Figure 5.

ONC201 disrupts integrated metabolic and epigenetic pathways. A, Schema of [13C₅]-glutamine tracing into the TCA cycle indicating predicted mass isotopes (m+4 or +5) for each metabolite. B, Bar graph demonstrating glutamine carbon incorporation in various metabolites (specific m+4 or +5 mass isotopes indicated) in H3.3K27M DIPG-007 cells treated with vehicle (blue) or 5 μmol/L ONC201 (red) for 48 hours (n = 3, each). C, Bar graph showing 2HG levels (Y-axis, Z-scores, measured by LC-MS) in H3.3K27M DIPGXIII*P, DIPG-007, and SF7761 cells treated with vehicle (blue) or 5 μmol/L ONC201 (red) for 48 hours (n = 3–4, each). D, Box and whisker plots showing metabolite measurements (Y-axis, ratio of metabolite pretreatment to posttreatment, a.u.) in CSF samples from ONC201-treated patients with H3K27M-DMG classified as responders and nonresponders (n = 4, each). Patients who showed a reduction in >50% tumor volume (per RANO criteria) on ONC201 were defined as responders. E, Tumor area (left Y-axis, purple line) and CSF 2HG levels (right Y-axis, red line) plotted against time (X-axis) from responder patient UMICH-006. F, Tumor area (left Y-axis, blue line) and CSF 2HG levels (right Y-axis, red line) plotted against time (X-axis) from nonresponder patient UMICH-022. Asterisks denote time points with metastatic disease. G, Bar plot showing D-2HG enantiomer-specific mass spectroscopy performed in H3.3K27M DIPG007, SF7761, and DIPGXIII*P cells treated with vehicle or ONC201 (5 μmol/L, 48 hours) and IDH1 R132H-mutant TB096 glioma cells. Quantification of D-2HG (μmol/mg protein, X-axis) is shown for ONC201-treated (brown) and DMSO-treated (green) cells. n = 4 for each condition. Data were analyzed using unpaired, two-sided, two-tailed, Student t test. H, Bar plot showing L-2HG enantiomer-specific mass spectroscopy performed in H3.3K27M DIPG007, SF7761, and DIPGXIII*P cells treated with vehicle or ONC201 (5 μmol/L, 48 hours) and IDH1 R132H-mutant TB096 glioma cells. Quantification of L-2HG (μmol/mg protein, X-axis) is shown for ONC201-treated (red) and DMSO-treated (blue) cells. n = 4 for each condition. Data were analyzed using an unpaired, two-sided, two-tailed, Student t test. I, Cartoon illustrating L-2HG inhibition of Jumonji C (JmjC) domain histone lysine demethylases resulting in increased histone methylation. J, H3.3K27M (DIPG007, DIPGXIII*P, H3.3K27M mNSC, and SF7761) and H3.1K27M (UON-JUMP4) cells were treated with 5 or 10 μmol/L ONC201 for 48 hours. Cells were probed for H3K27me3, H3K36me3, H3K9me3, H3K4me3, H3K27M, and total H3 (as loading control). The arrowhead indicates increased H3K27me3 and H3K36me3 across all cell lines. K, Cartoon illustrating L-2HG production from α-KG by “promiscuous” activity of LDHA and L-2HG-specific dehydrogenase L-2HGDH converting L-2HG back to α-KG. L, Left, H3.3K27M SF7761 cells transduced with or without shLDHA were probed for LDHA. Vinculin was used as a loading control. Right, SF7761 cells with or without LDHA knocked down were treated with vehicle or ONC201 (5 μmol/L, 48 hours) and probed for H3K27me3 and total H3 (as loading control). Arrowhead indicates H3K27me3. M, Left, L-2HGDH was overexpressed (o.e.) in H3.3K27M SF7761 cells. Cells were then probed for L-2HGDH. Vinculin was used as a loading control. Right, SF7761 cells with L-2HGDH overexpression were treated with vehicle or ONC201 (5 μmol/L, 48 hours) and probed for H3K27me3 and total H3 (as loading control). Arrowhead indicates H3K27me3. N, Left, D-2HGDH was overexpressed (o.e.) in H3.3K27M SF7761 cells. Cells were then probed for D-2HGDH. Vinculin was used as a loading control. Right, SF7761 cells with D-2HGDH overexpression were treated with vehicle or ONC201 (5 μmol/L, 48 hours) and probed for H3K27me3 and total H3 (as loading control). Arrowhead indicates H3K27me3.