PROJECT SUMMARY/ABSTRACT This proposal aims to identify the mechanisms by which ATM kinase suppresses lymphomagenesis. ATM is a tumor suppressor. Germline ATM defects cause Ataxia-Telangiectasia (A-T) syndrome with a 25% lifetime risk for lymphomas. ATM is also the 3rd most frequently germline mutated gene in cancers. Somatic inactivation of the 2nd allele is found in up to 50% of Mantel Cell Lymphomas and 4-8% of common epithelial cancers. In vitro, both DNA damage and reactive oxygen species (ROS) can activate ATM kinase. In the past two funding cycles, we investigated how ATM suppresses lymphomas via its role in DNA damage response (DDR). Using mouse models carrying ATM null, kinase-dead (KD), or missense mutations, we identified a structural function of ATM that is regulated by its kinase activity. As such, the expression of KD ATM leads to embryonic lethality, while complete loss of ATM (null) is compatible with development. Moreover, compared to A-T syndrome with little or no ATM expression, ~ 70% of ATM mutated cancer expresses inactive ATM proteins, imposing a different lymphoma risk than the null mutation. Mechanistically, we showed that ATM exchanges rapidly at DNA damage sites, and its exchange is coupled with catalysis. Thus, inactive ATM protein physically blocks DNA repair and causes hypersensitivity to TopoI inhibition. Several ongoing clinical trials are designed to target ATM-mutant cancers with TopoI inhibitors. During these studies, we found that Atm-R3008H mutation abolishes DSB-induced but not ROS-induced ATM activation and displays delayed lymphomagenesis, suggesting Atm’s role in oxidative stress response might also contribute to tumor suppression. Thus, we propose investigating how ATM regulates tumorigenesis by modulating oxidative stress responses. ROS activates purified ATM via intermolecular disulfide bonds. But whether ROS activation of ATM contributes to tumor suppression remains unknown due to 1) the lack of seperation-of-function mutation to study ATM ROS function during tumorigenesis; 2) ROS damages DNA, confounding DSB vs. ROS induced ATM function; 3) high oxygen in tissue culture stressed Atm-/- cells (20% O2 vs. 5% in blood). Here, we reported that loss of SLC7A11 (a system xC- antiporter critical for suppressing ferroptosis), but not Nrf2 (the master transcriptional regulator of oxidative response) delays lymphomagenesis in Atm-/- mice by >200 days without affecting chromosomal translocations, revealing a vulnerability of Atm-deficient cells to ferroptosis, non-apoptotic cell death by membrane lipids peroxidation and rupture. Moreover, we found that Slc7a11 is uniquely expressed among immature T cells. Based on these, we hypothesize that Atm regulates ferroptosis vulnerability to suppress immature T-cell malignancies. To test it, we will study how 1) SLC7A11 loss delays Atm-deficient thymic lymphomas, 2) ATM regulates ferroptosis sensitivity, and 3) whether ferroptosis can target ATM-deficient cancers and T-AL...