m⁶A-RNA Demethylase FTO Inhibitors Impair Self-Renewal in Glioblastoma Stem Cells

Abstract

<i>N</i>⁶-methyladenosine (m⁶A) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. m⁶A modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA m⁶A methylation is controlled by a multiprotein "writer" complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two "eraser" proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate m⁶A in transcripts. FTO can also demethylate <i>N</i>⁶,2'-<i>O</i>-dimethyladenosine (m⁶A_m), which is found adjacent to the m⁷G cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma <i>in vivo</i> models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC₅₀'s and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased m⁶A and m⁶A_m levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma.