Abstract Cancer stem cells exert enormous influence on neoplastic behavior, in part by governing asymmetric cell division and the balance between self-renewal & multipotent differentiation. Growth is favored by deregulated stem cell division, which enhances the self-renewing population & diminishes the differentiation program. To uncover mechanisms relevant to deregulated cell division in human glioma stem cells, we first developed a novel adult Drosophila brain tumor model, 100% enriched with tumor stem cells. Using this model, we performed a suppressor screen for kinases and found brain-specific dCdk5 partially reverses the tumor phenotype. dCdk5 and its human ortholog CDK5 (79% identity) are atypical protein kinases because they do not regulate cell cycle in normal cells, but are critical for neurogenesis & survival of mature neurons. Interestingly, many cancers show overactivation of CDK5 signaling, regulating cell cycle & leading to uncontrolled proliferation. Our analysis of TCGA data shows that IDH wt GBM has significantly high level of CDK5 compared to the IDH wt lower grade glioma. Additionally, we found a strong positive correlation between CDK5 & several stem cell markers in GBM. To date, not much is known about the role of CDK5 in glioblastoma GBM) & glioma stem cell self-renewal. Using patient-derived (GBM) neurosphere cultures & in vivo xenograft tumors in mice, we further demonstrated that a novel pharmaceutical suppressor of CDK5 signaling axis can suppress self-renewal properties through a new CDK5 phosphorylation site on CREB and Myc. Together, our results show that the CDK5 signaling pathway is important for glioma stem cell survival & that suppressing CDK5 could be a novel therapeutic approach to eliminate glioma stem cells. Citation Format: Subhas Mukherjee, Carol Tucker-Burden, Changming Zhang, Jun Kong, Monica Chau, Renee Read, Daniel J. Brat. Novel inhibitor of GBM stem cells targets unique CREB & Myc-mediated self-renewal pathways regulated by CDK5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2017-3897