Summary Glioblastoma (GBM, or astrocytoma grade IV) brain tumor remains one of the most lethal forms of human cancer and a major unmet need in current oncology. GBM is a highly heterogeneous and multifactorial disease characterized by the wide landscape of mutations and signaling alterations. Thus, most of the developing experimental therapies will likely only help a fraction of patients. We have discovered microRNA- 10b (miR-10b), a regulatory molecule whose transcriptional activation emerges as a unique mechanism shared by almost all gliomas, including both high-grade and low-grade, despite their heterogeneity. miR-10b regulates neoplastic transformation of normal glial cells and the growth of malignant gliomas. Moreover, it appears essential for the viability of heterogeneous glioma cells and glioma-initiating stem cells (GSC). Since miR-10b is highly expressed in practically all malignant gliomas, and its inhibition affects all glioma subtypes, miR-10b targeting represents a common therapeutic strategy for GBM. Despite the high levels and the critical role of miR-10b in GBM, how the expression of this molecule, which is silenced in the normal brain, gets activated in gliomagenesis is unknown. Based on our preliminary data, we hypothesize that various aberrations accumulating in the brain may converge on epigenetic alterations and reorganization of the three- dimensional structure of the miR-10b locus, resulting in its transcriptional activation. Such structural changes, primarily mediated by the CCCTC-binding factor (CTCF) and regulatory long non-coding RNA transcripts, will result in the exposure of miR-10b promoter to a corresponding enhancer, and thus miR-10b expression. In this R01 project, we will test our hypothesis, investigate the epigenetic mechanism underlying the activation of miR-10b locus in glioma, and establish the miR-10b-locus centered model of gliomagenesis. Specific Aim 1 will, therefore, provide the high-resolution analysis of the epigenetic landscape and three-dimensional chromatin conformation of miR-10b locus in normal neuroglial and glioma cells and tissues, and at the different stages of neoplastic transformation. It will also assess how generalizable such regulation is in glioma on a genomic scale. Specific Aim 2 will investigate functions of major regulatory DNA and RNA elements in the locus, including the promoter-associated and enhancer-associated RNAs, using a combination of biochemical, gene editing, and imaging-based approaches. Specific Aim 3 will model the process of the locus activation and neoplastic transformation of astrocytes and neuroprogenitors using cell and animal models of glioma. Discovery of the mechanism(s) converging on miR-10b expression in the brain cortex will shed light on the origin and etiology of ma...