Balancing growth: mechanisms underlying cerebellar development and medulloblastoma pathogenesis

Development of the nervous system requires precise regulation of proliferation to prevent neurological diseases such as microcephaly or cancer. Particularly, postnatal neurogenesis in the cerebellum gives rise to the largest population of neurons in the brain, cerebellar granule neurons (CGNs). Thus, cerebellar neurogenesis must be tightly regulated to prevent growth failure which could give rise to cerebellar hypoplasia or hyperproliferation, which can lead to the formation of tumors like medulloblastoma. While the mechanisms driving proliferation in the cerebellar granule neuron progenitors (CGNPs) is widely known to be mostly dependent on Sonic Hedgehog signaling (SHH), little is known about the mechanisms that oppose growth and promote differentiation. Furthermore, whether CGNP-derived medulloblastomas require similar mechanisms to promote growth remains unclear. To address this, in Chapter 2 we used a genetic knockout of both isoforms of glycogen synthase kinase (GSK-3), the main brake on WNT signaling in the brain, to determine the effect of WNT pathway activation in the cerebellum. We demonstrated that activation of the WNT signaling pathway through deletion of Gsk-3α and Gsk-3β in Math1+ cerebellar progenitors led to massive cerebellar hypoplasia by promoting cell cycle arrest but not cell death, which was driven by increased CDKN1A. Co-deletion of the main effector of the WNT signaling pathway, Ctnnb, was able to rescue proliferation in Gsk-3-deficient CGNPs. Moreover, we found that deletion of Gsk-3 was sufficient to block tumor growth in SHH-driven medulloblastomas in mice. Pharmacological inhibition of GSK-3 in vitro was sufficient to block CGNP and medulloblastoma tumor cell growth. Together, our research suggests that endogenous GSK-3 is necessary to prevent WNT signaling in the cerebellum and medulloblastoma to promote SHH-driven proliferation. In Chapter 3, we used single-cell transcriptomics analysis to probe the response of medulloblastoma tumor cells to a SHH pathway inhibitor, vismodegib. Administration of vismodegib produced a heterogenous response in SHH-driven tumor cells, leading to recurrence. We identified distinct cell types in untreated tumors, including stromal cells and tumor-derived cells in a spectrum of proliferative and differentiated states, through Drop-Seq. Vismodegib reduced a subset of proliferative tumor cells and enriched cells in a differentiated state. However, a subset of proliferative tumor cells, which expressed stem cell markers or persistent SHH-pathway activation remained after vismodegib treatment. Our findings demonstrate diversity within tumors that can confer inherent resistance to targeted inhibitor therapies. In Chapter 4, we dissected the role of the exon junction complex core component MAGOH in cerebellar development and medulloblastoma. Temporally controlled, conditional deletion of Magoh during cerebellar growth induced DNA damage and apoptosis within 48 hours, leading to massive depopulation of highly neurogenic regions, especially the external granule layer of the cerebellum. We found that deletion of trp53 was sufficient to block the DNA damage response in Magoh-deleted cells but was unable to inhibit Caspase-dependent cell death. Moreover, we explored the necessity of MAGOH in maintaining medulloblastoma tumor growth by conditionally deleting Magoh after tumor formation. As in CGNPs, Magoh loss was sufficient to induce DNA damage and cell death in tumor cells. Our studies show that MAGOH is necessary to promote survival in cerebellar progenitors and medulloblastoma tumor cells. Taken together, these studies may provide novel targets for SHH-driven medulloblastoma therapies.Doctor of Philosoph