scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth

TL;DR: It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.
Abstract: Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.

Content maybe subject to copyright    Report

Systemic depletion of serum L-Cyst(e)ine with an engineered
human enzyme induces production of reactive oxygen species
and suppresses tumor growth in mice
Shira L. Cramer
1,π
, Achinto Saha
2,π
, Jinyun Liu
3,π
, Surendar Tadi
4
, Stefano Tiziani
4
,
Wupeng Yan
5
, Kendra Triplett
1,5
, Candice Lamb
1,5
, Susan E. Alters
6
, Scott Rowlinson
6
, Yan
Jessie Zhang
5
, Michael J. Keating
7
, Peng Huang
3
, John DiGiovanni
2
, George Georgiou
1,5,*
,
and Everett Stone
5,*
1
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712,
United States
2
Division of Pharmacology and Toxicology and Dell Pediatric Research Institute, The University of
Texas at Austin, Austin, TX 78712, United States
3
Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer
Center, Houston, TX 77030, United States
4
Department of Nutritional Sciences, The University of Texas at Austin, Austin, TX 78712, United
States
5
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712,
United States
6
Aeglea Biotherapeutics, Austin, TX 78746, United States
7
Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
77030, United States
Abstract
Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than non-
malignant cells due to genetic alterations and abnormal growth and as a result, maintenance of the
anti-oxidant glutathione (GSH) is essential for their survival and proliferation
1–3
. Under elevated
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research,
subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
*
Correspondence to: gg@che.utexas.edu (G.G.); stonesci@utexas.edu (E.S.).
π
Equal contribution.
Accession codes: The Cyst(e)inase structure has deposited into the Worldwide Protein Data Bank with the PDB code of 5EIG.
Data Availability Statement: Data collection and refinement statistics for the Cyst(e)inase structure (PDB:5EIG) are summarized in
Supplementary Table 1.
Author Contributions: S.L.C., A.S., J.L., S. Tadi., W.Y., K.T., C.L., and Y.J.Z. performed key experiments; E.S., P.H., J.D. and G.G.
conceived and designed the research; S.L.C., A.S., J.L., S. Tadi., S. Tiziani, W.Y., K.T., S.E.A., S.R., Y.J.Z., M.J.K, P.H., J.D., G.G.,
and E.S. analyzed data; M.J.K. provided critical materials (CLL blood samples); S.L.C., A.S., J.L., G.G., J.D. and E.S. wrote the
manuscript.
Competing Financial Interests Statement: G. Georgiou, and E. Stone are inventors on intellectual property related to this work and
G. Georgiou, E. Stone, S. Rowlinson and S. Alters have an equity interest in Aeglea Biotherapeutics, a company pursuing the
commercial development of this technology.
HHS Public Access
Author manuscript
Nat Med
. Author manuscript; available in PMC 2017 May 21.
Published in final edited form as:
Nat Med
. 2017 January ; 23(1): 120–127. doi:10.1038/nm.4232.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

ROS conditions endogenous L-Cysteine (L-Cys) production is insufficient for GSH synthesis,
necessitating L-Cys uptake, predominantly in its disulfide form L-Cystine (CSSC) via the xCT(−)
transporter. Here we show that administration of an engineered, pharmacologically optimized,
human Cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC
pool in mice and non-human primates, selectively causes cell cycle arrest and death (PI and
Annexin-V staining) in cancer cells due to depletion of intracellular GSH and ensuing elevated
ROS, yet results in no apparent toxicities in mice even after months of continuous treatment.
Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in
prostate and breast cancer xenografts and doubled the median survival time of
TCL1
-Tg:
p53
−/−
mice that develop disease resembling human chronic lymphocytic leukemia. The observation that
enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple
tumors, yet is very well tolerated for prolonged periods suggests that Cyst(e)inase represents a safe
and effective therapeutic modality for inactivating anti-oxidant cellular responses in a wide range
of malignancies
4,5
.
As a precursor for the biosynthesis of GSH, L-Cys is essential for maintaining the
intracellular thiol redox potential. L-Cys is produced via the transsulfuration pathway
enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CGL), which are
widely expressed in numerous tissues in humans (Fig. 1a)
6–9
. In contrast, some tumors have
markedly lower expression of transsulfuration enzymes namely due to transcriptional
silencing
10,11
, or endogenous L-Cys synthesis is insufficient because rapid proliferation,
metabolic dysregulation, and oncogene expression markedly increase ROS levels.
Consequently, import of extracellular L-Cys becomes necessary to meet cellular GSH
requirements
12–16
. The main route for extracellular acquisition of L-Cys is transport of its
disulfide form, CSSC, through the xCT(−) CSSC/L-glutamate (L-Glu) antiporter (SLC7A11)
(Fig. 1a). Due to high demand for CSSC in numerous malignancies, xCT(−) represents a key
therapeutic target with small molecule inhibitors of the transporter displaying anti-tumor
effects
12–15
. As L-Cys is a non-essential amino acid in animals, eliminating L-Cys and CSSC
uptake should selectively impact tumors displaying increased ROS production and thus a
higher demand for anti-oxidants, without adversely affecting normal physiology. However,
inhibition of xCT(−) alone is insufficient because free L-Cys is imported via other
transporters (e.g. the system ASC transporters ASCT1 and ASCT2). Instead, a superior
approach to small molecule inhibition of multiple transporters is to eliminate the
extracellular pool of L-Cys and CSSC through the action of an enzyme that converts these
amino acids into non-toxic, readily metabolizable, products.
The pyridoxal phosphate (PLP)-dependent enzyme CGL (EC 4.4.1.1) catalyzes the last step
in
de novo
L-Cys synthesis, converting L-cystathionine (L-Cysta) into α-ketobutyrate, L-Cys,
and NH
4
+
. CGL can also accept L-Cys or CSSC as substrates producing pyruvate, NH
4
+
, and
H
2
S or thiocysteine respectively. Although CGL can degrade both CSSC and L-Cys, its
kinetics are too slow to be relevant for clinical applications given that dosing at >2 g/kg
would be required to achieve substantial reduction of serum L-Cys and CSSC levels in mice.
Structural analyses of CGL (PDB:3COG) suggested that amino acid substitutions of active
site residues E59 and E339, which are important for L-Cysta coordination, could enhance L-
Cys and CSSC degrading activity. Combinatorial saturation mutagenesis of these residues
Cramer et al. Page 2
Nat Med
. Author manuscript; available in PMC 2017 May 21.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

coupled with a high throughput assay for pyruvate production led to the isolation of CGL-
E59T-E339V, displaying 50- and 25-fold higher
k
cat
/
K
M
values for CSSC and L-Cys
respectively, than WT-CGL (Fig. 1b). Solving the X-ray structure of CGL-E59T-E339V in
the presence of 1 mM L-Cys at 2.7Å revealed an essentially identical structure to WT-CGL
with only minor perturbations at the mutation sites (0.66 Å RMS to PDB:2NMP); as well as
substrate complexes within the four active sites of the tetramer (Supplementary Table 1). In
two active sites, the amine of the substrate L-Cys forms a geminal diamine with the PLP
cofactor and the ε-amine of Lys212, representing a reaction intermediate just prior to
forming the L-Cys-external aldimine complex, which is further stabilized by a salt bridge to
the L-Cys α-carboxyl group and the guanidinium moiety of Arg375 (Fig. 1c). Collectively,
the structural and kinetic data indicate that the E339V substitution increases the
hydrophobicity of the active-site to enhance L-Cys binding
17,18
while the removal of the
E339 and E59 carboxyl groups better accommodates CSSC binding, a conclusion that is
further supported by molecular dynamic modeling (Supplementary Fig. 1).
To prevent renal clearance and thus impart long circulation persistence, CGL-E59T-E339V
was conjugated to methoxy PEG succinimidyl carboxymethyl ester, MW 5000 Da
(Supplementary Fig. 2) (conjugated protein henceforth referred to as Cyst(e)inase). Single
dose administration of Cyst(e)inase at 8 mg/kg to two cynomolgus monkeys resulted in a
marked reduction in total serum L-Cys content (Free+liberated protein-bound L-Cys and
CSSC) for 28 hours (Fig. 1d) with no observed toxicities (based on hematology, blood
chemistry and animal observations, see Supplementary Data Set 1), indicating that
Cyst(e)inase is able to modulate blood L-Cys and CSSC levels in primates.
Increased ROS production has been widely noted in human prostate carcinomas (PCa) with
high levels correlating with an aggressive phenotype
19–21
. We therefore evaluated the effect
of Cyst(e)inase on the human PCa cell lines PC3 and DU145; as well as the murine PCa cell
line, HMVP2
22
derived from the ventral prostate of HiMyc transgenic mice
23
. The HMVP2
murine PCa cell line constitutes a relevant model to human disease as it possesses
characteristics of cancer stem cells, expresses stem cell markers (such as LIN
neg
, Sca-1
high
,
CD49f
high
, CK14, and CD29), and produces spheroids
22
. Cyst(e)inase potently inhibited
survival of HMVP2 cells with an IC
50
of 23 ± 4 nM (Fig. 2a). A dose-dependent significant
increase in ROS (P<0.001) was evident by 4 hours after enzyme addition, with a
concomitant significant (P< 0.001) decrease in cellular GSH levels by 24 hours (Fig. 2b,c).
Consistent with the well-established effects of ROS accumulation and amino acid restriction
in eliciting autophagic responses
24–26
, Cyst(e)inase treatment increased AMPK
phosphorylation (pAMPK
Thr172
) (Fig. 2d), reduced phosphorylated mTOR (pmTOR
Ser2448
),
and increased autophagic responses manifested by changes in the phosphorylation levels of
mTORC1 and downstream genes such as p70S6K, ribosomal protein S6 and ULK1 as well
as LC-3 II formation (Fig. 2e,f). Similarly, Cyst(e)inase treatment of the human cell line
PC3 resulted in dose-dependent cell killing, a 4–6–fold increase of pAMPK
Thr172
and
increased autophagic responses (Supplementary Fig. 3a–d). AMPK and p70S6K can also
directly affect cell cycle progression
27,28
, and we observed a significant dose-dependent
arrest in G
0
/G
1
with a corresponding significant decrease of cells in S-phase and a non-
significant but slight increase in G
2
/M populations (Fig. 2g). The G
0
/G
1
arrest was
accompanied with a dose-dependent increase in protein levels of the cyclin-dependent kinase
Cramer et al. Page 3
Nat Med
. Author manuscript; available in PMC 2017 May 21.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

inhibitor p27 (> 7-fold, Fig. 2h), whose accumulation can be stabilized by pAMPK
27
. We
also detected decreased protein levels of c-Myc, the cyclin-dependent kinases CDK2 and
CDK4, phosphorylated retinoblastoma protein (pRB
Ser807/811
), E2F4, and cyclins A, D1 &
E1 (Fig. 2h,i). Conversely, and consistent with a ROS driven mechanism, we found that
treating cells with the ROS scavenger and L-Cys donor
N
-acetyl-L-cysteine, partially
restored survival (Fig. 2j and Supplementary Fig. 3e).
To assess Cyst(e)inase anti-tumor activity
in vivo
in murine models, we first established an
appropriate dosing schedule by measuring the pharmacokinetics of Cyst(e)inase and its
pharmacodynamics effect on thiol-containing metabolites in serum in mice. Following single
dose i.p. (50 mg/kg) administration of Cyst(e)inase in non-tumor bearing FVB mice, we
observed a clearance half-life of 25 ± 2 hrs with the data fitting well to an extravascular
clearance model
29
(Fig. 3a). Similar to the observations in cynomolgus monkeys, no weight
loss, adverse effects, nor organ abnormalities upon necropsy were observed in the mice (data
not shown). Time course serum metabolomics analyses revealed near complete elimination
of serum CSSC for 4 days, before recovering to pre-administration concentrations at day 6
(Fig. 3b). Free L-Cys levels were reduced by over 4-fold for 2 days before gradually
recovering (Fig. 3b). The impact of Cyst(e)inase on the concentration of other sulfur
containing metabolites was consistent with their respective steady-state serum
concentrations, enzyme kinetics, and the consequences of depleting extracellular L-Cys and
CSSC (Supplementary Fig. 4). From a clinical translation standpoint this treatment
corresponds to realistic clinical doses (based on allometric scaling, a 50 mg/kg dose in mice
corresponds 6.5 mg/kg in humans
30
).
We then treated (via i.p. injection) syngeneic male FVB/N mice bearing palpable HMVP2
tumors with either 50 or 100 mg/kg Cyst(e)inase, and PBS or 100 mg/kg heat-inactivated
Cyst(e)inase as controls every 4 days for 4 weeks. Tumors did not increase in size in either
Cyst(e)inase-treated group, while mice injected with heat-inactivated enzyme (a control for
the presence of residual impurities and endotoxin in active protein) showed identical tumor
growth to the PBS-treated group (Fig. 3c). Throughout the treatment period, no weight loss
or inappetence was observed (Fig. 3d), nor were any adverse effects as determined by
examination of tissues upon necropsy (data not shown).
Similarly, Cyst(e)inase treatment in male nude mice under the same dosing schedule
significantly blocked growth of both DU145 (P<0.0001 at 100 mg/kg, Fig. 3e) and PC3
xenograft tumors (P<0.0001 at 50 and 100 mg/kg; Fig. 3f). Once again no weight loss or
inappetance was observed, nor were changes in blood cell counts or blood chemistry
(Supplementary Figs. 5, 6). Macroscopically (at the time of sacrifice), the liver and other
major organs in treated mice looked similar to control with no obvious signs of toxicity (data
not shown). We also examined H&E stained sections of liver tissue from both control and
treated mice microscopically. The liver of Cyst(e)inase treated mice was normal in histologic
appearance, maintaining normal hepatic architecture with no obvious signs of cellular
toxicity (data not shown). Because ocular tissues are known to have a high demand for
CSSC
31,32
, we also performed histological examinations of ocular sections and found no
evidence of changes in tissue ultrastructure compared to control groups, further underscoring
that Cyst(e)inase treatment is well tolerated (data not shown). In addition to prostate
Cramer et al. Page 4
Nat Med
. Author manuscript; available in PMC 2017 May 21.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

carcinomas, Cyst(e)inase may be beneficial for the treatment of a wide range of cancers,
notably human breast carcinomas, which upregulate xCT(−)
12,33
. Accordingly, Cyst(e)inase
administration significantly abrogated the growth of MDA-MB-361 breast cancer xenografts
orthotopically implanted into female NOD SCID mice (P<0.0001 at 50 and 100 mg/kg;
Supplementary Fig. 7), suggestive of a common tumoral dependence on L-Cys and CSSC.
To further confirm Cyst(e)inase impacts tumors by affecting cellular thiol anti-oxidant
pathways, we investigated the effect of Cyst(e)inase in combination with well-established
drug candidates targeting the GSH or thioredoxin (TXN)- thioredoxin reductase (TXNR)
pathways. For these studies we used respectively, buthionine sulfoximine (BSO) which
inhibits GSH synthesis, and the natural product curcumin (which is the active component of
turmeric) that irreversibly inhibits TXNR
34
, and is being evaluated in numerous clinical
trials as a therapy for various neoplasias
35–37
. We compared the effects of Cyst(e)inase with
BSO or curcumin alone, and in combination on DU145 PCa cell viability using an MTT
assay. Cyst(e)inase was far more cytotoxic than BSO alone, but the combination showed a
synergistic effect (Supplementary Fig. 8a). Similarly, we observed that curcumin synergized
with Cyst(e)inase in DU145 cells and in the very aggressive, castrate resistant PCa cell line
22Rv1 (Supplementary Fig. 8b,c). Cyst(e)inase treatment in combination with either BSO or
curcumin led to a significant increase in cellular ROS levels (P<0.01 as compared to
treatment with individual agents) (Supplementary Fig. 8d–f).
In vivo
, and consistent with the
in vitro
results, only slight tumor inhibition was observed in male nude mice implanted with
22Rv1 cells when treated with moderate Cyst(e)inase (25 mg/kg) or curcumin (1% w/w in
diet) doses, whereas co-administration of Cyst(e)inase and curcumin significantly enhanced
tumor growth inhibition (Supplementary Fig. 8g). Notably, treatment with a higher dose of
Cyst(e)inase (100 mg/kg) as a single agent displayed analogous efficacy to the combination
treatment (Supplementary Fig. 8g), suggesting that Cyst(e)inase-treatment simultaneously
effects both the GSH and TXN anti-oxidant pathways.
Recently Huang
et al.
reported that primary leukemia cells isolated from chronic
lymphocytic leukemia (CLL) patients rely on cysteine uptake for GSH production, but have
low xCT(−) expression
16
. Consequently, CLL cell viability is dependent on stromal
protection, where surrounding bone marrow stromal cells (which highly express xCT(−))
uptake CSSC for conversion to L-Cys, which is then released into the microenvironment for
uptake by CLL cells (via system ASC transporters). The reliance of CLL cells on stromal
protection is further shown
in vitro
, where a high level of spontaneous apoptosis is observed
in CLL cells cultured alone, yet co-culture with stromal cells significantly enhances cell
viability
16
. Accordingly, we compared the effect of Cyst(e)inase to the CLL standard of care
drug fludarabine (or their combination) in mouse leukemic cells isolated from
TCL1
-
Tg:
p53
−/−
mice, a model with a
TCL1
transgenic and
p53
deletion genotype that exhibits
highly aggressive disease progression with 100% penetrance and a drug resistant phenotype
that mimics human CLL with a chromosomal 17p deletion (which is associated with poor
clinical outcomes)
38
.
TCL1
-Tg:
p53
−/−
mice develop splenomegaly, with IgM+CD5+ B
leukemic cells representing 80–90% of total cells in the spleen.
TCL1
-Tg:
p53
−/−
splenocytes
co-cultured with murine stromal Kusa-H cells (to mimic the protective CLL
microenvironment) were minimally affected by fludarabine, yet were potently killed by
Cyst(e)inase (Fig. 4a and Supplementary Fig. 9).
Cramer et al. Page 5
Nat Med
. Author manuscript; available in PMC 2017 May 21.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Citations
More filters
Journal ArticleDOI
05 Oct 2017-Cell
TL;DR: The mechanisms underlying ferroptosis are reviewed, connections to other areas of biology and medicine are highlighted, and tools and guidelines for studying this emerging form of regulated cell death are recommended.

3,356 citations


Cites background from "Systemic depletion of L-cyst(e)ine ..."

  • ...Likewise, direct depletion of cystine from plasma using an engineered cystine-degrading enzyme conjugate (i.e., cyst(e)inase) arrests tumor growth and triggers cell death (Cramer et al., 2017)....

    [...]

Journal ArticleDOI
01 May 2019-Nature
TL;DR: Investigation of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8 and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.
Abstract: Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.

1,222 citations

Journal ArticleDOI
TL;DR: The identification of FDA-approved drugs as ferroptosis inducers creates high expectations for the potential of ferroPTosis to be a new promising way to kill therapy-resistant cancers.

1,106 citations

Journal ArticleDOI
TL;DR: In this paper, the key molecular mechanisms of ferroptosis, including crosstalk with tumour-associated signalling pathways, and discuss potential therapeutic applications of the process are presented.
Abstract: The discovery of regulated cell death processes has enabled advances in cancer treatment. In the past decade, ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation, has been implicated in the development and therapeutic responses of various types of tumours. Experimental reagents (such as erastin and RSL3), approved drugs (for example, sorafenib, sulfasalazine, statins and artemisinin), ionizing radiation and cytokines (such as IFNγ and TGFβ1) can induce ferroptosis and suppress tumour growth. However, ferroptotic damage can trigger inflammation-associated immunosuppression in the tumour microenvironment, thus favouring tumour growth. The extent to which ferroptosis affects tumour biology is unclear, although several studies have found important correlations between mutations in cancer-relevant genes (for example, RAS and TP53), in genes encoding proteins involved in stress response pathways (such as NFE2L2 signalling, autophagy and hypoxia) and the epithelial-to-mesenchymal transition, and responses to treatments that activate ferroptosis. Herein, we present the key molecular mechanisms of ferroptosis, describe the crosstalk between ferroptosis and tumour-associated signalling pathways, and discuss the potential applications of ferroptosis in the context of systemic therapy, radiotherapy and immunotherapy. Ferroptosis is an iron-dependent form of regulated cell death driven by excessive lipid peroxidation. Pharmacological agents, ionizing radiation and cytokines can induce ferroptosis and thus suppress tumour growth, but ferroptosis can also trigger inflammation-associated immunosuppression. The authors describe the key molecular mechanisms of ferroptosis, including crosstalk with tumour-associated signalling pathways, and discuss potential therapeutic applications of ferroptosis.

793 citations

Journal ArticleDOI
03 Apr 2020-Science
TL;DR: This work shows that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality.
Abstract: Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC– subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

537 citations

References
More filters
Book ChapterDOI
TL;DR: The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
Abstract: Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.

31,667 citations

Journal ArticleDOI
TL;DR: Coot is a molecular-graphics program designed to assist in the building of protein and other macromolecular models and the current state of development and available features are presented.
Abstract: Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are `discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.

22,053 citations

01 Jan 2002

19,213 citations

Journal ArticleDOI
TL;DR: A description is given of Phaser-2.1: software for phasing macromolecular crystal structures by molecular replacement and single-wavelength anomalous dispersion phasing.
Abstract: Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods. The novel phasing algorithms implemented in Phaser have been developed using maximum likelihood and multivariate statistics. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise, and for single-wavelength anomalous dispersion experimental phasing, the new algorithms, which account for correlations between F+ and F−, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallographic community.

17,755 citations

Journal ArticleDOI
TL;DR: It is argued that modulating the unique redox regulatory mechanisms of cancer cells might be an effective strategy to eliminate these cells.
Abstract: Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells, and recent studies suggest that this biochemical property of cancer cells can be exploited for therapeutic benefits. Cancer cells in advanced stage tumours frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it might be possible to preferentially eliminate these cells by pharmacological ROS insults. However, the upregulation of antioxidant capacity in adaptation to intrinsic oxidative stress in cancer cells can confer drug resistance. Abrogation of such drug-resistant mechanisms by redox modulation could have significant therapeutic implications. We argue that modulating the unique redox regulatory mechanisms of cancer cells might be an effective strategy to eliminate these cells.

4,369 citations

Related Papers (5)