280491 Analysis of Myc-Driven Metabolic Reprogramming in B-Cells by Isotopically Nonstationary 13C Flux Analysis

Thursday, November 1, 2012: 10:36 AM
Somerset West (Westin )
Taylor A. Murphy1, Chi Dang2 and Jamey D. Young1, (1)Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, (2)Abramson Cancer Center

The transcription factor c-Myc is an oncoprotein whose expression is deregulated in over 30% of all cancers. A significant role of c-Myc (i.e., Myc) is its control of cell cycling and apoptosis, but it also exerts strong influence on central metabolism. Targeting metabolic phenotypes induced by Myc activation may offer a potential therapeutic strategy to selectively inhibit the proliferation of Myc-driven cancers. We have conducted 13C metabolic flux analysis (13C-MFA) studies on P493-6 B-cells to identify metabolic pathway alterations associated with Myc overexpression. These cells have a tetracycline-repressible genetic construct that enables three distinct c-Myc expression levels: High (oncogenic), Low (endogenous), and None. Using GC-MS profiling techniques on isotopically labeled protein and RNA samples, along with measurements of extracellular metabolite uptake and excretion fluxes, we have created comprehensive flux maps of the central carbon metabolism of P493-6 cells under both High and Low Myc conditions. These flux maps have revealed several key features of Myc metabolic regulation.

We observed that glycolysis is highly upregulated in both conditions, with lactate-to-glucose (L/G) ratios of 1.54 and 1.79 for High and Low Myc, respectively. However, 25% of the pyruvate synthesized via glycolysis in the High Myc condition entered the TCA cycle as compared to only 15% in the Low Myc cells. Most TCA cycle and amphibolic mitochondrial pathways exhibited 4-fold flux increases in High Myc cells, in contrast to modest increases in glucose uptake and lactate excretion. This was largely driven by increased catabolism of glutamine and other amino acids.  The oxidative pentose phosphate pathway exhibited minimal activity under both High and Low Myc conditions. Oxygen uptake was significantly increased in High Myc cells as compared to Low Myc, confirming the increase in mitochondrial activity detected by MFA. We are currently conducting studies to quantify how hypoxia modulates these metabolic phenotypes as well as examining siRNA knockdowns of specific enzymes that are activated in High Myc cells.

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