Clinically Relevant Oxygraphic Assay to Assess Mitochondrial Energy Metabolism in Acute Myeloid Leukemia Patients

Abstract

Resistant acute myeloid leukemia (AML) exhibits mitochondrial energy metabolism changes compared to newly diagnosed AML. This phenotype is often observed by evaluating the mitochondrial oxygen consumption of blasts, but most of the oximetry protocols were established from leukemia cell lines without validation on primary leukemia cells. Moreover, the cultures and storage conditions of blasts freshly extracted from patient blood or bone marrow cause stress, which must be evaluated before determining oxidative phosphorylation (OXPHOS). Herein, we evaluated different conditions to measure the oxygen consumption of blasts using extracellular flow analyzers. We first determined the minimum number of blasts required to measure OXPHOS. Next, we compared the OXPHOS of blasts cultured for 3 h and 18 h after collection and found that to maintain metabolic organization for 18 h, cytokine supplementation is necessary. Cytokines are also needed when measuring OXPHOS in cryopreserved, thawed and recultured blasts. Next, the concentrations of respiratory chain inhibitors and uncoupler FCCP were established. We found that the FCCP concentration required to reach the maximal respiration of blasts varied depending on the patient sample analyzed. These protocols provided can be used in future clinical studies to evaluate OXPHOS as a biomarker and assess the efficacy of treatments targeting mitochondria.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia

“…Genetic mutations in AML are heterogeneous, resulting in varying treatment responses and difficulty in identifying specific targets for complete remission [38]. Recent studies indicate that abnormal metabolism, attributed to proteins that modulate metabolic processes, contributes to the proliferation and progression of AML myeloblasts [3,4,6,7,14,26]. In the current study, we investigated the metabolic alterations in adult AML bone marrow and identified a key modulator protein involved in mitochondrial respiration and the glycolytic pathway.…”

“…AML cells have a higher demand for ATP to support their rapid proliferation and survival and possess a larger mitochondrial content compared to normal hematopoietic cells [3,6,27]. Mitochondrial dysfunction and defects in mitochondrial proteins are associated with poor prognosis and chemotherapy resistance in AML [30,39].…”

“…AML myeloblasts have a high demand for ATP to support their continuous proliferation and survival [4,26]. Metabolic alterations, including changes in mitochondrial bioenergetics and glycolysis, commonly occur in AML [3,4,6,7,14,30]. Previous studies have shown enhanced biogenesis of mitochondria and activity of mitochondria complexes II and V in the bone marrow of pediatric AML patients [14].…”

“…Myeloblasts show heightened glycolysis and ROS production, with increased intermediates of mitochondrial respiration to support their proliferation. In contrast, LSCs predominantly rely on oxidative phosphorylation rather than glycolysis or lactate production to obtain ATP [3,6,7].…”

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia

“…2 is highly dependent on the rapid replenishment of adenosine triphosphate (ATP), which fuels their metabolic needs and drives proliferation. Consequently, AML cells undergo metabolic reprogramming to meet these energy demands [3,4]. One significant metabolic alteration observed in the bone marrow of AML patients is increased mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) [3][4][5][6].…”