Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Abstract

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples.

Significance: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

Figure 1.

Ironomycin induces potent cell death in AML through a noncanonical cell death pathway. A, Half-maximal inhibitory concentration (IC₅₀) of AML cell lines with various genetic background after 72 hours of treatment with ironomycin using resazurin assay (n = 3 biological replicates). B, Proliferation curves of MV4;11, MOLM-13, and OCI-AML3 cell lines treated with ironomycin (n = 3 biological replicates, *, P < 0.05; **, P < 0.01; ***, P < 0.001). C, Protein expression of cleaved caspase-3 (cl. casp. 3) and cleaved caspase-7 shown by immunoblot. MV4;11 cells were treated with DMSO, 50 nmol/L venetoclax, or 500 nmol/L ironomycin. D, Effect on cell death of the pan-caspase inhibitor Z-VAD-fmk. MOLM-13 cells were pretreated with 50 μmol/L Z-VAD-fmk for 30 minutes and treated with 500 nmol/L ironomycin or 50 nmol/L venetoclax for 24 hours. Cell death was assessed by propidium iodide (PI) staining (n = 3 biological replicates; means ± SD; ***, P < 0.001). E, Effect on cell death of the necroptosis inhibitor necrostatin-1. MOLM-13 cells were pretreated with 10 μmol/L necrostatin-1 for 30 minutes and treated with 500 nmol/L ironomycin for 24 hours or 100 nmol/L birinapant plus 5 μmol/L IDN-6556 for 16 hours. Cell death was assessed by PI staining (n = 3 biological replicates; means ± SD; ***, P < 0.001). F, Visualization of the lysosomal localization of ironomycin using click chemistry in AML cells. Top, schematic illustration of the chemical labeling of ironomycin in cells. Bottom, fluorescence microscopy images of labeled ironomycin (Alexa Fluor 488, green), lysosome (lysotracker, red), and nucleus (DAPI, blue) in MOLM-13 cells after 2 hours of 10 μmol/L ironomycin treatment. Scale bar, 10 μm. G, Quantification of lysosomal Fe²⁺ using a lysosomal turn-on FACS probe in MOLM-13 cells after ironomycin (see Supplemental Data). Fe²⁺ specifically reduces Rhonox-M to a rhodamine B derivative, which fluoresces (n = 3 biological replicates; *, P < 0.05). H, FACS analysis of LMP using lysotracker (Lyso) and PI in the MOLM-13 cell line. We treated cells with DMSO or 500 nmol/L ironomycin. LMP is associated with a loss of lysotracker staining (n = 3 biological replicates; mean ± SD; *, P < 0.05). I, Analysis of lipid peroxidation by flow-cytometry staining of lipid ROS with the C11 BODIPY 581/591 (BODIPY C11) probe. MOLM-13 cells were treated with DMSO or 500 nmol/L ironomycin for 48 hours. The ratio of oxidized to total C11 median fluorescent intensity (MFI) is shown (n = 4 biological replicates; means ± SD; *, P < 0.05). J, Effect on cell viability of the ferroptosis inhibitor ferrostatin-1 used in combination with ironomycin. MOLM-13 cell line was pretreated with 20 μmol/L ferrostatin-1 for 30 minutes and treated with 500 nmol/L ironomycin or RSL-3 (30 nmol/L). Cell death was assessed by PI staining (n = 3 biological replicates; means ± SD; **, P < 0.01).

Figure 2.

Genome-wide CRISPR screen identifies cellular metabolism and mitochondrial homeostasis as key regulators of ironomycin activity. A, Schematic outline of the resistance genome-wide CRISPR/Cas9 loss-of-function screen in OCI-AML3 (n = 3 screens from two independent biological replicates). B, Venn diagram showing the common sgRNAs enriched in the ironomycin-resistant cells in the sequencing of the three screens (1a, 2a, and 1b). C, Table displaying the nine common genes from the three screens performed in the OCI-AML3 cell line (1a, 2a, and 1b). Genes related to glycolysis pathway are highlighted in blue and genes related to mitochondria homeostasis are highlighted in orange. D, Enrichment analysis showing GO terms significantly enriched in the top 100 genes from the OCI-AML3 screen. GO terms related to glycolysis are highlighted in blue, and GO terms related to mitochondria homeostasis are highlighted in orange. E, Bubble plots showing the top 1,000 enriched genes identified in the CRISPR screen (replicate 2a). The two hits selected for validation are colored in blue. Dotted line indicates Bonferroni-corrected significance threshold. F and G, Validation of the CRISPR screen in the MV4;11 cell line by competition assays using MV4;11 cells transfected with two independent sgRNAs targeting PGP (PGP.2 and PGP.4) or HK2 (HK2.2 and HK2.3) and a control nontargeted (NT) sgRNA. A 1:1 ratio of mCherry-positive sgRNA cells and mCherry-negative WT MV4;11 cells was treated with DMSO (F) or 500 nmol/L ironomycin (G), and the proportion of mCherry-positive sgRNAs cells was assessed by FACS (n = 2 biological replicates for each KO cell line; means ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Metabolic remodeling to reduce glycolytic flux and mitochondrial respiration protects against ironomycin. A, Heat map showing the differential abundance of metabolites in the MV4;11 WT cell line using mass spectrometry. We treated the cells with 500 nmol/L ironomycin or DMSO for 24 hours (n = 4 biological replicates). We selected the metabolites with a log₂ fold change >1 and a t test P < 0.05. B, Schematic representation of glycolysis and the branched PPP and function of the two metabolic enzymes hexokinase 2 (HK2) and phosphoglycolate phosphatase (PGP). DHAP, dihydroxyacetone phosphate; F6P, fructose-6-phosphate; F-1,6-BP, fructose-1,6-bisphophate; G3P, glyceraldehyde-3-phosphate; Ru5P, ribulose-5-phosphate; 6PG, 6-phospho-D-glycerate; TCA, tricarboxylic acid cycle. C, Bar graph showing the changes in metabolites expression in the sgHK2 cell line using mass spectrometry (n = 4 biological replicates; means ± SD; *, P < 0.05; ***, P < 0.001). D, Proportion of cell death of ironomycin-treated MV4;11 cells cultured in various glucose concentrations. We performed flow-cytometry analysis using PI. Cells were treated with 500 nmol/L ironomycin for 48 hours in RPMI medium (n = 3 biological replicates; means ± SD; *, P < 0.05). E, Bar graph showing the changes in metabolite expression in the sgPGP cell line. Metabolites downstream phosphofructokinase 2 (PFK2) are decreased such as F-1,6-BP and DHAP. Metabolites upstream PFK2 are increased such as G6P, 6PG, and G3P (n = 3 biological replicates; means ± SD; **, P < 0.01; ***, P < 0.001). F, Proportion of cell death of ironomycin-treated cells in combination with the PPP inhibitor 6AN. We performed flow-cytometry analysis of PI fluorescence in MOLM-13. Cells were pretreated with 10 μmol/L 6AN for 30 minutes and treated with 500 nmol/L ironomycin for 48 hours (n = 3 biological replicates; means ± SD; *, P < 0.05).

Figure 4.

Ironomycin induces mitochondrial stress through iron deprivation. A–C, RNA-seq analysis of MV4;11 cells treated with 6 hours of 500 nmol/L ironomycin or vehicle (DMSO, n = 3 biological replicates). A, Gene set enrichment analysis using the “mitostress” signature (30). B, KEGG pathways analysis (pathways upregulated are in red and pathways downregulated are in blue). C, Volcano plots showing the adjusted significance P value (−log₁₀P) versus the fold change (log₂). Genes that demonstrate a significant change in expression (P < 0.01) and a significant 2-fold downregulation (left) or 2-fold upregulation (right) are represented in red. Genes selected from the “mitostress” and mitoCarta 2.0 signatures are displayed. D, Validation of RNA-seq analyses by RT qPCR measuring mRNA expression of selected genes from the “mitostress” signature in AML cell lines 6 hours after exposure to 500 nmol/L ironomycin or DMSO. We used β2 m as a housekeeping gene (n = 3 technical replicates; means ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001). E, ATF4 protein expression by immunoblot after 500 nmol/L ironomycin treatment. F, Effect of high glucose culture condition (20 mmol/L) on RNA expression of selected genes from the “mitostress” signature upon ironomycin treatment in MV4;11 cells. We used β2 m as a housekeeping gene (n = 3 technical replicates; means ± SD; **, P < 0.01; ***, P < 0.001. G, Quantification of mitochondrial fe2+ by inductively couple plasma-mass spectrometry (ICP-MS) in MOLM-13 cells (n = 3 biological replicates; means ± SD; *, P < 0.05). H, Quantification of mitochondrial iron using a mitochondrial specific probe (see Supplemental Data) by flow cytometry on MOLM-13 cells treated with ironomycin (n = 3 biological replicates; means ± SD; *, P < 0.05). I, Seahorse assay measuring mitochondrial basal and maximal respiration in MV4;11 WT cells. We treated the cells for 6 hours with 500 nmol/L ironomycin (n = 3 biological replicates).

Figure 5.

Ironomycin-induced cell death is BAX/BAK dependent. A,TEM images of MV4;11 cells treated with vehicle (DMSO), 500 nmol/L ironomycin or 50 nmol/L venetoclax for 36 hours. Arrows, examples of apoptotic cells; V, vacuolization; N, chromatin condensation and nuclear fragmentation; white arrowheads, standard mitochondrial morphology; black arrowheads, changed mitochondrial morphology (cristae reduction and dilation, fragmentation, or dark condensed matrix). Scale bars, 20 μm, top; 2 μm, middle; 500 nm, bottom. B, Cell death assessed by PI exclusion using FACS in MV4;11 WT and BAX/BAK DKO cell lines after 48 hours of ironomycin (n = 3 biological replicates). C, Cell death assessed by PI exclusion using FACS in MEF cells treated for 48 hours with 2 μmol/L ironomycin. We compared a Bax^−/−Bak^−/− with a control Mcl1^−/− MEF cell line (n = 3 biological replicates; means ± SEM; *, P < 0.05). D, ATP luminescence measured by CellTiter-Glo in MV4;11 WT and BAX/BAK DKO cell lines after 48 hours of ironomycin (n = 3 biological replicates). E, ATP luminescence measured by CellTiter-Glo in MEF cells treated for 48 hours with 2 μmol/L ironomycin. We compared a Bax^−/−Bak^−/− with a control Mcl1^−/− MEF cell line (n = 3 biological replicates; means ± SEM; ***, P < 0.001). F, Seahorse assay measuring mitochondrial basal and maximal respiration in BAX/BAK DKO MV4;11 cells. We treated the cells for 6 hours with 500 nmol/L ironomycin or 50 nmol/L venetoclax (n = 3 biological replicates). G, Fractionation experiment showing BAX and COX1 protein expressions by immunoblot in total cell, cytosolic fraction, and mitochondrial membrane fraction. The nonheme protein VDAC1 was used as a loading marker of membrane fraction and HSP70 as a loading marker of cytoplasmic fraction. We used the Bax^−/−Bak^−/−Mcl1^−/− MEF cell line and treated cells with 500 nmol/L ironomycin.

Figure 6.

Ironomycin cell death is distinct from canonical apoptosis induced by BH3 mimetics. A, Confocal images of MCL1^−/− MEFs expressing tagged-OMM component TOMM20-Halo (JF646, red) with either IMM component TIMM23 (left; mNeonGreen, green) or MMX (right; tdTomato, green) after 24 hours untreated (top) or 3 μmol/L ironomycin (bottom). Insets highlight representative mitochondrial network morphology. B, Snapshots from long-term widefield imaging assay of MCL1^−/−Bax^−/−Bak^−/− MEFs reexpressing mNeonGreen-BAX and TOMM20-Halo, treated with 1 μmol/L ABT-737 + 20 μmol/L QVD-OPH or 500 nmol/L ironomycin, with insets highlighting BAX recruitment to mitochondria prior to cell death (n = 3 independent imaging experiments). C, Scatter plot displaying the time until the appearance of BAX foci, quantified manually, from long-term widefield imaging assay. Each data point represents a single cell (n = 3 biological replicates with >10 cells counted per experiment; means ± SD; **, P < 0.01; ***, P < 0.001). D, Fractionation experiment in MV4;11 cells treated with 500 nmol/L ironomycin showing cytochrome c (CYT C) and BAX protein expressions by immunoblot in total cell, cytosolic fraction, and mitochondrial membrane fraction. VDAC1 is used as a loading marker of mitochondrial membrane fraction and HSP70 as a loading marker of cytoplasmic fraction (n = 3; one representative experiment is shown). E, Cell viability assessed by FACS analysis of PI staining. We treated MV4;11 WT and MV4;11 cells with BCL2 overexpressed for 48 hours with ironomycin (top curve) and venetoclax (bottom curve, n = 3 biological replicates). F, Heat map showing the mRNA expression of BH3-only proteins in MV4;11 cells after 6 and 24 hours of ironomycin. G and H, Cell viability using PI FACS staining. We treated MV4;11 WT and MV4;11 cells with single knockout for the BH3-only proteins NOXA, PUMA, BIM, and BID (G) or double knockouts for BIM/BID and NOXA/PUMA (H) for 72 hours with ironomycin (top curves) and venetoclax (bottom curves; n = 3 biological replicates).

Figure 7.

Ironomycin shows marked synergy with BH3 mimetics and overcomes resistance to venetoclax. A, Heat maps showing the percentage of inhibition assessed by FACS (PI) using ironomycin and venetoclax as single agent and in combination in two AML cell lines (top) and Bliss calculation measuring synergy between the two drugs (bottom; n = 3 biological replicates). B, Analysis of mitochondrial membrane potential (Δ Ψm) using JC-1 staining assessed by FACS after 24 hours of low-dose venetoclax with or without low-dose ironomycin treatments. Loss of JC-1 staining is associated with a loss of Δ Ψm (n = 3 biological replicates, mean ± SEM; *, P < 0.05). C, Immunoblot showing cleaved caspase-3 after 24 hours of low-dose venetoclax with or without low-dose ironomycin treatments in OCI-AML3 cells. D,TEM images of mitochondria in MV4;11 WT (left) and BAX/BAK DKO cells (right) treated with low doses of ironomycin and venetoclax for 36 hours. Gray arrowheads, mitochondria in BAX/BAK DKO cells; black arrowheads, disrupted mitochondrial integrity. Scale bars, 2 μm, top; 500 nm, bottom. E, Kaplan–Meier analyses showing survival of NSG mice transplanted with MV4;11 cells (n = 5 mice per cohort) treated with ironomycin, venetoclax, and combination of the two drugs (*, P < 0.05; **, P < 0.01). F, Cell viability assessed by FACS analysis of PI staining. We treated MV4;11 WT and MV4;11 cells with TP53 KO for 72 hours with ironomycin (top) and venetoclax (bottom). G, Heat maps showing the effect on cell viability assessed by flow cytometry (PI) in response to escalating doses of ironomycin and venetoclax for 5 days as single agent and in combination in five patients known to be clinically resistant to venetoclax (top) and Bliss calculation measuring synergy between the two drugs (bottom; n = 1 representative experiment).