Abstract 452: Alpha-Ketoglutarate Dehydrogenase Inhibition by the Oncometabolite D2-HG Causes Proteome and Metabolome Remodeling in Myocytes

Abstract

Autophagy “scavenges” proteins and yields amino acids under conditions of metabolic stress to support cell survival and growth. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including dilated cardiomyopathy. Our recent in vivo work showed that increased D2-HG supply by IDH2-mutant hematopoetic stem cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. While heart failure in cancer is commonly associated with chemotherapy, cancer survivors have a five-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic changes and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase inhibition in myocytes results in metabolomic pertubations, proteomic remodeling, and increased autophagy. We measured autophagic flux and remodeling of the stable proteome upon D2-HG treatment in vivo using wild-type C57BL/J6 mice, and in vitro using both cultured L6 myocytes and adult mouse ventricular cardiomyocytes. We observed increases in the LC3-II/LC3-I ratio and p62 expression in heart and skeletal muscle from mice treated with D2-HG, indicating activation of autophagy. Live cell imaging with GFP-tagged LC3 indicated that D2-HG (1 mM) increased LC3-II lipidation and flux within 24 h. Furthermore, we observed increased phosphorylation and activation of AMPK, while phosphorylation of mTOR and p70S6K were decreased in presence of D2-HG. In vitro exposure to D2-HG resulted in the formation of a molecular complex between Sirt1 and LC3, indicating that increased NAD+ in presence of D2-HG promotes Sirt1 activation in myocytes. Finally, we used LC-MS/MS to assess the effect of D2-HG on the stable proteome and metabolome in heart and skeletal muscle. Myocytes exposed to D2-HG showed proteomic remodeling and metabolomic changes within 24 h. Integrating multi-omics data in a network-level context revealed upregulation of glycolysis and the pentose phosphate pathway. In short, autophagy activation may support proteome remodeling in muscle cells during IDH-mutant leukemia.