Despite aggressive therapy consisting of surgery followed by radio/chemotherapy GBM recurs in almost all patients with a progression-free survival of only 10 weeks. Currently there are no proven therapies to treat recurrent GBM largely due to our inadequate molecular understanding of the disease. Glioma stem-cells (GSCs) are critical determinants of intra-tumor heterogeneity, tumor propagation, therapeutic resistance, and recurrence following treatment. This project entitled Molecular determinants of cellular heterogeneity and therapeutic resistance in GBM brings together a cross-disciplinary team of experts using state-of-the art GBM cell models, single-cell genomics, and innovative gene-delivery technology to understand how GSC-driving mechanisms contribute to the generation of tumor-propagating and therapy resistant cells in GBM with the goal of defining novel therapeutic targets. Aim 1 of this proposal will seek to determine how stem-cell driving mechanisms induce therapy resistant GSCs. Mechanistically, we will test the hypothesis that Oct4 and Sox2 drive/maintain a therapy-resistant phenotype in GSCs by activating TGFBR2 expression and function via an Oct4/Sox2:ELF3:TGFBR2 axis. As a mid-term strategy to dissect the molecular mechanism driving therapy- resistance in GBM we will combine our validated cell systems and the state-of-the-art 10x Genomics Chromium pipeline to interrogate the transcriptome and chromatin state of patient-derived glioma cell lines their therapy- resistant counterparts at the single cell level. Aim 2 of this proposal will focus on developing novel molecular agents that target therapy-resistant GBM cell populations. We will build on the positive momentum of our recent novel developments in miRNA-based therapeutics to design molecular approaches to better inhibit tumor growth, prevent emergence of therapy-resistant cell subpopulations, and sensitize therapy-resistant cells to chemo/radiation in human xenograft models of GBM. Our preliminary data shows that miR-149-3p can inhibit 8 putative oncogenes simultaneously whose coordinate action drive tumor maintenance and therapeutic resistance. Aim 2.1 of this proposal will explore the mechanistic contribution of miR-149-3p to the therapy- resistant phenotype of GSCs. Aim 2.2 will investigate the pre-clinical translatability of our new-found concepts by testing novel combinations of miRNAs to more effectively normalize oncogenic networks dysregulated by stem-cell driving mechanisms in GBM. Completion of this study will: (i) Define the cell sub-populations capable of tumor propagation; (ii) define cell populations capable of transitioning to a therapy-resistant state; (iii) determine transcriptomic and chromatin changes associated with these cell populations that are amenable to therapeutic targeting; (iv) provide novel rational pre-clinical therapeutics to potentially treat recurrent GBM. Completion of this project will provide a roadmap to both understanding and more effect...