Analysis of Oncogenic Drivers in Supratentorial Brain Tumors

Pediatric brain tumors are a leading cause of cancer mortality among children and adolescents (age 0-19) because of the paucity of effective treatment regimens. Especially for ependymoma, surgical intervention combined with focal radiotherapy is the current standard of care in routine clinical practice while this regimen very often induces irreversible damage on the developing brain and patients frequently still suffer from tumor recurrence. Thus, generating de novo representative tumor models to decipher the underlying molecular mechanisms of tumorigenesis is imminent and crucial to provide more precise and mechanism-of-action based treatment plans. In my thesis, I applied various techniques to create in vivo models for several brain tumor types and identified potential therapeutic vulnerabilities. Chapter 2 focuses on dissecting the role of oncogenic fusion genes in C11orf95 fusion- positive supratentorial ependymoma (ST-EPN), a type of pediatric brain tumor with poor prognosis. C11orf95 is a zinc finger protein that binds to DNA but has not yet been well characterized. I performed in-utero electroporation in mouse embryos and found all tested C11orf95 fusion genes were able to drive malignant transformation in the cerebral cortex. The tumors faithfully recapitulated molecular characteristics of their human counterparts. The zinc finger domain and the fusion partners were essential for tumor formation. Cross-species genomic analyses demonstrated that C11orf95-related fusions can increase the expression of a sonic hedgehog mediator gene, GLI2. Targeting GLI2 with arsenic trioxide prolonged survival in mouse models, providing a basis for further preclinical studies for C11orf95 fusion-positive tumors. Based on these findings, C11orf95 is now officially designated as zinc finger translocation associated (ZFTA) by the HUGO Gene Nomenclature Committee. In the latest edition of the WHO classification of central nervous tumors, the group of ST-EPN with ZFTA fusion genes is now named as Supratentorial ependymoma, ZFTA fusion-positive (ST- EPN-ZFTA). In Chapter 3, I investigated on a novel group of neuroepithelial tumors harboring PLAGL1 fusion (NET_PLAGL1) that has been identified in 2021 only. Mouse model generation via in-utero electroporation unfortunately failed. However, after I had performed substantial methodological optimization, overexpression of PLAGL1 fusion gene through a doxycycline-mediated system in human induced pluripotent stem cell-derived neural stem cells, followed by in vivo orthotopic transplantation successfully led to brain tumor formation in mice. This inducible in vivo system offers a reliable model to study NET_PLAGL1 tumors as well as a versatile tool to answer various biological questions behind brain tumorigenesis.Array-based DNA methylation analysis to accurately classify tumors has been developed as a routine diagnostic tool for brain tumors and sarcomas. Since mouse models are the most widely used in vivo systems in pediatric cancer research, it is important to assess the molecular similarity across species based on the methylome. In Chapter 4, I describe the approach of generating a mouse model biobank for pediatric cancers. I collected and profiled 86 murine tumor models and 40 normal tissue controls. DNA methylation-based clustering showed that samples from the same model clustered together and the copy number alteration pattern of ependymoma and glioma (e.g TFG-MET fusion-driven) mouse models recapitulate their human counterparts. This validated biobank will serve as a beneficial resource for future developmental studies such as identifying cellular origin of the tumor and decoding the composition of tumor immune microenvironment