Epitranscriptomics is an emerging field that seeks to identify and understand chemical modifications in RNA; the enzymes that deposit, remove, and interpret the modifications (writers, erasers, and readers, respectively); and their effects on gene expression via regulation of RNA metabolism, function, and localization. Glioblastoma multiforme (GBM) is the deadliest brain tumor identified in both adults and children, with an average life expectancy of 15 months. GBM is characterized by high rates of both tumor infiltration and recurrence and is often resistant to treatment with radiation and chemotherapy. These characteristics have been attributed to the presence of undifferentiated glioblastoma tumor-initiating cells or glioblastoma stem-like tumor initiating cells (GSCs). Recent studies have shown that cells depleted in N6- methyladenosine (m6A) RNA modifications are resistant to differentiation, and it is suspected that misregulation of the reversible m6A pathway may play a role in generating tumor-initiating cells and promoting tumorigenesis. ALKBH5 expression is elevated in primary and established GBM cells enriched with GSCs, and high expression is correlated with poor prognosis in GBM patients. The objective of this proposal is to identify and develop novel inhibitors of the RNA demethylase alkylation repair homolog protein 5 (ALKBH5) as potential chemotherapeutics for glioblastoma multiforme. Our project has three aims. Aim 1: to optimize leads as potent and selective inhibitors of ALKBH5 with rational drug design. Aim 2: to establish the enzymatic and cellular mechanism of action of ALKBH5 inhibitors. Aim 3: to establish ALKBH5 inhibitors as effective antiproliferative agents in GSCs. The expected outcome of this work is a chemically diverse set of the first ALKBH5 inhibitors. This project will provide a clear positive impact by providing important groundwork for developing ALKBH5-targeted treatments of GBM.