Glioblastoma stem-like cells (or cancer stem cells, CSCs) maintain a capacity for multi-lineage differentiation and efficiently propagate tumor xenografts that accurately recapitulate the complex histopathology of clinical glioblastoma. Identifying and targeting the molecular mechanisms that regulate the GBM-CSC phenotype holds great promise for ultimately depleting tumors of their CSCs that are currently believed to have a major role in therapeutic resistance and tumor recurrence. Kruppel-like factor 9 (KLF9) is a poorly understood transcription factor with no significant previous link to cancer or stem cells. We recently found that KLF9 potently induces glioblastoma cancer stem cell (GBM-CSC) differentiation, inhibits GBM-CSC self-renewal, and suppresses the growth of GBM-CSC derived tumor xenografts. These tumor suppressing effects of KLF9 were found to result in part from the direct repression of Notch1 transcription. KLF9 is likely to modulate an extensive transcriptional network since it recognizes GC-GT BTE sites that are common in transcriptional promoters. This proposal is based on the general hypothesis that KLF9 regulates a transcriptional network that suppresses the oncogenic phenotype and therapeutic resistance of GBM-CSCs. The general goals of this proposal are to determine KLF9's therapeutic and tumor suppressing effects, its transcriptional targets, and to develop a direct cell-penetrating form of KLF9 for potential clinical translation. Aim #1 will use human GBM sphere-forming cells enriched in GBM-CSCs and their orthotopic tumor xenografts to determine how KLF9 modulates GBM-CSC responses to radiation and temozolomide chemotherapy, cytotoxic modalities that are currently the mainstay of GBM therapy. Aim #2 will identify the transcriptional networks regulated by KLF9 in GBM-CSCs using ChIP-Chip, gene expression array, and extensive bioinformatics analyses. Aim #3 will use the in vivo RCAS/tv-a system to determine if KLF9 modulates in vivo transformation of multipotent neural stem/progenitor cells and subsequent gliomagenesis. Aim #4 will use KLF9 modified with carboxy-terminal poly-arginines (KLF9-11R) to develop direct cell- penetrating KLF9 protein for targeting GBM-CSCs. Our discovery that KLF9 induction is differentiating, tumor suppressing, and radiation sensitizing in human GBM-CSCs is novel with broad biological and clinical translatable implications. Positive results from these experiments will significantly impact the goals to identify and understand molecular regulators of neoplastic stem cells and to ultimately control their growth, fate, and chemo/radiation sensitivity for brain cancer therapy.