Introduction: Since pioneering studies by Otto Warburg demonstrated that cancer cells had markedly reduced oxidative phosphorylation under conditions of high oxygen tension (Warburg Effect/aerobic glycolysis), metabolic reprogramming has become a well-recognized hallmark of cancer. While numerous discoveries have revealed highly specific metabolic differences between cancer cells, utilization of aerobic glycolysis as the primary means of ATP generation remains one of the most common metabolic alterations observed across a range of cancers. Gain-of-function mutations in p53 (mutp53) occur with a frequency of greater than 50% in many types of cancers and promote aerobic glycolysis by activation of glycolysis and inhibition of the tricarboxylic acid cycle. However, the manner by which these mutp53 regulates metabolic shifts in response to energy strain is only beginning to be elucidated. Hypothesis: Increased energy demand by cells caused by direct chelation of ATP by the novel, synthetic ATP-binding protein DX will be met by increases primarily in oxygen consumption. Methods: A cationic lipid mixture was complexed with active, purified DX protein to generate a DX/lipid complex. HeLa cells were incubated with the DX/lipid complex to allow efficient delivery of DX into the cytoplasm of the HeLa cells. The XF Real-Time ATP Rate Assay using Seahorse XF label-free technology was used to measure the rate of ATP production following DX expression and estimate the relative contributions of glycolysis and oxidative phosphorylation to the total ATP production rate. Results: HeLa cells transfected with DX had reduced viability, altered morphology, and decreased bioavailable intracellular ATP following DX expression. Unexpectedly, in response to DX expression and reduction in ATP, total ATP production rates in HeLa cells significantly decreased over time. Importantly, this drop in total ATP production was associated with a metabolic pathway utilization shift. Conclusion: The synthetically derived DX protein is a tool that allows for direct control of the ATP levels in cancer cells and has the potential to reveal novel metabolic adaptive responses to energy stress. Following regulated ATP reduction in HeLa cells, changes observed in the OXPHOS/glycolytic balance represent an overall cellular metabolic shift.

Citation Format: Ashley P. Brown, Taha Muhammad, Selina Martinez, Shaleen B. Korch, Jeffrey Norris. Analysis of metabolic adaptations in HeLa cells as a function of energy status using a novel, direct ATP chelation methodology [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-026.