Mitochondrial regulation of GPX4 inhibition-mediated ferroptosis in acute myeloid leukemia

Abstract

Resistance to apoptosis in acute myeloid leukemia (AML) cells causes refractory or relapsed disease, associated with dismal clinical outcomes. Ferroptosis, a mode of non-apoptotic cell death triggered by iron-dependent lipid peroxidation, has been investigated as potential therapeutic modality against therapy-resistant cancers, but our knowledge of its role in AML is limited. We investigated ferroptosis in AML cells and identified its mitochondrial regulation as a therapeutic vulnerability. GPX4 knockdown induced ferroptosis in AML cells, accompanied with characteristic mitochondrial lipid peroxidation, exerting anti-AML effects in vitro and in vivo. Electron transport chains (ETC) are primary sources of coenzyme Q₁₀ (CoQ) recycling for its function of anti-lipid peroxidation in mitochondria. We found that the mitochondria-specific CoQ potently inhibited GPX4 inhibition-mediated ferroptosis, suggesting that mitochondrial lipid redox regulates ferroptosis in AML cells. Consistently, Rho0 cells, which lack functional ETC, were more sensitive to GPX4 inhibition-mediated mitochondrial lipid peroxidation and ferroptosis than control cells. Furthermore, degradation of ETC through hyperactivation of a mitochondrial protease, caseinolytic protease P (ClpP), synergistically enhanced the anti-AML effects of GPX4 inhibition. Collectively, our findings indicate that in AML cells, GPX4 inhibition induces ferroptosis, which is regulated by mitochondrial lipid redox and ETC.

Figure 1.. GPX4 is a potential therapeutic target in AML

A Efficacy/selectivity plot for the whole genes analyzed on shinyDepMap. The top 10 ferroptosis suppressor genes on FerrDb are annotated. B Dependency scores of GPX4 in all cell lines are clustered based on their lineages. A lower dependency score indicates a higher dependency on GPX4. Leukemia cell lines are subdivided into AML, B-cell ALL, and T-cell ALL, based on the original DepMap annotations. C GPX4 protein levels in 11 AML cell lines were determined by immunoblotting. The relative expression levels of GPX4 were assessed with densitometry as a ratio of GPX4 to α-tubulin (the loading control) and normalized to that of OCI-AML3. D, E GPX4 protein levels in peripheral blood mononuclear cells from 16 AML patients and bone marrow mononuclear cells from 4 healthy bone marrow donors (HD) were determined by immunoblotting (D). The relative expression levels of GPX4 were assessed with densitometry as a ratio of GPX4 to Histon H3 (the loading control), normalized to that of AML patient #17 that serves also as inter-membrane control, and plotted (E). High and low GPX4 bands suggest posttranslational modification, part of which appear as smears possibly due to partial degradation. For quantification, all the bands detected are included in the densitometry analysis. F GPX4 protein expression measured by mass spectrometry with tandem mass tag (TMT) labeling is plotted for AML samples (n = 44) and lineage-depleted normal bone marrow (NBM) samples (n = 3). Original data was obtained from a previous study [29].

Figure 2.. The pharmacological inhibition of GPX4 induces ferroptosis in AML

A, C OCI-AML3 cells were treated with 0.5 μM ML210 with or without 1 μM liproxstatin-1 (Lip1), 100 μM α-tocopherol (aToc), or 4 μM deferoxamine (DFO) for 48 hours. Cell death (A) and lipid peroxidation (C) were determined by annexin V (AnnV) and/or DAPI positivity or by C11-BODIPY581/591 staining, respectively, on flow cytometry. B OCI-AML3 cells were treated with 1 μM venetoclax (Ven) plus 0.2 μM AMG-176 (MCL-1 inhibitor) to induce apoptosis [65] or 0.2 μM ML210, with or without 25 μM z-VAD-FMK (zVAD), for 24 hours. D Parental AML cells were treated with ML210 for 24 hours, and specific cell death was calculated as described in Supplemental information. X axis represents ML210 concentration on a logarithmic scale. E The area under the cell killing curve (AUC) for ML210 (Fig. 2D) was correlated with the relative GPX4 expression level (Fig. 1C). Correlation coefficient (r) and statistical significance (p) on Pearson correlation analysis were indicated. F MOLM-13 cells with wildtype TP53 (WT), TP53 knockout (KO), or the indicated TP53 mutations were treated with ML210 for 72 hours. G Kasumi-1 cells transfected with shRNA targeting TP53 (shp53) or with scramble control shRNA (shC) were treated with ML210 for 72 hours. H Peripheral blood mononuclear cells from AML patients (n = 14) or bone marrow mononuclear cells from healthy bone marrow donors (HD) (n = 12) were treated with 10 μM ML210 for 48 hours. Cell death was determined for CD45⁺ bulk hematopoietic cell populations. I AUC for ML210 in primary AML cells (Fig. S2O) was correlated with the GPX4 expression level (Fig. 1D) for matching samples (n = 13).

Figure 3.. The genetic knockdown of GPX4 induces ferroptosis in AML in vitro and in vivo

A OCI-AML3 cells transfected with shRNAs targeting GPX4 (shGPX4) or with control shRNA (shC) were treated with 1 μg/ml doxycycline (Dox) for 72 hours, and GPX4 protein levels were determined by immunoblotting. B OCI-AML3-shGPX4–1 cells were treated with 1 μg/ml Dox for 96 hours, and cell death (left panel) and lipid peroxidation (right panel) were determined by flow cytometry. C–E OCI-AML3-shGPX4 (C), MOLM-13-shGPX4 (D), and OCI-AML2-shGPX4 (E) cells were treated with Dox for 120 hours (C) or 96 hours (D, E). F OCI-AML3-shGPX4 cells were treated with 0.1 μg/ml Dox for 72 hours in combination with 0.1 μM ML210 for the last 24 hours. G Survival curves of the NSG mice transplanted with luciferase-labeled OCI-AML3-shGPX4–2 cells and treated with vehicle or tetracycline (n = 7 for each group) were plotted. H OCI AML3-shGPX4–2 cells were harvested from moribund mice after treatment with vehicle or tetracycline for 6–8 weeks (n = 3 for each group) and purified for AML cells as described in Supplemental information. GPX4 protein levels were determined by immunoblotting.

Figure 4.. Mitochondrial lipid peroxidation and electron transport chain complexes regulate AML cell ferroptosis

A OCI-AML3 cells were treated with 0.5 μM ML210 or with DMSO as a control for 6 hours and then subjected to transmission electron microscopy to assess the mitochondrial ultrastructure. B, C OCI-AML3 cells were treated with 0.5 μM ML210 with or without 1 μM liproxstatin-1 (Lip1) (B), 100 μM α-tocopherol (aToc) (B), or 4 μM deferoxamine (DFO) (C) for 48 hours. Mitochondrial superoxide production was determined by MitoSOX staining and flow cytometry. The median fluorescence intensity (MFI) of the treated cells was normalized to that of the untreated cells. D OCI-AML3 cells were treated with 0.5 μM ML210 for 48 hours, and mitochondrial lipid peroxidation was determined by MitoPerOx staining and flow cytometry. E OCI-AML3-shGPX4–1 cells were treated with 1 μg/ml doxycycline (Dox) for 96 hours, and mitochondrial superoxide (left) and lipid peroxidation (right) were determined by flow cytometry. MEFs (F) or OCI-AML3 cells (G) were treated with 2.0 μM ML210 in combination with increasing concentrations of MitoQ or DecylQ as indicated on the x axis for 24 hours. Cell viability was determined as the percentage of annexin V- and DAPI-negative cells on flow cytometry. H OCI-AML3 cells were treated with 0.5 μM ML210 with or without 0.1 μM MitoQ or 10 μM MitoTEMPO for 48 hours. I OCI-AML3-shGPX4–1 cells were treated with 1 μg/ml Dox with or without 0.1 μM MitoQ or 10 μM MitoTEMPO for 120 hours. J Lysates from HL-60-RhoWT cells or -Rho0 cells were subjected to immunoblotting for the indicated proteins. HL-60-Rho0 cells or -RhoWT cells were treated with ML210 for 48 (K) or 24 (L) hours.

Figure 5.. GPX4 inhibition–mediated ferroptosis is synergistically enhanced by ClpP hyperactivation

A OCI-AML3, MOLM-13, and OCI-AML2 cells were treated with ONC201 and/or ML210 for 72 hours, and cell death was determined by flow cytometry. A combination index (C.I.) less than 1.0 indicates synergistic effects. OCI-AML3 cells were treated with ONC201 and/or ML210 for 48 hours, and lipid peroxidation (B) and mitochondrial superoxide production (C) were determined by flow cytometry. D Primary cells from AML patients (n = 8) were treated with ML210 and/or ONC201 for 48 hours. Combination indexes were calculated based on specific cell death in CD45⁺ population upon the combinatorial treatment for each sample and were plotted. One sample was excluded from C.I. assessment because ONC201 was completely ineffective and thus C.I. calculations were not applicable. The sample from patient #12, which was resistant to a single treatment with ML210 on Figs. 2H and S2O, is indicated by arrows. E Primary cells from AML patients (n = 8) or healthy bone marrow donors (HD) (n = 8) were treated with 10 μM ML210 and 5 μM ONC201 for 48 hours, and cell death was determined for CD45⁺ populations. The sample from AML patient #12 is indicated by an arrow. F OCI-AML3-CLPP-Y118A cells were treated with doxycycline (Dox) for 144 hours in combination with ML210 for the last 72 hours. OCI-AML3-shGPX4–1 cells were treated with Dox for 120 hours in combination with ONC201 (G) or ONC212 (H) for the last 72 hours. I A genome-wide CRISPR knockout screening performed on NALM6 cells treated with ClpP agonists [46] were re-analyzed and sgRNA frequency scores (RANKS scores) were plotted. J OCI-AML3, OCI-AML2, and MOLM-13 cells were treated with 2 μM ONC201 for up to 48 hours, and GPX4 expression was determined by immunoblotting. K HL-60-Rho0 cells or -RhoWT cells were treated with ML210 with or without 0.25 μM ONC212 for 48 hours.