Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia
- Department of Physiology, Brody School of Medicine, East Carolina University, United States
- East Carolina Diabetes and Obesity Institute, East Carolina University, United States
- Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, United States
- Department of Cardiovascular Sciences, Brody School of Medicine, East Carolina University, United States
- Department of Surgery, Brody School of Medicine, East Carolina University, United States
- Department of Internal Medicine, Brody School of Medicine, East Carolina University, United States
Figures
Figure 1 with 1 supplement
Leukemia exhibits impaired cellular respiratory capacity amid an increased mitochondrial network.
All experiments were performed in intact cells. FCCP-stimulated flux normalized to cell count (A) and protein concentration (C) and represented as percentage of basal respiration (B). (D) Km of FCCP …
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Figure 1—source data 1
Raw values for 'Figure 1' and 'Figure 1—figure supplement 1'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Morphology of leukemia and respiratory flux stimulated by BAM15, a mitochondrial uncoupler.
(A) Comparison of cell size of leukemia cells and PBMC. Respiratory flux driven by BAM15 (B15), a mitochondrial uncoupler (B) and observed Km (C). (D) Cellular focal planes used to obtain confocal …
Figure 2 with 1 supplement
Impaired OXPHOS kinetics and ATP-dependent inhibition of ETS flux are unique phenotypes of leukemic mitochondria.
All experiments were performed using digitonin-permeabilized cells. (A) Schematic depicting changes in oxygen consumption (JO2) during an ETS capacity protocol (FCCP titration) where points 6–7 …
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Figure 2—source data 1
Raw values for 'Figure 2' and 'Figure 2—figure supplement 1'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Respiratory profile of healthy bone marrow mononuclear cells and primary human myoblasts.
(A–E) Permeabilized cells were used for all experiments. ETS capacity (A) and OXPHOS kinetic (B) protocols were performed in permeabilized BMHealthy. Data depict results from each replicate across …
Figure 3
In leukemic mitochondria ΔGATP regulates ETS flux independent of substrate condition.
(A) OXPHOS kinetics supported by P/M/G/S/O in mitochondria isolated from PBMC and leukemia cells. (B) Comparison of FCCP Effect calculated as the ratio of FCCP ΔGATP to JH+OXPHOS from B. (C) …
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Figure 3—source data 1
Raw values for 'Figure 3'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
Analysis of mitochondrial proteome reveal disparate expression of ANT isoforms in leukemia.
TMT-labeled nLC-MS/MS was performed on mitochondrial lysates from each cell type. (A) Volcano plot depicting changes in proteome between leukemia cell lines and PBMC with mitochondrial proteins …
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Figure 4—source data 1
Raw values for 'Figure 4' and 'Figure 4—figure supplement 1'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Heatmap analysis depicting abundance of subunits and assembly factors that comprise the ETS complexes.
Heatmap displaying relative protein abundance of the individual subunits and assembly factors belonging to (A) complex I (B) complex II (C) Complex III and (D) Complex IV.
Figure 5 with 1 supplement
ETS flux inhibition by ΔGATP links to matrix ATP consumption in leukemia.
(A–B) OXPHOS kinetics (via CK clamp) were assessed in the absence of adenylates or in the presence of minimal ΔGATP (−54.16), maximal ΔGATP (−61.49), or maximal ΔGATP + CAT (Carboxyatractyloside; …
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Figure 5—source data 1
Raw values for 'Figure 5' and 'Figure 5—figure supplement 1'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
ETS flux in the presence of ΔGATP is restored by ANT inhibition ANT.
(A) OXPHOS kinetics in permeabilized HL-60 cells in the presence of DMSO (vehicle), bongkrekic acid (20 µM; ANT inhibitor) or gamitrinib (1 µM; TRAP1 inhibitor with mitochondria-targeted moiety); n =…
Figure 6 with 1 supplement
17-AAG and gamitrinib increase fractional OXPHOS and restore ETS flux in the presence of ΔGATP.
(A) Log2 Abundance of TRAP1 in PBMC and leukemia cells. (B) Comparison of OXPHOS kinetics in presence of the TRAP1 inhibitor, 17-AAG (15 µM); n = 4 independent cell experiments. Comparison of (C) …
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Figure 6—source data 1
Raw values for 'Figure 6' and 'Figure 6—figure supplement 1'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Effects of 17-AAG on OXPHOS kinetics.
(A) Comparison of FCCP-stimulated respiration with the addition of 17-AAG (15 µM) in permeabilized MV-4–11 cells; n = 3 independent experiments. (B–D) OXPHOS kinetics in permeabilized HL-60 cells in …
Figure 7 with 1 supplement
Human primary leukemia is characterized by low Fractional OXPHOS.
(A) Basal respiration in intact cells – ‘PBMC’ (age-matched to the primary leukemia samples); ‘BMHealthy’ (bone marrow mononuclear cells); ‘CD34+’ (pure CD34+ cells not exposed to growth factors); …
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Figure 7—source data 1
Raw values for 'Figure 7' and 'Figure 7—figure supplement 1'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Effects of 17-AAG on OXPHOS kinetics and cell viability in PBMC and healthy bone marrow mononuclear cells.
(A) Comparison of OXPHOS kinetics in the presence of DMSO or 17-AAG (15 µM) in permeabilized PBMC from healthy donors; n = 5 independent experiments. (B) Comparison of OXPHOS kinetics in the …
Figure 8
OXPHOS power output is reduced in the setting of venetoclax resistance.
(A) Study schematic depicting bioenergetic characterization of OXPHOS power output in HL-60 cells either sensitive (HL60WT) or made resistant to venetoclax by continuous exposure to 1 µM venetoclax …
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Figure 8—source data 1
Raw values for 'Figure 8'.
- https://cdn.elifesciences.org/articles/63104/elife-63104-fig8-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Homo sapiens) | Peripheral Blood Mononuclear Cells; PBMC | Venous puncture | Freshly isolated from Homo sapiens, male and female, 18–75 years | |
| Biological sample (Homo sapiens) | Bone Marrow Aspirate; Primary Leukemia | Posterior Iliac Crest | Freshly isolated from Homo sapiens | |
| Biological sample (Homo sapiens) | Human Myoblast; HMB | Muscle biopsies from gastrocnemius muscle | Freshly isolated from Homo sapiens | |
| Biological sample (Homo sapiens) | CD34+ Cells | HemaCare | CAT#: BM34C | Isolated freshly from bone marrow (Homo sapien, M) |
| Biological sample (Homo sapiens) | CD34+ Cells | HemaCare | CAT#: BM34C | Isolated freshly from bone marrow (Homo sapien, M) |
| Biological sample (Homo sapiens) | CD34+ Cells | HemaCare | CAT#: BM34C | Isolated freshly from bone marrow (Homo sapien, F) |
| Biological sample (Homo sapiens) | Bone Marrow Aspirate; BM Healthy | HemaCare | CAT#: BM008F | Isolated freshly from bone marrow (Homo sapien, F) |
| Biological sample (Homo sapiens) | Bone Marrow Aspirate; BM Healthy | HemaCare | CAT#: BM008F | Isolated fresh (Homo sapien, M) |
| Biological sample (Homo sapiens) | Bone Marrow Aspirate; BM Healthy | HemaCare | CAT#: BM008F | Isolated fresh (Homo sapien, M) |
| Cell line (Homo sapien) | HL-60 | ATCC | CAT#: CCL-240 | |
| Cell line (Homo sapien) | MV-4–11 | ATCC | CAT#: CRL-9591 | |
| Cell line (Homo sapien) | KG-1 | ATCC | CAT#: CCL-246 | |
| Chemical compound, drug | Oligomycin; Oligo | Tocris | CAT#: 4110 | |
| Chemical compound, drug | FCCP | Millipore Sigma | CAT#: C2920 | |
| Chemical compound, drug | Rotenone; Rot | Millipore Sigma | CAT#: R8875 | |
| Chemical compound, drug | Antimycin; Ant | Millipore Sigma | CAT#: A8674 | |
| Chemical compound, drug | Digitonin; Digi | Millipore Sigma | CAT#: D1410599 | |
| Chemical compound, drug | Potassium Pyruvate; P; Pyr | Combi-Blocks | CAT#: QA-1116 | |
| Chemical compound, drug | L-Malic Acid; Malate; M | Alpha Aesar | CAT#: A13702 | |
| Chemical compound, drug | L-Glutamic Acid; Glutamate; G | RPI | CAT#: G25200 | |
| Chemical compound, drug | Succinic Acid; Succinate; S; Succ | Fisher | CAT#: BP336 | |
| Chemical compound, drug | Octanoyl-L-Carnitine; O | Millipore Sigma | CAT#: 50892 | |
| Chemical compound, drug | Cytochrome C | Millipore Sigma | CAT#: C2506 | |
| Chemical compound, drug | ATP | Ark Pharma | CAT#: AK54737 | |
| Chemical compound, drug | Creatine Kinase | Millipore Sigma | CAT#: C2506 | |
| Chemical compound, drug | Phosphocreatine; PCr | Millipore Sigma | CAT#: 237911 | |
| Chemical compound, drug | Carboxy Atractyloside; CAT | Millipore Sigma | CAT#: C4992 | |
| Chemical compound, drug | 17-AAG | Millipore Sigma | CAT#:100068 | |
| Chemical compound, drug | Gamitrinib TPP hexafluorophosphate; Gamitrinib | MedChem Express | CAT#: HY-102007A | |
| Chemical compound, drug | Curcumin | Millipore Sigma | CAT#: 239802 | |
| Chemical compound, drug | Venetoclax | Selleckchem | CAT#: S8048 | |
| Chemical compound, drug | Bongkrekic acid | Cayman Chemical | CAT#: 19079 | |
| Chemical compound, drug | ACK Lysis Buffer | Lonza | CAT#: BP10-548E | |
| Chemical compound, drug | Mitotracker Green-FM Dye; MTG-FM | Thermo Fisher | CAT#: M7514 | |
| Chemical compound, drug | Tetramethyl rhodamine methyl ester (TMRM) | Invitrogen | CAT#: 1924079 | |
| Chemical compound, drug | Pierce Lys-C Protease | Thermo Fisher | CAT#: 90307 | |
| Transfected construct (Homo sapien) | shRNA to TRAP1 | Origene | CAT#: TL300868V | shRNA lentiviral particles packaged from pGFP-C-shLenti vector |
| Sequence-based reagent | Primer for TRAP1 | Thermo Fisher | CAT#: 4331182 | |
| Sequence-based reagent | Primer for 18 s rRNA | Thermo Fisher | CAT#: 4319413E | |
| Peptide, recombinant protein | Seq Grade Trypsin | Millipore Sigma | CAT#: V5113 | |
| Peptide, recombinant protein | Human Stem Cell Factor; SCF | Gibco | CAT#: PHC2115 | |
| Peptide, recombinant protein | Human Thrombopoietin | Gibco | CAT#: PHC9514 | |
| Peptide, recombinant protein | FLT-3 Ligand | Sigma Aldrich | CAT#: SRP3044 | |
| Commercial assay or kit | RNeasy Midi kit | Qiagen | CAT#: 74124 | |
| Commercial assay or kit | Superscript IV reverse transcriptase | Invitrogen | CAT#: 18090010 | |
| Commercial assay or kit | TMT 10-plex | Thermo Fisher | CAT#: A34808 | |
| Software, algorithm | Prism 8.4 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | Proteome Discoverer 2.2 | Thermo Fisher | ||
| Software, algorithm | Mito Carta 2.0 | RRID:SCR_018165 |
Additional files
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Supplementary file 1
Mitochondrial proteome of AML cell lines, relative to PBMC.
(A) Exported results from PDv2.2. (B) Analyzed master protein expression by group.
- https://cdn.elifesciences.org/articles/63104/elife-63104-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/63104/elife-63104-transrepform-v2.pdf
