The majority of low-grade gliomas (70-90%) carry somatic mutations in the gene for isocitrate dehydrogenase 1 (IDH1). Wild-type IDH1 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) in the cytosol. Mutations in the gene result in the neomorphic ability to convert α-KG to 2-hydroxyglutarate (2-HG). 2-HG, via competitive inhibition of α-KG-dependent enzymes, has been shown to alter gene expression both via promoter methylation and via HIF-1α-dependent signaling. Selective inhibitors of the mutant IDH1 enzyme have been explored as therapeutic targets and one such inhibitor, AGI-5198, has been shown to cause some delay in the growth of IDH mutant gliomas¹. We recently showed that the IDH1 mutation leads to ¹H-MRS-detectable reduction in steady-state levels of glutamate, lactate and phosphocholine in glioma cells². The goal of the current study was to use ¹H-MRS to determine whether treatment with AGI-5198 reverses the MRS-detectable metabolic reprogramming induced by the IDH1 mutation in our glioma cells. Immortalized normal human astrocytes (NHA) stably expressing either IDH1 wild-type (NHAIDHwt) or mutant (NHAIDHmut) enzyme were generated and maintained as previously described². Cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. The effect of AGI-5198 on cell proliferation was assayed by measuring cell number following treatment with 10 μM AGI-5198 at various time points. The effect of AGI-5198 on clonogenicity was measured using a soft agar assay. To measure the effect of AGI-5198 on metabolite levels, cells (~10⁷) were treated with 10 μM AGI-5198 or DMSO (vehicle control) for 72 h and extracted using the dual-phase extraction method as previously described³. ¹H-MRS spectra (1D water pre-saturation ZGPR sequence, 90° pulse and 3 s relaxation delay) were acquired using a 500 MHz Avance spectrometer (Bruker) equipped with a Triple Resonance CryoProbe. Peak integrals were quantified using Mnova (Mestrelab Research) and normalized to a trimethylsilyl propanoic acid reference of known concentration and to cell number. All experiments were performed in triplicate (n=3) unless otherwise mentioned and statistical significance assessed using a two-tailed t-Test assuming unequal variance with p<0.05 considered significant.We used ¹H-MRS to compare metabolite levels between NHAIDHwt cells, and NHAIDHmut cells treated with 10 μM AGI-5198 or NHAIDHmut controls (Fig 1). Quantification of metabolite levels indicated that AGI-5198 reversed some, but not all, of the metabolic alterations induced by the IDH1 mutation (Fig. 2). 2-HG levels dropped by 102.8±1.9% (p<0.05) indicating that, as expected, AGI-5198 completely inhibited 2-HG production in NHAIDHmut cells (Fig. 2). The steady state glutamate concentration increased significantly (125.8±14.9%, p<0.05, Fig. 2) returning to the level observed in NHAIDHwt cells (p>0.05 when comparing treated NHAIDHmut to control NHAIDHwt). AGI-5198 treatment also increased phosphocholine (PC) levels in NHAIDHmut cells (Fig. 2, 219.5±77.2%, p<0.05), restoring them to NHAIDHwt levels (p>0.05 when comparing treated NHAIDHmut to control NHAIDHwt). However, there was no change in intracellular lactate levels in NHAIDHmut cells following AGI-5198 treatment, with lactate levels remaining significantly lower than controls (Fig. 2, p>0.05 when comparing treated NHAIDHmut to control NHAIDHmut and p<0.05 when compared to NHAIDHwt). We also tested the effect of AGI-5198 on cell proliferation and clonogenicity. While we did not observe an effect on cell proliferation upon treatment with 10 μM AGI-5198 (Fig. 3A), we did see a dose-dependent inhibition of clonogenicity in NHAIDHmut cells (Fig. 3B) indicating that the IDH1 mutation is essential for malignant transformation, but pointing to the limited impact of AGI-5198 on cell proliferation. In an effort to understand our findings, we considered our recent observations that the drop in glutamate in NHAIDHmut cells was due to a reduction in PDH activity mediated by HIF-1α stabilization⁴. We therefore, assessed PDH activity in NHAIDHmut cells following AGI-5198 treatment. Our results indicated that PDH activity was increased from 2.12x10^-9 OD/hr/cell in NHAIDHmut cells to 7.06x10^-9 OD/hr/cell in AGI-5198-treated NHAIDHmut cells, approaching the value of NHAIDHwt cells at 8.66x10^-9 OD/hr/cell (n=1). This observation suggests that inhibiting mutant IDH1 with AGI-5198 reversed HIF-1α-mediated metabolic alterations, some of which affect the clonogenic potential of our cells. However, other metabolic events are not altered and are likely dependent on promoter methylation, which cannot be readily reversed. Our findings highlight the complexity of the metabolic reprogramming that occurs in mutant IDH tumors and the value of MRS in helping to tease out this complexity.