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Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers. Significance: Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH–mediated transformation in AML and glioma.

Glioblastoma (GBM) is characterized by exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here, we found that the compositions of ribosomes of GBM cells in the tumour core and edge differ due to alternative RNA splicing. The acidic pH in the core switches before messenger RNA splicing of the ribosomal gene RPL22L1 towards the RPL22L1b isoform. This allows cells to survive acidosis, increases stemness and correlates with worse patient outcome. Mechanistically, RPL22L1b promotes RNA splicing by interacting with lncMALAT1 in the nucleus and inducing its degradation. Contrarily, in the tumour edge region, RPL22L1a interacts with ribosomes in the cytoplasm and upregulates the translation of multiple messenger RNAs including TP53. We found that the RPL22L1 isoform switch is regulated by SRSF4 and identified a compound that inhibits this process and decreases tumour growth. These findings demonstrate how distinct GBM cell populations arise during tumour growth. Targeting this mechanism may decrease GBM heterogeneity and facilitate therapy.

Analysis of tumor gene expression is an important approach for the classification and identification of therapeutic vulnerabilities. However, targeting glioblastoma (GBM) based on molecular subtyping has not yet translated into successful therapies. Here, we present an integrative approach based on molecular pathways to expose new potentially actionable targets. We used gene set enrichment analysis to conduct an unsupervised clustering analysis to condense the gene expression data from bulk patient samples and patient-derived gliomasphere lines into new gene signatures. We identified key targets that are predicted to be differentially activated between tumors and were functionally validated in a library of gliomasphere cultures. Resultant cluster-specific gene signatures associated not only with hallmarks of cell cycle and stemness gene expression, but also with cell type–specific markers and different cellular states of GBM. Several upstream regulators, such as PIK3R1 and EBF1 were differentially enriched in cells bearing stem cell like signatures and bear further investigation. We identified the transcription factor E2F1 as a key regulator of tumor cell proliferation and self-renewal in only a subset of gliomasphere cultures predicted to be E2F1 signaling dependent. Our in vivo work also validated the functional significance of E2F1 in tumor formation capacity in the predicted samples. E2F1 inhibition also differentially sensitized E2F1-dependent gliomasphere cultures to radiation treatment. Our findings indicate that this novel approach exploring cancer pathways highlights key therapeutic vulnerabilities for targeting GBM. Significance: Molecular classification of GBM has not yet resulted in the development of effective therapies. We have developed an integrative approach to identify molecular targets differentially utilized by individual tumors. This approach could lead to patient- and tumor-specific therapeutics.

Increased secretion of hyaluronic acid (HA), a glycosaminoglycan abundant in the brain extracellular matrix (ECM), correlates with worse clinical outcomes for glioblastoma (GBM) patients. GBM cells aggressively invade the brain parenchyma while encountering spatiotemporal changes in their local ECM, including HA concentration. To investigate how varying HA concentrations affect GBM invasion, we cultured patient-derived GBM cells within a soft, three-dimensional matrix in which HA concentration was precisely varied and cell migration observed. Data demonstrate that HA concentration can determine the invasive activity of patient-derived GBM cells in a biphasic and highly sensitive manner, where the absolute concentration of HA at which cell migration peaked was specific to each patient-derived line. Furthermore, we provide evidence that this response relies on phosphorylated ezrin, which interacts with the intracellular domain of HA-engaged CD44 to effectively link the actin cytoskeleton to the local ECM. Overall, this study highlights CD44-HA binding as a major mediator of GBM cell migration that acts independently of integrins and focal adhesion complexes and suggests that targeting HA-CD44-ezrin interactions represents a promising therapeutic strategy to prevent tumor cell invasion in the brain.

Inhibition of KDM5 enzymes by the (R)- and (S)-enantiomers of 2-hydroxyglutarate.

Supplementary Table S9 shows the gene set enrichment analysis gene set that corresponds to Figure S6A.

List of supplementary figures and tables.

Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers.Significance:Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH–mediated transformation in AML and glioma.

TCGA gene ontology analysis of clusters shows differentially enriched terms

GO terms and TF enriched in modules associated with GS clusters

Analysis of tumor gene expression is an important approach for the classification and identification of therapeutic vulnerabilities. However, targeting glioblastoma (GBM) based on molecular subtyping has not yet translated into successful therapies. Here, we present an integrative approach based on molecular pathways to expose new potentially actionable targets. We used gene set enrichment analysis to conduct an unsupervised clustering analysis to condense the gene expression data from bulk patient samples and patient-derived gliomasphere lines into new gene signatures. We identified key targets that are predicted to be differentially activated between tumors and were functionally validated in a library of gliomasphere cultures. Resultant cluster-specific gene signatures associated not only with hallmarks of cell cycle and stemness gene expression, but also with cell type–specific markers and different cellular states of GBM. Several upstream regulators, such as PIK3R1 and EBF1 were differentially enriched in cells bearing stem cell like signatures and bear further investigation. We identified the transcription factor E2F1 as a key regulator of tumor cell proliferation and self-renewal in only a subset of gliomasphere cultures predicted to be E2F1 signaling dependent. Our in vivo work also validated the functional significance of E2F1 in tumor formation capacity in the predicted samples. E2F1 inhibition also differentially sensitized E2F1-dependent gliomasphere cultures to radiation treatment. Our findings indicate that this novel approach exploring cancer pathways highlights key therapeutic vulnerabilities for targeting GBM.Significance:Molecular classification of GBM has not yet resulted in the development of effective therapies. We have developed an integrative approach to identify molecular targets differentially utilized by individual tumors. This approach could lead to patient- and tumor-specific therapeutics.

PDF File - (58K) - Secondary screen identified 168 GSC -effective compounds with selective inhibition profile

Canonical Pathways Associated (P

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

PDF File - (32K) - GSC-enriched GBM samples form tumors after transplantation into immunodeficient mice

PDF File - (874K) - A - Representative images demonstrating tumor formation success in an animal transplanted with serum (I) and sphere (II) derived GBM146 Ki67 and Human Nuclei (HuNu) immunostaining shows that virtually all transplanted cells died off in the animal transplanted with serum derived cells while an infiltrating tumor mass is visible in the animal transplanted with sphere derived cells of the same GBM sample (i - injection site, V- ventricle, SVZ - subventricular zone). B - Immunohistological sections for Human Nuclei (HuNu) and Ki67 demonstrating good survival of transplanted cells in animals that were sacrificed on the same day as well as 48h after transplantation (IT- injection tract, ST-striatum).

Supplementary Figure S1: Treatment with octyl-D-2-HG-induces MIR148A methylation; Supplementary Figure S2: Octyl-D-2-HG-induced MIR148A methylation status remains constant at late passages; Supplementary Figure S3: Octyl-D-2-HG induces CpG island methylation of the RBP1 promoter in 293T cells; Supplementary Figure S4: Octyl-D-2-HG induces CpG island methylation of the MIR148A promoter in normal human astrocytes (NHA); Supplementary Figure S5: MIR148A promoter CpG methylation in IDH1MUT-transfected 293T cells; Supplementary Figure S6: C227 prevents IDH1 mutant-induced MIR148A promoter methylation; Supplementary Figure S7: C227 prevents IDH1 mutant-induced RBP1 promoter methylation; Supplementary Figure S8: Schematic graph of MIR148A genomic location, CpG island, and promoter-reporter construct; Supplementary Figure S9: MIR148a expression is unchanged by octyl-D-2-HG treatment in early passages in 293T and NHA cells; Supplementary Figure S10: Octyl-D-2-HG withdrawal restores MIR148A promoter to an unmethylated state

PDF file - 212K, Fig. S2. Ligand-dependent PDGFRβ stimulation promotes the proliferation of erlotinib-treated glioblastoma cells.

PDF File - (67K) - Effects of OLDA, PALDA and NADA are not mediated via cannabinoid, vanilloid or dopamine receptors A - Exposure of GSC -enriched GBM 157 cells to CB1, CB2, TRPV1 receptor agonists or their combination, does not reproduce the phenotype seen after exposure to OLDA, PALDA or NADA. Graph showing ATP level expressed as % of DMSO treated controls. B - Exposure of OLDA, PALDA or NADA-pretreated GSC -enriched GBM 157 cells (at IC50 concentration) to antagonists of CB, TRPV and D receptors does not rescue the phenotype caused by OLDA, PALDA or NADA (B, C and D, respectively). The graphs are showing ATP levels expressed as % of the DMSO treated controls (100%). SE shown.

Supplementary Materials and Methods

PDF File - (61K) - OLDA, Emetine, 5256360 and 5560509 decrease clonal sphere formation in low passage GBM312 Graphs show a concentration-dependent reduction of clonal neurosphere size, neurosphere number as well as total cell mass. Error bars represent SEM.

Table legend for supplementary Table 1.

Glioblastoma stem cells (GSC) express both radial glial cell and neural crest cell (NCC)-associated genes. We report that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSCs. Serum-induced proliferative differentiation rapidly decreased EDN3 production and downregulated the expression of stemness-associated genes, and reciprocally, two glioblastoma markers, EDN1 and YKL-40 transcripts, were induced. Correspondingly, patient glioblastoma tissues express low levels of EDN3 mRNA and high levels of EDN1 and YKL-40 mRNA. Blocking EDN3/EDN receptor B (EDNRB) signaling by an EDNRB antagonist (BQ788), or EDN3 RNA interference (siRNA), leads to cell apoptosis and functional impairment of tumor sphere formation and cell spreading/migration in culture and loss of tumorigenic capacity in animals. Using exogenous EDN3 as the sole mitogen in culture does not support GSC propagation, but it can rescue GSCs from undergoing cell apoptosis. Molecular analysis by gene expression profiling revealed that most genes downregulated by EDN3/EDNRB blockade were those involved in cytoskeleton organization, pause of growth and differentiation, and DNA damage response, implicating the involvement of EDN3/EDNRB signaling in maintaining GSC migration, undifferentiation, and survival. These data suggest that autocrine EDN3/EDNRB signaling is essential for maintaining GSCs. Incorporating END3/EDNRB-targeted therapies into conventional cancer treatments may have clinical implication for the prevention of tumor recurrence. Mol Cancer Res; 9(12); 1668–85. ©2011 AACR.

PDF File - (39K) - Limiting dilution transplantation of GBM157 cells treated with OLDA, 5256360 or 5560509 shows decreased tumor formation frequency

PDF file - 199K, Fig. S1. EGFRvIII and wild type EGFR signaling regulate PDGFRβ in vitro.

PDF File - (523K) - OLDA, Emetine, 5256360 and 5560509 inhibit proliferation of sphere cultured GBM312 cells, using CFSE (carboxyfluorescein diacetate, succinimidyl ester) cell tracing experiments Proliferative inhibition is graphically represented by an increase in fluorescence or a shift to the right, away from that of the control. A proliferation index (PI) is a numerical value generated for each sample that measures the total increase in cell number divided by the original parent number. PI's are expressed as a percent of control: Emetine (0.2�M)- 18.6%, OLDA (4�M)- 16.0%, Compound 33 (2�M)- 31%, and Compound 62 (2�M)- 24.3%.

PDF File - (40K) - Growth kinetics of serum and sphere derived cells in screening (sphere) media reveals only minor differences in the degree of growth/proliferation between sphere and serum derived cultures of the same cell type Graph shows results of an ATP-based proliferation assay as measured 3 days after cell plating. Error bars depict SEM.

PDF File - (55K) - Sensitivity of GBM cells to OLDA, PALDA and NADA changes as a function of time, for which the cells were cultured in GSC -enriching media GBM107 cells were exposed to GSC -enriching media for increasing amounts of time and were then screened for sensitivity to dopaminergic cannabinoid compounds. While screening in serum-containing media requires a higher compound concentration due to the protective effect of serum in general, serum-derived cells become more and more sensitive towards GSC -specific compounds over time after transfer into sphere media (A, B, C). Such a difference is not present using non-GSC selective chemotherapeutics (D) or non-GBM control cells (E, F). ('ser in ser': serum derived cells screened in serum media; 'ser in sph': serum derived cells screened in sphere media; '1d sph': serum derived cells were cultured in sphere media for one day before the screen, etc.)

PDF file - 59K, Amino acid and ammonia changes with ASNase treatment in media and cell supernatants.

PDF file - 518K

Glioblastoma (GBM) and lower grade gliomas (LGG) are the most common primary malignant brain tumors and are resistant to current therapies. Genomic analyses reveal that signature genetic lesions in GBM and LGG include copy gain and amplification of chromosome 7, amplification, mutation, and overexpression of receptor tyrosine kinases (RTK) such as EGFR, and activating mutations in components of the PI3K pathway. In Drosophila melanogaster, constitutive co-activation of RTK and PI3K signaling in glial progenitor cells recapitulates key features of human gliomas. Here we use this Drosophila glioma model to identify death-associated protein kinase (Drak), a cytoplasmic serine/threonine kinase orthologous to the human kinase STK17A, as a downstream effector of EGFR and PI3K signaling pathways. Drak was necessary for glial neoplasia, but not for normal glial proliferation and development, and Drak cooperated with EGFR to promote glial cell transformation. Drak phosphorylated Sqh, the Drosophila ortholog of nonmuscle myosin regulatory light chain (MRLC), which was necessary for transformation. Moreover, Anillin, which is a binding partner of phosphorylated Sqh, was upregulated in a Drak-dependent manner in mitotic cells and colocalized with phosphorylated Sqh in neoplastic cells undergoing mitosis and cytokinesis, consistent with their known roles in nonmuscle myosin-dependent cytokinesis. These functional relationships were conserved in human GBM. Our results indicate that Drak/STK17A, its substrate Sqh/MRLC, and the effector Anillin/ANLN regulate mitosis and cytokinesis in gliomas. This pathway may provide a new therapeutic target for gliomas.Significance: These findings reveal new insights into differential regulation of cell proliferation in malignant brain tumors, which will have a broader impact on research regarding mechanisms of oncogene cooperation and dependencies in cancer.See related commentary by Lathia, p. 1036

This file contains all supplementary figures, including Supplementary Table 1 and Table 2 that list the Drosophila reagents used for genetic tests described in the manuscript. This file also contains Supplementary Figures S1, S2, S3, S4, and S5, which includes additional Drosophila phenotypic analysis, protein expression data and shRNA results from human cells, and TCGA data analysis.

The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform—microfluidic image cytometry (MIC)—capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. Cancer Res; 70(15); 6128–38. ©2010 AACR.

In this issue of Neuron, Bulstrode et al.

Non-enhancing (NE) infiltrating tumor cells beyond the contrast-enhancing (CE) bulk of tumor are potential propagators of recurrence after gross total resection of high-grade glioma. We leveraged single-nucleus RNA-sequencing to compare prospectively identified biopsy specimens from CE and NE regions. Recurrent gliomas recapitulate the previously reported glioma cellular states of primary gliomas. Tumor cells in NE regions are enriched in oligodendrocyte progenitor cell-like and neural progenitor cell-like cellular states, while CE regions are enriched for astrocyte-like and mesenchymal-like states. These NE glioma cells have similar copy number alterations and proportions of proliferative and putative glioma stem cells relative to CE regions. Tumor cells in NE regions exhibit upregulation of genes previously associated with lower grade gliomas. Cell-, gene-, and pathway-level analyses of the tumor microenvironment in the NE region reveal relative downregulation of tumor-mediated neovascularization and cell-mediated immune response, but increased glioma-to-neuron interactions. This comprehensive analysis illustrates differing tumor and non-tumor landscapes of CE and NE regions in high-grade gliomas, highlighting the NE region as an area harboring likely initiators of recurrence in a pro-tumor microenvironment and identifying possible targets for future design of NE-specific adjuvant therapy.

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

AimsThe goal of this study was to determine whether NADPH oxidase (NOX)-produced reactive oxygen species enhances brain tumor growth of glioblastoma (GBM) under hypoxic conditions and during radiation treatment.ResultsExogenous ROS promoted brain tumor growth in gliomasphere cultures that expressed functional PTEN, but not in tumors that were PTEN deficient. Hypoxia induced the production of endogenous cytoplasmic ROS and tumor cell growth via activation of NOX. NOX activation resulted in oxidation of PTEN and downstream Akt activation. Radiation also promoted ROS production via NOX which, in turn, resulted in cellular protection that could be abrogated by knockdown of the key NOX component, p22. Knockdown of p22 also inhibited tumor growth and enhanced the efficacy of radiation in PTEN-expressing GBM cells.InnovationWhile other studies have implicated NOX function in GBM models, these studies demonstrate NOX activation and function under physiological hypoxia and following radiation in GBM, two conditions that are seen in patients. NOX plays an important role in a PTEN-expressing GBM model system, but not in PTEN-non-functional systems and provide a potential, patient-specific therapeutic opportunity.ConclusionsThis study provides a strong basis for pursuing NOX inhibition in PTEN-expressing GBM cells as a possible adjunct to radiation therapy.