Role of the tRNA methyltransferase AlkBH8 in Colorectal Cancer

Project Summary: Reactive Oxygen Species (ROS) are produced as byproducts of oxidative phosphorylation and are found to be elevated in most cancers. In order to prevent ROS mediated cellular toxicity, cancer cells upregulate production of ROS detoxifying enzymes such as Glutathione Peroxidase 4 (GPx4). Essential to the catalytic activity of GPx4 is a catalytic selenocysteine residue, incorporated through specific reprogramming of the UGA-STOP codon by tRNA-selenocysteine (tRNASec). tRNASec requires chemical modification of its anticodon by the tRNA methyltransferase AlkBH8 for proper recognition and decoding ability of the UGA codon. Our preliminary data indicate that high levels of AlkBH8 are correlated with worse prognostic outcomes in colorectal cancer and that loss of AlkBH8 induces a growth arrest and reduces tumor forming ability in several colorectal cancer cell lines. However, at present almost nothing is known regarding the role of AlkBH8 and its tRNA modification in cancer cell growth and survival. Therefore, the studies proposed here are intended to fill a critical void in our understanding of tRNA modifications in cancer. First, we will use an inducible knockdown model of AlkBH8 to investigate the mechanisms following loss of AlkBH8 that lead to growth arrest and investigate whether this mechanism is conserved in in-vivo xenograft models. Second, we will utilize in-vitro analysis of recombinant AlkBH8 in order to deconvolute the contributions of each of its domains on its catalytic activity. The information generated through these studies will seek to establish a novel therapeutic target in one of the most frequently diagnosed and deadliest cancers across the world.