Figure 6. BCAT1i/AKG causes mTORC1 inactivation and protein depletion in IDH^WT GBM cells.

(A) Mass spectrometry analysis of AMP/ATP ratio in parental and BCAT1 KO#2 U251MG cells treated with vehicle or DMAKG (10 mM) for 24 hours (mean ± SEM, n = 5). *p < 0.05; ****p < 0.0001 by one-way ANOVA with Dunnett’s test. (B) Immunoblot analysis of mTORC1 activation in parental and BCAT1 KO#2 or KO#3 U251MG cells treated with vehicle (-) or DMAKG (10 mM) for 24 hours. Representative blots are shown in the left. Intensities of p-p70S6K and p70S6K bands are quantified and their ratio is shown in the right (mean ± SEM, n = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA with Tukey’s test. (C) Immunoblot analysis of mTORC1 activation in parental and BCAT1 KO#2 or KO#3 U251MG cells treated with vehicle (-), DMAKG (10 mM), KIC (1 mM), or both for 24 hours. (D) Analysis of protein synthesis in parental and BCAT1 KO#2 or KO#3 U251MG cells treated with vehicle (-), DMAKG (10 mM), KIC (1 mM), or both for 24 hours. (E) Analysis of protein synthesis in U251MG cells treated with vehicle (-), DMAKG (10 mM), gabapentin (Gaba, 20 mM), or both for 48 hours. (F) Analysis of protein synthesis in C116 cells treated with vehicle (-), DMAKG (10 mM), KIC (1 mM), or both for 24 hours. (G) Clonogenic assay in parental and BCAT1 KO#2 or KO#3 U251MG cells treated with vehicle, DMAKG (10 mM), or Rapamycin (1 nM) for 7 days. Representative images are shown at the left. The intensity of crystal violet is quantified at the right (mean ± SEM, n = 3). **p < 0.01; ***p < 0.001 by two-way ANOVA with Tukey’s test. (H) A proposed model of BCAT1i/AKG-induced synthetic lethality in IDH^WT GBM. BCAT1 inhibition causes a modest reduction of NADH, ATP, nucleotides, and proteins through loss of BCKAs, nitrogen donor glutamate, and/or mTORC1 activity in IDH^WT GBM cells. Likewise, AKG modestly reduces nucleotide biosynthesis and protein synthesis through mTORC1 inhibition, inhibits ATP production, and increases NADH oxidation into NAD⁺. While BCAT1 loss or AKG alone-induced metabolic alterations are not sufficient to kill cells, BCAT1i/AKG combination has a significant impact on mitochondrial dysfunction and depletion of ATP, nucleotides, and proteins, leading to IDH^WT GBM cell death. Gaba, gabapentin. Mito, mitochondrial. All immunoblot data are shown from one of three independent experiments.