Tissue Factor suppression in IDH1-mutant gliomas controls its malignant phenotype

Background: IDH1 mutations (IDH1mut) occur in 20-30% of gliomas, induce DNA hypermethylation, and are associated with a better prognosis than IDH1 wild-type (IDH1wt) gliomas, yet the basis for this remains unclear. Furthermore, venous thromboemboli (VTE) are a serious complication in glioma patients, and the mechanism for this is also unclear. We recently discovered that IDH1mut gliomas are much less likely to develop VTE compared to IDH1wt gliomas. Our data suggests that suppression of Tissue Factor (TF), the primary initiator of coagulation, is a key reason for this. TF also enhances tumor malignancy via protease-activated receptor 2 (PAR2). In this study, we further explored the significance of TF on malignancy and thrombosis in IDH1wt and IDH1mut gliomas. Methods: Assays were performed using 6 patient derived glioma cells, 2 IDH1wt (GBM6, GBM12) and 2 IDH1mut (TB09, GBM164). DNA methylation was analyzed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using preoperative glioma patient arterial plasma, conditioned medium, or cells. In vivo models of cancer induced thrombosis were created via surgical stenosis of the inferior vena cava of mice engrafted with IDH1wt or IDH1mut gliomas. In vitro markers of malignancy were measured by BrdU incorporation for cell proliferation, serum gradient Matrigel-coated transwell inserts for invasion, soft agar colony formation for clonogenicity. Nude mice (N≥6/group) were orthotopically transplanted with IDH1wt glioma cells and monitored for growth using bioluminescence imaging. Results: The TF gene, F3, was hypermethylated in IDH1mut cells compared to IDH1wt cells, and IDH1mut glioma cells had lower levels of TF protein and TF PCA compared to IDH1wt cells. Exogenous treatment with the product of IDH1mut enzyme, D-2-hydroxyglutarate, directly suppressed TF PCA by 38% in IDH1wt cells (P=0.03). Mice with IDH1mut glioma xenografts produced fewer and smaller IVC thrombi than mice with IDH1wt xenografts (0.9±0.9 vs 10.3±3.6 mg, P=0.02). Patients whose gliomas contained IDH1mut had lower TF PCA compared to IDH1wt (0.5±0.2 vs 1.2±0.2 pg/mL, P=0.04), and there was a strong link between levels of TF PCA and subsequent development of VTE (P=0.03). TF knockdown greatly reduced cell proliferation, invasion, and colony formation of IDH1wt cells. TF overexpression in IDH1mut cells increased cell proliferation, invasion, and colony formation. The PAR2 antagonist, GB83, inhibited IDH1wt cell proliferation by 54% (P<0.001) and reduced cell invasion by 25% (P<0.05). TF knockdown also reduced in vivo growth of IDH1wt gliomas by 96% at 28 days post engraftment (P=0.03), and extended median survival of engrafted mice by 86% (P=0.001). Conclusions: IDH1mut mediated suppression of TF may be a critical component of the less thrombogenic, and less malignant, IDH1mut phenotype. Targeting TF-PAR2 signaling may therefore represent a novel therapeutic strategy to reduce IDH1wt glioma malignancy.