Regulation of the Hedgehog pathway and Medulloblastoma response to radiochemotherapy

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Project Summary The Hedgehog (HH) pathway plays a pivotal role in diverse aspects of development and postnatal physiology. Perturbation of the HH pathway and activation of glioma-associated oncogene (GLI), a dedicated transcription factor in this pathway, is responsible for approximately 30% of medulloblastomas (MB), a common and aggressive type of pediatric brain tumor. Therefore, HH signaling has emerged as a therapeutic target for MB therapy. Despite the relevance of these insights to development and disease, substantial gaps still remain in our knowledge of the mechanisms involved in regulation of response to HH signaling and crosstalk with other pathways. Therefore, elucidating the molecular mechanisms of HH signaling is essential to advance our fundamental understanding of both developmental processes and HH-dependent MB. Combining a novel homemade Pan-anti-O-GlcNAc antibody with proteome-wide profiling of O-GlcNAcylated transcription factors by quantitative mass spectrometry, we identified a previously unknown mechanism by which the HH pathway is regulated by glucose-sensing O-GlcNAcylation. Specifically, the core component of the HH pathway (GLI1 and GLI2) is O-GlcNAcylated by O-GlcNAc transferase (OGT), which in turn regulates GLI transcriptional activity. Furthermore, GLI O-GlcNAcylation is regulated by HH ligands and the mTOR/S6K pathway. Importantly, both OGT and GLI O-GlcNAcylation are significantly elevated in MB. OGT inactivation renders MB cancer cells particularly sensitive to radiochemotherapy, suggesting that O-GlcNAcylation could serve as a novel potential therapeutic target for MB. Based on these preliminary findings, we hypothesize that (a) OGT-mediated GLI O- GlcNAcylation activates the HH pathway and promotes MB, and (b) dysregulation of O-GlcNAcylation could affect cancer cell sensitivity to radiochemotherapy. To test this hypothesis, in this application we propose to dissect the molecular mechanisms underlying O-GlcNAcylation-activated GLI in the HH pathway, determine how GLI O-GlcNAcylation is regulated by canonical and non-canonical HH signaling, and decipher the role of the OGT- GLI axis in MB growth in vivo. Our studies could address important questions regarding a novel role of O-GlcNAcylation in HH pathway and radiochemotherapy. Our multifaceted investigation is based on compelling premises and will be carried out with strong scientific rigor, thus promising to fill a major gap of knowledge and have a far-reaching conceptual advance in the field. From the translational perspective, elucidating the significance of O-GlcNAc dysregulation in MB could provide a novel biomarker and a potential alternative therapeutic strategy to treat patients with HH-dependent MB.