neuroblastoma tumors may be broadly sensitive to glutamine inhibitors. IMR-32 tumors were the most sensitive to DON treatment, leading to a 6-fold reduction in tumor size after 2.5 weeks of treatment. Tumor BrdU incorporation decreased following DON treatment, and IMR-32 tumors showed increased levels of cleaved caspase 3, a marker of apoptosis. At the higher 100mg/kg dose, DON treatment led to a more substantial decrease in BrdU incorporation, and at this dose BE2 tumors also showed significantly increased levels of cleaved caspase 3, which were not detected in AS tumors. These results suggest that MYCN-amplified tumors may be particularly sensitive to glutamine inhibition. To examine the mechanism of DON-induced cell death, we treated cells with the pan-caspase inhibitor QVD. QVD treatment for 48 or 72hr significantly rescued both MYCN-amplified cell lines (Kelly, IMR-32) and cells that express high levels of cMyc (SY5Y), while having minimal effect in the remaining neuroblastoma cell lines with low Myc levels. These results confirm that DON induces cell death partially by activating apoptosis in high Myc expressing cell lines. Next, we used shRNA to show that in Kelly cells DON-induced apoptosis occurs by signaling through Bax, but not Bak. Lastly, we tested DON in combination with pro-apoptotic compounds in vitro. We found that DON treatment combined with the Bcl-2 family inhibitor ABT-263 had synergistic effects on cell death in the MYCN-amplified cell lines Kelly and BE2. Our results suggest that combined targeting of glutamine metabolism while reducing the threshold for tumor cell apoptosis may be beneficial for neuroblastoma treatment. Citation Format: Rachelle R. Olsen, Michelle N. Mary-Sinclair, Zhirong Yin, Kevin W. Freeman. Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B37.

Abstract A06: N-MYC regulates mitochondrial dynamics in neuroblastoma via direct interaction with Drp1 and LRRC59

Abstract Background: Neuroblastoma (NB) is the most common extracranial tumor of childhood (1), accounting for approximately 7% of all cancer in children (2). The morbidity and mortality are affected by a number of factors – perhaps most significantly by amplification of the oncogene, N-MYC. The N-MYC gene product (N-Myc) regulates the transcription of a vast array of genes, and when overexpressed alters the regulation of essential cellular processes such as: the cell cycle, ribosome biogenesis and apoptosis (3). Although pathways such as these are believed to be contributory to tumorigenesis, the precise manner in which amplification of N-Myc contributes to the generation and maintenance of NB has not been fully elucidated. Objective: Many of the so-called Hallmarks of Cancers are phenotypes that are influenced by the mitochondria (4). As other members of the Myc family have been shown to significantly impact mitochondrial structure and function, we have hypothesized that that overexpression of N-Myc would alter mitochondrial dynamics in NB and speculate that these changes may contribute to tumorigenesis. Design/Methods: To perform these studies we utilized a cell line with endogenous levels of N-Myc (SK-N-SH), in which we have ectopically overexpressed N-Myc and examined the cells for changes in the mitochondrial structure with electron and confocal microscopy. Decay of fluorescence of a photoactivatable mitochondrially targeted GFP was used to measure rates of mitochondrial fusion. Western blots were used to assess differences in protein expression in response to N-Myc overexpression. Finally, the BIOID assay (5,6) was used to screen for proteins that interacted with the mitochondrial fusion protein, Drp1 and protein-protein interactions were confirmed with immunoprecipitation. Results: We found a two-fold increase in mitochondrial length in NB cells that overexpress N-Myc, which corresponded to a more tubular and elongated appearance of the organelle. We found this to be a result of an estimated two-fold increase in mitochondrial fusion in response to N-Myc overexpression. While the expression of mitochondrial fusion and fission proteins were altered in response to N-Myc overexpression, most of these changes were modest. In contrast, there was a greater than six-fold inhibition of mitochondrially-targeted Drp1 in N-Myc overexpressing NB. This led us to screen for changes in the Drp1 interactome in response to N-Myc overexpression, which resulted in the identification of LRRC59, an endoplasmic reticulum (ER) membrane protein which we found to directly interact with both Drp1 and N-Myc. In fact, we demonstrated that LRRC59 played a major role in the targeting of Drp1 to the mitochondrial surface and subsequent initiation of mitochondrial fission. Overexpression of N-Myc appeared to inhibit the Drp1-LRRC59 interaction, thereby decreasing mitochondrial localization of Drp1 and subsequently reducing fission. Conclusions: N-Myc amplification in NB increased mitochondrial length as a result of an increase in mitochondrial fusion. While there may be some contribution from transcriptional regulation of the genes involved in mitochondrial dynamics, it appeared that the most significant result of N-Myc overexpression is mislocalization of the fission protein, Drp1. This inability of Drp1 to properly translocate to the mitochondrial surface appeared to be a result of a non-transcriptional effect of N-Myc -- physically inhibiting the interaction of Drp1 with a newly identified protein partner, LRRC59 -- at mitochondrial-ER junctions. This novel mechanism of N-Myc is believed to be the primary mechanism underlying the appearance of elongated mitochondria in N-Myc overexpressing NB and we speculate that these changes are significant in the pathology of these tumors. REFERENCES 1. Siegel, R., Naishadham, D. &amp; Jemal, A. CA: a cancer journal for clinicians 63, 11–30 (2013). 2. Park, J. R., Eggert, A. &amp; Caron, H. Hematol. Oncol. Clin. North Am. 24, 65–86 (2010). 3. Bell, E. et al. Cancer Lett 293, 144–157 (2010). 4. Galluzzi, L. et al. Molecular Aspects of Medicine 31, 1–20 (2010) 5. Roux, K. J., Kim, D. I., Raida, M. &amp; Burke, B. J Cell Biol 196, 801–810 (2012). 6. Roux, K. J., Kim, D. I. &amp; Burke, B. Curr Protoc Protein Sci 74, 19.23.1–19.23.14 (2013) Citation Format: G. Casinelli, S. Banerjee, M. Sharma, S. Sims-Lucas, D. Stolz, J. A. Graves. N-MYC regulates mitochondrial dynamics in neuroblastoma via direct interaction with Drp1 and LRRC59. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A06.

Abstract 504: Glutamine metabolic inhibitors suppress growth and tumorigenicity in MYC and MYCN-driven pediatric malignancies

Abstract The MYC family of oncogenes is associated with aggressive pediatric cancers such as medulloblastoma and neuroblastoma. These transcription factors promote oncogenesis in part by altering cellular metabolism. In multiple types of cancer, high-level MYC expression drives increased glutamine metabolism. We hypothesized that MYC-driven medulloblastoma and MYCN amplified neuroblastoma would be sensitive to the glutamine metabolic inhibitors 6-diazo-5-oxo-l-norleucine (DON) and Acivicin. These drugs have previously be used in pediatric clinical trials, but never extensively clinically tested in MYC-driven malignancies. The high MYC medulloblastoma cell lines D425Med and D283Med experienced an 82% decrease in growth as measured by MTS assay when treated with Acivicin or DON (p&amp;lt;0.004). DON and Acivicin treatment also caused a 400% increase in apoptosis as measured by cleaved caspase-3 immunofluorescence (p&amp;lt;0.03). In human neural stem cells transformed with MYC, Acivicin treatment decreased proliferation by 50% as measured by BrdU incorporation (p&amp;lt;0.003) and increased apoptosis by over 100% as measured by cleaved caspase-3 immunofluoresence (p&amp;lt;0.0001). Human neural stem cells immortalized with SV40 were unaffected by Acivicin and DON treatment (p = 0.1). In mice with D283Med flank xenografts, Acivicin decreased the size of tumors by 70%. DON treatment decreased the volume of flank tumors by 94% in animals with D425Med xenografts (p = 0.001). In mice with D425Med cerebellar xenografts (p = 0.0018), DON tripled median survival from 26 to 90 days in animals with. The MYCN amplified neuroblastoma cell lines LAN5 and IMR32 were also sensitive to treatment with DON and Acivicin. DON and acivicin treatment significantly decreased proliferation as measured by BrdU incorporation (p&amp;lt;0.03) and significantly increased apoptosis as measured by cleaved caspase-3 immunofluroescence (p&amp;lt;0.003). The non-MYCN amplified neuroblastoma cell line SKNAS was resistant to treatment with both DON and Acivicin, suggesting that expression confers sensitivity to glutamine analogs. These data suggest glutamine metabolism is a therapeutic target in medulloblastoma and neuroblastoma with elevated MYC or MYCN levels, and potentially other pediatric cancer types associated with MYC oncogenes. Citation Format: Allison Hanaford, Catherine Guerra, Charles Eberhart, Eric Raabe, Antoinette Price. Glutamine metabolic inhibitors suppress growth and tumorigenicity in MYC and MYCN-driven pediatric malignancies. [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 504. doi:10.1158/1538-7445.AM2015-504

Abstract 3526: OTX015 effects in triple-negative breast cancer (TNBC) models are independent of hypoxia conditions and synergistic with other anticancer agents

Abstract Background: TNBC is an aggressive and heterogeneous subtype group of breast cancers clinically defined by the lack of estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (HER2). Few therapeutic options have shown clinical benefit beyond cytotoxic chemotherapy. Clinical studies have demonstrated that more than 50% of human breast cancers present a much lower median O2 partial pressure than normal breast tissue, correlating with chemo- and radio-resistance. We previously reported the in vivo effects of the BET-bromodomain inhibitor OTX015 (OncoEthix SA, Switzerland) in TNBC models (Vázquez et al.; EORTC-ENA 2014; #580). The aim of this work was to evaluate the in vitro antitumor activity of OTX015 both in normoxic and hypoxic environments, as well as in combination with antitumor agents. Materials and Methods: OTX015 growth inhibition concentrations 50% (GI50) values were determined in HCC197, MDA-MB-231 and MDA-MB-468 human-derived TNBC cell lines after 48 and 72 h in normoxia and hypoxia (0.1% atmospheric O2), employing the MTT assay and cell counting. RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, and after 24, 48 and 72 h of OTX015 treatments. OTX015 was combined with docetaxel or the mTOR inhibitor, everolimus, and the combination index (CI) determined by the Chou-Talalay method (CI &amp;lt; 0.9 synergy; CI = 0.9-1.1, additivity; CI &amp;gt; 1.1, antagonism). Results: OTX015 showed antiproliferative activity in the 3 cell lines after 48- and 72-h treatments in normoxia and hypoxia. MDA-MB-231 was the most sensitive in both conditions. Hypoxia significantly (p &amp;lt; 0.05) increased the antiproliferative activity of OTX015 in MDA-MB-468 cells. In addition, the antitumor effect of OTX015 was accompanied - in these cells only - with a marked (p &amp;lt; 0.05) reduction in the expression levels of c-MYC and n-MYC. The three cell lines presented a substantial increase in p21 expression following OTX015 exposure, correlating with G1 cell cycle arrest. Everolimus had an additive effect when simultaneously combined with OTX015 in HCC1937 and MDA-MB-231 cells (CI = 1.02 and 0.94, respectively), and was antagonistic in MDA-MB-468 cells (CI = 1.60). Likewise, docetaxel also had an additive effect when simultaneously combined with OTX015 in HCC1937 and MDA-MB-231 cells (CI = 1.03 and 0.95 respectively), and showed a slight synergism in MDA-MB-468 cells (CI = 0.87). Conclusion: These preclinical data support the further development OTX015 in the clinical setting for TNBC, alone or in combination with mTOR inhibitors. A single agent Phase Ib study in solid tumor patients including TNBC is underway. Citation Format: Ramiro Vázquez, Lucile Astorgues-Xerri, María E. Riveiro, Luca Beltrame, Sergio Marchini, Francesco Bertoni, Ivo Kwee, Mohamed Mohamed, Esteban Cvitkovic, Roberta Frapolli, Maurizio D'Incalci. OTX015 effects in triple-negative breast cancer (TNBC) models are independent of hypoxia conditions and synergistic with other anticancer agents. [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 3526. doi:10.1158/1538-7445.AM2015-3526

Abstract 1218: A biological crosstalk between p53 and MYCN/MYC in neuroblastoma

Abstract We previously showed that TSA (an HDAC inhibitor) and Epoxomycin (a proteasome inhibitor) as single agents and in combination significantly suppressed growth of MYCN-amplified neuroblastoma cells. However, these compounds had contrasting effects on MYCN expression. TSA down-regulated MYCN expression, but Epoxomycin and the TSA/Epoxomycin combination led to MYCN hyper-expression (defined as markedly increased expression beyond that observed in the untreated cells). The expression of p53 was also increased in MYCN-amplified cells treated with Epoxomycin or the TSA/Epoxomycin combination. In an independent study, we also found that treatment of neuroblastoma cells (MYCN-amplified or non-MYCN amplified) with 17-DMAG and S(+) Ibuprofen resulted in an increase in p53 expression and a reduction in MYCN or MYC expression. In this study, we examined (i) the pattern of gene expression induced by MYCN hyper-expression in MYCN-amplified cells, and (ii) a potential functional relationship between p53 and MYCN/MYC in neuroblastoma. Transient transfection of MYCN and TP53 into neuroblastoma cells was done by electroporation. Gene expression profiling, TaqMan real-time PCR, and Western blot assays were used to detect expression patterns of genes and proteins. It was observed that ectopic over MYCN expression in MYCN-amplified IMR5 cells resulted in growth suppression. Gene expression profiling analysis revealed that the hyper-expression of MYCN in the MYCN-transfected IMR5 cells led to an increased expression of genes involved in growth suppression and apoptosis, including EGR1, EPHA2, KLF2, PERP and SEL1L. The expression of PERP and EPHA2 was confirmed by TaqMan real-time PCR and Western blot assay, respectively. Interestingly, co-transfection of TP53 and MYCN in IMR5 cells led to high p53 expression but a reduction in MYCN expression (below the levels of endogenous MYCN). Transfection of TP53 into IMR5, SY5Y, and SKNAS reduced endogenous MYCN and MYC expression in these cells. Consistent with these observations, treatment of IMR5 and SY5Y cells with known p53-inducers, Doxorubicin and CoCl2, resulted in an increased p53 expression and a reduction of MYCN and MYC expression. Although high MYCN expression sustains growth of MYCN-amplified neuroblastoma, the hyper-expression of MYCN is deleterious to survival of these cells. In addition, augmented p53 expression may elicit a negative feedback regulation of MYCN/MYC expression in neuroblastoma. Citation Format: Naohiko Ikegaki, Xao Tang. A biological crosstalk between p53 and MYCN/MYC in neuroblastoma. [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 1218. doi:10.1158/1538-7445.AM2015-1218