Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Malignant gliomas have a very poor prognosis. The current standard of care for these cancers consists of extended adjuvant treatment with the alkylating agent temozolomide after surgical resection and radiotherapy. Although a statistically significant increase in survival has been reported with this regimen, nearly all gliomas recur and become insensitive to further treatment with this class of agents. We sequenced 500 kb of genomic DNA corresponding to the kinase domains of 518 protein kinases in each of nine gliomas. Large numbers of somatic mutations were observed in two gliomas recurrent after alkylating agent treatment. The pattern of mutations in these cases showed strong similarity to that induced by alkylating agents in experimental systems. Further investigation revealed inactivating somatic mutations of the mismatch repair gene MSH6 in each case. We propose that inactivating somatic mutations of MSH6 confer resistance to alkylating agents in gliomas in vivo and concurrently unleash accelerated mutagenesis in resistant clones as a consequence of continued exposure to alkylating agents in the presence of defective mismatch repair. The evidence therefore suggests that when MSH6 is inactivated in gliomas, alkylating agents convert from induction of tumor cell death to promotion of neoplastic progression. These observations highlight the potential of large scale sequencing for revealing and elucidating mutagenic processes operative in individual human cancers. (Cancer Res 2006; 66(8): 3987-91)

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IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.

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Brain metastases from lung adenocarcinoma (BM-LUAD) cause significant patient mortality. To identify genomic alterations that promote brain metastases, we performed whole-exome sequencing of 73 BM-LUAD cases. Using case-control analyses, we discovered candidate drivers of brain metastasis by identifying genes with more frequent copy-number aberrations in BM-LUAD compared to 503 primary lung adenocarcinomas. We identified three regions with significantly higher amplification frequencies in BM-LUAD, including MYC (12% vs 6%), YAP1 (7% vs 0.8%), and MMP13 (10% vs 0.6%) and significantly more frequent deletions in CDKN2A/B (27% vs 13%). We confirmed that amplification frequencies of MYC and YAP1 / MMP13 were elevated in an independent cohort of 105 patients. Functional assessment in patient-derived xenograft mouse models validated that MYC , YAP1 or MMP13 overexpression increased the brain metastasis incidence. These results demonstrate that somatic alterations contribute to brain metastases and that genomic sequencing of a large number of metastatic tumors can reveal novel metastatic drivers.

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Background.Patients with malignant gliomas present with variation in neurocognitive function (NCF) not attributable to lesion size or location alone. A potential contributor is the rate at which tumors grow, or "lesion momentum." Isocitrate dehydrogenase 1 wild type (IDH1-WT) are more proliferative and aggressive than IDH1-mutant (IDH1-M) tumors. We hypothesized that patients with IDH1-WT would exhibit worse NCF than patients with IDH1-M tumors. Methods.Comprehensive NCF testing was completed in 119 patients with malignant glioma prior to surgical resection. IDH1 status was determined with immunohistochemistry and sequencing. Rates of impairment and mean test performances were compared by IDH1.Results. NCF impairment was significantly more frequent in patients with IDH1-WT tumors in memory, processing speed, visuoconstruction, language, executive functioning, and manual dexterity. Mean performances of patients with IDH1-WT were also significantly lower than those with IDH1-M tumors on measures of learning and memory, processing speed, language, executive functioning, and dexterity. Lesion volume was not statistically different between IDH1-WT and IDH1-M tumors. Tumor and lesion volume on T1-weighted and fluid attenuated inversion recovery MRI were significantly associated with most NCF tests in patients with IDH1-WT, but only significantly associated with a single measure in patients with IDH1-M tumors. Conclusion.Patients with IDH1-WT show reduced NCF compared with those with IDH1-M malignant gliomas. Lesion volume is inversely associated with NCF for patients with IDH1-WT, but not IDH1-M tumors. These findings are consistent with the hypothesis that patients with IDH1-WT tumors present with more severe NCF impairment due to greater lesion momentum, which may impede compensatory neuroplasticity and cerebral reorganization.