Project description:We perform high-throughput single-nucleus RNA and ATAC sequencing (snRNA- and snATAC-seq) on primary IDH mutant gliomas and snRNA-seq on a cohort of primary and recurrent IDH mutant glioma pairs to comprehensively resolve tumor diversity and TAM states. Our results resemble previously described differentiation hierarchies, but also reveal a novel group of epigenetically and transcriptionally distinct non-cycling stem-like tumor cells in diffuse gliomas. Further, we identify significant transcriptional differences in TAM states between oligodendrogliomas and astrocytomas, with a notable interaction between immunosuppressive TAMs and astrocytic tumor subpopulations in astrocytomas. These results suggest that TAM-tumor interactions may contribute to the clinical course of oligodendrogliomas and astrocytomas.
Project description:Gene expression data generated for the purpose of correlating differentially-expressed genes between IDH1 mutant and IDH1 wild-type high grade gliomas with differential hydroxymethylcytosine profiles as determined using Illumina EPIC BeadChip platform.
Project description:The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA-cycle are associated with IDH1 mut. Here we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, in order to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism and the TCA-cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA cycle activity in IDH1 mut gliomas.
Project description:The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA-cycle are associated with IDH1 mut. Here we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, in order to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism and the TCA-cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA cycle activity in IDH1 mut gliomas.
Project description:Gliomas harboring mutations in isocitrate dehydrogenase 1/2 (IDH1/2) have the CpG island methylator phenotype (CIMP) and significantly longer patient survival time than wild-type IDH1/2 tumors. Although there are many factors underlying the differences in survival between these two tumor types, immune-related differences in cell content are potentially important contributors. In order to investigate the role of IDH mutations in immune response, we created a syngeneic pair mouse model for mutated IDH1 (mutIDH1) and wild-type IDH1 (wtIDH1) gliomas and demonstrated that muIDH1 mice showed many molecular and clinical similarities to muIDH1 human gliomas, including a 100-fold higher concentration of 2-hydroxygluratate (2-HG), longer survival time, and higher CpG methylation compared to wtIDH1. Also, we showed that IDH1 mutations caused downregulation of leukocyte chemotaxis, resulting in repression of the tumor-associated immune system. Given that significant infiltration of immune cells such as macrophages, microglia, monocytes, and neutrophils is linked to poor prognosis in many cancer types, these reduced immune infiltrates in muIDH1 glioma tumors may contribute in part to the differences in aggressiveness of the two glioma types.
Project description:IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite D-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild-type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients, mutant IDH1 and 2HG are not required for later clonal expansions.
Project description:Understanding the glioma stem cell (GSC) heterogeneity within IDH1 and TP53 mutant tumors may elucidate possible targets for astrocytoma treatment. We performed single-nucleus transcriptomics of 6 mutant and wild-type glioma samples sorted for Sox2 stem cell marker. Malignant states of different clusters were evaluated by the expression of the normal and hematopoietic markers. We found that mutant GSCs were characterized by collagen synthesis and CD44-high phenotype prone to migration, while wild-type GSCs had vulnerability points in ATP synthesis. Additionally, mutant GSCs displayed altered lipogenesis probably attributed to the low NADPH consumed by mutant IDH1. The collagen and lipid biosynthesis represent possible target pathways for prospective tackling GSCs with IDH1 R132H and TP53 point mutations, uncouplers of oxidative phosphorylation are promising to address wild-type GSC proliferation
Project description:As part of a multi-omics and CRISPR/Cas9-screen based study to extensively characterize the molecular basis of diffuse hemispheric gliomas, H3G34-mutant (DHG-H3G34), we applied single-cell/single-nucleus RNA-sequencing to a patient cohort of 9 fresh and frozen untreated DHG-H3G34.
Project description:Mutations in IDH1 and IDH2 genes are very common in low grade gliomas and secondary GBM. They induce hypermethylation of DNA and histones in tumor cells, and generate a specific epigenetic phenotype. Therefore, we decided to perform a chemical screen consisting of inhibitors of epigenetic regulators on patient-derived IDH mutant cell lines. Dual combinations of hit inhibitors in low doses were also analyzed to increase treatment efficiency. Individual and combinatorial effects of these inhibitors were also analyzed in wild type and IDH mutant pair of A172 GBM cell line that was derived by ectopic overexpression of mutant IDH1. GSK-J4 and Belinostat combination was highly effective on both patient-derived and engineered IDH mutant cells compared to wild type cells. Interestingly, sensitized IDH mutant cells were recovered via mutant IDH inhibitor, GSK864, which is thought as a candidate drug for IDH mutant glioma. On the other hand, viability of non-malignant fibroblasts were not affected from these drugs in these doses. GSK-J4 and Belinostat combination induce apoptosis which was shown by increased Caspase 3/7 activity, PARP cleavage and recovery with general caspase inhibitor (Z-VAD-FMK). Change in histone modifications such as H3K4me3, H3K9me3, H3K27me3 and H3K27ac were observed after mutant IDH overexpression and also drug treatments. RNA-seq and GSEA analysis revealed activation of unfolded protein and viral response pathways in IDH mutant cells which indicate increased cell stress. GSKJ4 and Belinostat induces cell cycle arrest and apoptosis by further increasing cell stress probably because of dramatic changes in chromatin structure.