Gene expression profiling of MYCN-driven neuroblastoma tumor development in the TH-MYCN mouse model.
Ontology highlight
ABSTRACT: Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system progenitor cells. MYCN and ALK are driver oncogenes both of which are specifically expressed during early neurogenesis. This is in line with the assumption that neuroblastoma arises through disruption of normal developmental processes. MYCN has a broad impact on the tumor phenotype; however, the details of the MYCN driven oncogenic program are far from clear. In order to gain further insight into the role of gene expression during neuroblastoma initiation and progression, we evaluated gene expression profiles of hyperplastic ganglia and tumors isolated from MYCN transgenic mice.
Project description:Cancer genomic studies that rely on analysis of diagnostic biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic neuroblastoma model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in 9 hyperplastic ganglia harvested at 3 time points, and 4 tumor cohorts of progressively larger size (n=6 each group) in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We manually curated the 24 candidates and prioritized 3 targets (Cenpe, Gpr49, Impdh2) with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell line models we further prioritized CENPE due to potent inhibition of cellular proliferation. Targeting of CENPE with the selective small molecular inhibitor GSK923295 showed potent inhibition of in vitro proliferation of 19 neuroblastoma derived cell lines (median IC50=41 nM; range 27-266 nM), and significantly delayed tumor growth kinetics in 3 xenograft models (p-values ranged from p<0.0001 to p=0.018). Here we provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets, and identify CENPE as a novel therapeutic candidate in neuroblastoma. set 1: 24 tumor samples analyzed set 2: 9 hyperplastic ganglia harvested at 3 time points (day 0, day 7 and day 14) and 2 tumors "small" size were analysed
Project description:MYCN amplification in neuroblastoma leads to aberrant expression of MYCN oncoprotein, which binds active genes promoting transcriptional amplification. Yet how MYCN coordinates transcription elongation to meet productive transcriptional amplification and which elongation machinery represents MYCN-driven vulnerability remain to be identified. We conducted a targeted screen of transcription elongation factors and identified the super elongation complex (SEC) as a unique vulnerability in MYCN-amplified neuroblastomas. MYCN directly binds EAF1 and recruits SEC to enhance processive transcription elongation. Depletion of EAF1 or AFF1/AFF4, another core subunit of SEC, leads to a global reduction in transcription elongation and elicits selective apoptosis of MYCN-amplified neuroblastoma cells. A combination screen reveals SEC inhibition synergistically potentiates the therapeutic efficacies of FDA-approved BCL2 antagonist ABT-199, in part due to suppression of MCL1 expression, both in MYCN-amplified neuroblastoma cells and in patient-derived xenografts. These findings identify disruption of the MYCN-SEC regulatory axis as a promising therapeutic strategy in neuroblastoma.
Project description:miRNAs which are differentially expressed upon MYCN overexpression (1h, 4h and 24h after induction) in a neuroblastoma line were detected using deep sequencing.
Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive âtargetâ genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ChIP-Seq in SHEP21, BE2C, KELLY, and NGP neuroblastoma cell lines for H3K27ac, H3K4me3, RNA PolII, MYCN, BRD4, or TWIST1
Project description:To identify the MYCN transcription factor binding sites across the genome, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-MYCN and anti-IgG antibodies on a MYCN-amplified NB cell line, SK-N-BE(2)-C. Identification of MYCN binding in neuroblastoma cells.
Project description:The effect of the GSK3 inhibitor Azakenpaullone (Azak) and the differentiation agent retinoic acid (RA) was studied in low and high MYCN levels in the MYCN inducible neuroblastoma cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN). Azak was dissolved in DMSO in order to apply it to the cells. Therefore a vehicle control consisting of SY5Y-MYCN cells treated with 24h 1 ul/ml DMSO only was used in duplicates. Doxycycline (Sigma) dissolved in water was used at a final concentration of 1ug/ml to induce MYCN expression in SY5Y-MYCN. A co-treatment study with Dox and Azak was conducted. SY5Y-MYCN cells were treated with 24h Azak, 24h Azak & 48h Dox and 48h Dox, with biological duplicates. 1 uM RA (dissolved in DMSO) and 1 ug/mL Doxycycline were given individually and in combination. SY5Y-MYCN cells were treated with 24h RA, 24h RA & 48h Dox, and 48h Dox and RNA was extracted in biological duplicates. For the 24h RA & 48h Dox co-treatment cells were treated with Dox for 24h and then with RA and fresh Dox for a further 24h.
Project description:Inducible MYCN-knockdown, followed by RNA-seq analysis in the MYCN-amplified neuroblastoma cell line IMR5-75, reveals profound time-dependent transcriptome changes. For modulation of MYCN levels, stable neuroblastoma cell models were used where MYCN can be downregulated via vector-based hairpin RNA induction upon addition of 1µg/ml tetracycline (IMR5-75-shMYCN. From cells treated either with tetracycline or solvent (ethanol), RNA was isolated at time points 6 hours, 12 hours and 24 hours. Experiments were done in duplicates. RNA was sequenced.
Project description:mRNA profiles of thousands of human tumors are available, but methods to deduce oncogenic signaling networks from these data lag behind. It is especially challenging to identify main-regulatory routes, and to generalize conclusions obtained from experimental models. We designed the bioinformatic platform R2 (http://r2.amc.nl) in parallel with a wet-lab approach of neuroblastoma. Here we demonstrate how R2 facilitates an integrated analysis of our neuroblastoma data. Analysis of the MYCN pathway suggested important regulatory connections to the polyamine synthesis route, the Notch pathway and the BMP/TGFβ pathway. A network of genes emerged connecting major oncogenes in neuroblastoma. Genes in the network carried strong prognostic values and were essential for tumor cell survival. Time-course experiments in triplicate of MYCN induction after doxycycline addition, with 7 timepoints.
Project description:Bromodomain-containing protein 4 (BRD4) functions as an epigenetic reader and binds to so-called super-enhancer regions of driving oncogenes such as MYC in cancer. We investigated the possibility to target super-enhancer regulated genes in neuroblastoma and in MYCN amplified disease in particular. We used OTX015, the first small-molecule BRD4 inhibitor to enter clinical phase I/II trials in adults, to test the feasibility to specifically target super-enhancer regulated gene-expression in neuroblastoma. BRD4 inhibition lead to significant transcriptional down-regulation of genes that were associated with super-enhancers, supporting the notion that BRD4 preferentially acts at these chromatin sites. BRD4 inhibition not only attenuated MYCN transcription but most significantly affected MYCN-regulated transcriptional programs.