<p>Mechanisms of reduced cell viability induced by OTX015 are cell line dependent. OTX015 reduces cell proliferation over time and induces cell cycle changes as well as apoptosis. (a) Representative photomicrographs of neuroblastoma cell lines treated with OTX015 for 72 h. Scale bar=200 µm. (b) The slope (1/h) of cell proliferation over time measured using xCELLigence of cell lines treated with OTX015. Significance was measured with the Student's t test. (c) Cell proliferation monitored using the xCELLigence system over time in neuroblastoma cell lines treated with 500 nM OTX015 (black) or DMSO control (grey). (d) Cell lines were treated 72 h with 500 nM OTX015 or DMSO, then fixed and stained with propidium iodide for flow cytometry. The distribution of OTX015-treated cells in different stages of the cell cycle is shown. (e) Fraction of apoptotic cells measured in the cell death ELISA assay after 72 h treatment with 250 nM or 500 nM OTX015 or DMSO control. Significant differences between treated cells versus controls were calculated by Student's t test. * p < 0.05, ** p < 0.01. (f) Cell proliferation measured in the BrdU ELISA after 72 h of treatment with 250 nM or 500 nM OTX015 or DMSO. Significant differences between treatment groups and the controls were calculated by Student's t test.</p>
Immune checkpoint inhibitors have significantly improved the treatment of several cancers. T-cell infiltration and the number of neoantigens caused by tumor-specific mutations are correlated to favorable responses in cancers with a high mutation load. Accordingly, checkpoint immunotherapy is thought to be less effective in tumors with low mutation frequencies such as neuroblastoma, a neuroendocrine tumor of early childhood with poor outcome of the high-risk disease group. However, spontaneous regressions and paraneoplastic syndromes seen in neuroblastoma patients suggest substantial immunogenicity. Using an integrative transcriptomic approach, we investigated the molecular characteristics of T-cell infiltration in primary neuroblastomas as an indicator of pre-existing immune responses and potential responsiveness to checkpoint inhibition. Here, we report that a T-cell-poor microenvironment in primary metastatic neuroblastomas is associated with genomic amplification of the MYCN (N-Myc) proto-oncogene. These tumors exhibited lower interferon pathway activity and chemokine expression in line with reduced immune cell infiltration. Importantly, we identified a global role for N-Myc in the suppression of interferon and pro-inflammatory pathways in human and murine neuroblastoma cell lines. N-Myc depletion potently enhanced targeted interferon pathway activation by a small molecule agonist of the cGAS-STING innate immune pathway. This promoted chemokine expression including Cxcl10 and T-cell recruitment in microfluidics migration assays. Hence, our data suggest N-Myc inhibition plus targeted IFN activation as adjuvant strategy to enforce cytotoxic T-cell recruitment in MYCN-amplified neuroblastomas.
Amplification or overexpression of MYCN is involved in development andmaintenance of multiple malignancies.A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma.In order to model the spectrum of MYCNdriven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells.However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland.Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas.Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon-and GFAP-positive islet cells.Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system.Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype.MYCN-directed treatment by AuroraA-or Brd4-inhibitors resulted in significantly
// Anneleen Beckers 1,* , Gert Van Peer 1,* , Daniel R. Carter 2 , Evelien Mets 1 , Kristina Althoff 3,4 , Belamy B. Cheung 2 , Johannes H. Schulte 3,4,5, 6 , Pieter Mestdagh 1 , Jo Vandesompele 1 , Glenn M. Marshall 2,7 , Katleen De Preter 1 and Frank Speleman 1 1 Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium 2 Children's Cancer Institute, University of New South Wales, Sydney, Australia 3 Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Essen, Germany 4 German Cancer Consortium (DKTK), Germany 5 German Cancer Research Center (DKFZ), Heidelberg, Germany 6 Translational Neuro-Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany 7 Kids Cancer Centre, Sydney Children's Hospital, Sydney, Australia * These authors contributed equally to this work Correspondence: Frank Speleman, email: // Keywords : MYCN, microRNA, neuroblastoma, feedback regulation, cross-species Received : July 01, 2014 Accepted : September 15, 2014 Published : September 16, 2014 Abstract MYCN is a transcription factor that plays key roles in both normal development and cancer. In neuroblastoma, MYCN acts as a major oncogenic driver through pleiotropic effects regulated by multiple protein encoding genes as well as microRNAs (miRNAs). MYCN activity is tightly controlled at the level of transcription and protein stability through various mechanisms. Like most genes, MYCN is further controlled by miRNAs, but the full complement of all miRNAs implicated in this process has not been determined through an unbiased approach. To elucidate the role of miRNAs in regulation of MYCN, we thus explored the MYCN-miRNA interactome to establish miRNAs controlling MYCN expression levels. We combined results from an unbiased and genome-wide high-throughput miRNA target reporter screen with miRNA and mRNA expression data from patients and a murine neuroblastoma progression model. We identified 29 miRNAs targeting MYCN, of which 12 miRNAs are inversely correlated with MYCN expression or activity in neuroblastoma tumor tissue. The majority of MYCN-targeting miRNAs in neuroblastoma showed a decrease in expression during murine MYCN-driven neuroblastoma tumor development. Therefore, we provide evidence that MYCN-targeting miRNAs are preferentially downregulated in MYCN-driven neuroblastoma, suggesting that MYCN negatively controls the expression of these miRNAs, to safeguard its expression.
