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APR-246 induced ferroptosis in AM
Results
APR-246 induces ferroptosis in acute myeloid leukemia cells
In order to determine the activity of APR-246 in AML, we assayed a set of 12 AML cell lines carrying diverse and representative molecular abnormalities, and five primary AML samples (Online Supplementary Table S1). Most of the AML cell lines and primary AML cells were sensitive to cell death induction by APR-246 (Figure 1A and B). The IC50 of APR-246 at 24 h for each cell line ranged from 11 mM to more than 200 mM, independently of their TP53 mutational status (Figure 1C). Previous in vivo human data have shown that the plasma concentrations of APR-246 in the 12 h following its intravenous administration range from 50 to 500 mM, suggesting that concentrations above 50 mM are suitable for in vitro studies of the early effects of APR-246.19 In our subsequent experiments, we selected five AML cell lines sensitive to APR-246 in these concen- tration ranges, with or without TP53 mutations.
In order to investigate the mechanisms underlying APR-246 activity against AML cells, we exposed the cells treated with APR-246 to inhibitors of various cell death pathways (Figure 2A). The decrease in cell viability was almost completely rescued by either iron chelation via deferoxamine (DFO) or by the lipophilic antioxidant fer- rostatin-1 (Fer1), demonstrating that cell death is both iron and reactive- oxygen species (ROS) dependent. Necrostatin-1 (Nec1) also consistently prevented cell death induced by APR-246 after short-term incubation in all AML cells lines. Nec1 has been used to define necrop- totic cell death, but can also protect against ferroptosis through a target which is as yet unknown.20 Induction of necroptosis after treatment with APR-246 in our cells was excluded due to the absence of protection by more specif- ic necroptosis inhibitors (Necrostatin 1s and Necrosulfonamide),21,22 and due to the absence of MLKL phosphorylation, a key marker in necroptosis21 (Online Supplementary Figure S1A and B). Inhibitors of autophagy (chloroquine) or apoptosis (QVD-OPH) failed to block APR-246-induced cell death. We confirmed that the mechanism of APR-246 induced cell death is distinct from apoptosis, as evidenced by the absence of caspases 3/8 or PARP1 cleavage, including in TP53 mutated AML cell lines (Figure 2B; Online Supplementary Figure S2A to C). Notably, the protection against cell death observed with Fer1, DFO or Nec1 was partially lost at higher doses of APR-246 (Figure 2C) and at later time points (Figure 2D). However, apoptosis did not appear to be the mechanism of this late death, since caspases 3/8 and PARP1 were not cleaved and QVD-OPH was still unable to prevent the cell death (Online Supplementary Figure S2A to E). Examination of the ultrastructural changes induced by APR-246 treatment did not reveal any characteristic fea- tures of apoptosis (i.e., no plasma membrane blebbing, chromatin condensation or nuclear fragmentation) or autophagy (absence of autophagolysosomes). Necrotic cells were rare, and some mitochondria showed mem- brane rupture and reduced cristae (Figure 3A; Online Supplementary Figure S3). We analyzed the levels of endogenous lipid peroxidation – a hallmark of ferroptosis induction – following APR-246 treatment by flow cytom- etry analysis with C11-BODIPY staining. We observed a high induction of lipid ROS in AML cell lines and primary AML cells from two patients treated with APR-246 ex
vivo. This increase in lipid ROS was fully reversed by Fer1, indicating that lipid peroxides had been newly generated (Figure 3B and C). All these results allowed to conclude that APR-246 induces early cell death by ferroptosis, a recently described non-apoptotic form of regulated cell death that links together membrane lipid peroxidation, cysteine and iron metabolism, glutathione peroxidase activity and oxidative stress (as summarized in Figure 4A23–25). As previously reported,14,16 we observed that APR-246 treatment induced a dramatic decrease in GSH levels in AML cell lines (Figure 4B). Cysteine is the main biosynthetic precursor of GSH. Cysteine can be trans- ported into cells via membrane transporters for neutral amino acids. However, in the extracellular space, cysteine is rapidly reduced to cystine. Thus the main source of intracellular cysteine comes from the entry of cystine into the cell via system xc-.26 b-mercaptoethanol (b-ME) can promote cystine uptake through an alternative pathway.27 b-ME was able to completely rescue the cell death and GSH depletion induced by APR-246 (Figure 4C and D). Cysteine treatment showed similar results as Fer1 treat- ment (Figure 4C and D). Altogether, these results demon- strate that APR-246 induces GSH depletion which induces ferroptosis in AML cells irrespective of their TP53 mutational status, and that ferroptosis induction is the main mechanism of cell death after early exposure to APR-246.
Cystine uptake determined the sensitivity of acute myeloid leukemia cells to APR-246
Since cysteine is a biosynthetic precursor of GSH, we asked whether the ability of cells to provide cysteine for GSH synthesis underlies the sensitivity to APR-246. Using FITC-labeled cystine, we showed that after expo- sure to APR-246, AML cells increased their uptake of cys- tine from the extracellular space (Figure 5A). Western blot analysis of the protein levels of SLC7A11 showed an increased amount of SLC7A11 (Figure 5B). This suggests that the cells enhanced cystine uptake by increasing SLC7A11 protein levels to maintain intracellular GSH lev- els after exposure to APR-246. We modulated the cystine uptake through SLC7A11 overexpression or inhibition. SLC7A11 overexpression in MOLM-14 and OCI-AML2 (Online Supplementary Figure S4) decreased the cell death and prevented the depletion of GSH induced by APR-246 (Figure 5C and D). Then we showed that targeting the SLC7A11 cystine transporter by RNA interference reduced the basal uptake of cystine (Online Supplementary Figure S4B and C) and had a very little effect on cell death (Online Supplementary Figure S4D), but strongly reduced cell proliferation in AML cells in vitro (Online Supplementary Figure S4E). Inhibition of SLC7A11 with RNA interference increased cell death and viability impairment, GSH depletion, and the accumulation of lipid peroxides induced by APR-246 (Figure 6A to C; Online Supplementary Figure S4F). Interestingly, basal GSH levels were not consistently affected by b−ME addition, cysteine addition, or the overexpression or inhibition of SLC7A11, which suggests that the amount of GSH in basal conditions is not a reliable marker of cell cystine uptake ability (Figures 4D, 5D and 6C). Finally, targeting SLC7A11 with erastin, a potent inhibitor of system xc-28 that showed variable sensitivity in our AML cell lines (Online Supplementary Figure S4G), had synergistic activity with APR-246 both on cell death and on cell viability
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