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Simple Summary Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10–15% of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease, of which nearly 50% relapse and die of their disease. In addition, standard therapies cause serious lifelong side effects and increased risk for secondary tumors. Further research is crucial to better understand the molecular basis of neuroblastomas and to identify novel druggable targets. Neuroblastoma tumorigenesis has to this end been modeled in both mice and zebrafish. Here, we present a detailed dissection of the gene expression patterns that underlie tumor formation in the murine TH-MYCN-driven neuroblastoma model. We identified key factors that are putatively important for neuroblastoma tumor initiation versus tumor progression, pinpointed crucial regulators of the observed expression patterns during neuroblastoma development and scrutinized which factors could be innovative and vulnerable nodes for therapeutic intervention. Abstract Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module—centered around a TEAD4–MYCN positive feedback loop—emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo. Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4–MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582–99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517

Introduction: Neuroblastoma (NB) is a pediatric malignancy arising from peripheral neuronal sympathoblasts and exhibiting remarkable clinical and genetic heterogeneity. Patients older than 18 months have a poor prognosis with tumors presenting with highly recurrent segmental copy number alterations and MYCN amplification in half of these high-risk cases. The mechanism by which MYCN contributes to the development of neuroblastoma is unresolved and direct targeting of this key oncogene is not currently possible. Experimental Procedures: Our discovery efforts focused on identifying cooperating interactors and vulnerabilities in the MYCN regulatory network. MYCN-driven NBs can be modeled in mice with morphologic and genomic features that recapitulate human MYCN amplified NBs. Thus, this model serves as a valid tool for cross-species genomic analysis. Using this model, we performed a time-resolved analysis of the dynamic transcriptional changes of protein coding genes during murine TH-MYCN driven neuroblastoma development, focusing on timepoints representing tumor initiation and early tumor growth. We triangulated expression changes of key genes with publicly available exome-wide CRISPR-cas9 knockout analyses on a panel of human neuroblastoma cell lines and patient survival data. This unique data resource uncovered the relevance of MEIS2 as putative early cooperating initiating factor for neuroblastoma. Analysis of the genome-wide binding profile of MEIS2 in MYCN-amplified NB cell lines showed a striking overlap with enhancer-driven gene expression in regions of open chromatin, providing evidence that MEIS2 is a novel member of the adrenergic neuroblastoma core-regulatory circuitry. CRISPR-Cas9 mediated deletion of MEIS2 in animal models suppresses establishment of neuroblastoma tumors, indicating its putative requirement for tumor initiation. MEIS2, as a member of the CRC binds to several master regulators of gene expression, including the FOXM1 locus. Summary and conclusion: In conclusion, we present an in-depth characterization of the dynamic transcriptome profiles of TH-MYCN driven murine premalignant and established tumors and integrate with both primary human neuroblastoma tumor expression data, epigenetic and functional genomics data to identify and validate candidate cooperating dependencies suitable for targeting as a precision medicine approach in neuroblastoma. Citation Format: Kaat Durinck, Mark Zimmerman, Nina Weichert-Leahey, Jolien Dewyn, Wouter Van Loocke, Carolina Nunes, Anneleen Beckers, Bieke Decaesteker, Pieter-Jan Volders, Christophe Van Neste, Belamy Cheung, Daniel Carter, Thomas A. Look, Glenn Marshall, Katleen De Preter, Adam Durbin, Franki Speleman. Time-resolved transcriptome analysis of murine TH-MYCN driven neuroblastoma identifies MEIS2 as early initiating factor and novel core gene regulatory circuitry constituent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2481.

Contains fulltext : 177057.pdf (Publisher’s version ) (Closed access) A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dbetah promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Beisi Xu, Shizhen Zhu, Xiaoling Zhang, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. MYC activation through enhancer hijacking or focal enhancer amplification drives a subset of high-risk pediatric neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA19.

Childhood neuroblastomas with MYCN gene amplification form a particularly high-risk subset of this disease and are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that reflect important pathways in tumorigenesis, and thus could provide valuable targets for the development of novel therapeutics. Using genome-scale CRISPR-Cas9 approaches that allow unbiased detection of genes critically involved in tumor cell growth and survival, we identified 147 candidate genes associated with selective vulnerabilities in nine MYCN-amplified neuroblastoma cell lines, compared to findings in over 300 other human cancer cell lines representing multiple tumor cell types. We then used genome-wide ChIP-seq analysis to test the hypothesis that a small number of transcription factors - MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, all represented in the selective dependency group - are members of the transcriptional core regulatory circuitry (CRC) that underlies cell state in MYCN-amplified neuroblastoma. We show that these transcription factors bind as dense clusters at defined epicentres within the enhancers of their own genes, as well as those of the other CRC transcription factor genes, creating a positive feed-forward autoregulatory loop that establishes and maintains high levels of gene expression. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which we postulated would act synergistically by targeting both transcriptional initiation and elongation required to synthesize regulatory transcription factors. MYCN-amplified neuroblastoma cells treated with both drugs were killed synergistically, in vitro and in vivo, and accompanied by rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma, a subset of which comprises the oncogenic transcriptional regulatory circuitry that underlies cell state and survival in this tumor. Citation Format: Adam D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia, Brian J. Abraham, Brian J. Abraham, Amanda Balboni-Iniguez, Nina Weichert-Leahey, Shuning He, John M. Krill-Burger, David E. Root, Francisca Vazquez, Aviad Tsherniak, William C. Hahn, Todd R. Golub, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry [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 2062.

The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that c-MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Xu Beisi, Shizhen Zhu, Xiaoling Zhang, Zhaodong Li, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. c-MYC drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification [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 4135.