Acute myeloid leukemia mitochondria hydrolyze ATP to support oxidative metabolism and resist chemotherapy

Abstract

OxPhos inhibitors have struggled to show a clinical benefit because of their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to acute myeloid leukemia (AML) mitochondria. Unlike healthy cells that couple respiration to ATP synthesis, AML mitochondria support inner-membrane polarization by consuming ATP. Matrix ATP consumption allows cells to survive bioenergetic stress. Thus, we hypothesized AML cells may resist chemotherapy-induced cell death by reversing the ATP synthase reaction. In support, BCL-2 inhibition with venetoclax abolished OxPhos flux without affecting mitochondrial polarization. In surviving AML cells, sustained mitochondrial polarization depended on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to down-regulations in the endogenous F₁-ATPase inhibitor ATP5IF1. Knockdown of ATP5IF1 conferred venetoclax resistance, while ATP5IF1 overexpression impaired F₁-ATPase activity and heightened sensitivity to venetoclax. These data identify matrix ATP consumption as a cancer cell-intrinsic bioenergetic vulnerability actionable in the context of BCL-2 targeted chemotherapy.

Fig. 1.. Intrinsic lesions in respiratory complex IV limit OxPhos flux in AML cells.

All experiments were performed using whole cells and digitonin-permeabilized cells. (A) Comparison of fractional OxPhos of normal blood cells, patient AML, AML cell lines, chemotherapy refractory AML cell lines, and mouse AML cells calculated as the ratio of JH⁺_OXPHOS to JH⁺_Total (n = 3 to 30 replicates) and represented as a percentage of total respiratory capacity. Skeletal muscle (SkM) progenitors refer to myoblasts isolated from human skeletal muscle biopsy. (B) Volcano plot comparing mitochondrial and non-mitochondrial proteome of AML cells and healthy bone marrow mononuclear cells (BMMCs). Log₂FC, log₂ fold change. (C) Comparison of mitochondrial content in AML cells and healthy BMMCs (n = 3 to 30 replicates). (D) Comparison of whole-cell respiratory capacity in healthy BMMCs, MV4-11 cells, and OCI-AML2 cells normalized to milligrams of protein (n = 3 to 5 replicates). n.s., not significant. (E) Comparison of the OxPhos proteome between healthy BMMCs and AML cells (n = 3 replicates). (F) Comparison of whole-cell respiratory capacity in healthy BMMCs, MV4-11 cells, and OCI-AML2 cells normalized to milligrams of mitochondrial protein (n = 3 to 5 replicates). (G) Schematic depicting complex IV lesions and reduced respiration of individual AML mitochondria. Data are presented as means ± SEM and analyzed by two-way ANOVA [(E) and (F)] and one-way ANOVA [(A), (C), and (D)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NAD⁺, nicotinamide adenine dinucleotide (oxidized form); NADH (reduced form); FAD⁺, flavin adenine dinucleotide (oxidized form); FADH₂ (reduced form). (A) and (G) created using BioRender.com.

Fig. 2.. Mitochondria in AML cells consume cellular ATP to sustain mitochondrial polarization.

All experiments were performed using whole intact cells. (A) Schematic depicting strategy to determine functional OxPhos in ΔΨ_m assays. (B) Representative image from flow cytometric analysis of intact cell ΔΨ_m in AML2_Rho0 cells. (C) Flow cytometric analysis of intact cell ΔΨ_m in AML2_Rho0 cells (n = 4 replicates). (D) Flow cytometric analysis of intact cell ΔΨ_m in healthy BMMCs (n = 3 replicates). (E) Flow cytometric analysis of intact cell ΔΨ_m in myeloid and lymphoid populations sorted from PBMCs (n = 4 to 6 replicates). (F) Flow cytometric analysis of intact cell ΔΨ_m in AML cell lines (n = 3 to 4 replicates). (G). Flow cytometric analysis of intact cell ΔΨ_m in patient AML (n = 4 patients). (H) Flow cytometric analysis of intact cell ΔΨ_m in pooled MV411_WT, HL60_WT, and AML2_WT cells (n = 1 replicate per cell type). (I) Representative trace of permeabilized cell ΔΨ_m assay in AML2_WT cells. (J) Comparison of oligomycin-induced depolarization in AML2_WT cells in the presence of increasing doses of rotenone (n = 4 replicates). (K) Schematic of mitochondrial inhibitors and their targets. (L) Flow cytometric analysis of intact cell ΔΨ_m in MOLM13_WT, AML2_WT, and patient AML cells exposed to the following inhibitors: oligomycin (10 μM), BA (50 μM), CPI-613 (200 μM), CB-839 (5 μM), IACS (0.5 μM), and FCCP (10 μM). Data are presented as means ± SEM and analyzed by two-way ANOVA (F), one-way ANOVA [(C), (D), (G), (H), (J), and (L)], or unpaired t test (E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (A) and (K) created using BioRender.com.

Fig. 3.. F₁-ATPase activity supports both oxidative metabolism and survival in AML cells.

All experiments were performed using whole intact cells, digitonin-permeabilized cells, or alamethicin-permeabilized mitochondria. (A) Effect of ΔG_ATP on maximal NADH (complex I) or succinate/rotenone (complex II) supported respiration in alamethicin-permeabilized MV411_WT mitochondria (n = 4 replicates). (B) Effect of oligomycin on inhibition of uncoupled respiration by ΔG_ATP in permeabilized AML2_WT cells (n = 3 replicates). (C) Effect of oligomycin on uncoupled respiration in intact AML2_WT cells (n = 3 replicates). (D) Effect of ΔG_ATP on polarization by pyruvate/malate (Pyr/Mal) in the presence of oligomycin and in the presence or absence of carboxyatractyloside (CAT; 5 μM), in permeabilized AML2_WT cells (n = 3 replicates). (E) Change in TMRM fluorescence of individual mitochondria in mitochondrial populations isolated from mouse heart or HL60 cells before and after oligomycin addition during flow cytometric analysis of the ΔΨ_m. MFI, mean fluorescence intensity. (F) Cell viability, assessed via trypan blue, in AML cell lines and patient AML cells exposed to vehicle or BA (50 μM) for 48 hours (n = 3 replicates). (G) Effect of increasing doses of BAM15 uncoupler on AML2_WT, MV411_WT, and HL60_WT cell viability. Viability was measured using PI (n = 4 to 5 replicates). (H) Effect of IACS, antimycin A, or oligomycin on AML cell viability. Viability was measured using trypan blue (n = 3 replicates). (I) Effect of IACS, antimycin A, or oligomycin on AML2_WT, MV411_WT, and HL60_WT colony formation. Colonies were manually counted using light microscopy (n = 5 replicates). (J) Colony-forming unit assay in patient AML cells exposed to DMSO, IACS, or oligomycin (n = 6). Colonies quantified using CellTiter-Glo. Data are presented as means ± SEM and analyzed by two-way ANOVA [(A) to (D), (G), and (H)], one-way ANOVA [(I) and (J)], or unpaired t test (F). *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 4.. Venetoclax exposure collapses mitochondrial respiration, but polarization is sustained via F₁-ATPase activity.

(A) Effect of 1 hour exposure to venetoclax (0.1 and 1 μM doses combined) on AML cell viability (n = 9 replicates). (B and C) Effect of 1 hour exposure to 1 μM venetoclax on respiratory capacity (B) or OxPhos capacity (C) in permeabilized AML cells (n = 3 to 4 replicates). (D) Effect of 1 hour exposure to 100 nM venetoclax on polarization induced by complex I substrates (P/M/G), complex II substrates (S/R), or complex V substrates (ATP) in permeabilized AML cells (n = 4 to 6 replicates). (E) Flow cytometric analysis of intact MOLM-13_WT cell ΔΨ_m in response to single-agent venetoclax, and venetoclax in combination with IACS and antimycin A, and those in combination with oligomycin (n = 3 replicates). (F) Fluorescent microscopy analysis of intact MV411_WT cell ΔΨ_m in response to single-agent venetoclax, and venetoclax in combination with rotenone and antimycin A, and those in combination with oligomycin (n = 100 to 311 cells). (G) Volcano plot depicting changes in intracellular metabolites in OCI-AML2 cells exposed to DMSO or venetoclax (100 nM) for 1 hour (n = 3). CTP, cytidine 5′-triphosphate; UTP, uridine 5′-triphosphate. (H) Effect of single-agent venetoclax, venetoclax in combination with IACS and oligomycin, or IACS in combination with oligomycin on HL60_WT cell viability. Viability was measured using CellTiter-Glo (n = 4 replicates). (I) Effect of single-agent venetoclax or venetoclax in combination with either oligomycin or IACS on MOLM-13_WT cell viability. Viability was measured using CellTiter-Glo (n = 4 to 8 replicates). (J) Bliss Synergy analysis of cell viability in HL60_WT cells exposed venetoclax (0, 1, 10, and 100 nM) in the absence and presence of BAM15 (5 μM). Data are presented as means ± SEM and analyzed by two-way ANOVA [(C) to (E) and (H)], one-way ANOVA [(B) and (F)], or unpaired t test [(A) and (I)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.. Chemoresistant AML mitochondria present with a phenotypic shift toward enhanced ATP consumption.

(A) Schematic depicting generation of venetoclax-resistant AML cell lines. (B) Effect of venetoclax on treatment naïve AML and chemoresistant AML cell viability. Viability was measured using PI (n = 3 to 6 replicates). Created using BioRender.com. (C) Comparison of OxPhos proteome in permeabilized treatment naïve AML cells and venetoclax-resistant AML cells (n = 3 replicates). (D) Comparison of ATP5IF1 expression in mitochondria isolated from MV411_WT and MV411_Vclax cells (n = 3 replicates). (E) Comparison of respiratory capacity in permeabilized treatment naïve AML cells and venetoclax-resistant AML cells (n = 3 to 4 replicates). (F) Comparison of OxPhos capacity in permeabilized treatment naïve AML cells and venetoclax-resistant AML cells (n = 3 to 4 replicates). (G) Comparison of oligomycin-induced depolarization in permeabilized treatment naïve AML cells relative to permeabilized venetoclax-resistant AML cells (n = 3 to 6 replicates). (H) Flow cytometric analysis of intact cell ΔΨ_m in venetoclax-resistant AML cells (n = 4 replicates). (I) Confocal microscopy images and analysis of MV411_Vclax cells in the presence or absence of oligomycin. (J) Comparison of OxPhos kinetics in treatment naïve MV411_WT cells and MV411_Vclax cells grown in venetoclax or after venetoclax has been removed from culture medium for 24 hours (n = 4 replicates). (K) Effect of IACS and venetoclax or oligomycin in the presence or absence of venetoclax on MOLM-13_Vclax cell viability. Viability was measured using CellTiter-Glo (n = 4 to 8 replicates). (L) Effect of IACS and venetoclax, BAM15 and venetoclax, or oligomycin in the presence or absence of venetoclax on MV411_Vclax cell viability. Viability was measured using trypan blue (n = 3 replicates) Data are presented as means ± SEM and analyzed by two-way ANOVA [(B) and (J)], one-way ANOVA [(E) to (G)], or unpaired t test [(C), (D), (H), (K), and (L)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6.. Knockdown of ATP5IF1 confers resistance to BCL-2 targeted therapy.

(A) Expression of ATP5IF1 in isolated mitochondria (n = 3 replicates). (B) Impact of ATP5IF1 knockdown on the inhibition of respiration by ΔG_ATP in permeabilized scrambled short hairpin RNA (shRNA) control AML cells and permeabilized ATP5IF1 knockdown AML cells (n = 4 to 8 replicates). (C) Impact of ATP5IF1 knockdown on intact cell respiration of scrambled shRNA control AML cells and ATP5IF1 knockdown AML cells (n = 8 to 15 replicates). (D) Impact of ATP5IF1 knockdown (KD) on respiratory capacity in permeabilized scrambled shRNA control AML cells and permeabilized ATP5IF1 knockdown AML cells (n = 8 to 13 replicates). (E) Impact of ATP5IF1 knockdown on OxPhos capacity (n = 8 to 14 replicates). (F) Impact of ATP5IF1 knockdown on fractional OxPhos in permeabilized cells (n = 8 to 14 replicates). (G) Flow cytometric analysis of intact cell ΔΨ_m in MV411_shCtrl and MV411_shIF1 cells (n = 3 replicates). (H) Effect of increasing doses of venetoclax on viability of scrambled shRNA control AML cells and ATP5IF1 knockdown AML cells after 48 hours, or the effect of venetoclax on MV411_shCtrl and MV411_shIF1 viability after 7 days of exposure to venetoclax. Viability measured using trypan blue viable cell count (n = 4 replicates). (I and J) Effect of venetoclax on colony formation of MV411_shCtrl cells and MV411_shIF1 cells. Colony formation was quantified using CellTiter-Glo (n = 3 replicates). Representative images of colony formation of MV411_shCtrl cells and MV411_shIF1 cells in the presence or absence of venetoclax in (I). Data are presented as means ± SEM and analyzed by two-way ANOVA [(B) to (D) and (G)], one-way ANOVA [(A) and (E)], or unpaired t test [(F), (H), and (J)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 7.. Overexpression of ATP5IF1 enhances sensitivity to BCL-2 targeted therapy.

(A) Expression of ATP5IF1 in isolated mitochondria (n = 3 replicates). (B) Expression of ATP5IF1 in isolated mitochondria derived from lentivirus control HCT116 cells and ATP5IF1-overexpressing HCT116 cells (n = 3 replicates). (C) ATP5IF1 expression represented as fold change (FC) from lentivirus control AML and HCT116 cells (n = 3 replicates). (D) Impact of ATP5IF1 overexpression on the inhibition of respiration by ΔG_ATP in permeabilized lentivirus control AML cells and permeabilized ATP5IF1-overexpressing AML cells (n = 3 to 13 replicates). (E) Impact of ATP5IF1 overexpression on intact cell respiration in AML2_Ctrl cells and AML2_IF1 cells (n = 5 replicates). (F) Impact of ATP5IF1 overexpression on respiratory capacity in permeabilized AML2_Ctrl cells and permeabilized AML2_IF1 cells (n = 4 replicates). (G) Impact of ATP5IF1 overexpression on OxPhos capacity in permeabilized AML2_Ctrl cells and permeabilized AML2_IF1 cells. (H) Flow cytometric analysis of ΔΨ_m using TMRM fluorescence in AML2_Ctrl, AML2_shIF1, and AML2_IF1. Data expressed as a percentage of maximal TMRM fluorescence across all conditions (n = 5 replicates). (I) Effect of venetoclax on viability of AML2_Ctrl cells and AML2_IF1 cells. Viability measured using trypan blue viable cell count (n = 5 replicates). (J to K) Effect of venetoclax on colony formation of AML2_Ctrl cells and AML2_IF1 cells. Colony formation measured using CellTiter-Glo (n = 7 to 16 replicates). Representative image of colony formation of AML2_Ctrl cells and AML2_IF1 cells in the presence or absence of venetoclax depicted in (J). (L) Schematic depicting mechanism of death of ATP5IF1-overexpressing AML cells in response to venetoclax. Created using BioRender.com. Data are presented as means ± SEM and analyzed by two-way ANOVA [(D) to (F) and (H)], one-way ANOVA (C), or unpaired t test [(A), (B), (G), (I), and (K)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.