Project Summary Phospholipids are the major component of biological membranes and are essential for all cellular life. Phospholipid metabolism is altered in tumor cells. However, there are currently no approved anti-cancer drugs that target phospholipid metabolic enzymes. Inhibition of survival-promoting phospholipid metabolic enzymes would present a novel strategy to induce cell death in cancer cells. Ferroptosis is a form of cell death that is dependent on membrane phospholipid composition. Ferroptosis is caused by direct damage to membrane phospholipids by reactive oxygen species (ROS). Our group and others have found that the tail groups of phospholipids can determine a cell’s sensitivity to ferroptosis. Tail groups with multiple double-bonds (polyunsaturated fatty acids, PUFAs) are more likely to be oxidized than those with only one double bond (monounsaturated fatty acid, MUFA). Therefore, cells with greater levels of PUFAs are more susceptible to death by ferroptosis, whereas cells with greater levels of MUFAs resist ferroptosis. This is particularly relevant in cancer treatment because induction of ferroptosis is emerging as an effective strategy to kill cancer cells. In preliminary studies, I find that Membrane-Bound O-Acyltransferase 1 (MBOAT1) is a novel ferroptosis regulating gene. MBOAT1 is a lipid metabolic gene that incorporates MUFAs into phospholipids. MBOAT1 gene expression or copy number is elevated in multiple tumor types, and higher expression of MBOAT1 is correlated with poor prognosis in pancreatic cancer patients. I find that inhibiting MBOAT1 increases cancer cells’ susceptibility to ferroptosis. Therefore, MBOAT1 represents a novel target for cancer treatment. Based on these findings, I hypothesize that inhibition of MBOAT1 sensitizes cells to ferroptosis by altering phospholipid metabolism. In Aim 1, I will elucidate the role of MBOAT1 in ferroptosis sensitivity and in phospholipid metabolism. Then in Aim 2, I will test the combination of MBOAT1 inhibition and ferroptosis induction as a novel strategy for the treatment of cancer in vivo. Overall, this work will have biological significance by characterizing the function and regulation of MBOAT1, and clinical significance by establishing MBOAT1 as a novel target to induce ferroptosis in cancer cells.