Article Figures & Data
Figures
- Figure 1.
Continuous treatment with 1 or 10 μmol/L AGI-5198 rapidly decreases 2-HG levels in E6/E7 and E6/E7/hTERT cell lines expressing mutant IDH1. Intracellular 2-HG levels were determined by 1H-NMR mass spectroscopy after continuous AGI-5198 exposure for 72 hours (A), 14 days (B), or 28 days (C). 2-HG levels in AGI-5198–treated cells are expressed as the percentage of 2-HG levels in corresponding controls cells receiving vehicle (DMSO) only.
- Figure 2.
A, schematic overview of the experimental timeline for the different E6/E7/hTERT experimental cell groups (numbered 1–9) following addition (small downward arrowheads) or removal (small upward arrowheads) of 1 μmol/L AGI-5198 in temporal relation to the introduction of a blank construct (large open triangle) or a construct encoding mutant IDH1 (large black triangles) and final analysis of histone modifications and clonogenicity. B, immunoblotting of mutant IDH1 (top) and various histone H3 methylation levels (bottom) in the different E6/E7/hTERT experimental cell groups at the time endpoints indicated in A. GAPDH and histone H3 were used as loading controls for whole-cell and nuclear protein lysates, respectively. C, soft agar colony formation as a measure of in vitro cellular transformation in the nine different E6/E7/hTERT cell groups shown in A. **, P < 0.01; ***, P < 0.001.
- Figure 3.
Growth of the different E6/E7 and E6/E7/hTERT cell lines in response to continuous treatment with drug vehicle (DMSO) or 1 or 10 μmol/L AGI-5198 over a time period of 28 days. No reduction in cell proliferation was observed in immortalized E6/E7/hTERT cells expressing WT IDH1 (A), E6/E7/hTERT cells transformed independently of mutant IDH1 by mutant V12H-Ras (B), mutant IDH1–driven transformed E6/E7/hTERT cells (C), untransformed E6/E7 cells expressing WT IDH1 (D), untransformed E6/E7 cells expressing mutant IDH1 (precrisis cells; E), or mutant IDH1–driven transformed E6/E7 cells (postcrisis cells; F).
- Figure 4.
Immunoblot analysis of lysine di- and trimethylation in histone H3 in E6/E7 and E6/E7/hTERT cells expressing either WT or mutant IDH1 after 28 days of continuous treatment with vehicle or 1 or 10 μmol/L AGI-5198. Untreated E6/E7/IDH1WT cells were used as a control for baseline histone H3 methylation levels. Total levels of histone H3 were used as loading control. No significant change in H3K4me3, H3K9me2, H3K9me3, or H3K27me3 levels were seen between control and AGI-5198–treated cells in the E6/E7/IDH1WT (A), E6/E7/IDH1mut precrisis (B), E6/E7/IDH1mut postcrisis (C), E6/E7/hTERT/IDH1WT (D), or E6/E7/hTERT/IDH1mut (E) cells. *, P < 0.05.
- Figure 5.
Colony formation in soft agar as a measure of anchorage-independent growth in response to 28-day treatment with drug vehicle or 1 or 10 μmol/L AGI-5198. No significant reduction in colony formation was noted in E6/E7/hTERT cells expressing WT IDH1 (A) or in cells transformed by expression of mutant V12H-Ras (B). High-dose AGI-5198 (10 μmol/L) moderately reduced colony formation in mutant IDH1–driven transformed E6/E7 cells (C) and in mutant IDH1–driven transformed E6/E7 cells (postcrisis, D). *, P < 0.05.
- Figure 6.
Potential therapeutic implications for the administration of the mutant IDH1–specific inhibitor AGI-5198. Mutant IDH1–driven transformation of immortalized p53/pRb–deficient human astrocytes can be blocked by AGI-5198 administration prior to or concurrent with the oncogenic insult made by the introduction of mutant IDH1 (1). However, cells already transformed by mutant IDH1 become rapidly insensitive to AGI-5198 (2). This suggests that the temporal window in which effective pharmacologic inhibition of mutant IDH1 can be achieved appears very limited.
Volume 14, Issue 10
