SUMMARY/ABSTRACT Glioblastoma multiforme (GBM), the most common brain cancer, is universally fatal with median survival of ~ 18 months. New therapies are desperately needed. GBM is heterogeneous at the cellular level with small populations of tumor propagating stem-like cells (aka, GBM stem cells, GSCs) that contribute disproportionately to GBM growth, therapeutic resistance and recurrence. Substantial evidence indicates that GBM cells are highly plastic and dynamically transition between the stem-like/tumor propagating phenotype and more differentiated/non-tumor propagating phenotype in response to contextual epigenetic events. Elucidating the epigenetic mechanisms involved in the generation and maintenance of GSCs (i.e. repression of differentiating and activation of dedifferentiation transcriptional programs) is critical to understanding the molecular underpinnings of glioblastoma malignancy and will further the development of more effective therapeutics. We have found that the reprogramming transcription factors Oct4 and Sox2 (Oct4/Sox2) phenotypically reprogram non-GSCs to tumor-propagating GSCs through the silencing of miRNAs that cooperatively inhibit the GSC phenotype. We now hypothesize that the repression of specific miRNAs that target (i) the non-histone DNA-binding protein HMGA1, and (ii) components of the polycomb repressor complex 2 (PRC2) that catalyzes histone3K27 trimethylation (H3K27me3) plays an essential role in the generation of GSCs by Oct4/Sox2. This proposal focuses on how HMGA1 and PRC2 cooperatively regulate chromatin structure and function and gene expression during the induction of tumor propagating GSCs by Oct4/Sox2 and on testing the therapeutic efficacy of inhibiting these Oct4/Sox2-regulated miRNA circuits. These goals will be achieved via three specific aims- (1) Determine the role of Oct4/Sox2-repressed miRNAs in the up-regulation of HMGA1, PRC2, H3K27me3 and GSC induction by Oct4/Sox2; (2) Determine how HMGA1 and H3K27me3 co-regulate chromatin function and transcriptional networks in response to Oct4/Sox2; (3) Develop novel GBM treatment strategies based on inhibiting these mechanism of GSC induction by Oct4/Sox2. This research will fill critical gaps in our understanding of how reprogramming transcriptions factors induce GSCs and GBM propagation. We will specifically identify how the “structural transcription factor” HMGA1 and the PRC2-dependent H3K27me3 cooperate to regulate the GSC-inducing transcriptome. We will also evaluate the potential to therapeutically target these novel GSC-drivers using innovative nanoparticle- based and pharmacologic therapeutics.