Abstract
Abstract 1364
Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in AML and AML stem cells over normal hematopoietic cells through the inhibition of mitochondrial translation. This heightened sensitivity was due to increased mitochondrial mass and reliance on oxidative metabolism in the AML cells compared to normal hematopoietic cells. Here, we sought to better understand the mechanisms of sensitivity and resistance to inhibitors of mitochondrial translation.
To establish cells resistant to tigecycline, we exposed TEX leukemia cells to increasing concentrations of tigecycline over 4 months and selected a population of TEX cells resistant to tigecycline (RTEX+TIG) with an IC50 > 24 μM (versus an IC50 of 2.8 + 0.4 μM in wild type cells). We then profiled oxidative metabolism in the resistant cells. In RTEX+TIG cells, levels of Cox-1 and Cox-2, subunits of respiratory complex IV in the electron transport chain that are translated by mitochondrial ribosomes, were undetectable. In contrast, Cox-4 that is part of the same respiratory chain, but translated in the cytoplasm, was only slightly reduced. RTEX+TIG cells also had undetectable levels of oxygen consumption and increased rates of glycolysis compared to wild type cells. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia, thus demonstrating the functional importance to the change in their metabolic status. RTEX+TIG cells also had reduced mitochondrial membrane potential by 44.4 + 7.2% and reduced mitochondrial mass compared to wild type cells. Morphologically, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures.
To understand the molecular basis for the metabolic changes in the RTEX+TIG cells, we performed RNA sequencing of the RTEX+TIG cells and wild type TEX cells. Unbiased analysis, by two independent approaches, of the promoter sequences of transcripts upregulated 1.5-fold or greater in RTEX+TIG cells demonstrated a significant over-representation of binding sites for the hypoxia-inducible factor 1 alpha HIF1α :HIF1β transcription factor complex. Specifically, a subset of HIF1α target genes involved in energy balance and cellular metabolism were coordinately upregulated in RTEX+TIG cells, corresponding with our phenotypic observations of the metabolic state of these cells. We validated the upregulation of HIF1α mRNA and protein by Q-RTPCR and immunoblotting.
Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels. However, HIF1α remained elevated. Upon re-treatment with tigecycline, the cells remained resistant and the glycolytic phenotype was re-established.
TEX cells display features of leukemia stem cells, including differentiation, self-renewal and hierarchical organization. Interestingly, RTEX+TIG cells were more differentiated and had reduced stemness compared to the wild type TEX cells. By immunohistochemistry, RTEX+TIG had increased non-specific esterase activity (NSE). In addition, RTEX+TIG cells had reduced clonogenic growth and ability to engraft immune deficient mice compared to wild type cells. Moreover, RNA sequencing data showed reduced expression of stem cell maintenance genes in RTEX+TIG cells.
Depletion of mitochondrial DNA via prolonged exposure of parental cell lines to cationic lipophilic agents such as ethidium bromide produces rho-zero cells that have irreversibly lost mitochondrially translated proteins. These cells lack a functional respiratory chain and cannot derive energy from oxidative phosphorylation. Instead, these cells rely on glycolysis for their energy supply. Here, we have produced a reversible rho-zero like metabolic phenotype by sustained inhibition of mitochondrial translation. This work, therefore, highlights mechanisms of metabolic adaption to inhibition of oxidative phosphorylation. Finally, these data suggest a unique role for metabolism in differentiation and stemness in AML.
Disclosures:
No relevant conflicts of interest to declare.
Increased cyclooxygenase-2 expression and prostaglandin E2production in pressurized renal medullary interstitial cells
