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Abstract

Pediatric cancer arises in developing tissues, typically from early progenitor cells. The tumor cells maintain features of their developmental origin, and fail to complete normal differentiation, a phenomenon known as developmental block. The mechanisms underlying this block are diverse, and remain largely unknown. Recently, central nervous system (CNS) embryonal tumors (ET) with aberrant activations of developmental transcription factors of the PLAG-gene family, including PLAG1, have been described. However, no model systems of PLAG-overexpressing brain tumors have been developed so far, making it hard to investigate if and how aberrant PLAG-gene activation contributes to developmental block and tumorigenesis. To overcome this issue, I generated two distinct mouse models of PLAG1-overexpressing brain tumors; a genetically engineered mouse model (GEMM) and an in utero electroporation (IUE) mouse model, and additional in vitro tumor spheroid cultures derived from those. After in-depth characterization of both models, I discovered an unexpected copy number variation (CNV), encompassing the Nras locus, in the GEMM model, which can be attributed to a bystander effect of Cre-mediated recombination of the LoxP-stop-loxP (LSL) PLAG1 transgene. This CNV compromises the model’s fidelity by affecting the transcriptome and genetic dependencies. Its unexpected generation therefore represents an important finding for the field, especially when working with LSL-transgenes, or the LSL-PLAG1 mouse line specifically. The IUE model, on the other side, appeared more credible in reflecting the human tumors. Next, I integrated RNA sequencing (RNA-seq) data of both brain tumor models, and extracted a set of commonly overexpressed genes, which surprisingly included several muscle-related genes. I validated expression of some of those in the human CNS ET PLAG1-fused tumors, supporting the robustness of this gene set. Furthermore, both models exhibited remarkable signs of a developmental block, as assessed by bulk and snRNA-seq. Using whole genome CRISPR-Cas9 screening in the tumor spheroid cultures, I discovered novel genetic dependencies. Finally, I integrated these CRISPR screen dependencies with bulk RNA-seq and ChIP-seq data of the IUE model, and RNA-seq data of human tumors, and hypothesize about a possible regulatory axis including PLAG1, Zfp423, Notch1 and Ccnd3, that could be involved in developmental block and tumorigenesis. Given the established role of Zfp423 in retinoic acid-induced differentiation and treatment of neuroblastoma, the findings of my thesis motivate the investigation of similar differentiation-inducing effects in the PLAG1- overexpressing preclinical in vivo and in vitro brain tumor models I generated, which may enhance treatment efficacy in PLAG1-overexpressing pediatric brain tumors