MUTANT IDH1 SUPPRESSES APOPTOSIS REGULATOR PAR-4 IN GLIOMAS

Abstract

BACKGROUND: Glioma-associated mutations in isocitrate dehydrogenase 1 (IDH1) alter its catalytic properties, leading to the production of D-2-hydroxyglutarate (D-2-HG). This promotes hypermethylation and inhibits differentiation, but its full range of effects remains unclear. In pediatric acute myeloid leukemias, the presence of mutant IDH1 is associated with upregulation of Prostate apoptosis response-4 (Par-4), which promotes cancer-selective apoptosis. Because of this, and because very little work has been done studying Par-4 in gliomas, we tested the effect of mutant IDH1 on Par-4 in these tumors. METHODS: Par-4 expression was measured via western immunoblots in cell cultures and immunohistochemistry in paraffin-embedded tumor tissue microarrays. Par-4 mRNA levels and stability were measured using RT-PCR, cycloheximide, and actinomycin D assays.Par-4 promoter activity was studied with a luciferase reporter assay. Glioblastoma (GBM) cell lines and patient-derived stem cells were infected via lentivirus with either the Selective for Apoptosis induction in Cancer cells (SAC) domain of Par-4 or with control plasmid, for in vitro studies as well as prior to intracerebral injection in nude mice. External validation datasets included The Cancer Genome Atlas (TCGA) and the REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT). RESULTS: In vitro treatment with exogenous unmodified D-2-HG or expression of R132H IDH1 downregulates Par-4 in GBM cells. This occurs by inhibition of Par-4 promoter activity as well as by acceleration of Par-4 mRNA degradation, but not direct promoter hypermethylation. In patients, Par-4 expression is markedly lower in IDH1-mutant high grade gliomas, both in our cohort (P = 0.003) as well as in GBMs from TCGA (P = 0.008). Expression of the SAC domain of Par-4 induces apoptosis in GBM cells. When GBM stem cell intracranial xenografts express SAC, mice have longer median survival compared to controls (30 vs 23 days, P = 0.016). Among high-grade gliomas that are IDH1 wild-type, those that express Par-4 have significantly longer median survival (18.4 versus 8.0 months, P = 0.002), a finding confirmed in external GBM cohorts. CONCLUSIONS: These data suggest that a.) Mutant IDH1 and D-2-HG can affect protein expression independent of direct gene hypermethylation, b.) Suppression of Par-4 may contribute to IDH1-mutant tumorigenesis; c.) While this might seem to be a paradoxical effect of mutant IDH1 given its association with better prognosis, not all its activities will be “favorable” or the mutation would not be so prevalent in gliomas; d.) Regardless of IDH1 status, Par-4 may be an effective sensitizer of gliomas to apoptosis. SECONDARY CATEGORY: Tumor Biology.