Glioblastoma (GBM) is the most common and lethal brain tumor in adults, claiming about 14,000 lives annually in the U.S. alone. GBM is resistant to radiation and chemotherapy and this appears due partly to genetic diversity and partly to a sub-population of stem-like cells (GSCs) that resist standard therapy and repopulate the tumor. Several groups have recently identified MBNL1 as a key mediator of cellular differentiation of embryonic stem cells. In our preliminary work we show that MBNL1 inhibits glioblastoma stem cell self-renewal in vitro and tumor initiation and growth in vivo. Because GSC have been shown to be intrinsically more resistant to ionizing radiation than their differentiated progenies, we propose to test if MBNL1 may sensitize GSC to ionizing radiation by promoting their differentiation. If true, these results should have strong impact. Our long-term goal is to develop pharmacological means to activate MBNL1 protein as effective new therapies for GBM and other malignant brain tumors. Our findings will likely not be limited to brain tumors as MBNL1 inactivation is the mechanism in the pathophysiology of myotonic dystrophy. Therefore, our studies should have cross-disease implications. The central hypothesis of this proposal is that the hypoxic microenvironment inactivates MBNL1 to drive aggressive tumor behavior (proliferation, self-renewal, invasion, and survival) and that the mechanism is a switch towards fetal-like splicing patterns of pre-mRNA targets (Aim1). Because GSC have been previously shown to be intrinsically resistant to radiation, we also propose that inducing MBNL1 in preformed tumors will promote adult- like (differentiation) splicing patterns and will sensitize GBM to standard of care radiation (Aim2). Our proposal contains two aims: Specific Aim 1: To determine the effects of the hypoxic microenvironments on MBNL1 and to define GSC vulnerability to MBNL1 activity under these conditions. Specific Aim 2: To exploit GSC vulnerability to MBNL1 activity as a therapeutic strategy for GBM. MBNL1 expression is commonly repressed in hypoxic elements of GBM and hypoxia has been linked to negative prognosis for survival in GBM patients. We expect our studies to establish this developmentally-relevant, key differentiation inducing splicing-factor, as a novel therapeutic target in GBM. These results are of high clinical relevance, and will provide the rationale for the development of small molecules that can increase MBNL1 activity. Successful completion of the proposed studies will further illuminate the biology of MBNL1 in GBM and establish MBNL1 as a target with multiple mechanisms of action against cancer, with a strong likelihood for therapeutic impact in patients with GBM.