Project description:Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II-III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.
Project description:Genomic DNA from IDH1 mutant and WT tumors were used to detect genome wide methylation using Agilent SureSelect XT Mouse Methyl-Seq Enrichment System capture kit following manufacturer’s recommendations.
Project description:BackgroundATRX inactivation occurs with IDH1R132H and p53 mutations in over 80% of Grades II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1R132H/p53mut glioma cells and xenografts.MethodsIsogenic astrocytoma cells (+/-IDH1R132H/+/-ATRXloss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline-inducible IDH1R132H, ATRX shRNA, or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1R132H+/-ATRXloss on cell migration, growth, DNA repair, and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays, and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization, and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1R132H/p53mut/ATRXloss cells.ResultsAdding ATRX loss to the IDH1R132H/p53mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune-suppressive transcriptome consisting of up-regulated immune checkpoints (e.g., PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g., IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1R132H/p53mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune-suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition.ConclusionsATRX loss activates a BRD-dependent immune-suppressive transcriptome and immune escape mechanism in IDH1R132H/p53mut astrocytoma cells.
Project description:Resistance to apoptosis and uncontrolled proliferation are two hallmarks of cancer cells. p53 is crucial for apoptosis triggered by a broad range of stresses and a well-known gatekeeper for neoplastic transformation. Here we show that oncogenic IDH1 R132H/R132Q mutants robustly inhibit p53 expression and such an effect is attributed to 2-HG production. Mechanistically, 2-hydroxyglutarate (2-HG) stabilizes hypoxia-inducible factor-2α, which in turn activates the expression of miR-380-5p, a characterized microRNA against p53 expression. Rescue expression of p53 can inhibit the proliferation rate and impair the resistance of apoptosis induced by doxorubicin in IDH1 R132Q mouse embryonic fibroblast cells. Furthermore, p53 protein levels correlates negatively with IDH1 R132H levels in human glioma samples. Our results thus shed a new light on how p53 is down-regulated by 2-HG and suggests that impairment of p53-mediated apoptosis contributes to the tumorigenesis driven by IDH1 mutants.
