Academic Writing

Nucleotide synthesis is a metabolically demanding process essential for DNA replication and other processes in the cell. Several anticancer drugs that inhibit nucleotide metabolism induce apoptosis. How inhibition of nucleotide metabolism impacts non-apoptotic cell death is less clear. Here, we report that inhibition of nucleotide metabolism by the p53 pathway is sufficient to suppress the non-apoptotic cell death process of ferroptosis. Mechanistically, stabilization of wild-type p53 and induction of the p53 target gene CDKN1A (p21) leads to decreased expression of the ribonucleotide reductase (RNR) subunits RRM1 and RRM2 RNR is the rate-limiting enzyme of de novo nucleotide synthesis that reduces ribonucleotides to deoxyribonucleotides in a glutathione-dependent manner. Direct inhibition of RNR results in conservation of intracellular glutathione, limiting the accumulation of toxic lipid peroxides and preventing the onset of ferroptosis in response to cystine deprivation. These results support a mechanism linking p53-dependent regulation of nucleotide metabolism to non-apoptotic cell death.

Ferroptosis is a form of iron-dependent, non-apoptotic cell death that occurs due to the collapse of the glutathione peroxidase antioxidant system and the lethal accumulation of lipid peroxides. Selectively inducing ferroptosis in cancer cells represents a promising therapeutic strategy. However, it remains unclear howcanonical tumor suppressors and oncogenes influence cell susceptibility to ferroptosis. The p53 tumor suppressor gene is the most commonly mutated gene in human cancers and serves as a master regulator of cell stress responses, metabolic adaptation, DNA damage repair, and cell death. Using a time-lapse microscopy system to study cell death kinetics over time, I found that activation of wild-type p53 suppresses the onset of ferroptosis in human cancer cells, and that deletion of p53 results in heightened ferroptosis sensitivity. The transcriptional

activity of p53 is required for this function, in part through transactivation of CDKN1A (encoding p21). Activation of the p53-p21 pathway suppresses the accumulation of lipid peroxides and promotes the conservation of glutathione, an essential antioxidant required for the reduction of lipid peroxides. p53-p21 pathway activation results in decreased expression of the enzyme ribonucleotide reductase (RNR), which uses glutathione as a cofactor in the reduction of ribonucleotides to deoxyribonucleotides. Genetic and chemical inhibition of RNR is sufficient to suppress ferroptosis and conserve glutathione similar to chemical activators of the p53-p21 pathway. These results suggest that the ability of the p53-p21 pathway to

downregulate RNR may allow cells to conserve glutathione and direct it towards the management of lipid peroxides during ferroptosis. This work supports the known role for wild-type p53 in regulating cell survival under conditions of metabolic stress and identifies nucleotide metabolism as a new regulator of ferroptosis in cancer cells. These findings have important implications for the development of ferroptosis-based therapies for human disease.

Ferroptosis is an iron-dependent form of non-apoptotic cell death that is characterized by the accumulation of toxic lipid reactive oxygen species (ROS). The execution of ferroptosis does not appear to rely on particular protein effectors. Rather, the initiation and execution of this process depends upon modulation of intracellular metabolism. In particular, the process of lipid peroxidation is essential for ferroptosis. While the central role of lipid peroxidation in ferroptosis is clear, the precise lipid peroxide species formed during ferroptosis, as well as how and where they are generated, remain poorly understood. This chapter explores these questions and describes the role of lipids and lipid metabolism in the initiation, execution, and suppression of ferroptosis.

Ferroptosis is an iron-dependent form of non-apoptotic cell death that is characterized by the accumulation of toxic lipid reactive oxygen species (ROS). The execution of ferroptosis does not appear to rely on particular protein effectors. Rather, the initiation and execution of this process depends upon modulation of intracellular metabolism. In particular, the process of lipid peroxidation is essential for ferroptosis. While the central role of lipid peroxidation in ferroptosis is clear, the precise lipid peroxide species formed during ferroptosis, as well as how and where they are generated, remain poorly understood. This chapter explores these questions and describes the role of lipids and lipid metabolism in the initiation, execution, and suppression of ferroptosis.