Elevated FSP1 protects KRAS-mutated cells from ferroptosis during tumor initiation.

Oncogenic KRAS is the key driver oncogene for several of the most aggressive human cancers. One key feature of oncogenic KRAS expression is an early increase in cellular reactive oxygen species (ROS) which promotes cellular transformation if cells manage to escape cell death, mechanisms of which remain incompletely understood. Here, we identify that expression of oncogenic as compared to WT KRAS in isogenic cellular systems renders cells more resistant to ferroptosis, a recently described type of regulated necrosis. Mechanistically, we find that cells with mutant KRAS show a specific lack of ferroptosis-induced lipid peroxidation. Interestingly, KRAS-mutant cells upregulate expression of ferroptosis suppressor protein 1 (FSP1). Indeed, elevated levels of FSP1 in KRAS-mutant cells are responsible for mediating ferroptosis resistance and FSP1 is upregulated as a consequence of MAPK and NRF2 pathway activation downstream of KRAS. Strikingly, FSP1 activity promotes cellular transformation in soft agar and its overexpression is sufficient to promote spheroid growth in 3D in KRAS WT cells. Moreover, FSP1 expression and its activity in ferroptosis inhibition accelerates tumor onset of KRAS WT cells in the absence of oncogenic KRAS in vivo. Consequently, we find that pharmacological induction of ferroptosis in pancreatic organoids derived from the LsL-KRASG12D expressing mouse model is only effective in combination with FSP1 inhibition. Lastly, FSP1 is upregulated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) as compared to the respective normal tissue of origin and correlates with NRF2 expression in PDAC patient datasets. Based on these data, we propose that KRAS-mutant cells must navigate a ferroptosis checkpoint by upregulating FSP1 during tumor establishment. Consequently, ferroptosis-inducing therapy should be combined with FSP1 inhibitors for efficient therapy of KRAS-mutant cancers.

Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes.

Loss of TP53 and RB1 in treatment-naïve small cell lung cancer (SCLC) suggests selective pressure to inactivate cell death pathways prior to therapy. Yet, which of these pathways remain available in treatment-naïve SCLC is unknown. Here, through systemic analysis of cell death pathway availability in treatment-naïve SCLC, we identify non-neuroendocrine (NE) SCLC to be vulnerable to ferroptosis through subtype-specific lipidome remodeling. While NE SCLC is ferroptosis resistant, it acquires selective addiction to the TRX anti-oxidant pathway. In experimental settings of non-NE/NE intratumoral heterogeneity, non-NE or NE populations are selectively depleted by ferroptosis or TRX pathway inhibition, respectively. Preventing subtype plasticity observed under single pathway targeting, combined treatment kills established non-NE and NE tumors in xenografts, genetically engineered mouse models of SCLC and patient-derived cells, and identifies a patient subset with drastically improved overall survival. These findings reveal cell death pathway mining as a means to identify rational combination therapies for SCLC.

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics.

Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca2+ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways.

Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown to block transferrin receptor endocytosis, can protect from ischemia/reperfusion injury as well as neuronal cell death following spinal cord injury. Yet, it is unknown how dynasore exerts these cell death-protective effects. Using small interfering RNA suppression, lipid reactive oxygen species (ROS), iron tracers and bona fide inducers of ferroptosis, we find that dynasore treatment in lung adenocarcinoma and neuronal cell lines strongly protects these from ferroptosis. Surprisingly, while the dynasore targets dynamin 1 and 2 promote extracellular iron uptake, their silencing was not sufficient to block ferroptosis suggesting that this route of extracellular iron uptake is dispensable for acute induction of ferroptosis and dynasore must have an additional off-target activity mediating full ferroptosis protection. Instead, in intact cells, dynasore inhibited mitochondrial respiration and thereby mitochondrial ROS production which can feed into detrimental lipid peroxidation and ferroptotic cell death in the presence of labile iron. In addition, in cell free systems, dynasore showed radical scavenger properties and acted as a broadly active antioxidant which is superior to N-acetylcysteine (NAC) in blocking ferroptosis. Thus, dynasore can function as a highly active inhibitor of ROS-driven types of cell death via combined modulation of the iron pool and inhibition of general ROS by simultaneously blocking two routes required for ROS and lipid-ROS driven cell death, respectively. These data have important implications for the interpretation of studies observing tissue-protective effects of this dynamin inhibitor as well as raise awareness that off-target ROS scavenging activities of small molecules used to interrogate the ferroptosis pathway should be taken into consideration.