Glioblastoma multiforme (GBM) is a highly aggressive brain tumor associated with extremely poor prognosis and survival. Although relatively rare in the US general population, GBM is the third most common cause of cancer death in the US active-duty military. The relatively transient nature of clinical responses to currently available therapies underlines the urgency for the development of additional therapeutic strategies. Multiple studies have demonstrated the existence of aggressive cancer stem cell (CSC) subpopulations in GBM and characterized their contribution to GBM progression, therapeutic resistance and recurrence; however, efforts to eliminate or functionally modulate these therapy-refractory subpopulations have thus far met with limited success. ATP-binding cassette, sub-family B (MDR/TAP), member 5 (ABCB5), an integral plasma membrane protein first cloned and characterized by the applicant (Frank et al. J Biol Chem. 2003), is highly expressed by normal tissue-specific stem cells and CSCs in several malignancies, including GBM. ABCB5 is expressed in primary GBM tumors, in which its expression is significantly correlated with the CSC marker CD133 and with overall poor survival (Lee et al. J Biol Chem. 2020). In GBM-CSCs, ABCB5 has been shown to mediate clinically relevant drug resistance to temozolomide (TMZ). ABCB5 blockade inhibited CD133-positive GBM- CSC self-renewal and abrogated TMZ-induced G2/M arrest. Recently, the applicant identified a novel critical anti-apoptotic function of ABCB5 required for normal stem cell maintenance (Ksander et al. Nature 2014) and, as a corollary, for CSC-driven tumor growth, invasion and therapeutic resistance, involving, in part, ABCB5- dependent regulation of signal transduction of the RTK AXL (Guo et al., J Biol Chem. 2018). Importantly, the applicant’s most recent preliminary studies further revealed that ABCB5 serves as a novel receptor for Phosphatidylinositol 4,5-bisphosphate (PIP2), with ABCB5/PIP2 binding shown to be required for PIP3 phosphorylation. PIP2-derived PIP3 serves as a critical mediator of receptor tyrosine kinase (RTK) signaling, and hence activation of the downstream PI3K/AKT signaling cascade. Remarkably, inhibition of ABCB5-PIP2 binding through ABCB5 monoclonal antibody blockade or ABCB5 gene knockout (KO) inhibits PIP2 phosphorylation, blocks PIP3 production and interrupts down-stream pAKT activation of the PI3K/pAKT signaling pathway, impairing RTK signal transduction on ABCB5-positive GBM-CSC. Based on this newly identified central mechanism, we hypothesize that ABCB5 is critically required for multiple RTK-dependent functions in human GBM, including stem cell-intrinsic self-renewal, anti-apoptotic, pro-angiogenic and invasive capacities, and stem cell-driven epigenetic evolution and therapeutic resistance associated cell cycle arrest (Lee et al. J Biol Chem. 2020). The proposed studies will further support the development of ABCB5 as a novel therapeutic target in GBM and sh...