Illuminating the subcellular localization and regulation of D-2-hydroxyglutarate using a novel genetically encoded fluorescent biosensor

PROJECT SUMMARY We propose to employ our recently developed, novel and innovative tool, D2HGlo, to quantify intracellular concentrations of the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG is present at strikingly high levels in isocitrate dehydrogenase (IDH) mutant cancers, including glioma and acute myeloid leukemia. It is widely accepted that D-2-HG accumulation is the initial driver of tumorigenesis in the early stages of these cancers, as D-2-HG antagonizes α-ketoglutarate-dependent proteins, causing widespread DNA hypermethylation. D-2-HG is produced as a direct result of the noncanonical activity of cytosolic IDH1 and mitochondrial IDH2 enzymes. Mutations that emerge in the active sites of IDH1 and IDH2 bestow the ability of both enzymes to convert α-ketoglutarate to D-2-HG. There is no debating that mutant IDH1 and IDH2 drive massive intracellular changes in D-2-HG concentration. However, the subcellular distribution between the cytosol and mitochondria (sites of D-2-HG production) and the nucleus (site of D-2-HG interference) has not been elucidated. Currently accepted practices for D-2-HG detection and quantitative measurements include liquid/gas chromatography coupled with tandem mass spectrometry, magnetic resonance spectroscopy and biochemical assays. These methods are prohibitive for determination of spatiotemporal determination of D-2- HG concentration as they destroy the cell during analysis. This significantly impedes the study of D-2-HG biology in situ. Our research team at Northern Michigan University engineered the genetically encoded fluorescent biosensor D2HGlo to address the need for a tool capable of specific D-2-HG detection in intact cells. D2HGlo has demonstrated efficacy in determining D-2-HG concentration in subcellular compartments, allowing us to interrogate the role of D-2-HG in cancer progression. We have preliminary data that demonstrates that D2HGlo responds to physiologically relevant D-2-HG concentrations and is ten-fold more selective for D-2-HG compared with its enantiomer L-2-HG. The objective of this project is to elucidate the molecular mechanisms governing D-2-HG homeostasis in living cells using the D2HGlo platform. The aims of this proposal are to optimize our genetically encoded fluorescent biosensor D2HGlo for noninvasive quantitative and specific subcellular measurements of D-2-HG in living cells and to investigate how the mutational status of IDH1 and IDH2 shapes the subcellular D-2-HG landscape. Utilizing established glioma cells lines as well as primary cell lines derived from patient tumors, we intend to utilize D2HGlo to interrogate the spatiotemporal distribution of D-2-HG to advance our understanding of D-2-HG driven oncogenesis. With such a poor current understanding of the cellular dynamics associated with D-2-HG production and localization, D2HGlo is primed to answer fundamental biological questions regarding the pleiotropic roles of D- 2-HG in cancer biology.