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Abstract

Despite advancements in the treatment of acute myeloid leukemia (AML), the 5-year survival rate of AML patients remains poor. The majority of patients face disease recurrence partly due to acquired resistance against classical cell death programs. Therefore, exploiting alternative forms of cell death, such as ferroptosis, may allow to design novel therapies to limit survival or occurrence of such resistant AML clones. Ferroptosis is a recently identified iron-dependent and non-apoptotic form of cell death. It is characterized by an excessive ROS-induced peroxidation of poly-unsaturated fatty acids (PUFAs) within cell membranes and its chemical induction has shown promising therapeutic potential in multiple solid cancer entities. The work provided here aimed to investigate the role of ferroptosis in AML and explore its therapeutic possibilities for the treatment of AML. In this thesis, I first investigated the overall dependency of AML on ferroptosis-related genes (FRGs) using publicly available data sets. Secondly, I evaluated the ferroptosis sensitivity of various AML cell lines through chemical inhibition and genetic deletion of ferroptosis regulators. With these insights, I demonstrated the essentiality of the master ferroptosis suppressor GPX4 in AML cell lines in vitro as well as in vivo and revealed high sensitivity of AML cell lines to chemical ferroptosis induction with Imidazole Ketone Erastin (IKE). In addition, I described the prognostic value of several FRGs and generated a 7-gene ferroptosis signature as a powerful predictor of patient survival. By performing a comprehensive drug screen aimed at assessing the ferroptosis-inducing capacity of the drugs, I identified arsenic trioxide (ATO) as a potent inducer of ferroptosis in AML cells. Notably, the detailed characterization of the drug’s mechanism of action on the RNA level using SLAM-Sequencing revealed the activity of the iron-metabolism gene HMOX1 as being essential for ATO-induced ferroptosis. Mechanistically, we showed that ATO directly interacts with GPX4, one of the major ferroptosis-suppressive regulators, resulting in its inhibition and degradation by the proteasomal system. These findings position ATO as a new class II ferroptosis-inducing agent directly acting on GPX4, for which no clinically usable inhibitor exists to date. By performing targeted mass spectrometry and metabolite tracing experiments, we highlighted the importance of glutaminolysis for glutathione (GSH) synthesis. I further demonstrated that ATO treatment results in upregulation of metabolic pathways providing cysteine for GSH synthesis; namely SLC7A11-mediated cystine-uptake and the transsulfuration pathway. Chemical inhibition as well as genetic deletion of these pathways combined with ATO treatment showed strong synergistic effects marking these pathways as new targetable vulnerabilities in AML to enhance ATO-induced ferroptosis. By combining these findings, I designed a novel triple combination therapy (ATO+ CB-839+PPG) targeting the glutaminolysis and transsulfuration pathways in combination with ATO-mediated GPX4 inhibition and degradation. I demonstrated that the triple combination therapy resulted in high efficacy in killing AML cell lines as well as primary AML patient samples at diagnosis and relapse. Moreover, this triple combination therapy was effective in venetoclax + azacytidine (VenAza) resistant AML cell lines, providing a potential means to overcome VenAza resistance. Moreover, I demonstrated that the ferroptosis inducers ATO and IKE synergize with VenAza treatment, offering additional promising combination therapies. In summary, the results in this thesis classify AML as one of the most ferroptosis-sensitive cancer entities. Moreover, we revealed the ferroptosis-inducing capacity of ATO as a novel GPX4 inhibitor and degrader. Detailed characterization of the drug’s mechanism on the genetic and metabolic level enabled me to design new effective combination therapies exploiting newly identified vulnerabilities in AML. These results provide a rational basis for a pre-clinical and subsequent clinical evaluation of ferroptosis-inducing therapies in AML patients.