APR-246 induces early cell death by ferroptosis in acute myeloid leukemia

Abstract

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Figure 1.

APR-246 induces cell death in acute myeloid leukemia cells irrespective of their TP53 mutational status. (A) Viability curves for the indicated cells at 24 hours (h) post APR-246 treatment. Error bars, ± standard deviation. (B) Viability curves for the indicated primary acute myeloid leukemia (AML) cells at 24 h post APR-246 treatment. Patient characteristics are provided in the Online Supplementary Table S1. (C) Half maximal inhibitory concentration percentage (IC₅₀%) of APR- 246 treatment for 24 h across a panel of AML cell lines based on cell viability (n=3). In our subsequent experiments, we selected five AML cell lines sensitive to APR- 246 in these concentration ranges, with or without TP53 mutations (highlighted in bold font). wt: wild-type; fs: frameshift; del: deletion.

Figure 2.

APR-246 induces ferroptosis in acute myeloid leukemia cells. (A) Cell viability (%) for the indicated cells at 16 hours (h) post-APR-246 treatment (60 mM) with or without ferrostatin-1 (10 mM), deferoxamine (DFO) (100 mM), necrostatin-1 (20 mM), chloroquine (20 mM) or QVD-OPH (25 mM) (n=3). Error bars ± standard error of the mean [SEM]. All compounds were added 2 h (h) prior to APR-246 in the medium. Statistics, 2-way ANOVA; *P<0.05, **P<0.01, ***P<0.0001. (B) Immunoblotting analysis of PARP, caspase 8 and caspase 3 in MOLM-14 cells treated for 16 h with dimethyl sulfoxide (DMSO), APR-246 (60 μM) or puromycin (1 mg/mL). b-actin was used as a loading control (n=2). (C) Viability curves for the indicated cells at 16 h post APR-246 treatment with or without ferrostatin-1 (10 mM), DFO (100 mM), necrostatin-1 (20 mM), chloroquine (20 mM) or QVD-OPH (25 mM) (n=3). Error bars ± SEM. (d) Cell death (%) of the indicated cells at 16 h and 24 h post-APR-246 treatment (50 mM) with or without ferrostatin-1 (10 mM) (n=3). Error bars ± standard deviation.

Figure 3.

APR-246 induces ferroptosis in acute myeloid leukemia cells. (A) Electron microscopy analysis of MOLM-14 cells treated with or without APR-246 (60 mM, H16). The white arrowhead indicates a mitochondrion showing membrane rupture and reduced cristae. (B and C) Detection of lipid peroxidation using C11-BODIPY and flow cytometry (FCM) at 14 hours post APR-246 treatment in acute myeloid leukemia (AML) cell lines (B) and in primary AML cells (C). APR-246 was used at a 100 mM concentration for MOLM-14 and 50 μM for other AML cell lines. Left panels show representative FCM quantification (n=3). Error bars ± standard deviation.

Figure 4.

APR-246 induces glutathione depletion in acute myeloid leukemia cells. (A) Summary of the cellular pathways involved in ferroptosis. Ferroptosis execution is triggered by an iron-catalyzed excessive peroxidation of polyunsaturated fatty acids (PUFA)-containing phospholipids (PL-PUFA). Glutathione (GSH) and glutathione peroxidase 4 (GPX4) are the two key elements controlling the elimination of lipid peroxides. Solute carrier family 7 member 11 (SLC7A11) encodes the transporter subunit of the heterodimeric cystine-glutamate antiporter named system xc-. System xc- mediates cystine entry into the cell in exchange for glutamate. Once inside the cell, cystine is rapidly reduced to cysteine which is the limiting amino acid for GSH synthesis. SLC7A11 inhibition results in cellular cysteine depletion, which leads to the exhaustion of intracellular pool of GSH. GPX4 is a pleiotropic selenoprotein that uses GSH to selectively reduce lipid hydroperoxides to lipid alcohols, in order to protect the cells against membrane lipid peroxidation. GPX4 inhibition is either due to its direct inhibition or downregulation, or to GSH depletion via direct or indirect processes. The inhibition of GPX4 results in uncontrolled polyunsaturated fatty acid phospholipid (PL-PUFA) oxidation and fatty acid radical generation, leading to ferroptotic cell death. ACSL4: acyl-CoA synthetase long chain family member 4; LPCAT 3: lysophosphatidylcholine acyltransferase 3; ALOX: arachidonate lipoxygenase; PUFA: polyunsaturated fatty acid; PL: phospholipids; PE: phosphatidylethanolamine; GPX4: glutathione peroxidase 4. (B) GSH (mBCI) measurement in acute myeloid leukemia (AML) cell lines by flow cytometry (FCM) at 14 hours (h) post APR-246 and fer1 (10 mM) treatment. APR-246 was used at 100 mM for MOLM-14 and 50 mM for other AML cell lines. Fer1 was associated to prevent cell death and allowed analysis of GSH depletion. Left panels show representative FCM quantification. n=3. Error bars + standard deviation. Statistics by t-test. *P<0.05, **P<0.01, ***P<0.0001. (C and D) Cell death (%) (C) and GSH (mBCI) measurement (D) for MOLM-14 at 24 h post-APR-246 treatment (60 mM) with or without B-ME (50 mM), cysteine (50 mM) or Fer1 (10 mM). Error bars + standard deviation. Statistics by t-test; *P<0.05, **P<0.01, ***P<0.0001.

Figure 5.

SLC7A11 overexpression prevents glutathione depletion and cell death following APR-246 exposure. (A) Cystine uptake in MOLM-14 and OCI-AML2 cells lines at 16 hours (h) post-APR-246 (100 mM) and Fer1 (10 mM) treatment. Fer1 was associated to prevent cell death and allowed analysis of cystine uptake. (B) Immunoblotting analysis of SLC7A11 in MOLM-14 cells treated for 16 h with dimethyl sulfoxide (DMSO) or APR-246 (n=2). b-actin was used as a loading control. (C and D) Cell death (%) (C) and glutathione (GSH) (mBCI) measurement (D) of the indicated cells at 20 h post-APR-246 treatment (n=3). For GSH measurement, Fer1 was associated to prevent cell death and allowed analysis of GSH depletion. Error bars ± standard deviation. Statistics by t-test. *P<0.05, **P<0.01, ***P<0.0001.

Figure 6.

SLC7A11 inhibition sensitizes cells to APR-246. (A) Viability curves for MOLM-14 and OCI-AML2 with small hairpin RNA control (shSCR) or doxycyclineinducible shRNA (shSLC7A11) cells at 16 hours (h) post APR-246 treatment. Prior to adding APR-246, the cells were treated for 3 days with doxycycline (n=3). Error bars ± standard deviation (SD). (B and C) Cell death (%) (B) and glutathione (GSH) (mBCI) measurement (C) of the indicated cells at 20 h post-APR-246 (MOLM-14 30 mM, OCI-AML2 10 mM) treatment (n=3). For GSH measurement, Fer1 was associated to prevent cell death and allowed analysis of GSH depletion. Prior to adding APR-246, the cells were treated for 3 days with doxycycline (n=3). Error bars ± SD. Statistics by t-test. *P<0.05, **P<0.01, ***P<0.0001. (D) Cell death (%) for the indicated cell types at 24 h post APR-246 (MOLM-14 30 mM, OCI-AML2 10 μM) and or erastin (MOLM-14 100 nM, OCI-AML2 1 mM). Error bars ± SD. Statistics by t-test; *P<0.05, **P<0.01. (E) Illustrative synergy map (left panel) of 24 h co-treatment of MOLM-14 cells with APR-246 and erastin. The mean cell viability of three independent experiments was used. Mean synergy scores of the most synergistic area of 24 h co-treatment of AML cell lines with APR-246 and erastin (n=3). (F) Mean synergy score of the 48 h co-treatment of primary AML cells with APR-246 and erastin (n=1).

Figure 7.

The combination of APR-246 with ferroptosis inducers has synergistic anti-leukemic effects in acute myeloid leukemia in vitro. (A) Viability curves for MOLM-14 and OCI-AML2 cells with or without GPX4 inducible small hairpin RNA (shRNA) at 16 hours (h) post APR-246 treatment. Prior to adding APR-246, the cells were treated for 2 days with doxycycline (n=3). Error bars ± standard deviation. (B) Illustrative synergy map (left panel) of 24 h co-treatment of MOLM-14 cells with APR-246 and RSL3. The mean cell viability of three independent experiments was used. Mean synergy scores of the most synergistic area of 24 h co-treatment of acute myeloid leukemia (AML) cell lines with APR-246 and RSL3 (n=3).(C) Illustrative synergy map (left panel) of 24 h co-treatment of MOLM-14 cells with APR-246 and FINO2. The mean cell viability of three independent experiments was used. Mean synergy scores of the most synergistic area of 24 h co-treatment of AML cell lines with APR-246 and FINO2 (n=3).

Figure 8.

The combination of APR-246 with SLC7A11 inhibition has synergistic anti-leukemic effects in acute myeloid leukemia in vivo. (A) Design of the in vivo experiment. (B) Representative flow cytometry analysis of bone marrow cells marked with mCD45.1 and hCD45 from each treatment subgroup. (C) Viable mCD45.1- hCD45⁺ hCD33⁺ cell counts in the bone marrow of the different treatment subgroups. Bars represent mean of all experiments and errors denote ± standard deviations. Statistics by t-test; *P<0.05, **P<0.01, ***P<0.0001.