Ferroptosis-mediated Cell Death in Erythroblasts Treated with ATR Inhibitor Camonsertib (RP-3500)

1) Camonsertib directly induces cell death, accumulation of ROS, labile iron, and lipid peroxidation in erythroblasts in vitro. 2) Ferrostatin-1 reverses ferritinophagy and ferroptosis in camonsertib-treated erythroblasts. ABSTRACT Ataxia telangiectasia mutated and Rad3-related kinase (ATR) mediates cellular responses to replication stress and DNA damage. Camonsertib is a potent and selective ATR inhibitor with promising clinical activity (NCT04497116) in cancer patients, but severe anemia is a common side effect of treatment. Iron is essential for heme synthesis during erythropoiesis but increases erythroblast vulnerability to iron-mediated oxidative damage. Iron accumulation in ferritin and nuclear coactivator 4 (NCOA4) mediated ferritinophagy are essential for normal erythropoiesis while during oxidative stress, iron release from ferritin promotes ferroptosis, a form of cell death resulting from iron-mediated lipid peroxidation. We hypothesize that camonsertib-induced anemia is due to increased ferroptosis occurring during terminal erythropoiesis. We previously showed that camonsertib leads to bone marrow erythroblast depletion in mice with reversal upon drug withdrawal. Currently, using primary cultures of human erythroblasts, we demonstrate that camonsertib induces a dose-dependent decrease in erythroblast number and an increase in reactive oxygen species (ROS) in erythroblasts. We also demonstrate that in camonsertib-treated erythroblasts, ferritin decreases while NCOA4 increases, consistent with enhanced ferritinophagy. Furthermore, camonsertib-treated erythroblasts exhibit a dose-dependent increase in lipid peroxidation and cellular iron accumulation. Finally, the expression of glutathione peroxidase 4 ( GPX4 ), a key regulator of ferroptosis, is decreased in camonsertib-treated erythroblasts. Camonsertib-induced increase in ROS, lipid peroxidation, and NCOA4 expression are ameliorated by ferrostatin-1 (a ferroptosis inhibitor) and N-acetylcysteine (an antioxidant agent). Taken together, our results demonstrate ferroptosis induction in camonsertib-treated erythroblasts and may represent the underlying cause of ATRi-induced anemia in patients.