Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality1,2. Here, using loss-of-function screening based on RNA interference, we show that environmental oxygen levels are a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3–8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron–sulfur cluster biosynthetic enzyme NFS1. Mammary or subcutaneous tumours grow despite suppression of NFS1, whereas metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the iron–sulfur co-factors present in multiple cell-essential proteins upon exposure to oxygen compared to other forms of oxidative damage. Furthermore, insufficient iron–sulfur cluster maintenance robustly activates the iron-starvation response and, in combination with inhibition of glutathione biosynthesis, triggers ferroptosis, a nonapoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage.

Intracellular iron levels are strictly regulated to support homeostasis and avoid iron-mediated ROS production. Loss of iron-sulfur cluster (ISC) synthesis can increase iron loading and promote cell death by ferroptosis. Iron-responsive element-binding proteins IRP1 and IRP2 posttranscriptionally regulate iron homeostasis. IRP1 binding to target mRNAs is competitively regulated by ISC occupancy. However, IRP2 is principally thought to be regulated at the protein level via E3 ubiquitin ligase FBXL5–mediated degradation. Here, we show that ISC synthesis suppression can activate IRP2 and promote ferroptosis sensitivity via a previously unidentified mechanism. At tissue-level O2 concentrations, ISC deficiency enhances IRP2 binding to target mRNAs independent of IRP1, FBXL5, and changes in IRP2 protein level. Deletion of both IRP1 and IRP2 abolishes the iron-starvation response, preventing its activation by ISC synthesis inhibition. These findings will inform strategies to manipulate ferroptosis sensitivity and help illuminate the mechanism underlying ISC biosynthesis disorders, such as Friedreich’s ataxia.

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The ER-bound kinase/endoribonuclease (RNase), inositol-requiring enzyme-1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1's one known, specific RNA target, X boxbinding protein-1 (XBP1) or the RNA substrates of IRE1-dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide-derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross-talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1's RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P 3) 5phosphatase-2 (INPPL1) is a direct target of miR-2137, which controls PI(3,4,5)P 3 levels in macrophages. The modulation of cellular PI(3,4,5)P 3 /PIP 2 ratio and anabolic mTOR signaling by the IRE1induced miR-2137 demonstrates how the ER can provide a critical input into cell growth decisions.

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The authors regret to report that errors in data reporting were introduced after peer review during finalization of data for publication.In Fig. 2b, the data points in the "Poor" and "Moderate" groups are shifted by approximately -30 units on the y axis. The original Fig. 2b is shown next to the corrected panel with the correct values (Supplementary Fig. 1).In the Source Data for Fig. 3b, a column is partially duplicated. These duplicated data points were erroneously reported on the panel for Fig. 3b. The original Fig. 3b is shown next to the corrected panel without the erroneous replicates (Supplementary Fig. 2). The source data for the original Fig. 3b have also been corrected and are provided. In the text, the Fig. 3 legend should have read "Five-day proliferation assay, MDA-MB-231 cells, 3% O 2 (n = 4) or 21% O 2 and with trolox (100 μM, n = 5), Fer-1 (1 μM, n = 3) or N-acetylcysteine (NAC, 750 μM, n = 3)."Also, in Extended Data Fig. 4h, the two representative images shown on the top right, while independently acquired, are from partially overlapping areas of the same tumour. We have replaced the partially duplicated image with an independent representative image (Supplementary Fig. 3).Additional supplementary files are now provided (Supplementary Figs. 1-3). Amended source data are included for Fig. 3b. Additional source data are now provided for all images (Figs. 2c and 4e and Extended Data Figs. 3b, 3c, 3f, 4c, 4h, 6i, 7a and 7b).

Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality. Here, using RNAi-based loss of function screening, we identify environmental oxygen level as a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3-8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron-sulfur cluster (ISC) biosynthetic enzyme NFS1. Accordingly, mammary or subcutaneous tumours grow despite NFS1 suppression, while metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the ISC cofactors present in multiple cell-essential proteins upon exposure to O2 compared to other forms of oxidative damage. Additionally, insufficient ISC maintenance robustly activates the iron-starvation response and, in combination with glutathione biosynthesis inhibition, triggers ferroptosis, a non-apoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage. These observations lead to the tantalizing hypothesis that one can trick cancer cells into taking up large quantities of iron and releasing intracellular iron stores via modulation of NFS1 or downstream effectors, such as IRPs, leaving them at increased risk for ROS-mediated cell death mechanisms such as ferroptosis. Citation Format: Samantha W. Alvarez, Vladislav O. Sviderskiy, Erdem M. Terzi, Thales Papagiannakopoulos, Andre L. Moreira, Sylvia Adams, David M. Sabatini, Kıvanç Birsoy, Richard L. Possemato. NFS1 undergoes positive selection in lung tumors and protects cells from ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr NG02.