MULTI-FACETED INHIBITION OF TET PATHWAY WITH CELL-PERMEABLE 2HG AND BOBCAT 339 REDUCES PROLIFERATION AND INDUCES APOPTOSIS IN DIPG

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is one of the worst forms of pediatric brain tumors, with a 100% mortality-rate. A prominent mutation observed in them is a lysine to methionine change in histone3 which causes trapping of the repressive-chromatin-complex, leading to genome-level hypomethylation, affecting global epigenetic-regulation. Ten-Eleven Translocation (TET) proteins are alpha-ketoglutarate (α-KG) dependent dioxygenases that mediate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), a key-step in removing DNA methylation-marks. We previously identified increased levels of 5hmC and TETs in DIPG. In IDH mutant glioma, the R132H-IDH produces an oncometabolite, 2-hydroxyglutarate (2HG), instead of α-KG. 2HG competitively inhibits TET function, resulting in a hypermethylated genome. Bobcat339, a cytosine-analog targets TETs by blocking the cytosine binding-site on TETs. We hypothesized that cell-permeable 2HG and Bobcat339 would synergize to block TET activity in DIPG, restore epigenetic-balance by increasing genomic methylation, and induce cell-death. 2HG induced apoptosis in DIPG cells at concentrations ranging from 100-300uM, as measured by cleaved-PARP western and cleaved-caspase3 immunofluorescence (2-7fold increase in CC3, depending on cell line). Cell-permeable 2HG also decreased proliferation, measured by phospho-RB western in DIPG cells (30-50% reduction in pRB band intensity, depending on cell line). Similarly, Bobcat339 suppressed proliferation at concentrations from 10-50uM, measured by BrdU incorporation (25% reduction in BrdU+ at 50uM, p= 0.02 compared to control). Bobcat339 induced apoptosis, measured by cPARP western and CC3 immunofluorescence (4-10fold more of CC3+ compared to control p< 0.0004). In combination, cell-permeable 2HG (100-200uM) and Bobcat339 (10-20uM) combined to suppress cell-viability, measured by CellTiterBlue assay, producing ZIP scores of ~20 in DIPG cells (indicating high-level synergy). In combination, 2HG and Bobcat339 increased apoptosis in DIPG (3-fold increase in cleaved-PARP band intensity in JHH-DIPG1 cells treated with 20uM Bobcat339 + 100uM 2HG, compared to single-treated cells). Our results may lead to new approaches that target TET pathway in DIPG tumors.