Abstract 5472: Inhibiting the TET pathway in diffuse midline glioma reduces levels of 5-hydroxymethylcytosine and increases levels of 5-methylcytosine

Abstract

Abstract Diffuse Midline Glioma, DMG, is one of the worst forms of pediatric brain tumors and is associated with a 100% mortality rate. DMG, unlike other types of brain tumors, exists in a hypomethylated epigenetic state. In this study, we investigate the contribution of the Ten Eleven Translocation (TET) enzymes towards this unique epigenetic landscape. The TET enzymes which reduce 5methylcytosine (5mC) to 5hydroxymethylcytosine (5hmC) are essential for removing the methylation marks in the genome. In our previous studies, we saw that there is an increased TET expression in DMG, while 5hmC was not globally decreased. We hypothesized that inhibiting the TET enzymes would restore the epigenetic balance in DMG. We targeted the TET pathway with two different TET inhibitors, 2-hydroxyglutarate (2HG), a competitive inhibitor of αKG dependent dioxygenases, as well as Bobcat 339, a cytosine based TET inhibitor. We found that 2HG, when used in concentrations from 100µM to 300µM, had a dose dependent effect in reducing the levels of 5hmC and increasing the levels of 5mC. We performed a dot blot assay and observed a ~70% reduction in 5hmC intensity and 2 fold increase in the 5mC intensity, depending on the cell line. This change in the genomic 5hmC and 5mC levels was associated with reduced proliferation (~96% reduction in the phospho-RB band intensity as measured by a western blot in HSJD007) and induction of apoptosis (7 fold increase in cleaved caspase3 signal as measured by immunofluorescence in HSJD007, P<0.0001 compared to control). 2HG also reduced the levels of the stem cell marker Nestin by ~62% in JHHDIPG1 as measured by western blot. Similarly, Bobcat 339, when used in concentrations from 10µM to 50µM, also had a dose dependent effect in reducing 5hmC and increasing 5mC, with ~94% reduction in 5hmC levels and up to a 6 fold increase in the 5mC levels, depending on the cell line. This change in the genomic 5hmC and 5mC levels was also associated with reduced proliferation (up to a ~70% reduction in phospho-RB band intensity as measured by a western blot in HSJD007) and induction in apoptosis (up to 8 fold increase in cleaved caspase3 signal as measured by immunofluorescence in HSJD007, P<0.0001 compared to control) Additionally, we found that 2HG and Bobcat 339 synergize to further reduce the levels of 5hmC. The combination reduced the levels of 5hmC by ~60% in cells treated with 100µM of 2HG and 20µM of Bobcat 339 in JHHDIPG1 when compared to the 5hmC levels in cells treated with either 2HG or Bobcat 339. We also observed that a ~3 fold increase in cPARP band intensity in the combination treated cells as compared to the controls. These results indicate that pharmacologic inhibition of the TET pathway increases 5mC and decreases 5hmC, restoring epigenetic balance, decreasing proliferation and inducing cell death in DMG. Targeting TET enzymes may be of value therapeutically in epigenetically driven diseases like DMG. Citation Format: Akhila Parthasarathy, Antje Arnold, Charles Eberhart, Eric Raabe. Inhibiting the TET pathway in diffuse midline glioma reduces levels of 5-hydroxymethylcytosine and increases levels of 5-methylcytosine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5472.