PROJECT SUMMARY Medulloblastoma (MB) is a highly malignant tumor of the cerebellum that occurs most frequently in children. Despite advances in treatment – including surgery, radiation, and chemotherapy – approximately one-third of MB patients still die from the disease and survivors suffer severe side effects as a result of treatment. Genomic profiling and bioinformatic analyses of patient samples have identified four subgroups of MB – WNT, Sonic hedgehog (SHH), Group 3 (MYC-driven) and Group 4. These subgroups differ in terms of mutations, gene expression profiles and patient outcomes, and patients can be stratified using relevant genetic and immunohistochemical markers. Approximately one-quarter of MBs are Group 3 (G3) tumors, which exhibit overexpression or amplification of the c-Myc (MYC) oncogene. Patients with G3 MB are more likely to present with metastatic disease at time of diagnosis, have a higher incidence of recurrence and the poorest survival rate. Thus, more effective therapies for G3 MB are critically needed. To identify MYC inhibitors that would be effective in G3 MB, we developed a phenotypic, target-agnostic assay using disease-relevant cells isolated from G3 MB orthotopic patient-derived xenografts (PDXs). The assay was designed to identify small molecules that reduce endogenous MYC levels – the signature molecular marker of G3 MB – in 4 hours, to preferentially modulate targets directly affecting MYC stability and avoid indirect or off- target effects at later time points. We applied this assay to screen a 100,000 compound collection and identified small molecule scaffolds that robustly decrease cellular MYC levels. Hits were validated in a rigorous testing funnel designed to avoid undesired mechanisms of action, and initial SAR was explored for several scaffolds. From these studies the most promising series was prioritized with activities in the 100 nM potency range as well as compound properties indicative of good BBB penetration. We further confirmed that the compounds in this series decrease cell viability after 48 hours exposure and that this effect correlates with potency in the MYC assay. Preliminary data for the most active compound of this series, SBI1242, indicate that this decrease in cell viability of G3 MB patient cells is selective over iPSC-derived neurons, suggesting a possible therapeutic window. The goal of this proposal is to further optimize SBI1242 for preclinical in vivo testing and use the best analog to test our hypothesis that inhibitors of the G3 MB signature biomarker MYC identified in a highly disease-relevant context can safely and effectively arrest or reverse tumor growth in our G3 MB-specific orthotopic PDX mouse model. We anticipate that successful completion of these studies will be a significant step towards our long-term goal of identifying novel, safe, and effective treatments for MB and other MYC-driven cancers.