Tumor progression is independent of tumor-associated macrophages in cell lineage-based mouse models of glioblastoma.

Macrophage targeting therapies have had limited clinical success in glioblastoma (GBM). Further understanding the GBM immune microenvironment is critical for refining immunotherapeutic approaches. Here, we use genetically engineered mouse models and orthotopic transplantation-based GBM models with identical driver mutations and unique cells of origin to examine the role of tumor cell lineage in shaping the immune microenvironment and response to tumor-associated macrophage (TAM) depletion therapy. We show that oligodendrocyte progenitor cell lineage-associated GBMs (Type 2) recruit more immune infiltrates and specifically monocyte-derived macrophages than subventricular zone neural stem cell-associated GBMs (Type 1). We then devise a TAM depletion system that offers a uniquely robust and sustained TAM depletion. We find that extensive TAM depletion in these cell lineage-based GBM models affords no survival benefit. Despite the lack of survival benefit of TAM depletion, we show that Type 1 and Type 2 GBMs have unique molecular responses to TAM depletion. In sum, we demonstrate that GBM cell lineage influences TAM ontogeny and abundance and molecular response to TAM depletion.

Quiescent human glioblastoma cancer stem cells drive tumor initiation, expansion, and recurrence following chemotherapy.

We test the hypothesis that glioblastoma harbors quiescent cancer stem cells that evade anti-proliferative therapies. Functional characterization of spontaneous glioblastomas from genetically engineered mice reveals essential quiescent stem-like cells that can be directly isolated from tumors. A derived quiescent cancer-stem-cell-specific gene expression signature is enriched in pre-formed patient GBM xenograft single-cell clusters that lack proliferative gene expression. A refined human 118-gene signature is preserved in quiescent single-cell populations from primary and recurrent human glioblastomas. The F3 cell-surface receptor mRNA, expressed in the conserved signature, identifies quiescent tumor cells by antibody immunohistochemistry. F3-antibody-sorted glioblastoma cells exhibit stem cell gene expression, enhance self-renewal in culture, drive tumor initiation and serial transplantation, and reconstitute tumor heterogeneity. Upon chemotherapy, the spared cancer stem cell pool becomes activated and accelerates transition to proliferation. These results help explain conventional treatment failure and lay a conceptual framework for alternative therapies.

Cell-of-origin susceptibility to glioblastoma formation declines with neural lineage restriction.

The contribution of lineage identity and differentiation state to malignant transformation is controversial. We have previously shown that adult neural stem and early progenitor cells give origin to glioblastoma. Here we systematically assessed the tumor-initiating potential of adult neural populations at various stages of lineage progression. Cell type-specific tamoxifen-inducible Cre recombinase transgenes were used to target glioblastoma-relevant tumor suppressors Nf1, Trp53 and Pten in late-stage neuronal progenitors, neuroblasts and differentiated neurons. Mutant mice showed cellular and molecular defects demonstrating the impact of tumor suppressor loss, with mutant neurons being the most resistant to early changes associated with tumor development. However, we observed no evidence of glioma formation. These studies show that increasing lineage restriction is accompanied by decreasing susceptibility to malignant transformation, indicating a glioblastoma cell-of-origin hierarchy in which stem cells sit at the apex and differentiated cell types are least susceptible to tumorigenesis.

EGFR and PDGFRA co-expression and heterodimerization in glioblastoma tumor sphere lines.

Concurrent amplifications of EGFR and PDGFRA have been reported in up to 5% of glioblastoma (GBM) and it remains unclear why such independent amplification events, and associated receptor overexpression, would be adaptive during glioma evolution. Here, we document that EGFR and PDGFRA protein co-expression occurs in 37% of GBM. There is wide cell-to-cell variation in the expressions of these receptor tyrosine kinases (RTKs) in stable tumor sphere lines, frequently defining tumor cell subpopulations with distinct sensitivities to growth factors and RTK inhibitors. We also find evidence for functional transactivation of PDGFRA by EGFR and EGF-induced receptor heterodimerization, both of which are abolished by EGFR inhibitors. These results indicate that GBM growth responses to targeted therapies previously tested in clinical trials are strongly influenced by the balance of EGFR and PDGFRA activation in individual cells, which is heterogeneous at baseline.

Loss of the tyrosine phosphatase PTPRD leads to aberrant STAT3 activation and promotes gliomagenesis.

PTPRD, which encodes the protein tyrosine phosphatase receptor-δ, is one of the most frequently inactivated genes across human cancers, including glioblastoma multiforme (GBM). PTPRD undergoes both deletion and mutation in cancers, with copy number loss comprising the primary mode of inactivation in GBM. However, it is unknown whether loss of PTPRD promotes tumorigenesis in vivo, and the mechanistic basis of PTPRD function in tumors is unclear. Here, using genomic analysis and a glioma mouse model, we demonstrate that loss of Ptprd accelerates tumor formation and define the oncogenic context in which Ptprd loss acts. Specifically, we show that in human GBMs, heterozygous loss of PTPRD is the predominant type of lesion and that loss of PTPRD and the CDKN2A/p16(INK4A) tumor suppressor frequently co-occur. Accordingly, heterozygous loss of Ptprd cooperates with p16 deletion to drive gliomagenesis in mice. Moreover, loss of the Ptprd phosphatase resulted in phospho-Stat3 accumulation and constitutive activation of Stat3-driven genetic programs. Surprisingly, the consequences of Ptprd loss are maximal in the heterozygous state, demonstrating a tight dependence on gene dosage. Ptprd loss did not increase cell proliferation but rather altered pathways governing the macrophage response. In total, we reveal that PTPRD is a bona fide tumor suppressor, pinpoint PTPRD loss as a cause of aberrant STAT3 activation in gliomas, and establish PTPRD loss, in the setting of CDKN2A/p16(INK4A) deletion, as a driver of glioma progression.

Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates.

Intragenic deletion is the most common form of activating mutation among receptor tyrosine kinases (RTK) in glioblastoma. However, these events are not detected by conventional DNA sequencing methods commonly utilized for tumor genotyping. To comprehensively assess the frequency, distribution, and expression levels of common RTK deletion mutants in glioblastoma, we analyzed RNA from a set of 192 glioblastoma samples from The Cancer Genome Atlas for the expression of EGFRvIII, EGFRvII, EGFRvV (carboxyl-terminal deletion), and PDGFRAΔ8,9. These mutations were detected in 24, 1.6, 4.7, and 1.6 % of cases, respectively. Overall, 29 % (55/189) of glioblastomas expressed at least one RTK intragenic deletion transcript in this panel. For EGFRvIII, samples were analyzed by both quantitative real-time PCR (QRT-PCR) and single mRNA molecule counting on the Nanostring nCounter platform. Nanostring proved to be highly sensitive, specific, and linear, with sensitivity comparable or exceeding that of RNA seq. We evaluated the prognostic significance and molecular correlates of RTK rearrangements. EGFRvIII was only detectable in tumors with focal amplification of the gene. Moreover, we found that EGFRvIII expression was not prognostic of poor outcome and that neither recurrent copy number alterations nor global changes in gene expression differentiate EGFRvIII-positive tumors from tumors with amplification of wild-type EGFR. The wide range of expression of mutant alleles and co-expression of multiple EGFR variants suggests that quantitative RNA-based clinical assays will be important for assessing the relative expression of intragenic deletions as therapeutic targets and/or candidate biomarkers. To this end, we demonstrate the performance of the Nanostring assay in RNA derived from routinely collected formalin-fixed paraffin-embedded tissue.

Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine.

Mutation in the IDH1 or IDH2 genes occurs frequently in gliomas and other human malignancies. In intermediate grade gliomas, IDH1 mutation is found in over 70% of tumors. These mutations impart the mutant IDH enzyme with a neomorphic activity - the ability to synthesize 2-hydroxyglutarate (2-HG). This ability leads to a reprogramming of chromatin state, a block in differentiation, and the establishment of the glioma hypermethylator phenotype (G-CIMP). It has been hypothesized but not proven that the extensive DNA methylation that occurs in G-CIMP tumors helps maintain and "lock in" glioma cancer cells in a dedifferentiated state. Here, we tested this hypothesis by treating patient derived IDH1 mutant glioma initiating cells (GIC) with non-cytotoxic, epigenetically targeted doses of the DNMT inhibitor decitabine. Global methylome analysis of treated IDH1 mutant GICs showed that DAC treatment resulted in reversal of DNA methylation marks induced by IDH and the re-expression of genes associated with differentiation. Accordingly, treatment of IDH1 mutant glioma cells resulted in a dramatic loss of stem-like properties and efficient adoption of markers of differentiation, effects not seen in decitabine treated IDH wild-type GICs. Induction of differentiation was much more efficient than that seen following treatment with a specific inhibitor of mutant IDH enzyme (Agios). Decitabine also decreased replicative potential and tumor growth in vivo. Reexpression of polycomb regulated genes accompanied these DAC-induced phenotypes. In total, our data indicates that targeting the pathologic DNA methylation in IDH mutant cells can reverse mutant IDH induced hypermethylation and block in differentiation and promote tumor control. These findings have substantial impact for exploring new treatment strategies for patients with IDH mutant gliomas.

CSF-1R inhibition alters macrophage polarization and blocks glioma progression.

Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ), facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM.

An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells.

The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant--but not IDH1-wild-type--glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

Differential sensitivity of glioma- versus lung cancer-specific EGFR mutations to EGFR kinase inhibitors.

UNLABELLED: Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. SIGNIFICANCE: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain.

miR-34a repression in proneural malignant gliomas upregulates expression of its target PDGFRA and promotes tumorigenesis.

Glioblastoma (GBM) and other malignant gliomas are aggressive primary neoplasms of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed distinct disease subclasses within malignant gliomas whose defining genomic features highlight dysregulated molecular networks as potential targets for therapeutic development. The "proneural" designation represents the largest and most heterogeneous of these subclasses, and includes both a large fraction of GBMs along with most of their lower-grade astrocytic and oligodendroglial counterparts. The pathogenesis of proneural gliomas has been repeatedly associated with dysregulated PDGF signaling. Nevertheless, genomic amplification or activating mutations involving the PDGF receptor (PDGFRA) characterize only a subset of proneural GBMs, while the mechanisms driving dysregulated PDGF signaling and downstream oncogenic networks in remaining tumors are unclear. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate gene expression by binding loosely complimentary sequences in target mRNAs. The role of miRNA biology in numerous cancer variants is well established. In an analysis of miRNA involvement in the phenotypic expression and regulation of oncogenic PDGF signaling, we found that miR-34a is downregulated by PDGF pathway activation in vitro. Similarly, analysis of data from the Cancer Genome Atlas (TCGA) revealed that miR-34a expression is significantly lower in proneural gliomas compared to other tumor subtypes. Using primary GBM cells maintained under neurosphere conditions, we then demonstrated that miR-34a specifically affects growth of proneural glioma cells in vitro and in vivo. Further bioinformatic analysis identified PDGFRA as a direct target of miR-34a and this interaction was experimentally validated. Finally, we found that PDGF-driven miR-34a repression is unlikely to operate solely through a p53-dependent mechanism. Taken together, our data support the existence of reciprocal negative feedback regulation involving miR-34 and PDGFRA expression in proneural gliomas and, as such, identify a subtype specific therapeutic potential for miR-34a.

Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response.

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity, which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications, clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however, the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs, most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification, revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well.

PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas.

Gene rearrangement in the form of an intragenic deletion is the primary mechanism of oncogenic mutation of the epidermal growth factor receptor (EGFR) gene in gliomas. However, the incidence of platelet-derived growth factor receptor-α (PDGFRA) gene rearrangement in these tumors is unknown. We investigated the PDGFRA locus in PDGFRA-amplified gliomas and identified two rearrangements, including the first case of a gene fusion between kinase insert domain receptor (KDR) (VEGFRII) and the PDGFRA gene, and six cases of PDGFRA(Δ8, 9), an intragenic deletion rearrangement. The PDGFRA(Δ8, 9) mutant was common, being present in 40% of the glioblastoma multiformes (GBMs) with PDGFRA amplification. Tumors with these two types of PDGFRA rearrangement displayed histologic features of oligodendroglioma, and the gene products of both rearrangements showed constitutively elevated tyrosine kinase activity and transforming potential that was reversed by PDGFR blockade. These results suggest the possibility that these PDGFRA mutants behave as oncogenes in this subset of gliomas, and that the prevalence of such rearrangements may have been considerably underestimated.

Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations.

BACKGROUND: Glioblastoma multiforme (GBM) is an umbrella designation that includes a heterogeneous group of primary brain tumors. Several classification strategies of GBM have been reported, some by clinical course and others by resemblance to cell types either in the adult or during development. From a practical and therapeutic standpoint, classifying GBMs by signal transduction pathway activation and by mutation in pathway member genes may be particularly valuable for the development of targeted therapies. METHODOLOGY/PRINCIPAL FINDINGS: We performed targeted proteomic analysis of 27 surgical glioma samples to identify patterns of coordinate activation among glioma-relevant signal transduction pathways, then compared these results with integrated analysis of genomic and expression data of 243 GBM samples from The Cancer Genome Atlas (TCGA). In the pattern of signaling, three subclasses of GBM emerge which appear to be associated with predominance of EGFR activation, PDGFR activation, or loss of the RAS regulator NF1. The EGFR signaling class has prominent Notch pathway activation measured by elevated expression of Notch ligands, cleaved Notch receptor, and downstream target Hes1. The PDGF class showed high levels of PDGFB ligand and phosphorylation of PDGFRbeta and NFKB. NF1-loss was associated with lower overall MAPK and PI3K activation and relative overexpression of the mesenchymal marker YKL40. These three signaling classes appear to correspond with distinct transcriptomal subclasses of primary GBM samples from TCGA for which copy number aberration and mutation of EGFR, PDGFRA, and NF1 are signature events. CONCLUSIONS/SIGNIFICANCE: Proteomic analysis of GBM samples revealed three patterns of expression and activation of proteins in glioma-relevant signaling pathways. These three classes are comprised of roughly equal numbers showing either EGFR activation associated with amplification and mutation of the receptor, PDGF-pathway activation that is primarily ligand-driven, or loss of NF1 expression. The associated signaling activities correlating with these sentinel alterations provide insight into glioma biology and therapeutic strategies.

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: role of alphaGMR.

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with alphaGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the alphaGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.

Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase beta.

Reactive oxygen species (ROS) are key intermediates in cellular signal transduction pathways whose function may be counterbalanced by antioxidants. Acting as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). We discovered that DHA directly inhibits IkappaBalpha kinase beta (IKKbeta) and IKKalpha enzymatic activity in vitro, whereas AA did not have this effect. When cells were loaded with AA and induced to generate DHA by oxidative stress in cells expressing a constitutive active IKKbeta, NF-kappaB activation was inhibited. Our results identify a dual molecular action of vitamin C in signal transduction and provide a direct linkage between the redox state of vitamin C and NF-kappaB signaling events. AA quenches ROS intermediates involved in the activation of NF-kappaB and is oxidized to DHA, which directly inhibits IKKbeta and IKKalpha enzymatic activity. These findings define a function for vitamin C in signal transduction other than as an antioxidant and mechanistically illuminate how vitamin C down-modulates NF-kappaB signaling.

Vitamin C suppresses TNF alpha-induced NF kappa B activation by inhibiting I kappa B alpha phosphorylation.

Extracellular stimuli signal for activation of the transcription factor NFkappaB, leading to gene expression regulating processes involved in immune responses, inflammation, and cell survival. Tumor necrosis factor-alpha (TNFalpha) activates NFkappaB via a well-defined kinase pathways involving NFkappaB-inducing kinase (NIK), which activates downstream multisubunit IkappaB kinases (IKK). IKK in turn phosphorylates IkappaB, the central regulator of NFkappaB function. We found that intracellular vitamin C inhibits TNFalpha-induced activation of NFkappaB in human cell lines (HeLa, monocytic U937, myeloid leukemia HL-60, and breast MCF7) and primary endothelial cells (HUVEC) in a dose-dependent manner. Vitamin C is an important antioxidant, and most cells accumulate ascorbic acid (AA) intracellularly by transporting the oxidized form of the vitamin, dehydroascorbic acid (DHA). Because ascorbic acid is a strong pro-oxidant in the presence of transition metals in vitro, we loaded cells with vitamin C by incubating them with DHA. Vitamin C-loaded cells showed significantly decreased TNFalpha-induced nuclear translocation of NFkappaB, NFkappaB-dependent reporter transcription, and IkappaBalpha phosphorylation. Our data point to a mechanism of vitamin C suppression of NFkappaB activation by inhibiting TNFalpha-induced activation of NIK and IKKbeta kinases independent of p38 MAP kinase. These results suggest that intracellular vitamin C can influence inflammatory, neoplastic, and apoptotic processes via inhibition of NFkappaB activation.