ETMR-07. OVEREXPRESSION OF THE PLAG-GENE FAMILY MEMBER PLAG1 DURING MURINE BRAIN DEVELOPMENT LEADS TO ABERRANT EXPRESSION OF IMPRINTED GENES AND THE FORMATION OF MIDLINE BRAIN TUMORS

Abstract

Abstract BACKGROUND The transcription factor PLAG1, ordinarily active during development, has been identified as overexpressed in several pediatric brain tumor entities, including H3 K27-altered diffuse midline glioma. Additionally, our group recently identified a novel type of embryonic CNS tumors with amplification of the related genes PLAGL1 and PLAGL2. How this aberrant PLAG family gene activation drives tumor formation is unknown. We hypothesize that aberrant activity of these transcription factors impedes normal differentiation, thereby fostering neoplastic transformation. METHODS To investigate this, we utilize transgenic mouse models engineered to exhibit targeted PLAG1 overexpression and loss of Trp53 during brain development. Following, we use bulk and single-cell RNA-seq to examine underlying mechanisms and are working on whole genome CRISPR-Cas9 screens to detect genetic dependencies in derived tumorsphere cultures. RESULTS We found that transgenic mice develop brain tumors in the midline. RNA-seq of these tumors suggests the activation of developmental (homeobox transcription factors) and imprinted genes e.g. H19 and Dlk1, the latter being similarly activated in pediatric CNS tumors with PLAGL1/2 amplification. Using single-cell RNA-seq of the resultant tumors, we compared the gene expression profiles of the tumor cells to a developmental mouse brain atlas. Tumor cells best correlated to radial glia stem cells and glioblasts, supporting the hypothesis of a developmental block caused by PLAG1 overexpression. A subpopulation of the tumor cells seemed to escape the radial glia cell state but were then ultimately locked in the OPC state. A similar mechanism was observed in H3 K27M diffuse midline gliomas. CONCLUSION Collectively, we provide evidence that PLAG1 overexpression drives pediatric brain tumor formation by inhibiting normal cellular differentiation. The ongoing CRISPR screen in PLAG1 and PLAGL1 overexpressing murine cell lines, ChIP-seq analysis of the murine tumors, and comparisons with human data and hNSC models aims to unveil the specific molecular mechanisms underlying tumor progression.