ATR inhibitor camonsertib (RP-3500) suppresses early-stage erythroblasts by mediating ferroptosis

Maayan LEVY¹, Adrian FRETLAND⁴, Francesca CARLOMAGNO⁵, Anne ROULSTON⁴, Yelena GINZBURG¹, Gino FERRARO², Li LI², Yongshuai HAN³, Giorgia FEDERICO⁵, Lilian VARRICCHIO¹, Sara FOURNIER², Xiuli AN³, Ronald HOFFMAN¹

¹Icahn School Of Medicine At Mount Sinai, New York, United States
²Repare Therapeutics Inc, Saint-Laurent, Quebec, Canada
³New York Blood Center, New York, United States
⁴Repare Therapeutics Inc, Cambridge, Massachusetts, United States
⁵University of Naples, Naples, Italy

Ataxia telangiectasia mutated and Rad3-related kinase (ATR) mediates cellular response to replication stress and DNA damage. Camonsertib is a potent and selective ATR inhibitor (ATRi) with strong pre-clinical efficacy and promising clinical activity (NCT04497116) in cancer patients but leads to severe anemia. High iron requirements are essential for heme synthesis during erythropoiesis, specifically increasing erythroblast vulnerability to iron-mediated oxidative damage. We evaluate intracellular iron homeostasis by measuring effects of camonsertib on iron uptake (TFR1), storage (ferritin), and transport (PCBP1/2 and NCOA4). Iron accumulation in ferritin and NCOA4-mediated ferritinophagy is essential for normal erythropoiesis while unmitigated iron release from ferritin increases the sensitivity of cells to ferroptosis, a non-apoptotic iron-dependent form of cell death resulting from iron-mediated lipid peroxidation. We hypothesize that camonsertib induces ferroptosis in erythroblasts, causing anemia. We demonstrate that erythroblast depletion occurs following camonsertib in mice and is reversible with erythroblast regeneration during dose interruption. In addition, using human CD34+ cells induced to differentiate to erythroblasts in vitro, cells treated with camonsertib demonstrate a dose-dependent decrease in proliferation and differentiation and increase in apoptosis and reactive oxygen species (ROS). Specifically, decreased TFRC and PCBP1/2 as well as increased NCOA4 and FTH expression is consistent with enhanced ferritinophagy. In addition, we demonstrate a dose-response increase in lipid peroxidation and 4HNE in camonsertib-treated erythroblasts. Finally, consistent with increased ferroptosis, GPX4 expression decreased and the addition of ferristatin-1 prevents increased ROS in camonsertib-treated erythroblasts. Taken together, we demonstrate ferroptosis induction in camonsertib-treated erythroblasts as a potential underlying cause of ATRi-induced anemia.