PROPOSAL ABSTRACT This proposal will develop CDK4/6 inhibitor therapy for medulloblastoma, using a novel, nanoparticle formulation of palbociclib, studied in vivo in transgenic medulloblastoma-prone mice and combining with the OLIG2 inhibitor CT-179. Medulloblastoma is the most common malignant pediatric brain tumor. New medulloblastoma treatments are needed because current therapy with surgery radiation and chemotherapy fails 20% of patients and leaves survivors at risk for neurocognitive injury, growth defects, and psychosocial impairment. While medulloblastoma is a heterogenous disease with four subgroups, all subgroups have intact RB and require CDK4/6 activity. The CyclinD1/CDK4/6/Retinoblastoma pathway is therefore a druggable target shared amongst the medulloblastoma subgroups. I have found that a nanoparticle formulation of the FDA- approved CDK 4/6 inhibitor palbociclib shows reduced systemic toxicity compared to the parent drug and can extend the survival of transgenic mice with endogenous, SHH-driven medulloblastoma. Here, I show that palbociclib produces both the expected effect of reduced RB phosphorylation and also unexpected effects, including a durable prolongation of S phase and an increase in OLIG2-expressing stem cells. I now propose in SA1 to define the mechanism of S phase alterations and identify sensitive and resistant populations of medulloblastoma cells, using single-cell transcriptomic analysis (scRNA-seq), western blot and immunohistochemistry. These studies will demonstrate canonical and non-canonical mechanisms of action, and identify mechanisms of resistance that can be targeted in future studies. In SA2, I propose to test the therapeutic efficacy of combining palbociclib therapy with the OLIG2 inhibitor CT-179. Clinical practice has shown that no drug used as a single agent is curative for medulloblastoma, and all current treatments depend on combinations of agents. The combination of palbociclib and CT-179 is rationally chosen based on my preliminary data. Completing my Research Proposal and Training Plan will provide me with didactic and experiential learning opportunities in a diverse range are approaches, from mouse genetics, to multi-dimensional cytometric assays, to computational analysis of scRNA-seq data. This training will allow me to develop basic and translational research skills and build a foundation for a career as an innovative, independent research scientist.