Nature | Vol 585 | 24 September 2020 | 603Article
Plasticity of ether lipids promotes
ferroptosis susceptibility and evasion
Yilong Zou1,2,8 ✉, Whitney S. Henry3,8, Emily L. Ricq1,2,8, Emily T. Graham^1 , Vaishnavi V. Phadnis^3 ,
Pema Maretich^4 , Sateja Paradkar^3 , Natalie Boehnke^5 , Amy A. Deik^1 , Ferenc Reinhardt^3 ,
John K. Eaton^1 , Bryan Ferguson^1 , Wenyu Wang^1 , Joshua Fairman^3 , Heather R. Keys^3 ,
Vlado Dančík^1 , Clary B. Clish^1 , Paul A. Clemons^1 , Paula T. Hammond5,6, Laurie A. Boyer4,7,
Robert A. Weinberg^3 ✉ & Stuart L. Schreiber1,2 ✉Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in
various degenerative diseases and represents a targetable susceptibility in certain
cancers^1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise
from certain lineages or be acquired during cell-state transitions^2 –^5. However,
precisely how susceptibility to ferroptosis is dynamically regulated remains poorly
understood. Here we use genome-wide CRISPR–Cas9 suppressor screens to identify
the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity
in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that
peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether
phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in
turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive
to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is
associated with extensive downregulation of PUFA-ePLs. We further find that the
pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other
cell types, including neurons and cardiomyocytes. Together, our work reveals roles
for the peroxisome–ether-phospholipid axis in driving susceptibility to and evasion
from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is
dynamically regulated during cell-state transitions, and suggests multiple regulatory
nodes for therapeutic interventions in diseases that involve ferroptosis.The molecular and metabolic basis that underlies the dynamic regu-
lation of ferroptosis sensitivity is poorly understood. To identify fac-
tors that modulate susceptibility to ferroptosis, we performed two
independent genome-wide CRISPR–Cas9 suppressor screens in the
ferroptosis-susceptible clear-cell renal cell carcinoma (ccRCC) model
786-O^3 and the high-grade serous ovarian carcinoma model OVCAR-8
(Fig. 1a). In both models, ferroptosis is induced through inhibition
of the lipid peroxidation repair enzyme glutathione peroxidase 4
(GPX4) using either ML210 or 1S,3R-RSL3 (RSL3)^3 ,^6 ,^7 (Supplementary
Video 1). Both screens revealed known ferroptosis regulators—includ-
ing acyl-CoA synthetase long-chain family member 4 (ACSL4)^8 (Fig. 1b,
Extended Data Fig. 1a, b, Supplementary Data 1)—confirming the robust-
ness of our screens for identifying mediators of ferroptosis sensitivity.
Among the previously uncharacterized pro-ferroptotic genes, per-
oxisome components emerged as the most enriched gene cluster using
both STRING (a protein network database^9 ) and a pathway analysis
algorithm we developed named Gene-List Network Enrichment Analysis
(GeLiNEA) (Extended Data Fig. 1a–e, Supplementary Data 2). Peroxiso-
mal genes identified in both screens include peroxisomal biogenesis
genes PEX10 and PEX3^10 ,^11 , and those encoding the peroxisomal enzymes
alkylglycerone phosphate synthase (AGPS) and fatty acyl-CoA reductase
1 (FAR1)^12 ; other peroxisomal genes—including glyceronephosphate
O-acyltransferase (GNPAT), PEX12 and PEX7—were significantly enriched
hits at each individual time point (Fig. 1b, Extended Data Fig. 1a, b, f, g).
Because peroxisomes had not previously been implicated in fer-
roptotic cell death, we focused on elucidating their possible role in
this process. We found that depleting genes encoding PEX3, PEX10 and
PEX12 in both OVCAR-8 and 786-O cells using CRISPR–Cas9 reduced
the abundance of peroxisomes and diminished the sensitivity of the
cells to ferroptosis induced by GPX4 inhibition (Fig. 1c, Extended
Data Fig. 2a–d). Conversely, experimental overexpression of mouse
Pex3 or Pex10 cDNAs resistant to inactivation by the corresponding
human single-guide RNAs (sgRNAs) restored sensitivity to ferroptosis
(Extended Data Fig. 2e, f ), supporting a role of peroxisomes as contribu-
tors to ferroptosis susceptibility in renal and ovarian carcinoma cells.
Among other biochemical functions, peroxisomes detoxify
cytosolic reactive oxygen species and initiate the degradation of
very-long-chain and branched-chain fatty acids^11. In addition, per-
oxisomes participate in the biosynthesis of ether-linked glycerolip-
ids that, unlike ester-linked diacyl glycerolipids (R^1 CH 2 CO 2 CH 2 R^2 ),https://doi.org/10.1038/s41586-020-2732-8
Received: 26 November 2019
Accepted: 24 June 2020
Published online: 16 September 2020
Check for updates(^1) Broad Institute, Cambridge, MA, USA. (^2) Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. (^3) Whitehead Institute for Biomedical Research, Cambridge,
MA, USA.^4 Department of Biology, MIT, Cambridge, MA, USA.^5 Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA.^6 Department of Chemical Engineering, MIT, Cambridge,
MA, USA.^7 Department of Biological Engineering, MIT, Cambridge, MA, USA.^8 These authors contributed equally: Yilong Zou, Whitney S. Henry, Emily L. Ricq. ✉e-mail: [email protected];
[email protected]; [email protected]