Amplification of N-Myc is associated with a T-cell-poor microenvironment in metastatic neuroblastoma restraining interferon pathway activity and chemokine expression

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. N-Myc is the key oncogenic transcription factor amplified in high-risk neuroblastomas and associated with a poor outcome. New treatment approaches including immunotherapy are needed for this disease. To explore the role of N-Myc in interferon immune signaling, we depleted N-Myc by two independent siRNAs (siMYCN1, siMYCN2) in the human neuroblastoma cell line SK-N-BE as well as in the murine N-Myc transgenic (carrying a human MYCN transgene) neuroblastoma cell line mNB-A1 and exposed these cells to interferon gamma or TNF-alpha. Cells transfected with non-targeting control siRNAs (siNT) and cultured in regular medium served as experimental references for the respective stimulations. In addition we exposed the mouse neuroblastoma cell line mNB-A1 to the small molecule STING agonist DMXAA (25microM and 50microM) for targeted and cell intrinsic activation of the interferon system. We performed 3'mRNA-Seq transcriptome analyses on the Illumina HiSeq2500 sequencing platform.