Ferroptosis, Cellular Metabolism, and Cancer Abstract Ferroptosis is a form of non-apoptotic cell death driven by cellular metabolism and iron-dependent lipid peroxidation. Although the physiological role of ferroptosis remains elusive, mounting evidence has established that ferroptosis impacts various pathological processes, including cancer. This competitive renewal proposal is built upon what we have achieved during the previous funding cycle and aims to further elucidate the molecular basis of ferroptosis, its interplay with metabolism, and its role in cancer. In the previous funding period, we found that multiple cellular metabolic pathways, such as autophagy, glutaminolysis, and strikingly, the normal metabolic activity of mitochondria, contribute to ferroptotic death. We also found that the CDH1-NF2-Hippo-YAP and PI3K- AKT-mTOR-SREBP signaling pathways, both highly relevant to cancer, regulate ferroptosis through modulating cellular iron homeostasis and lipid metabolism. Moreover, via a whole genome CRISPR/cas9-activation screen, we identified several lipid modifying enzymes as novel ferroptosis suppressors, further underscoring the intimate relationship between lipid metabolism and ferroptosis. Importantly, our TCGA analysis indicates overexpression of one of these enzymes, MBOAT2, predicts poor prognosis in multiple cancer types, including liver cancer, bladder cancer, and pancreatic ductal adenocarcinoma (PDAC). Based on these preliminary results, the central hypothesis of the grant is that lipid modification regulates cancer cell metabolism, invasiveness, and ferroptosis, through modulating cellular lipid storage and membrane composition; and targeting MBOAT2 in combination with ferroptosis induction holds cancer therapeutic potential. To investigate this hypothesis and to define the underlying mechanisms, we will tackle following questions. First, what is the mechanism by which these lipid modifiers protect cells fromferroptosis,do they dictate lipid peroxidation viaaltering specific phospholipidspecies, and do they communicate with SREBP, a master transcriptional regulator of lipogenesis and a potent ferroptosis suppressor (Aim-1)? Second, do these lipid modifiers modulate cellular properties such as cellular storage of lipids as energy source and plasma membrane plasticity? As these cellular properties impact cancer cell invasive/metastatic capability, metabolism, and likely redox homeostasis, is there a functional interplay between ferroptosis and these cancer-relevant cellular processes (Aim-2)? Third and directly relevant to cancer treatment (Aim-3), by using patient-derived tumor organoids, xenograft mouse models, and genetically engineered mouse models (GEMM), we will investigate how our newly-identified ferroptosis suppressors modulate tumorigenesis, metastasis and the responsiveness of cancer cells to ferroptosis induction, and assess whether the combination of MBOAT2 inhibition with ferroptosis induction can be an effective...