Periodic Reporting for period 3 - IRONDEATH (The advent of the Iron Age in cell death)
Periodo di rendicontazione: 2023-11-01 al 2025-04-30
Gaining control over life and death decisions in cells either affords to prevent (neuro)degenerative diseases or to combat cancer. In this context, ferroptosis, a recently described non-apoptotic cell death modality characterized by an iron-dependent overwhelming lipid peroxidation, is emerging as the root cause of numerous degenerative diseases including neurodegeneration and tissue ischemia/reperfusion injury. Conversely, ferroptosis has been proposed as a pharmacologically tractable vulnerability in therapy-resistant and de-differentiating tumors. Yet, years before the term “ferroptosis” was coined in 2012 our laboratory had shown for the first time that loss of a distinct redox enzyme, namely glutathione peroxidase 4 (GPX4), causes a novel form of non-apoptotic cell death in cells and mice, now generally known as ferroptosis. Meanwhile, we and others have shown that GPX4, along with its cofactor glutathione, is the guardian of ferroptosis. By using genome-wide genetic and pharmacological screens, our group has made several landmark discoveries unraveling all key regulatory nodes of ferroptosis, including acyl-CoA synthetase long-chain family member 4 (ACSL4) and ferroptosis suppressor protein-1 (FSP1). Furthermore, we also introduced the first in vivo efficacious ferroptosis inhibitor liproxstatin-1. Despite these key discoveries, numerous open questions have remained. For instance, it remains unclear whether there are yet-unrecognized powerful ferroptosis suppressive systems beyond GPX4 and FSP1. Besides, there are numerous conflicting reports regarding the subcellular site of an initial ferroptotic death signal. Finally, the genetic and metabolic determinants that eventually decide the cell vulnerability to ferroptosis or other forms of oxidative cell death have remained unclear. Therefore, the research program IRONDEATH aims to tackle these key issues by taking advantage of (i) genome-wide genetic suppressor screens and next-generation antibody-derived tools, (ii) a chemogenetic approach to site-specifically generate oxidative stress signals within cells, and (iii) non-targeted metabolomics and targeted oxidative lipidomics studies in representative transgenic mouse models. Findings obtained in the IRONDEATH program are expected to generate breakthrough discoveries in ferroptosis and cell death in general as they will provide novel concepts for the development of target-based in vivo applicable therapies based on ferroptosis modulation.
For the identification of yet-unrecognized ferroptosis suppressors, a genome-wide screen was conducted using a commercially available cDNA library containing about 17,000 open reading frames (ORF) of the mammalian genome. Upon ferroptosis induction and double selection of the survival cells expressing the library, a number of antiferroptotic candidate genes were revealed. Among them, FSP1 was obtained, thereby validating our screen, though GPX4 was not retrieved as the ORF library does not contain the 3’ untranslated regions, which are necessary elements for expression of selenoproteins including GPX4. Further analysis of the identified genes, however, failed to validate their potential anti-ferroptotic roles, indicating that besides GPX4 and FSP1 there are perhaps no other key ferroptosis regulators that robustly protect against ferroptosis. In parallel to these studies, four selected ferroptosis players were recombinantly expressed in Escherichia coli and used for the immunization of alpacas. For two of them nanobodies were successfully obtained which are currently being further validated for their specificity and functionality. To address which subcellular compartment is responsible for the initial ferroptotic death signal, we take advantage of a chemogenetic approach that allows the scalable generation of hydroperoxides in a time-resolved manner. Targeting this generator to different subcellular sites allowed us to pinpoint the intracellular localization that plays a decisive role in ferroptosis initiation. Based on these results, a transgenic mouse model with a spatiotemporal expression of this generator was developed that will be used to interrogate the cell-based findings in an in vivo context. For a long time, reactive oxygen species (ROS) have been considered to impact different cell death routes. To disentangle the genetic and metabolic determinants that decide upon how cells will die, a series of mouse models with tissue-specific ablation of enzymes involved in redox control are being generated that will be subjected to metabolomics and (epi)lipidomics studies under normal housing conditions and upon manipulating the antioxidant status of the mouse chow. In addition, treatment of these mouse models with next generation liproxstatins is conceived to elucidate whether these in vivo efficacious ferroptosis inhibitors are truly specific for ferroptosis or also impact on other ROS-dependent cell death pathways.
The genome-wide genetic suppressor screen failed to identify new ferroptosis suppressors, suggesting either the absence of additional antiferroptotic systems or insufficient representation of the genes in the library. Nonetheless, the nanobody-based approach allowed us to identify several highly potent nanobodies against ferroptosis regulators that will be fully validated until the end of this research program. We expect to determine whether these novel tools are suitable not only for the detection of respective target proteins, but also for the modulation of their functions. After a comprehensive cell-based characterization, these nanobodies will be applied to animal models of cancer, aiming to develop a new therapeutic approach. Upon successful identification of the subcellular site that is most relevant in triggering ferroptosis in response to a peroxide challenge, a genome-wide reverse CRISPR/Cas9 screen will be performed. This set of the studies will answer whether there are additional yet-unknown ferroptosis modulators. Furthermore, the established mouse models with spatiotemporal expression of the ROS generator will be thoroughly analyzed enduring the second term of the project with the ultimate goal to identify the most ferroptosis-prone tissue(s). Besides, we are currently cross-breeding these mice with a ferroptosis sentinel mouse line to phenocopy certain degenerative disease states associated with the aberrant activation of ferroptosis. Such models might be useful to assess the pharmacological potential of novel ferroptosis inhibitors as potential future drugs. To disentangle metabolic and genetic decision-maker determinants in cell death, a series of animal models with targeted deficiencies in enzymes of cellular redox metabolism are being generated. They will be used for non-targeted metabolomics and epilipidomics studies and a series of experiments with an altered antioxidant dietary status along with the use of ferroptosis inhibitors. The comparative and longitudinal analysis of the respective samples completed until the end of the project will allow us (i) to explore a role of these enzymes in cell metabolism and cell death, (ii) to identify potential cell death biomarkers and (iii) to assess the possible effect of ferroptosis inhibitors on other forms of oxidative cell death. In conclusion, all of the different subprojects are on track and are projected to be accomplished by the end of the IRONDEATH project.