Hyperpolarized [1-13C] glutamate: a metabolic imaging biomarker of IDH1 mutational status in glioma

Abstract

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Noninvasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use (13)C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-(13)C] α-KG to hyperpolarized [1-(13)C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-(13)C] α-KG, hyperpolarized [1-(13)C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-(13)C] glutamate was significantly lower in mutant IDH1 cells and tumors compared with their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-(13)C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-(13)C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production.

Figure 1. IDH1 mutation is associated with a drop in BCAT1 activity and expression in our cell model

(A) BCAT1 enzymatic activity, as measured by a spectrophotometric assay, is significantly decreased in mutant IDH1 cells as compared to wild-type cells (*p<0.002; n=4 per cell line). (B) Western blots of the BCAT1 enzyme, showing the drop in BCAT1 protein levels in U87IDHmut cells when compared to U87IDHwt.

Figure 2. Hyperpolarized [1-¹³C] glutamate formation from hyperpolarized [1-¹³C] α-KG can be detected in live cells, and is decreased in U87IDHmut cells as compared to U87IDHwt

Stack plots of dynamic ¹³C MR spectra acquired at 11.7 Tesla following injection of hyperpolarized [1-¹³C] α-KG in live U87IDHwt (A) and U87IDHmut (B) perfused cells (temporal resolution 9 seconds), showing the formation of hyperpolarized [1-¹³C] glutamate in U87IDHwt cells. Note the absence of detectable hyperpolarized [1-¹³C] glutamate in U87IDHmut cells. (C) Intensities of hyperpolarized [1-¹³C] glutamate in U87IDHwt (□) and U87IDHmut ( ) perfused cells, showing the significantly higher level of glutamate in U87IDHwt cells as compared to U87IDHmut. Also of importance is the delayed formation of hyperpolarized [1-¹³C] glutamate versus the time of maximum hyperpolarized [1-¹³C] α-KG (vertical dashed line), as expected when metabolism occurs. The fit derived from the gamma-variate analysis (GVA) is displayed as a continuous line for U87IDHwt and as a dashed line for U87IDHmut perfused cells.

Figure 3. Hyperpolarized [1-¹³C] glutamate from hyperpolarized [1-¹³C] α-KG can be detected in vivo in normal brain and in U87IDHwt tumors only

T2-weighted MR images of (A) a U87IDHwt and (B) a U87IDHmut tumor-bearing animals overlaid with tumor (red) and normal brain (blue) voxels from the 2D CSI acquisition. The tumors appear as hypersignals and are circled with dotted lines. The corresponding hyperpolarized ¹³C MR spectra from tumor and normal brain voxels are shown for both animals. The observed resonances are: (1) hyperpolarized [1-¹³C] glutamate (green arrow, δ_GLU=177.5ppm); (2) hyperpolarized [1-¹³C] α-KG (δ_α-KG=172.6ppm); (3) hyperpolarized [1-¹³C] α-KG hydrate (δ_α-KG-Hyd=180.9ppm). Note the presence of hyperpolarized [1-¹³C] glutamate in normal brain for both animals, but only in the tumor tissue of the U87IDHwt animal. (C) T2-weighted MR images (first column) of the head of a U87IDHwt (top row) and a U87IDHmut (bottom row) tumor-bearing animal overlaid with the grid used for 2D ¹³C CSI acquisition. Corresponding heatmaps of hyperpolarized [1-¹³C] α-KG and hyperpolarized [1-¹³C] glutamate acquired 25 seconds post injection of hyperpolarized [1-¹³C] α-KG. These heatmaps illustrate the presence of hyperpolarized [1-¹³C] glutamate in normal brain and tumor in U87IDHwt animals, and in normal brain only in U87IDHmut animals. (D) Ratio of hyperpolarized [1-¹³C] glutamate to hyperpolarized [1-¹³C] α-KG (integral values) expressed as a percent of the same ratio in normal brain. The normalized glutamate/α-KG ratio is lower in U87IDHmut tumors as compared to U87IDHwt (n=2 per tumor type).

Figure 4. In addition to BCAT1, the presence of IDH1 mutation is associated with decreased activities and protein levels of additional enzymes catalyzing α-KG-to-glutamate conversion

(A) Schematic of [1-¹³C] α-KG metabolism illustrating four enzymatic pathways through which α-KG can be metabolized to glutamate: BCAT1 (branched chain aminotransferase 1; BCAAs: branched-chain aminoacids; BCKAs: branched-chain ketoacids), AST1 and AST2 (aspartate aminotransferase 1 and 2), GDH1 and GDH2 (glutamate dehydrogenase 1 and 2) and ALT1 and ALT2 (Alanine Aminotransferase 1 and 2). The reactions catalyzed by wild-type and mutant IDH1 and IDH2 are also shown (For all enzymes: 1=cytoplasmic isoform; 2=mitochondrial isoform). The ¹³C label at the C1 position of α-KG is highlighted in bold. (B) AST and (C) GDH enzymatic activities as measured by spectrophotometric assays. The activities of both enzymes are significantly decreased in mutant IDH1 cells as compared to wild-type cells (*p<0.05; **p<0.01; n=4 per cell line and per enzyme). (D) Western blots for the AST1, AST2, GDH2, ALT1 and ALT2 enzymes. BCAT1 and AST1 protein levels were significantly decreased in U87IDHmut cells, whereas the rest of the enzymes levels were unchanged. β-Actin was used as a loading control. (Note: GDH1 was below detection level).