Abstract Improving the survival of children with central nervous system (CNS) cancers, especially brain cancers such as medulloblastoma (MB), the most frequent CNS cancer in children, will require overcoming two therapeutic barriers: the blood brain barrier (BBB) and blood tumor barrier (BTB). Medulloblastoma (MB), a malignant pediatric brain tumor of the cerebellum, is the most common brain tumor and leading cause of non-accidental death in children and adults, especially children under 5 years. Overall the Group 3 (G3) subtype of MB has the worst overall prognosis, expresses the ABC transporter, ABCG2, and are the most difficult to treat. High expression of ABCG2 in G3 MB is associated with poor overall survival. We have shown in a pre-clinical murine model of G3 MB (that faithfully recapitulates the human disease) that ABCG2 in the tumor (the BTB) remains an obstacle to effective therapy. ABCG2's high expression at the BBB also limits the therapeutic effectiveness of many active CNS medications. Overcoming ABCG2 activity by more than competitive inhibition is a desirable strategy and likely to provide a more durable suppression of ABCG2 activity and consequently improvement in chemotherapeutic efficacy. Our promising preliminary data shows that we have developed a novel ABCG2 pharmacophore model and used this to identify a new ABCG2 inhibitor that promotes ABCG2 loss of function by activating retrieval from the membrane. The membrane retrieval of ABCG2 appears to be initiated by a specific protein interaction that is driven by the inhibitor because removal of the inhibitor results in loss of the protein interaction. We propose three complementary Aims that encompass studies that will mechanistically elucidate how this new inhibitor not only initiates the retrieval of ABCG2 from the membrane, but also determines its efficacy compared to a conventional ABCG2 inhibitor. The aims will: 1) test the hypothesis that ABCG2 inhibition can be driven by loss of membrane localization. 2) elucidate the genetic and molecular pathway by which the inhibitor promotes ABCG2 membrane retrieval. 3) determine if the novel ABCG2 inhibitor is more effective than conventional inhibitors in improving the chemotherapeutic efficacy in G3 MB. The knowledge from this project will have important therapeutic implications and promote improved strategies for treating children with G3 MB.