Identification of Let-7 miRNA Activity as a Prognostic Biomarker of SHH Medulloblastoma

Medulloblastoma (MB) is the most common pediatric embryonal brain tumor. The current consensus classifies MB into four molecular subgroups: sonic hedgehog-activated (SHH), wingless-activated (WNT), Group 3, and Group 4. MYCN and let-7 play a critical role in MB. Thus, we inferred the activity of miRNAs in MB by using the ActMiR procedure. SHH-MB has higher MYCN expression than the other subgroups. We showed that high MYCN expression with high let-7 activity is significantly associated with worse overall survival, and this association was validated in an independent MB dataset. Altogether, our results suggest that let-7 activity and MYCN can further categorize heterogeneous SHH tumors into more and less-favorable prognostic subtypes, which provide critical information for personalizing treatment options for SHH-MB. Comparing the expression differences between the two SHH-MB prognostic subtypes with compound perturbation profiles, we identified FGFR inhibitors as one potential treatment option for SHH-MB patients with the less-favorable prognostic subtype.

Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells

Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

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

Background: 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 mTOR pathway are up-regulated 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. Methods: Several independent NB patient cohorts with corresponding mRNA expression data were analyzed for EIF4EBP1 expression. An institutional NB cohort consisting of 69 prospectively collected tumors was employed to immunohistochemically analyze expression of EIF4EBP1-encoded protein (4EBP1). In addition, we performed an in vitro luciferase reporter gene assay with an episomal EIF4EBP1 promoter and genetically modulated MYCN expression in NB cells. Findings: EIF4EBP1 mRNA expression was positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. High EIF4EBP1 mRNA expression was 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. High levels of 4EBP1 were significantly associated with prognostically unfavorable NB histology. Functional analyses in vitro revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter. Interpretation: High EIF4EBP1 expression is associated with poor prognosis in NB patients and may serve to stratify patients with high-risk NB.

MYCN in Neuroblastoma: “Old Wine into New Wineskins”

MYCN Proto-Oncogene, BHLH Transcription Factor (MYCN) has been one of the most studied genes in neuroblastoma. It is known for its oncogenetic mechanisms, as well as its role in the prognosis of the disease and it is considered one of the prominent targets for neuroblastoma therapy. In the present work, we attempted to review the literature, on the relation between MYCN and neuroblastoma from all possible mechanistic sites. We have searched the literature for the role of MYCN in neuroblastoma based on the following topics: the references of MYCN in the literature, the gene’s anatomy, along with its transcripts, the protein’s anatomy, the epigenetic mechanisms regulating MYCN expression and function, as well as MYCN amplification. MYCN plays a significant role in neuroblastoma biology. Its functions and properties range from the forming of G-quadraplexes, to the interaction with miRNAs, as well as the regulation of gene methylation and histone acetylation and deacetylation. Although MYCN is one of the most primary genes studied in neuroblastoma, there is still a lot to be learned. Our knowledge on the exact mechanisms of MYCN amplification, etiology and potential interventions is still limited. The knowledge on the molecular mechanisms of MYCN in neuroblastoma, could have potential prognostic and therapeutic advantages.

An E2F5-TFDP1-BRG1 Complex Mediates Transcriptional Activation of MYCN in Hepatocytes

Liver regeneration is characterized by cell cycle reentrance of hepatocytes. N-Myc, encoded by MYCN, is a member of the Myc family of transcription factors. Elevation of MYCN expression has been noted in the course of liver regeneration whereas the underlying mechanism remains unclear. Here we describe that up-regulation of MYCN expression, as measured by quantitative PCR, Western blotting, and immunohistochemical staining, paralleled liver regeneration in animal and cell models. MYCN expression was up-regulated as a result of transcriptional activation. Ingenuity pathway analysis (IPA) revealed several up-stream transcriptional regulators for MYCN and RNA interference validated E2F5 and TFDP1 as essential for hepatocyte growth factor (HGF)-induced MYCN trans-activation. Further examination showed that deficiency of BRG1, a chromatin remodeling protein, attenuated MYCN induction during liver regeneration. BRG1 interacted with and was recruited by E2F5/TFDP1 to the MYCN promoter. Mechanistically, BRG1 might play a role regulating histone H3 acetylation and H3K4 trimethylation and facilitating/stabilizing the binding of RNA polymerase II surrounding the MYCN promoter. Over-expression of ectopic MYCN in BRG1-null hepatocytes overcame deficiency of proliferation. Importantly, a positive correlation between MYCN expression and BRG1/E2F5/TFDP1 expression was observed in human liver specimens. In conclusion, our data identify a novel epigenetic pathway where an E2F5-TFDP1-BRG1 complex regulates MYCN transcription to promote liver regeneration.

The Oncogene MYCN Modulates Glycolytic and Invasive Genes to Enhance Cell Viability and Migration in Human Retinoblastoma

Retinoblastoma is usually initiated by biallelic RB1 gene inactivation. In addition, MYCN copy number alterations also contribute to RB pathogenesis. However, MYCN expression, its role in disease progression and correlation with RB histological risk factors are not well understood. We studied the expression of MYCN in enucleated RB patient specimens by immunohistochemistry. MYCN is overexpressed in RB compared to control retina. Our microarray gene expression analysis followed by qRT-PCR validation revealed that genes involved in glucose metabolism and migration are significantly downregulated in MYCN knockdown cells. Further, targeting MYCN in RB cells using small molecule compounds or shRNAs led to decreased cell survival and migration, increased apoptosis and cell cycle arrest, suggesting that MYCN inhibition can be a potential therapeutic strategy. We also noted that MYCN inhibition results in reduction in glucose uptake, lactate production, ROS levels and gelatinolytic activity of active-MMP9, explaining a possible mechanism of MYCN in RB. Taking clues from our findings, we tested a combination treatment of RB cells with carboplatin and MYCN inhibitors to find enhanced therapeutic efficacy compared to single drug treatment. Thus, MYCN inhibition can be a potential therapeutic strategy in combination with existing chemotherapy drugs to restrict tumor cell growth in RB.

BIOL-11. THE ROLE OF ABERRANT EXPRESSION OF PRDM6 IN THE DEVELOPING CEREBELLUM AND IN GROUP 4 MEDULLOBLASTOMA

Group 4 medulloblastoma is the most common medulloblastoma subgroup with an intermediate prognosis and a high incidence of metastasis and late-onset relapse cases. Despite several comprehensive genomic studies in medulloblastoma, Group 4 medulloblastomas lack a unifying oncogenic driver and treatment targets. This subgroup is characterized by recurrent genetic alterations in chromatin modifiers, amplification of stemness genes, and enhancer hijacking events. 17% of Group 4 medulloblastoma cases are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. PRDM6 amplified medulloblastoma cases show additional mutations in other chromatin regulators, such as KDM6A, KMT2C and KMT2D, ZMYM3, and high MYCN expression. In this project, we investigate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and the developing mouse cerebellum. We drive expression of PRDM6 in human iPSC-derived neuroepithelial stem cells (NESCs) with and without high MYCN expression to study its implications in tumorigenesis. To test for tumor growth in vivo and changes in tumor progression as a function of PRDM6 activity, NESCs are injected into the cerebellum of adult mice. In order to elucidate impact of PRDM6 activity during embryonic cerebellar development, we also introduce PRDM6 expression into mouse embryonic stem cells (ESCs) for analysis via a new, in vivo cerebellar blastocyst complementation model. The latter approach is designed to ablate and repopulate early granule neural precursor cells in the embryonal cerebellum with progenitors derived from injected PRDM6-ESCs and thus to recapitulate pre- and postnatal cerebellar development in vivo. Together, our studies aim to understand the role of PRDM6 during normal cerebellar development and tumorigenesis and advance the understanding of the genetic drivers for Group 4 medulloblastoma.

The MicroRNA Landscape of MYCN-Amplified Neuroblastoma

MYCN gene amplification and upregulated expression are major hallmarks in the progression of high-risk neuroblastoma. MYCN expression and function in modulating gene synthesis in neuroblastoma is controlled at virtually every level, including poorly understood regulation at the post-transcriptional level. MYCN modulates the expression of various microRNAs including the miR-17-92 cluster. MYCN mRNA expression itself is subjected to the control by miRNAs, most prominently the miR-17-92 cluster that balances MYCN expression by feed-back regulation. This homeostasis seems disturbed in neuroblastoma where MYCN upregulation coincides with severely increased expression of the miR-17-92 cluster. In the presented study, we applied high-throughput next generation sequencing to unravel the miRNome in a cohort of 97 neuroblastomas, representing all clinical stages. Aiming to reveal the MYCN-dependent miRNome, we evaluate miRNA expression in MYCN-amplified as well as none amplified tumor samples. In correlation with survival data analysis of differentially expressed miRNAs, we present various putative oncogenic as well as tumor suppressive miRNAs in neuroblastoma. Using microRNA trapping by RNA affinity purification, we provide a comprehensive view of MYCN-regulatory miRNAs in neuroblastoma-derived cells, confirming a pivotal role of the miR-17-92 cluster and moderate association by the let-7 miRNA family. Attempting to decipher how MYCN expression escapes elevated expression of inhibitory miRNAs, we present evidence that RNA-binding proteins like the IGF2 mRNA binding protein 1 reduce miRNA-directed downregulation of MYCN in neuroblastoma. Our findings emphasize the potency of post-transcriptional regulation of MYCN in neuroblastoma and unravel new avenues to pursue inhibition of this potent oncogene.

A Chemo-Genomic Approach Identifies Diverse Epigenetic Therapeutic Vulnerabilities in MYCN-Amplified Neuroblastoma

Although a rare disease, neuroblastoma accounts for the highest proportion of childhood cancer deaths. There is a lack of recurrent somatic mutations in neuroblastoma embryonal tumours, suggesting a possible role for epigenetic alterations in driving this cancer. While an increasing number of reports suggest an association of MYCN with epigenetic machinery, the mechanisms of these interactions are poorly understood in the neuroblastoma setting. Utilising chemo-genomic approaches we revealed global MYCN-epigenetic interactions and identified numerous epigenetic proteins as MYCN targets. The epigenetic regulators HDAC2, CBX8 and CBP (CREBBP) were all MYCN target genes and also putative MYCN interactors. MYCN-related epigenetic genes included SMARCs, HDACs, SMYDs, BRDs and CREBBP. Expression levels of the majority of MYCN-related epigenetic genes showed predictive ability for neuroblastoma patient outcome. Furthermore, a compound library screen targeting epigenetic proteins revealed broad susceptibility of neuroblastoma cells to all classes of epigenetic regulators, belonging to families of bromodomains, HDACs, HATs, histone methyltransferases, DNA methyltransferases and lysin demethylases. Ninety-six percent of the compounds reduced MYCN-amplified neuroblastoma cell viability. We show that the C646 (CBP-bromodomain targeting compound) exhibits switch-like temporal and dose response behaviour and is effective at reducing neuroblastoma viability. Responsiveness correlates with MYCN expression, with MYCN-amplified cells being more susceptible to C646 treatment. Thus, exploiting the broad vulnerability of neuroblastoma cells to epigenetic targeting compounds represents an exciting strategy in neuroblastoma treatment, particularly for high-risk MYCN-amplified tumours.