LAUSR

Cancer metabolism is highly heterogenous and flexible with the Warburg effect or oxidative phosphorylation (OXPHOS) prevailing in a cancer type- and context-dependent manner. Past studies have demonstrated that targeting OXPHOS robustly inhibits glycolysis-deficient cancer cell viability and tumorigenicity. However, the therapeutic potential of OXPHOS inhibition in metabolically flexible glycolysis-competent cancers is unclear. Furthermore, whether the depletion of OXPHOS-derived ATP or the abolition of OXPHOS-supported biosynthesis is the major determinant of cancer cell susceptibility remains obscure. To address these questions, we exposed a panel of metabolically flexible glycolysis-competent cancer cell lines to OXPHOS inhibitors and tested cell survival and proliferation. We monitored metabolic phenotypes and changes in metabolites using seahorse metabolic flux assays and targeted metabolomics, respectively. Stable isotope-tracing was carried out with uniformly labelled [15N]-/[13C]-aspartate. Patient-derived xenograft (PDX) models of colorectal cancer in NSG mice were used for in vivo validation. Here we provide evidence that OXPHOS inhibition potently diminishes metabolically flexible glycolysis-competent cancer cell proliferation and tumorigenicity without causing devastating energy stress. The inhibition of cell proliferation by OXPHOS inhibitors is associated with S-phase cell cycle arrest and the enrichment of the G2/M DNA-damage check point regulation pathway, suggestive of replication stress. Indeed, IACS treatment significantly reduces the purine/pyrimidine nucleotide pools, which is primarily caused by aspartate deficiency resulting from a shortage in the electron acceptor NAD+. The supplementation of exogenous nucleosides, aspartate, or pyruvate that can accept electron generating NAD+, into the culture medium rescues cells from IACS-induced cell cycle arrest. Instructively, inhibition of GOT1, which catalyzes cytosolic aspartate biosynthesis when mitochondrial aspartate production is dampened, renders cancer cells grown in two- and three-dimensional cultures more susceptible to OXPHOS inhibition. Collectively, these results indicate that 1) disruption of nucleotide homeostasis is a major determinant of cancer cell susceptibility to OXPHOS inhibition; 2) OXPHOS inhibition is a promising avenue for the treatment of cancers that are metabolic flexible and glycolysis competent; and 3) GOT1 targeting is potentially a useful approach to improve the therapeutic efficacy of OXPHOS inhibition for cancer treatment. Citation Format: Xiaohong Zhao, Yuan Yuan Zhang, Liang Xu, Ting La, Yu Chen Feng, Hai Jie Tang, Ran Xu, Vinod K. Narayana, David P. De Souza, Lake-Ee Quek, Jeff Holst, Rick F. Thorne, Mark Baker, Tao Liu, Lei Jin, Xu Dong Zhang. Disruption of nucleotide homeostasis confers cancer cell susceptibility to oxidative phosphorylation inhibition independently of energy depletion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3687.