Title: Targeting Histone Modifications in Isocitrate Dehydrogenase-1 R132H Mutated Glioma and Oligodendrocyte Progenitor Cells
Description: Isocitrate Dehydrogenase-1 has been found to be mutated in over 70% of lower grade gliomas and has become an important diagnostic tool for tumor prognosis, however its role in glioma development, and its impact on response to therapy, is still not fully understood. Unmutated IDH1 functions to convert isocitrate to alpha-ketoglutarate (a-KG) in the tricarboxylic acid (TCA) cycle. Mutated IDH1R132H changes the enzymatic equilibrium and converts a-KG to 2-hydroxyglutarate (2-HG), an oncometabolite. IDH1R132H mutated tumors show an elevated production of 2-HG and epigenetic alterations in DNA and histone methylation. This mutation is predominantly seen in the proneural glioma subtype in which oligodendrocytes progenitors (OPCs) are considered the cell of origin due to phenotypic similarities. The effect of IDH1R132H mutation in cellular transformation has not been fully established. Epigenetic modifications connect genotype to phenotype by genetic expression alterations and epigenetic modifications are necessary for OPC differentiation.

Tri-methylation of lysine residue K27 on histone H3 (H3K27me3) is a repressive mark associated with cell pluripotency. H3K27me3 is trimethylated by Enhancer of Zeste 2 (EZH2) and demethylated by the a-KG-dependent demethylases UTX/KMD6A and JMJD3/KDM6B. 2-HG is a competitive inhibitor of a-KG-dependent demethylases, providing a mechanistic link between IDH mutations and increases H3K27me3 by inhibiting demethylation. In this thesis, we evaluated the epigenetic changes in mouse models of IDH mutant and wildtype glioma and genetically-transformed OPCs and tested the effects of drugs that target specific epigenetic marks. We developed a mouse model of glioma to compare IDH1R132H cells to wildtype glioma cells and found that although there was no difference in survival, IDH1R132H gliomas have increased levels of 2-HG by MALDI-IMS and metabolomic analysis. Interestingly, based on RNA-sequencing analysis our IDH1R132H model has a more OPC-restricted expression profile compared the wildtype glioma model which have higher enrichment of genes from other cell lineages, including neurons, astrocytes, myelinating oligodendrocytes and microglia.

We used the EZH2 inhibitor (Tazemetostat, EPZ-6438) and found that this treatment was not cytotoxic or cytostatic to our cells although H3K27me3 was reduced. Interestingly, Tazemetostat treatment increased the expression of non-OPC genes (genes normally expressed by other lineages as assessed using the Barres transcriptomic database). To better understand how IDH1R132H influences OPC transformation, we transformed OPCs in vitro. OPCs were isolated from floxed p53 postnatal day 5 mice from and retrovirally infected with viruses to delete p53 alone or to also express IDH1R132H. OPCs that express IDH1R132H had increased levels of 2-HG by metabolomics and showed alteration in H2K27 methylation and acetylation that resembled those seen in glioma cells. Standard methods of western blot analysis consist of analyzing whole cell lysate, cytoplasmic and nuclear fractionation, or histone acid extraction. To analyze both the cytoplasmic fraction as well as histone modification, I developed a cellular extraction method in which cells were fractionated and the nuclear fraction was acid extracted.

This method allows for the analysis of both cytoplasmic proteins as well as histone modifications by western blot. Using this method, we found that treating glioma cells or OPCs with synthetic cell permeable octyl-2HG, or expressing IDH1R132H, caused cells to have increased H3K27me3, while treatment with Tazemetostat caused a decrease in H3K27me3. Based on the RNA-sequencing data we found that increased H3K27me3 (ID1R132H mutation) express more OPC-like phenotype while reduced H3K27me3 (Tazemetostat treatment) induced an upregulation of genes associated with other lineages making them less restricted to the OPC transcriptional phenotype. We found that in both the glioma cells and OPCs, Tazemetostat treatment decreased H3K27me3 and increased H3K27ac. Based on the increase of H3K27ac after Tazemetostat treatment, we hypothesized that a Histone deacetylase inhibitor (HDACi) would be synergistic. We found that although the HDACi Panobinostat was less cytotoxic to IDH1R132H mutated glioma cells and OPCs, co-treatment with Tazemetostat is synergistic in mutant and wildtype models.

We also saw that in IDH1R132H ex vivo slices, the co-treatment reduced tumor marker composition. These findings point to a novel therapeutic strategy for IDH1-mutated proneural gliomas that targets the specific epigenetic alteration in these tumors.