Overcoming Therapy Resistance in Hedgehog Pathway-Driven Medulloblastoma - ABSTRACT Medulloblastoma (MB) is the most common malignant pediatric brain cancer. A third of MB, called Group 2 or SHH MB, arises from the overactivation of a highly conserved cell-cell communication circuit called the Hedgehog (Hh) pathway. Several FDA-approved drugs target oncogenic Hh signaling by inhibiting SMOOTHENED (SMO), the key switch that regulates Hh pathway activity. Group 2 MB initially respond to SMO inhibitors, but these molecules ultimately lose their effectiveness due to secondary mutations within SMO. Clearly, new strategies are needed to prevent or overcome SMO-mediated drug resistance to improve patient outcomes. In this proposal, our goal is to harness new biochemical discoveries of SMO activation from our labs that identified 1) a new ligand-binding pocket deep within the SMO seven-transmembrane (7TM) region that explains how resistance emerges in SMO and suggests strategies to minimize it in the future and 2) G protein- coupled receptor kinases 2 and 3 (GRK2/3) as essential for SMO to activate downstream glioma-associated (GLI) transcription factors and stimulate expression of pro-oncogenic genes, ultimately driving tumorigenesis. Our findings immediately suggest two clear therapeutic strategies to combat chemotherapy resistance in SMO- driven malignancies. First, we hypothesize that a distinct class of SMO inhibitors which bind deep within the 7TM pocket and overlap extensively with the endogenous cholesterol ligand will be less prone to resistance than existing agents. Second, we hypothesize that GRK2/3 inhibitors will block Hh signaling arising from all existing oncogenic or therapy-resistant forms of SMO. Furthermore, we expect that combining SMO and GRK2/3 inhibitors will be even more effective than either inhibitor alone. We provide significant published and preliminary data to support these hypotheses and now propose to evaluate the extent to which SMO deep-pocket inhibitors and/or GRK2/3 inhibitors generate less tumor resistance than conventional therapies by rigorously evaluating these inhibitors using multiple assay platforms and species (fish, mice, human) of Group 2 MB. We will generate highly scalable in vivo brain tumor models in zebrafish to rapidly interrogate the impact of SMO and GRK2/3 inhibitors and leverage this knowledge to selectively test single or combination strategies as effective treatments that minimize drug resistance in genetically engineered mouse and human cell transplant models of Group 2 MB. We have assembled a team of experts covering the spectrum from biochemical/structural mechanisms of Hh pathway activation, animal models of MB and pre-clinical human cell-based in vivo models. Success in these studies will establish deep-pocket SMO and GRK2/3 inhibitors, alone or in combination, as new viable therapeutic strategies to treat Group 2 MB that forestalls or overcomes resistance more effectively than conventional SMO inhibitors. These studies have the potential to transform current treatment paradigms for Group 2 MB and other Hh-driven brain tumors and provide justification to design future clinical trials in children.
