Heme metabolism plays a critical role in sensitizing cells to ferroptosis

Anand RUBAN AGARVAS1,2, Marlène LE TERTRE1,2, Veronica FIORITO3, Lianghao MAO4, Emanuela TOLOSANO3, Ashok Kumar JAYAVELU4,5, Martina MUCKENTHALER1,2,5

11Department of Pediatric Hematology, Oncology and Immunology, University Hospital Heidelberg, Heidelberg, Germany
2Molecular Medicine Partnership Unit, Heidelberg, Germany
33Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Turin, Italy
4Proteomics and Cancer Cell Signaling, German Cancer Research Center (DKFZ), Heidelberg, Germany
5Hopps Children's Cancer Center (KiTZ), Heidelberg, Germany

Iron plays a well-established role in ferroptosis, but the contribution of heme-bound iron remains unclear. Heme oxygenase 1 (HMOX1) mediated heme catabolism can release free iron, however, both pro-and anti-ferroptotic roles of HMOX1 have been reported. Here, we investigate how heme and its metabolism affect ferroptosis sensitivity. To induce ferroptosis, we deprived HT1080 cells of cysteine (either by switching to a cysteine-deficient medium or via treatment with Erastin, an inhibitor of cystine importer SLC7A11) and show that intracellular heme levels are increased 4h later. Both, inhibition of heme breakdown via HMOX1 inhibitors as well as inhibition of two enzymes (Aminolevulinic acid synthase 1 or Ferrochelatase) required for heme biosynthesis protected from ferroptosis. Interestingly, inhibition of heme synthesis further prevents the sensitization to ferroptosis by exogenous iron. These results strongly suggest that heme-bound iron plays an essential role in ferroptosis. To test the requirements for heme and iron in a time-dependent manner we induced ferroptosis and added either an HMOX1 inhibitor (Zinc Protoporphyrin IX), an iron chelator (Desferroxamine) or a ferroptosis inhibitor (Liproxstatin-1; lipid peroxide scavenger) at different time points. Intriguingly, we found that heme degradation is an early event that precedes the requirement for iron or lipid peroxidation during ferroptosis. Our results reveal important insights into how various iron sources influence ferroptosis and show that iron derived from heme is essential in the earliest phases of ferroptosis induction. Experiments are ongoing to understand the regulation of players of iron and heme metabolism during ferroptosis.