bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis
Kenji M. Fujihara1,2, Bonnie Zhang1,2, Thomas D. Jackson3, Brunda Nijiagel4, Ching-Seng Ang3, Iva Nikolic5, Viv Sutton1,2, Joe Trapani1,2, Kaylene J. Simpson5, Diana Stojanovski3, Silke Leimuehler6, Sue Haupt1,2, Wayne A. Phillips1,2,7, Nicholas J. Clemons1,2,*
1Division of Cancer Research, Peter MacCallum Cancer Centre 2Sir Peter MacCallum Department of Oncology, The University of Melbourne 3Bio21 Institute of Biotechnology, The University of Melbourne 4Metabolomics Australia 5Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre 6Department for Molecular Enzymology, University of Potsdam 7Department of Surgery, St. Vincent’s Hospital *Lead Contact
Corresponding Author: Associate Professor Nicholas J. Clemons Group Leader, Tumourigenesis and Cancer Therapeutics Laboratory Peter MacCallum Cancer Centre Email: [email protected] Tel: 03 8559 5273
Declaration: The authors disclose that there are no conflicts of interest in the work that contributed towards this manuscript. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
ABSTRACT
The mechanisms by which cells respond and adapt to oxidative stress are largely
unknown but are key to developing a rationale for cancer therapies that target antioxidant
pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation
in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia1. Whilst the mechanism
of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent
modification of cysteine residues in the core domain of mutant-p53 protein2,3, here we report
that the anti-neoplastic capacity of APR-246 lies predominantly in the conjugation of free
cysteine. Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics
in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of GSH and
mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was
increased through loss of key enzymes in mitochondrial one-carbon metabolism, SHMT2 and
MTHFD1L, due to diminished glycine supply for de novo GSH synthesis. Critically, we show
that APR-246 induces iron-dependent, apoptotic machinery-independent cell death,
ferroptosis. Whole-cell proteomics analyses indicated an upregulation of proteins involved in
iron-sulfur cluster biogenesis (eg. FDX1). GSH, acetyl-CoA and NADH levels were also
depleted in APR-246 treated cells. Importantly, we found that APR-246 inhibits iron-sulfur
cluster biogenesis in the mitochondria of cancer cells through cysteine conjugation. This work
not only details novel determinants of APR-246 activity in cancer cells, but also provides a
clinical roadmap for targeting antioxidant pathways in tumours – beyond targeting mutant-p53
tumours.
Mito1C SHMT2 MTHFD1L
Gly GCLC GCLM GSS APR-246 Cys GSH Ferroptosis
Loss of essential Cellular Fe2 + [Fe-S] NFS1 cell functions proliferation bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
MAIN
To distinguish the mechanism of action of APR-246, we designed and executed a set
of complimentary genome-wide CRISPR perturbation screens (Figure 1A). We transduced
OACM5.1 oesophageal cancer cells expressing Cas9-mCherry with the Brunello genome-
wide CRISPR knockout (CRISPRko) library4 and challenged these cells with a sublethal dose
of APR-246 for 8 days. In parallel, we transduced OACM5.1 cells expressing dCas9-VP64
with the Calabrese Set A genome-wide CRISPR activation (CRISPRa) library5 and challenged
these cells with a lethal dose of APR-246 for 28 days. The CRISPRko screen aimed to identify
genetic deletions that ‘drop-out’ in the APR-246 treatment cells compared to vehicle treated
cells. Meanwhile, the CRISPRa screen aimed to identify gene overexpression that protects
cells from APR-246. In this way, the CRISPRa screens has the advantage of findings genetic
modulators of APR-246 sensitivity that may not be revealed in the CRISPRko screen as
essential genes drop-out during the puromycin selection.
Consistent with our and others prior observations that APR-246 triggers GSH depletion in
cancer cells6-9, the CRISPRko screen identified de novo GSH synthesis genes SLC7A11 and
GCLM whilst GCLC was the top enriched gene in the CRISPRa screen (Figure 1B). Notably,
two mitochondrial one-carbon (mito-1C) metabolism genes, SHMT2 and MTHFD1L, and the
s-formylglutathione hydrolase gene, ESD, involved in GSH-mediated formaldehyde
detoxification were also identified10-12. We confirmed that two independent sgRNA guides
targeting GCLM, SLC7A11, SHMT2, MTHFD1L and ESD, and one sgRNA guide for GCLC
activation, modulated sensitivity to APR-246 as expected (Figure 1C). Consistent with this,
gene dependency on GCLC correlated with cancer cell line (CCL) sensitivity to APR-246
analogue, APR-017 (PRIMA-1), across 456 CCLs of diverse lineage (Figure 1D).
We next investigated involvement of mito-1C metabolism in APR-246 sensitivity. Of note, mito-
1C metabolism intersects with de novo GSH synthesis through SHMT2-mediated production
of glycine from serine13, and GSH-mediated formaldehyde detoxification through MTHFD1L- bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
mediated production of formate10 (Figure 1E). First, we attempted to rescue the increased
sensitivity to APR-246 in mito-1C or ESD deficient cells with exogenous formate, however,
formate supplementation did not alter sensitivity to APR-246 (Figure 1F). Previous studies
have shown that mito-1C deficient cells have altered dependency on exogenous glycine due
to the loss of the glycine production through SHMT2 when mito-1C is perturbed14. Indeed,
cells expressing SHMT2 and MTHFD1L sgRNAs displayed increased glycine auxotrophy
whilst cells transduced with control vector or ESD sgRNAs did not (Figure 1G). Meanwhile,
all cells displayed sensitivity to serine deprivation and simultaneous serine and glycine
deprivation. Importantly, the glycine auxotrophy in mito-1C deficient cells could be reversed
with the exogenous supply for cell permeable GSH (Figure 1H) – in agreement with previous
studies14. This highlights that mito-1C deficient cells have a higher demand for exogenous
glycine for de novo GSH synthesis. As a result, the increased sensitivity to APR-246 mito-1C
deficient cells can be reversed by supplementation of additional glycine to the cell media
(Figure 1I). Together, these data demonstrate that mito-1C metabolism and de novo GSH
synthesis interplay to regulate the mechanism of action of APR-246.
Given the involvement of mito-1C and GSH metabolism in regulating sensitivity to ARP-246
in cancer cells, we sought to investigate whether limiting supply of serine and glycine (SG) or
cystine (CySS) to cancer cells increased sensitivity to APR-246. Indeed, both SG and CySS
depletion increased FLO-1 LM cells, a model of spontaneous metastatic oesophageal
cacner15, to APR-246 in cell culture (Figure 2A). Restriction of dietary serine and glycine has
previously been shown to reduce tumour growth in xenograft and genetically engineered
mouse models16,17. Whilst cystine depletion in vitro has been widely demonstrated to induce
ferroptosis18,19, to our knowledge, cystine restriction has not be previously shown to inhibit
tumour growth. Using the FLO1 LM xenograft model in immunocompromised mice to access
the effects of SG or CySS diet restriction on APR-246 efficacy, we found that SG restriction
significantly enhanced the efficacy of APR-246 (Figure 2B). Interestingly, CySS restriction
had no effect on tumour, whilst SG restriction alone had greater efficacy than APR-246 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
monotherapy. SG and CySS restriction in combination with APR-246 were well tolerated
(Figure 2C) and chow consumption remained consistent across the different diet interventions
(Figure 2D). These results demonstrate, for the first time, that SG restriction can inhibit the
growth of oesophageal cancer tumours and synergies with APR-246.
Returning to our investigation the mechanism of action of APR-246, we next examined
whether APR-246 induces ferroptosis. To do this, we utilised three distinct inhibitors of
ferroptosis – N-acetylcysteine (NAC), ferrostain-1 (Fer-1) and ciclopirox olamine (CPX)
(Figure 3A). Indeed, we found that APR-246-induced cell death, as measured by propidium
iodine (PI) uptake, could be reversed by all the ferroptosis inhibitors, but not by the pan-
caspase inhibitor, Z-VAD-FMK in both p53-null H1299 lung cancer cells and mut-p53
expressing FLO-1 oesophageal cancer cells (Figure 3B - top). Importantly however, the
cellular proliferation defect induced by APR-246 was only reversed in cell co-treated with NAC
(Figure 3B - bottom). This indicates that the mechanism of action of APR-246 likely diverts
at the level of conjugating to cysteine to induce cell death through GSH depletion and another
perturbed pathway that blocks cell proliferation. This phenomenon may also provide an
explanation for why previous studies of APR-246-mediated cell death that relied on long term
exposures (> 48 hr) with APR-246 and the use of cellular metabolism assays as surrogates
for cellular viability failed to demonstrate rescue with ferroptosis inhibitors6. Furthermore, this
is in keeping with recent findings that distinguish erastin, which block cystine import into cells,
as maintaining cell proliferation activity in the presence of ferroptosis inhibitors20. Extending
this, given that the proposed mechanism of action of APR-246 is to induce apoptotic cell death,
we also genetically engineered the Mc38 mouse colon adenocarcinoma model to be
completely deficient in the intrinsic apoptotic machinery (Bax/Bak/Bid/Casp3/Casp7 KO). We
found that APR-246 was equally as efficacious in both apoptosis proficient cells and apoptosis
deficient cells (Figure 3C). Furthermore, apoptosis deficient cells underwent cell death
following exposure to APR-246 (Figure 3D - left). Conversely, staurosporine (STS) failed to bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
induce cell death in these cells (Figure 3D - right). Together, this proves that APR-246
induces ferroptosis and does not require apoptotic machinery in order to elicit cell death.
Next, we sought to explain the cell proliferation defect induced by APR-246. To this end, we
performed label-free proteomics and untargeted metabolomics in OACM5.1 cells treated with
APR-246 prior to the onset of ferroptosis (Figure 4A,B). In response to APR-246 treatment,
cells upregulated mitochondrial protein ferredoxin 1 (FDX1), a critical component of
mitochondrial iron-sulfur cluster biosynthesis, as well as two other protein, DNAJA3 and
ABCF2, with reported roles in iron metabolism21,22. As expected, APR-246 treatment reduced
GSH levels and simultaneously increased cystine levels likely as an adaptive response to
APR-246 treatment. Meanwhile, mitochondrial metabolism altered as demonstrated by the
downregulation of acetyl-CoA and NADH. As a result of these investigations, we hypothesised
that APR-246 may be inhibiting mitochondrial iron-sulfur cluster biogenesis likely through
binding to free cysteine and limiting the cysteine desulferase activity of NFS1 (Figure 4C). We
first interrogated the publicly available gene essentiality data, correlating CCL dependency on
NFS1 and the activity of the 480 compounds across over 800 CCLs. Supporting our
hypothesis, the top compound correlated to NFS1 dependency was APR-246 analogue, APR-
017 (Figure 4D). We also confirmed, in line with previous studies23, that MQ is able to bind
directly to free cysteine (Figure 4E). Importantly, we shown a cell free NFS1 activity assay
and in intact cells that MQ is able to inhibit the desulfurase activity of NFS1 in a dose
dependent fashion (Figure 4F,G). Furthermore, MQ also reduced aconitase activity, a
surrogate marker for iron-sulfur stability24 (Figure 4H). Together, these results demonstrate
that APR-246 inhibits iron-sulfur cluster biogenesis through cysteine conjugation – providing
a novel explanation for the cell proliferation defect induced by APR-246 in the presence of
ferroptosis inhibitors.
In sum, we show that APR-246 induces ferroptosis and inhibits iron-sulfur cluster biogenesis
through cysteine conjugation. This has broad implications for the clinical utility of APR-246. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
This proposed mechanism of action highlights that APR-246 should be expanded to beyond
mutant-p53 cancers, which we have illustrated in previous studies25. In the context of TP53-
mutated MDS, APR-246 is currently only indicated in ~7% of all MDS cases26. Dysregulated
iron metabolism is a hallmark of a subset of MDS with ringed sideroblasts, characterised by
aberrant iron accumulation in erythroblast mitochondria27. The aberrant iron accumulation in
MDS with ringed sideroblasts likely increases erythroblasts sensitivity to ferroptosis.
Importantly, the most frequent genomic alteration associated with MDS with ringed
sideroblasts are SF3B1 mutations, however these are mutually exclusive with TP53
mutations28. Therefore, our work reveals that the clinical utility of APR-246 could be potentially
expanded to include MDS cases with aberrant iron accumulation in order to leverage its
capacity to induce ferroptosis.
METHODS SUMMARY
CRISPR screens performed in human OACM5.1 cells. Validation was performed on polyclonal
OACM5.1 cells following selection with puromycin. Formate and glycine rescue experiments
were performed in conditions matching the CRISPR screening procedure scaled down to T25
flask. Serine/Glycine auxotrophy experiments were perform in 96-wells plates utilising
dialyzed FBS and media reconstituted without serine and glycine. Cancer dependency data
and APR-017 cancer cell line sensitivity data was obtained from the DepMap web-portal
(www.depmap.org) and correlation analyses were performed on R studio using the cor.test
function. For diet intervention studies, ~6-week-old female NSG mice were sub-cutaenously
injected with 5´106 FLO1 LM cells and randomised to SG-deplete, CySS-deplete or control
chow (AIN93G Rodent Diet) and dosed with 0.9% saline or 100 mg/kg APR-246 (Aprea) once
tumours reach 100mm3. For ferroptosis rescue experiments, cell death and proliferation were
quantified utilising IncuCyte FLR (Essen BioSciences). Cell viability was determined by
alamarBlue assay (Life Technologies) using Cytation 3 (BioTek). Mc38 apoptosis-deficient
cells were generated using CRISPR-Cas9 technology. Label-free proteomics (LC-MS/MS)
was performed at the Mass Spectrometry and Proteomics Facility at Bio21 Institute and bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
untargeted metabolomics (LC-MS) in collaboration with Metabolomics Australia. NFS1 cell-
free and in cell assays and aconitase assays were performed as previously described29.
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FIGURE LEGENDS bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 1. Genome-wide CRISPR screens highlight the role of de novo GSH synthesis
and mito-1C metabolism in APR-246 mechanism of action
(A) Schematic diagram details the protocols utilised for the CRISPRko and CRISPRa screens
in OACM5.1 cells. Sublethal dose = 10 µM. Lethal dose = 15 µM. (B) Mageck scores (negative
indicating ‘dropout’ and positive indicating ‘enrichment’) from CRISPR screens plots in order
to magnitude. (C) Cell viability following 72 h exposure with APR-246 at indicated doses to
validation of hits. (D) Scatterplot correlating the expression of GCLC gene dependency with
APR-017 (APR-246 analogue) activity (area-under the curve, AUC). (E) Schematic diagram
illustrating the connections between de novo GSH synthesis and mito-1 metabolism. (F)
Relative cell numbers of OACM5.1 cells with indicated vectors treated with 10 µM APR-246 ±
1 mM sodium formate supplementation for 4 days. (G) Cell viability following 72 h of serine,
glycine or serine and glycine (SG) deprivation compared to complete media (CM). (H) Cell
viability following 72 h of glycine deprivation ± 1 mM GSH-monoethyl ester (GSH-MEE). (I)
Relative cell number following treatment with 10 µM APR-246 ± 1 mM glycine supplementation
for 4 days. (D) Pearson’s correlation. (G) one-way ANOVA with Dunnet’s multiple comparison
post-test. (H,I) unpaired two-tailed t-test. Error bars = SEM. (B) N=2 for CRISPRko screen,
N=1 for CRISPRa. (C,F,G) N=3. (H,I) N=5.
Figure 2. SG-free diet enhances APR-246 efficacy in vivo
(A) Cell viability in FLO1 LM cell following 72 h exposure to APR-246 at indicated doses in the
absence of SG or cystine (CySS) compared to CM. (B) Growth curves of FLO1 LM tumours
treated with APR-246 (100 mg/kg, daily) on either control, SG-free, CySS-free diets for 35
days. (C) Body weight as a percentage from baseline. Ethically acceptable weight loss is
defined by Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee as
<20% compared to pre-treatment days. (D) Average chow consumption over 7 days per box
of mice per mouse. (B) one-way ANOVS with Dunnet’s multiple comparison post-test. Error
bars = SEM. (A,D) N=3. (B,C) N=8 for all groups. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 3. APR-246 triggers ferroptosis
(A) Schematic diagram illustrating the ferroptosis inhibitors mechanisms. (B) Percentage of
dead cells (as determined by percentage of PI+ cells) and cell proliferation (as determined by
cell confluency) induced by 50 µM APR-246 co-treated with 50 µM Z-VAD-FMK (pan-caspase
inhibitor), 12.5 µM Ferrostain-1 (Fer-1, lipophilic antioxidant), 6.25 µM ciclopirox olamine (CPX,
iron chelator) and 2.5 mM N-acetyl-cysteine (NAC, cysteine supplement) in H1299 and FLO1
cells. (C) Cell viability in Mc38 apoptosis-proficient (black, WT) and apoptosis-deficient (red,
Bax/Bak/Bid/Casp3/Casp7 KO) cells. (D) Cell death induced in WT and apoptosis deficient
Mc38 cells by 50 μM APR-246 or 12.5 μM Staurosporine (STS). Error bars = SEM. (B,C,D)
N=3.
Figure 4. APR-246 inhibits iron-sulfur cluster biogenesis
(A) Changes in proteins determined by label-free quantitative proteomics and (B) untargeted
LC-MS metabolomics following 50 μM APR-246 for 12 h compared to vehicle. (C) Schematic
diagram illustrating mitochondrial iron-sulfur cluster biogenesis and its role in mitochondrial
metabolism. (D) Box-and-whisker plot (1st-99th percentile) of Fischer’s transformed z- scored
Pearson correlation strength of NFS1 gene dependency and the 481 Cancer Therapeutic
Response Portal v2 (CTRPv2) compounds. Red dot indicates APR-017 (APR-246 analogue)
is the top positive correlated compound to NFS1 gene dependency. (E) Mass spectrometry
pattern of 10 μM pre-heated APR-246 mixed with 10 μM cysteine in H2O showing MQ
conjugated to cysteine (MQ-Cys, m/z = 259.111 [m + H]). (F) Cell-free and (G) in-cell (in HEK-
239T cells) L-cysteine desulfurase assay measuring NFS1 activity at indicated doses of MQ
following 1 h incubation. (H) Aconitase activity assay in HEK-239T cells following 1 h
incubation with MQ at indicated doses. Error bars = SEM. (A) N=4. (B) N=6. (F,G,H) N=3. Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C CRISPR-Cas9 CRISPR-dCas9 OACM5.1 pLentiPuro Control 125 sgGCLM_1 treatment 4 5 sgGCLM_2 GCLC 100 sgSLC7A11_1 75 sgSLC7A11_2 8 Days sgRNA barcode sgSHMT2_1 Genome-wide sequencing ) CRISPR KO 2 50 sgSHMT2_2 sgRNA Library sgMTHFD1L_1
e sgMTHFD1L_2 r 25 OACM5.1 cells ehicle + Cas9
Sublethal o sgESD_1 Cas9-mCherry V APR-246 c 0 sgESD_2 S 0 0 0 100 101 102 Control ck treatment e 125 pXPR_502 sgGCLC iability (x 100 V Mag
sgRNA barcode % 75 28 Days -2 SLC7A11 Genome-wide sequencing MTHFD1L CRISPR Activation 50 sgRNA Library SHMT2 GCLM OACM5.1 cells ESD 25 dCas9-p65 HSF Lethal + dCas9 APR-246 -4 -5 0 0 100 101 102 D F H APR-246 ( M) DepMap Formate Rescue Glutathione Rescue P=0.0005 P=0.0128 P=0.0018 P=0.0176
1.0 R = 0.3375 Vehicle APR-246 APR-246 + Formate
P < 0.0001 1.0 ) 100 CM 0.5 -Glycine ) -Glycine + GSH ) 0.0
ES 0.5 50 ehicle R V
E -0.5 iability (x CM ( V (C Gene Dependency 0.0 % 0 C -1.0 Relative Cell Number _1 _2 _1 _2 _1 _2 _1 _2 _1 _2
GCL -1.5 ESD ESD SHMT2 SHMT2 5 10 15 20 SHMT2 SHMT2 pLentiPuro pLentiPuro + sg + sg MTHFD1LMTHFD1L APR-017 Activity (AUC) MTHFD1LMTHFD1L + sg + sg + sg + sg + sg + sg + sg + sg E G Serine/Glyine Auxotrophy I Glycine Rescue Cystine Glutamate P<0.0001 P=0.0009 P=0.0001 P=0.0003 Vehicle APR-246 APR-246 + Glycine
P=0.0015 P=0.0061 P=0.0947 P=0.0163 ) 100 CM 1.0 -Serine
SLC7A11 -Glycine ) -SG
Serine 50 0.5 ehicle
Cystine Glutamate Glycine V
SHMT2 iability (x CM (
THF V Cysteine % MTHFD1L 0 0.0
GCLC GCLM Relative Cell Number Glycine _1 _2 _1 _2 _1 _2 Formate ESD ESD GSH ESD Formate SHMT2 SHMT2 pLentiPuro pLentiPuro + sg + sg MTHFD1LMTHFD1L + sgSHMT2_1+ sgSHMT2_2 Formaldehyde + sg + sg + sg + sg + sgMTHFD1L_1+ sgMTHFD1L_2 Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Cell Culture ) FLO1 LM 3
150 CM -SG -CySS m 1500 Vehicle ) APR-246 125 -CySS/Vehicle 1000 100 -CySS/APR-246 P=0.0024 -SG/Vehicle
iability 75 olume (m V
V -SG/APR-246 ehicle CM 50 500 V % 25 ( 0 0 umour
0 10-0.5 100.5 101.5 T -30 7 14 21 28 35 APR-246 ( M) Days
Body Weight Chow Consumption 120 4
t e ) 110 100 3 eigh Acceptable
W 90 weight loss 2 y Change
80
od 80 1 (% Vehicle -CySS/Vehicle -SG/Vehicle B
APR -CySS/APR -SG/APR g/D per mous 0 0 0 7 14 21 28 35 Time (Days) -SG Control -CySS Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A Cysteine NAC
GSH GPX4 Fer-1 GSSG
Lipid Peroxidation Ferroptosis
Fe 2+ CPX
B H1299 FLO-1 C ) Mc38 125 WT 100 50 Vehicle Bax/Bak/Bid/Casp3/Casp7 KO APR-246 40 + Z-VAD ehicle 100
75 V 30 + Fer-1 75 50 + CPX 20 + NAC 50 25 25
10 iability (x % Dead Cells V 0 0 0 0 1 2 % 0 10 10 10 0 12 24 0 12 24 D APR-246 ( M) APR-246 STS 40 40 40 60
y 30 30 30 40 20 20 20 20 10 10 10 % Confleunc % Dead Cells 0 0 0 0 0 12 24 0 12 24 0 12 24 0 12 24 Time (h) Time (h) Time (h) Time (h) Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.398867; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B D Proteomics Metabolomics DepMap 5 4 3 APR-017 DNAJA3 FDX1 h
) 4 ) ABCF2 2
2 alue alue 3 Acetyl-CoA 0
V GSH V
P P (
( NADH
0 L-Cystine 0 2 1 -2 1 g g 1 -lo Gene Dependency -lo 1 -4 Correlation Strengt
0 0 NFS1 -6 -2 -1 0 1 3 -2 -1 0 1 2 Compounds log (FC) log2(FC) 2 C Cys E [Fe-S] ETC OrbiTrap SFXN? 156.1019 e 100 259.1111
ABCB7 80
Cys NFS1 [Fe-S] 60 122.0272 Abundac
Acetyl CoA
Citric Oxaloacetate NADH acid NAD+ 40 192.1595 Isocitric 2+ Malate acid
CO2 + 107.0567 138.0914 Fe TCA NAD 174.1125 NADH H2 O 200.1281 α-Ketoglutaric Fumarate Cycle acid CO2
+ FADH2 NAD
FAD Succinyl NADH Succinate CoA 20 SLC25A28/ 38 ATP ADP
Relative 0 100 120 140 160 180 200 220 240 260 280 Fe 2+ m/z
F G H )
293T
In Vitro NFS1 Activity mg 293T /
0.020 (u 0.0010 ) 0.4 ) Vehicle ty i v mg mg 0.5 mM MQ 0.015 i / 0.3 1 mM MQ / (u (u 2 mM MQ Act 0.010 0.0005 ty ty 0.2 3 mM MQ e i i
4 mM MQ s v v i i
5 mM MQ a 0.1 0.005 t Act Act
0.0 0.000 coni 0.0000 0 A L-Cysteine Desulfurase L-Cysteine Desulfurase 0 200 400 50 0 100 500 20 50 Cysteine ( M) 100020004000 100 MQ ( M) MQ ( M)