C19MC ONCOMIRS CONVERGE ON MULTIPLE CELL CYCLE REGULATORS TO MODULATE NEURAL DIFFERENTIATION AND THE TUMOR EPIGENOME

Abstract

BACKGROUND: Childhood CNS-PNETs comprise a heterogeneous spectrum of diseases with poorly defined biology. The embryonic stem cell enriched C19MC OncomiR cluster is frequently amplified in one sub-group of CNS-PNETs (group 1 CNS-PNETs) with distinctly aggressive clinic-pathologic features. However, the specific oncogenic role of C19MC in group 1 CNS-PNETs, and mechanisms by which C19MC effects cellular transformation remains unknown. In this study we used exome and RNA-sequencing of C19MC associated tumors, and functional studies of the C19MC OncomiRs in human neural stem cell to define oncogenic partners and downstream effectors of the C19MC locus. METHODS: Whole exome and RNA seq analyses were performed respectively on 25 and 10 group 1 CNS-PNETs with known amplification of the C19MC locus to identify C19MC cooperating loci. The Illumina 450K methylation arrays were used to analyse the epigenomic of 30 CNS-PNETs ad C19MC transformed human neural stem cells. A “maxi gene” comprised of 5 C19MC OncomiRs most highly expressed in primary group 1 CNS-PNETs was constructed and stably expressed in a panel of normal human neural stem cells, human tumor and murine fibroblast line, and used to identify C19MC target genes. C19MC target genes were identified by combining gene expression profiling with target prediction programs. Target genes were validated using reporter gene assays, immuno-histochemical and miRNA in-situ hybridization analyses. RESULTS: Exome sequencing revealed few recurrent genetic alterations other than the C19MC amplicon in group 1 CNS-PNETs. Unexpectedly, RNAseq analyses also uncovered recurrent fusion events of the C19MC locus to TTHY1–a neural developmental locus, thus indicating C19MC as a major oncogenic driver in these tumors. Studies in human neural stem cells identified p21, p27 and RBL2 as highly conserved direct gene targets of the C19MC oncoMiRs. Remarkably, experimental studies uncovered a C19MC-RBL2-DNMT3b regulatory axis that was associated with distinct methylation signatures in primary human CNS-PNETs and C19MC transformed human neural stem cells. CONCLUSIONS: Our results suggest that C19MC which are normally expressed only in a restricted developmental window, become potent drivers of cellular transformation and tumour development when de-regulated, and act by modulating the cell cycle and global epigenomic landscape to effect C19MC tumourigenesis. SECONDARY CATEGORY: Tumor Biology.