Direct Transcriptional Target Of MYCN Research Articles

Abstract 280: Targeting mRNA pseudouridylation to suppress neuroblastoma metabolism

Abstract Pseudouridylation of mRNA is a common epigenetic modification that reduces RNA conformational flexibility and increases its structural stability and may increase its availability for translation. RNA pseudouridylation is catalyzed by the family of pseudouridine synthases, including dyskerin pseudouridine synthase 1 (DKC1). Here we report the DKC1 gene is a direct transcriptional target of MYCN, which is a major oncogene in driving the development of high-risk neuroblastoma, a childhood cancer with a poor prognosis. The transcriptional upregulation of DKC1 expression in MYCN-amplified cells is of functional significance, as DKC1 depletion by RNA interference inhibits the proliferation of neuroblastoma cell lines. Furthermore, gene expression profiling reveals that DKC1 has a key role in the regulation of amino acid biosynthesis enzymes. Genetic and pharmacological inhibition of DKC1 reduces the expression of ATF4 and its downstream target genes involved in amino acid synthesis and transport. These findings reveal a critical role of DKC1 in sustaining the proliferation of neuroblastoma cells by promoting amino acid metabolism, which could be exploited as a therapeutic strategy against high-risk neuroblastoma. Citation Format: Anamika Gupta, Mohit Bansal, Han-Fei Ding. Targeting mRNA pseudouridylation to suppress neuroblastoma metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 280.

EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma

Neuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mechanistic target of rapamycin (mTOR) pathway are upregulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients. Using publicly available data sets, we found that EIF4EBP1 mRNA expression is positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. In addition, high EIF4EBP1 mRNA expression is associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. This was confirmed by monitoring the clinical value of 4EBP1 protein expression, which revealed that high levels of 4EBP1 are significantly associated with prognostically unfavorable NB histology. Finally, functional analyses revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter in NB cells. Our data highlight that EIF4EBP1 is a direct transcriptional target of MYCN whose high expression is associated with poor prognosis in NB patients. Therefore, EIF4EBP1 may serve to better stratify patients with NB.

A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma.

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

Glycine decarboxylase is a transcriptional target of MYCN required for neuroblastoma cell proliferation and tumorigenicity.

Genomic amplification of the oncogene MYCN is a major driver in the development of high-risk neuroblastoma, a pediatric cancer with poor prognosis. Given the challenge in targeting MYCN directly for therapy, we sought to identify MYCN-dependent metabolic vulnerabilities that can be targeted therapeutically. Here, we report that the gene encoding glycine decarboxylase (GLDC), which catalyzes the first and rate-limiting step in glycine breakdown with the production of the one-carbon unit 5,10-methylene-tetrahydrofolate, is a direct transcriptional target of MYCN. As a result, GLDC expression is markedly elevated in MYCN-amplified neuroblastoma tumors and cell lines. This transcriptional upregulation of GLDC expression is of functional significance, as GLDC depletion by RNA interference inhibits the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines by inducing G1 arrest. Metabolomic profiling reveals that GLDC knockdown disrupts purine and central carbon metabolism and reduces citrate production, leading to a decrease in the steady-state levels of cholesterol and fatty acids. Moreover, blocking purine or cholesterol synthesis recapitulates the growth inhibitory effect of GLDC knockdown. These findings reveal a critical role of GLDC in sustaining the proliferation of neuroblastoma cells with high-level GLDC expression and suggest that MYCN amplification is a biomarker for GLDC-based therapeutic strategies against high-risk neuroblastoma.

SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma

SKP2 is the substrate recognition subunit of the ubiquitin ligase complex which targets p27KIP1 for degradation. Induced at the G1/S transit of the cell cycle, SKP2 is frequently overexpressed in human cancers and contributes to malignancy. We previously identified SKP2 as a possible MYCN target gene and hence hypothesise that SKP2 is a potential therapeutic target in MYCN amplified disease. A positive correlation was identified between MYCN activity and SKP2 mRNA expression in Tet21N MYCN-regulatable cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to E-boxes within the SKP2 promoter and induced transcriptional activity which was decreased by the removal of MYCN and E-box mutation. Although SKP2 knockdown inhibited cell growth in both MYCN amplified and non-amplified cells, cell cycle arrest and apoptosis were induced only in non-MYCN amplified neuroblastoma cells. In conclusion these data identify SKP2 as a direct transcriptional target of MYCN and supports SKP2 as a potential therapeutic target in neuroblastoma.

TWIST1 is a direct transcriptional target of MYCN and MYC in neuroblastoma

In neuroblastoma, MYCN amplification is associated with a worse prognosis and is a criterion used in the clinic to provide intensive treatments to children even with localized disease. In correlation with MYCN amplification, upregulation of TWIST1, a transcription factor playing a crucial role in inhibition of apoptosis and differentiation, was previously reported. Clinical data set analysis of MYCN, MYC and TWIST1 expression permits us to confirm that TWIST1 expression is upregulated in MYCN amplified neuroblastoma but also in a subset of neuroblastoma harboring high expression of MYCN or MYC without gene amplification. In silico analyses reveal the presence of several MYC regulatory motifs (E-Boxes and INR) within the TWIST1 promoter. Using gel shift assay and reporter activity assays, we demonstrate that both N-Myc and c-Myc proteins can bind and activate the TWIST1 promoter. Therefore, we propose TWIST1 as a direct MYC transcriptional target.

Abstract LB-68: MYCN-induced TRPM7 expression and channel activity occurs through a mechanism that involves ornithine decarboxylase

Abstract Neuroblastoma (NB) is an extra-cranial solid cancer in children. MYCN is a transcription factor that regulates the expression of genes that are involved in cell proliferation, growth, cell migration, etc. MYCN gene amplification is a prognostic indicator of poor outcome in NB. Recent studies have shown that intracellular free calcium regulates multiple steps involved in cell migration. Although significant advances have been made in understanding the role of calcium during migration, little has been achieved towards understanding its impact on the malignant progression of NB. Our results showed that MYCN gene amplification or over-expression of MYCN in NB cells increased TRPM7 expression, increased peak TRPM7 current, induced constitutive TRPM7 channel activity, enhanced calcium signaling, and promoted cell migration, implicating TRPM7 as a potential regulator of calcium signals that promote NB cell migration and invasion. Recent results suggest that MYCN may regulate TRPM7 expression through a process that involves ornithine decarboxylase (ODC), a rate limiting enzyme in the biosynthesis of polyamines, and a direct transcriptional target of MYCN. Treatment of NB cells with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, or transfection of ODC specific siRNA effectively decreased TRPM7 expression. This effect was attenuated by supplementing the culture media with spermidine, confirming that ODC may function downstream of MYCN to regulate TRPM7 expression. Interestingly, western blot analysis of NB cell lysates showed that MYCN induced the cleavage of the TRPM7 kinase domain, a post-translational modification that has been shown to increase TRPM7 currents and induce constitutive TRPM7 channel activity. Additionally, DFMO inhibited TRPM7 cleavage, an effect that was reversed by spermidine supplementation. Taken together, these results suggest that MYCN may regulate TRPM7 expression and channel activity through a mechanism that involves ODC-mediated polyamine biosynthesis. Citation Format: Dana-Lynn T. Koomoa, Ingo Lange. MYCN-induced TRPM7 expression and channel activity occurs through a mechanism that involves ornithine decarboxylase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-68. doi:10.1158/1538-7445.AM2014-LB-68

ALK is a MYCN target gene and regulates cell migration and invasion in neuroblastoma

Human anaplastic lymphoma kinase (ALK) has been identified as an oncogene that is mutated or amplified in NBLs. To obtain a better understanding of the molecular events associated with ALK in the pathogenesis of NBL, it is necessary to clarify how ALK gene contributes to NBL progression. In the present study, we found that ALK expression was significantly high in NBL clinical samples with amplified MYCN (n = 126, P < 0.01) and in developing tumors of MYCN-transgenic mice. Indeed, promoter analysis revealed that ALK is a direct transcriptional target of MYCN. Overexpression and knockdown of ALK demonstrated its function in cell proliferation, migration and invasion. Moreover, treatment with an ALK inhibitor, TAE-684, efficiently suppressed such biological effects in MYCN amplified cells and tumor growth of the xenograft in mice. Our present findings explore the fundamental understanding of ALK in order to develop novel therapeutic tools by targeting ALK for aggressive NBL treatment.

Abstract 789: GSTP1 expression is regulated by MycN and is a marker of poor outcome in childhood neuroblastoma

Abstract 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

MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63

MYCN amplification is a major biomarker of poor prognosis, occurring in 25-30% of neuroblastomas. MYCN plays contradictory roles in promoting cell growth and sensitizing cells to apoptosis. We have recently shown that p53 is a direct transcriptional target of MYCN in neuroblastoma and that p53-mediated apoptosis may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14ARF pathway is often inactivated through MDM2 amplification or p14ARF inactivation. We hypothesised that reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis especially in MYCN amplified cells. Using the SHEP Tet21N MYCN regulatable system, MYCN(−) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared to MYCN(+) cells and siRNA mediated knockdown of MYCN in 4 MYCN amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of 18 neuroblastoma cell lines treated with Nutlin-3 and MI-63, the sub-set amplified for MYCN had a significantly lower mean GI50 value and increased caspase 3/7 activity compared to the non MYCN amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. We conclude that amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wildtype p53 to MDM2-p53 antagonists and that these compounds may therefore be particularly effective in treating high risk MYCN amplified disease.

P53 Is a Direct Transcriptional Target of MYCN in Neuroblastoma

MYCN amplification occurs in approximately 25% of neuroblastomas, where it is associated with rapid tumor progression and poor prognosis. MYCN plays a paradoxical role in driving cellular proliferation and inducing apoptosis. Based on observations of nuclear p53 accumulation in neuroblastoma, we hypothesized that MYCN may regulate p53 in this setting. Immunohistochemical analysis of 82 neuroblastoma tumors showed an association of high p53 expression with MYCN expression and amplification. In a panel of 5 MYCN-amplified and 5 nonamplified neuroblastoma cell lines, and also in the Tet21N-regulatable MYCN expression system, we further documented a correlation between the expression of MYCN and p53. In MYCN-amplified neuroblastoma cell lines, MYCN knockdown decreased p53 expression. In Tet21N MYCN+ cells, higher levels of p53 transcription, mRNA, and protein were observed relative to Tet21N MYCN- cells. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to a Myc E-Box DNA binding motif located close to the transcriptional start site within the p53 promoter, where it could initiate transcription. E-Box mutation decreased MYCN-driven transcriptional activation. Microarray analysis of Tet21N MYCN+/- cells identified several p53-regulated genes that were upregulated in the presence of MYCN, including MDM2 and PUMA, the levels of which were reduced by MYCN knockdown. We concluded that MYCN transcriptionally upregulates p53 in neuroblastoma and uses p53 to mediate a key mechanism of apoptosis.

MYCN-Directed Centrosome Amplification Requires MDM2-Mediated Suppression of p53 Activity in Neuroblastoma Cells

The MYC family oncogenes cause transformation and tumor progression by corrupting multiple cellular pathways, altering cell cycle progression, apoptosis, and genomic instability. Several recent studies show that MYCC (c-Myc) expression alters DNA repair mechanisms, cell cycle checkpoints, and karyotypic stability, and this is likely partially due to alterations in centrosome replication control. In neuroblastoma cell lines, MYCN (N-Myc) expression induces centrosome amplification in response to ionizing radiation. Centrosomes are cytoplasmic domains that critically regulate cytokinesis, and aberrations in their number or structure are linked to mitotic defects and karyotypic instability. Whereas centrosome replication is linked to p53 and Rb/E2F-mediated cell cycle progression, the mechanisms downstream of MYCN that generate centrosome amplification are incompletely characterized. We hypothesized that MDM2, a direct transcriptional target of MYCN with central inhibitory effects on p53, plays a role in MYC-mediated genomic instability by altering p53 responses to DNA damage, facilitating centrosome amplification. Herein we show that MYCN mediates centrosome amplification in a p53-dependent manner. Accordingly, inhibition of the p53-MDM2 interaction with Nutlin 3A (which activates p53) completely ablates the MYCN-dependent contribution to centrosome amplification after ionizing radiation. We further show that modulating MDM2 expression levels by overexpression or RNA interference-mediated posttranscriptional inhibition dramatically affects centrosome amplification in MYCN-induced cells, indicating that MDM2 is a necessary and sufficient mediator of MYCN-mediated centrosome amplification. Finally, we show a significant correlation between centrosome amplification and MYCN amplification in primary neuroblastoma tumors. These data support the hypothesis that elevated MDM2 levels contribute to MYCN-induced genomic instability through altered regulation of centrosome replication in neuroblastoma.

Cellular Retinoic Acid–Binding Protein II Is a Direct Transcriptional Target of MycN in Neuroblastoma

Neuroblastoma is a heterogeneous disease in which 22% of tumors show MycN oncogene amplification and are associated with poor clinical outcome. MycN is a transcription factor that regulates the expression of a number of proteins that affect the clinical behavior of neuroblastoma. We report here that cellular retinoic acid-binding protein II (CRABP-II) is a novel MycN target, expressed at significantly higher levels in primary neuroblastoma tumors with mycN oncogene amplification as compared with non-MycN-amplified tumors. Moreover, regulated induction and repression of MycN in a neuroblastoma-derived cell line resulted in temporal and proportionate expression of CRABP-II. CRABP-II is expressed in several cancers, but its role in tumorigenesis has not been elucidated. We show that MycN binds to the promoter of CRABP-II and induces CRABP-II transcription directly. In addition, CRABP-II-transfected neuroblastoma cell lines show an increase in MycN protein levels resulting in increased cell motility. Gene expression profiling of CRABP-II-expressing cell lines uncovered increased expression of the HuB (Hel N1) gene. Hu proteins have been implicated in regulating the stability of MycN mRNA and other mRNAs by binding to their 3' untranslated regions. We did not, however, observe any change in MycN mRNA stability or protein half-life in response to CRABP-II expression. In contrast, de novo MycN protein synthesis was increased in CRABP-II-expressing neuroblastoma cells, thereby suggesting an autoregulatory loop that might exacerbate the effects of MycN gene amplification and affect the clinical outcome. Our findings also suggest that CRABP-II may be a potential therapeutic target for neuroblastoma.

The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma.

The MYCN oncogene is the major negative prognostic marker in neuroblastoma with important roles in both the pathogenesis and clinical behavior of this aggressive malignancy. MYC oncogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis. To identify novel transcriptional targets mediating the MYCN-dependent phenotype, we screened a MYCN-amplified neuroblastoma cell line by using chromatin immunoprecipitation (ChIP) cloning. We identified the essential p53 inhibitor and protooncogene MDM2 as a putative target. MDM2 has multiple p53-independent functions modulating cell cycle and transcriptional events. Standard ChIP with MYCN antibodies established the binding of MYCN to a consensus E-box within the human MDM2 promoter. Oligonucleotide pull-down assays further established the capacity of MYCN to bind to this promoter region, confirming the ChIP results. Luciferase reporter assays confirmed the E-box-specific, MYCN-dependent regulation of the MDM2 promoter in MYCN-inducible neuroblastoma cell lines. Real-time quantitative PCR and Western blot analysis demonstrated a rapid increase in endogenous MDM2 mRNA and MDM2 protein upon induction of MYCN. Targeted inhibition of MYCN in a MYCN-amplified neuroblastoma cell line resulted in decreased MDM2 expression levels with concomitant stabilization of p53 and induction of apoptosis. Our finding that MYCN directly modulates baseline MDM2 levels suggests a mechanism contributing to the pathogenesis of neuroblastoma and other MYC-driven malignancies through inhibition of MYC-stimulated apoptosis.