Mentor Name: Robert Wechsler-Reya

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, with ~30% of patients presenting with leptomeningeal dissemination at diagnosis. Metastasis is particularly prevalent in Group 3 (MYC-driven) MB, contributing to poor patient outcomes. Effective treatment requires a deeper understanding of metastatic mechanisms and potential therapeutic targets. The leptomeningeal microenvironment differs significantly from the primary tumor, presenting distinct vascular structures, lower nutrient availability, and unique surrounding cell types. Tumor cells must detach, survive in circulation, adhere to new sites, and adapt to these conditions—processes likely regulated by changes in gene expression. My project aims to investigate these changes in MB. Previous work in the Wechsler-Reya lab using patient-derived xenografts (PDXs) and cell lines identified cFOS as consistently upregulated in metastatic MB samples. cFOS encodes a transcription factor involved in diverse cellular processes, necessitating further study to determine its role in MB metastasis. To explore this, this project will first involve culturing the D283 Group 3 MB cell line in vitro. Next, we will use a lentiviral vector to introduce a doxycycline-inducible short hairpin RNA (shRNA) to silence cFOS expression. This system allows for controlled knockdown, where cFOS expression remains intact until doxycycline is added to the culture medium. Once knockdown is induced, we will validate its effectiveness by assessing cFOS levels using quantitative PCR (qPCR) at the RNA level and Western blot analysis at the protein level. Following successful knockdown, we will perform RNA sequencing on cFOS-silenced cells and compare the results with control cells with normal cFOS expression. This analysis will identify which genes are upregulated or downregulated as a result of cFOS depletion, shedding light on the pathways it regulates in MB metastasis. By understanding these downstream targets of cFOS, we aim to identify key genes that could serve as novel targets for therapy. To further validate our findings, we will also employ CUT&RUN sequencing, a technique that provides a direct method of mapping transcription factor binding sites on DNA. This will help confirm which genes are directly regulated by cFOS, complementing the RNA sequencing data and giving a clearer picture of its role in metastatic progression. By systematically dissecting the role of cFOS in Group 3 MB metastasis, this project will contribute to a deeper understanding of the molecular mechanisms that drive tumour spread. The insights gained may help identify targets for therapeutic intervention, ultimately improving treatment.