Itaconic acid, or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR based metabolomics and 13C-labeling, we have detected itaconic acid in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased itaconic acid production and secretion. Crude cell extracts synthesize itaconic acid via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.

Many metastatic cancers are derived from compromised respiration in tissue cells of myeloid origin (e.g., macrophages), which use glutamine as a major energy substrate. Although impaired respiration can render tumor cells dependent on glycolysis for energy in the presence of oxygen (Warburg effect), it has not been determined if glutamine alone could provide enough energy (ATP) through substrate level phosphorylation in the TCA (Krebs) cycle to maintain tumor cell viability. Using a bioluminescent-based ATP assay, we determined the viability of metastatic mouse VM-M3 tumor cells grown in serum free medium in the presence of glucose alone (25 mM), glutamine alone (4 mM), or in glucose + glutamine. The VM-M3 cells could not survive on glucose alone, but could survive in glutamine alone indicating an absolute requirement for glutamine in these metastatic tumor cells. Glucose + glutamine acted synergistically in providing growth and viability. Lactic acid production was 10-fold lower in the cells grown in glutamine alone than in glucose alone. Glutamine could also maintain viability in the absence of glucose and in the presence of the F1 ATPase inhibitor oligomycin. Glutamine could not maintain viability in the presence of the TCA cycle enzyme inhibitor, 3-Nitropropionic acid. NMR analysis indicated that glutamine metabolism via the TCA cycle was active resulting in accumulation of both succinate and aspartate. The data indicate that glutamine can provide ATP for viability in the metastatic VM-M3 cells through Krebs cycle phosphorylation in the absence of energy from either glycolysis or oxidative phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 53.

Upregulation of the AKT pathway leads to a variety of human cancers. Phosphatidylinositol ether analogs, or PIAs, are small molecules designed to inhibit the Ser/Thr kinase AKT, by binding to its pleckstrin homology (PH) domain and preventing translocation from the cytoplasm to the nucleus. D‐3′‐deoxy‐diC8‐phosphatidylinositol is a soluble PIA that has an IC50 of 40 µM in U937 human lymphoma cells, which predominantly express Akt1. This compound (and not related further deoxygenated inositol lipids or L‐inositol species) does not appear to affect activation and growth of cells that have other Akt isoforms as well as Akt1 (e.g., B cells). We have shown that D‐3′‐deoxy‐diC8PI inhibits phosphorylation of proteins downstream of Akt1. However, near normal levels of phosphorylation of Akt1 strongly indicate that plasma membrane translocation of Akt1 has not been significantly inhibited. A GFP‐Akt1 fusion protein was expressed in U937 and MCF‐7 cell lines, and the movement of Akt1 when treated with D‐3′‐deoxy‐diC8PI was tracked using confocal microscopy. Treatment with D‐3′‐deoxy‐diC8PI did not inhibit plasma membrane or nuclear translocation, indicating cytotoxicity occurs by a different mechanism. Binding of the Akt1 PH domain in the absence and presence of the short‐chain PIA has also been examined to confirm the in vivo results.(This work is supported by N.I.H. GM60418).