Search

To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.

An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)-associated factor ( PAF ) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumor-initiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM)., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood-brain/blood-tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM., (© 2015 Kouri et al.; Published by Cold Spring Harbor Laboratory Press.)

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼ 7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity., (© 2015 Kim et al.; Published by Cold Spring Harbor Laboratory Press.)

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

DNA sequencing offers a powerful tool in oncology based on the precise definition of structural rearrangements and copy number in tumor genomes. Here, we describe the development of methods to compute copy number and detect structural variants to locally reconstruct highly rearranged regions of the tumor genome with high precision from standard, short-read, paired-end sequencing datasets. We find that circular assemblies are the most parsimonious explanation for a set of highly amplified tumor regions in a subset of glioblastoma multiforme samples sequenced by The Cancer Genome Atlas (TCGA) consortium, revealing evidence for double minute chromosomes in these tumors. Further, we find that some samples harbor multiple circular amplicons and, in some cases, further rearrangements occurred after the initial amplicon-generating event. Fluorescence in situ hybridization analysis offered an initial confirmation of the presence of double minute chromosomes. Gene content in these assemblies helps identify likely driver oncogenes for these amplicons. RNA-seq data available for one double minute chromosome offered additional support for our local tumor genome assemblies, and identified the birth of a novel exon made possible through rearranged sequences present in the double minute chromosomes. Our method was also useful for analysis of a larger set of glioblastoma multiforme tumors for which exome sequencing data are available, finding evidence for oncogenic double minute chromosomes in more than 20% of clinical specimens examined, a frequency consistent with previous estimates.

An immature state of cellular differentiation--characterized by stem cell-like tendencies and impaired differentiation--is a hallmark of cancer. Using glioblastoma multiforme (GBM) as a model system, we sought to determine whether molecular determinants that drive cells toward terminal differentiation are also genetically targeted in carcinogenesis and whether neutralizing such genes also plays an active role to reinforce the impaired differentiation state and promote malignancy. To that end, we screened 71 genes with known roles in promoting nervous system development that also sustain copy number loss in GBM through antineoplastic assay and identified A2BP1 (ataxin 2 binding protein 1, Rbfox1), an RNA-binding and splicing regulator that is deleted in 10% of GBM cases. Integrated in silico analysis of GBM profiles to elucidate the A2BP1 pathway and its role in glioma identified myelin transcription factor 1-like (Myt1L) as a direct transcriptional regulator of A2BP1. Reintroduction of A2BP1 or Myt1L in GBM cell lines and glioma stem cells profoundly inhibited tumorigenesis in multiple assays, and conversely, shRNA-mediated knockdown of A2BP1 or Myt1L in premalignant neural stem cells compromised neuronal lineage differentiation and promoted orthotopic tumor formation. On the mechanistic level, with the top-represented downstream target TPM1 as an illustrative example, we demonstrated that, among its multiple functions, A2BP1 serves to regulate TPM1's alternative splicing to promote cytoskeletal organization and terminal differentiation and suppress malignancy. Thus, in addition to the activation of self-renewal pathways, the neutralization of genetic programs that drive cells toward terminal differentiation may also promote immature and highly plastic developmental states that contribute to the aggressive malignant properties of GBM.

With the advent of high-throughput sequencing technologies, much progress has been made in the identification of somatic structural rearrangements in cancer genomes. However, characterization of the complex alterations and their associated mechanisms remains inadequate. Here, we report a comprehensive analysis of whole-genome sequencing and DNA copy number data sets from The Cancer Genome Atlas to relate chromosomal alterations to imbalances in DNA dosage and describe the landscape of intragenic breakpoints in glioblastoma multiforme (GBM). Gene length, guanine-cytosine (GC) content, and local presence of a copy number alteration were closely associated with breakpoint susceptibility. A dense pattern of repeated focal amplifications involving the murine double minute 2 (MDM2)/cyclin-dependent kinase 4 (CDK4) oncogenes and associated with poor survival was identified in 5% of GBMs. Gene fusions and rearrangements were detected concomitant within the breakpoint-enriched region. At the gene level, we noted recurrent breakpoints in genes such as apoptosis regulator FAF1. Structural alterations of the FAF1 gene disrupted expression and led to protein depletion. Restoration of the FAF1 protein in glioma cell lines significantly increased the FAS-mediated apoptosis response. Our study uncovered a previously underappreciated genomic mechanism of gene deregulation that can confer growth advantages on tumor cells and may generate cancer-specific vulnerabilities in subsets of GBM.

Large-scale cancer genomics efforts are identifying hundreds of somatic genomic alterations in glioblastoma (GBM). Distinguishing between active driver and neutral passenger alterations requires functional assessment of each gene; therefore, integrating biological weight of evidence with statistical significance for each genomic alteration will enable better prioritization for downstream studies. Here, we demonstrate the feasibility and potential of in vitro functional genomic screens to rapidly and systematically prioritize high-probability candidate genes for in vivo validation. Integration of low-complexity gain- and loss-of-function screens designed on the basis of genomic data identified 6 candidate GBM oncogenes, and RINT1 was validated as a novel GBM oncogene based on its ability to confer tumorigenicity to primary nontransformed murine astrocytes in vivo. Cancer genomics-guided low-complexity genomic screens can quickly provide a functional filter to prioritize high-value targets for further downstream mechanistic and translational studies.

Purpose: NovoTTF-100A is a portable device delivering low-intensity, intermediate frequency electric fields via non-invasive, transducer arrays. Tumour Treatment Fields (TTF), a completely new therapeutic modality in cancer treatment, physically interfere with cell division., Methods: Phase III trial of chemotherapy-free treatment of NovoTTF (20-24h/day) versus active chemotherapy in the treatment of patients with recurrent glioblastoma. Primary end-point was improvement of overall survival., Results: Patients (median age 54 years (range 23-80), Karnofsky performance status 80% (range 50-100) were randomised to TTF alone (n=120) or active chemotherapy control (n=117). Number of prior treatments was two (range 1-6). Median survival was 6.6 versus 6.0 months (hazard ratio 0.86 [95% CI 0.66-1.12]; p=0.27), 1-year survival rate was 20% and 20%, progression-free survival rate at 6 months was 21.4% and 15.1% (p=0.13), respectively in TTF and active control patients. Responses were more common in the TTF arm (14% versus 9.6%, p=0.19). The TTF-related adverse events were mild (14%) to moderate (2%) skin rash beneath the transducer arrays. Severe adverse events occurred in 6% and 16% (p=0.022) of patients treated with TTF and chemotherapy, respectively. Quality of life analyses favoured TTF therapy in most domains., Conclusions: This is the first controlled trial evaluating an entirely novel cancer treatment modality delivering electric fields rather than chemotherapy. No improvement in overall survival was demonstrated, however efficacy and activity with this chemotherapy-free treatment device appears comparable to chemotherapy regimens that are commonly used for recurrent glioblastoma. Toxicity and quality of life clearly favoured TTF., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally deleted gene is a member of a functionally redundant family of genes carrying out an essential function. The glycolytic gene enolase 1 (ENO1) in the 1p36 locus is deleted in glioblastoma (GBM), which is tolerated by the expression of ENO2. Here we show that short-hairpin-RNA-mediated silencing of ENO2 selectively inhibits growth, survival and the tumorigenic potential of ENO1-deleted GBM cells, and that the enolase inhibitor phosphonoacetohydroxamate is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger-deleted genes encoding functionally redundant essential activities and provide an effective treatment strategy for cancers containing such genomic events.

Unlabelled: Leveraging The Cancer Genome Atlas (TCGA) multidimensional data in glioblastoma, we inferred the putative regulatory network between microRNA and mRNA using the Context Likelihood of Relatedness modeling algorithm. Interrogation of the network in context of defined molecular subtypes identified 8 microRNAs with a strong discriminatory potential between proneural and mesenchymal subtypes. Integrative in silico analyses, a functional genetic screen, and experimental validation identified miR-34a as a tumor suppressor in proneural subtype glioblastoma. Mechanistically, in addition to its direct regulation of platelet-derived growth factor receptor-alpha (PDGFRA), promoter enrichment analysis of context likelihood of relatedness-inferred mRNA nodes established miR-34a as a novel regulator of a SMAD4 transcriptional network. Clinically, miR-34a expression level is shown to be prognostic, where miR-34a low-expressing glioblastomas exhibited better overall survival. This work illustrates the potential of comprehensive multidimensional cancer genomic data combined with computational and experimental models in enabling mechanistic exploration of relationships among different genetic elements across the genome space in cancer., Significance: We illustrate here that network modeling of complex multidimensional cancer genomic data can generate a framework in which to explore the biology of cancers, leading to discovery of new pathogenetic insights as well as potential prognostic biomarkers. Specifically in glioblastoma, within the context of the global network, promoter enrichment analysis of network edges uncovered a novel regulation of TGF-β signaling via a Smad4 transcriptomic network by miR-34a.

Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFβR2 and the TGFβ signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFβ pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.

Glioblastoma is both the most common and lethal primary malignant brain tumor. Extensive multiplatform genomic characterization has provided a higher-resolution picture of the molecular alterations underlying this disease. These studies provide the emerging view that "glioblastoma" represents several histologically similar yet molecularly heterogeneous diseases, which influences taxonomic classification systems, prognosis, and therapeutic decisions.

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.

Although GBP1 (guanylate binding protein 1) was among the first interferon-inducible proteins identified, its function is still largely unknown. Epidermal growth factor receptor (EGFR) activation by amplification or mutation is one of the most frequent genetic lesions in a variety of human tumors. These include glioblastoma multiforme (GBM), which is characterized by independent but interrelated features of extensive invasion into normal brain parenchyma, rapid growth, necrosis, and angiogenesis. In this study, we show that EGFR activation promoted GBP1 expression in GBM cell lines through a signaling pathway involving Src and p38 mitogen-activated protein kinase. Moreover, we identified YY1 (Yin Yang 1) as the downstream transcriptional regulator regulating EGFR-driven GBP1 expression. GBP1 was required for EGFR-mediated MMP1 (matrix metalloproteinase 1) expression and glioma cell invasion in vitro. Although deregulation of GBP1 expression did not affect glioma cell proliferation, overexpression of GBP1 enhanced glioma cell invasion through MMP1 induction, which required its C-terminal helical domain and was independent of its GTPase activity. Reducing GBP1 levels by RNA interference in invasive GBM cells also markedly inhibited their ability to infiltrate the brain parenchyma of mice. GBP1 expression was high and positively correlated with EGFR expression in human GBM tumors and cell lines, particularly those of the neural subtype. Together, these findings establish GBP1 as a previously unknown link between EGFR activity and MMP1 expression and nominate it as a novel potential therapeutic target for inhibiting GBM invasion.

Glioblastoma multiforme (GBM) is a fatal primary brain tumor harboring myriad genetic and epigenetic alterations. The recent multidimensional analysis of the GBM genome has provided a more complete view of the landscape of such alterations and their linked pathways. This effort has demonstrated that certain pathways are universally altered, but that the specific genetic events altered within each pathway can vary for each particular patient's tumor. With this atlas of genetic and epigenetic events, it now becomes feasible to assess how the patterns of mutations in a pathway influence response to drugs that are targeting such pathways. This issue is particularly important for GBM because, in contrast to other tumor types, molecularly targeted therapies have failed to alter overall survival substantially. Here, we combined functional genetic screens and comprehensive genomic analyses to identify CDK6 as a GBM oncogene that is required for proliferation and viability in a subset of GBM cell lines and tumors. Using an available small molecule targeting cyclin-dependent kinases (CDKs) 4 and 6, we sought to determine if the specific pattern of retinoblastoma pathway inactivation dictated the response to CDK4/6 inhibitor therapy. We showed that codeletion of CDKN2A and CDKN2C serves as a strong predictor of sensitivity to a selective inhibitor of CDK4/6. Thus, genome-informed drug sensitivity studies identify a subset of GBMs likely to respond to CDK4/6 inhibition. More generally, these observations demonstrate that the integration of genomic, functional and pharmacologic data can be exploited to inform the development of targeted therapy directed against specific cancer pathways.

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis, EGFR targeted therapies have achieved limited clinical efficacy. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII, an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state, which contributes to its plasticity and therapeutic resistance. Here, integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells., ((c) 2010 Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is the most common type of primary brain cancer and carries a dismal prognosis primarily due to the emergence of resistance towards extant radiation, conventional and targeted chemotherapies. Although GBM resists therapy-induced apoptosis, tumors show a seemingly paradoxical propensity for florid intratumoral necrogenesis. This necrosis manifests pathologically as microscopic foci or confluent expanses of necrotic tumor. While it is now well recognized that necrosis is an active cell death process and that apoptosis and necrosis death modalities are intertwined on multiple levels, the precise molecular mechanisms and genetic elements underlying these forms of cell death in GBM remain areas of active investigation. In recent oncogenomic studies, we identified a novel GBM oncoprotein, Bcl2-Like 12 (Bcl2L12), which is significantly expressed in the majority of primary GBM tumor specimens and distantly related to canonical Bcl-2 proteins. Due to its distinctive impact on cell death signaling, Bcl2L12 phenocopies pro-necrotic and anti-apoptotic propensities of high grade glioma: Mechanistically, we determined that unlike prototypic Bcl-2 family members, Bcl2L12 does not safeguard mitochondrial membrane integrity, but instead potently inhibits apoptosis at the level of post-mitochondrial effector caspase-3/7 activation. A combination of enforced expression, RNAi-mediated extinction, co-localization and protein interaction studies revealed that Bcl2L12 inhibits caspases 3 and 7 via distinct mechanisms. Direct physical interaction underlies Bcl2L12's inhibition of caspase-7 processing, whereas Bcl2L12-induced transcriptional upregulation of the small heat shock protein alpha B-crystallin is instrumental to neutralization of caspase-3 activation. Mirroring the cellular phenotype elicited by energy depletion, genetic or pharmacologic inhibition of post-mitochondrial apoptosis signaling molecules, Bcl2L12 promotes necrogenesis in glial cells in the context of a proapoptotic stimulus establishing that it represents a novel regulator of the balance between apoptosis and necrosis in GBM.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer that is characterized by the paradoxical features of intense apoptosis resistance yet a marked propensity to undergo necrosis. Bcl2L12 (for Bcl2-Like12) is a nuclear and cytoplasmic oncoprotein that is universally overexpressed in primary GBM and functions to block postmitochondrial apoptosis signaling by neutralizing effector caspase-3 and caspase-7 maturation. This postmitochondrial block in apoptosis engenders the alternate cell fate of cellular necrosis, thus providing a molecular explanation for GBM's classical features. Whereas Bcl2L12-mediated neutralization of caspase-7 maturation involves physical interaction, the mechanism governing Bcl2L12-mediated inhibition of caspase-3 activity is not known. The nuclear localization of Bcl2L12 prompted expression profile studies of primary astrocytes engineered to overexpress Bcl2L12. The Bcl2L12 transcriptome revealed a striking induction of the small heat shock protein alpha-basic-crystallin (alphaB-crystallin/HspB5), a link reinforced by robust alphaB-crystallin expression in Bcl2L12-expressing orthotopic glioma and strong coexpression of alphaB-crystallin and Bcl2L12 proteins in human primary GBMs. On the functional level, enforced alphaB-crystallin or Bcl2L12 expression enhances orthotopic tumor growth. Conversely, RNAi-mediated knockdown of alphaB-crystallin in Bcl2L12-expressing astrocytes and glioma cell lines with high endogenous alphaB-crystallin showed enhanced apoptosis, yet decreased necrotic cell death with associated increased caspase-3 but not caspase-7 activation. Mirroring this specific effect on effector caspase-3 activation, alphaB-crystallin selectively binds pro-caspase-3 and its cleavage intermediates in vitro and in vivo. Thus, alphaB-crystallin is a Bcl2L12-induced oncoprotein that enables Bcl2L12 to block the activation of both effector caspases via distinct mechanisms, thereby contributing to GBM pathogenesis and its hallmark biological properties.

We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18(INK4C) and p16(INK4A) codeletion. Functional reconstitution of p18(INK4C) in GBM cells null for both p16(INK4A) and p18(INK4C) resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18(INK4C) in p16(INK4A)-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16(INK4A) in primary astrocytes induced a concomitant increase in p18(INK4C). Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18(INK4C) in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

Glioblastoma (GBM) is a highly lethal primary brain cancer with hallmark features of diffuse invasion, intense apoptosis resistance and florid necrosis, robust angiogenesis, and an immature profile with developmental plasticity. In the course of assessing the developmental consequences of central nervous system (CNS)-specific deletion of p53 and Pten, we observed a penetrant acute-onset malignant glioma phenotype with striking clinical, pathological, and molecular resemblance to primary GBM in humans. This primary, as opposed to secondary, GBM presentation in the mouse prompted genetic analysis of human primary GBM samples that revealed combined p53 and Pten mutations as the most common tumor suppressor defects in primary GBM. On the mechanistic level, the "multiforme" histopathological presentation and immature differentiation marker profile of the murine tumors motivated transcriptomic promoter-binding element and functional studies of neural stem cells (NSCs), which revealed that dual, but not singular, inactivation of p53 and Pten promotes cellular c-Myc activation. This increased c-Myc activity is associated not only with impaired differentiation, enhanced self-renewal capacity of NSCs, and tumor-initiating cells (TICs), but also with maintenance of TIC tumorigenic potential. Together, these murine studies have provided a highly faithful model of primary GBM, revealed a common tumor suppressor mutational pattern in human disease, and established c-Myc as a key component of p53 and Pten cooperative actions in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential.

Targeted therapies that inhibit receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol 3-kinase (PI3K) signaling pathway have shown promising anticancer activity, but their efficacy in the brain tumor glioblastoma multiforme (GBM) and other solid tumors has been modest. We hypothesized that multiple RTKs are coactivated in these tumors and that redundant inputs drive and maintain downstream signaling, thereby limiting the efficacy of therapies targeting single RTKs. Tumor cell lines, xenotransplants, and primary tumors indeed show multiple concomitantly activated RTKs. Combinations of RTK inhibitors and/or RNA interference, but not single agents, decreased signaling, cell survival, and anchorage-independent growth even in glioma cells deficient in PTEN, a frequently inactivated inhibitor of PI3K. Thus, effective GBM therapy may require combined regimens targeting multiple RTKs.

Glioblastoma (GBM) is an astrocytic brain tumor characterized by an aggressive clinical course and intense resistance to all therapeutic modalities. Here, we report the identification and functional characterization of Bcl2L12 (Bcl2-like-12) that is robustly expressed in nearly all human primary GBMs examined. Enforced Bcl2L12 expression confers marked apoptosis resistance in primary cortical astrocytes, and, conversely, its RNA interference (RNAi)-mediated knockdown sensitizes human glioma cell lines toward apoptosis in vitro and impairs tumor growth with increased intratumoral apoptosis in vivo. Mechanistically, Bcl2L12 expression does not affect cytochrome c release or apoptosome-driven caspase-9 activation, but instead inhibits post-mitochondrial apoptosis signaling at the level of effector caspase activation. One of Bcl2L12's mechanisms of action stems from its ability to interact with and neutralize caspase-7. Notably, while enforced Bcl2L12 expression inhibits apoptosis, it also engenders a pronecrotic state, which mirrors the cellular phenotype elicited by genetic or pharmacologic inhibition of post-mitochondrial apoptosis molecules. Thus, Bcl2L12 contributes to the classical tumor biological features of GBM such as intense apoptosis resistance and florid necrosis, and may provide a target for enhanced therapeutic responsiveness of this lethal cancer.

Glioblastoma is classified into two subtypes on the basis of clinical history: "primary glioblastoma" arising de novo without detectable antecedent disease and "secondary glioblastoma" evolving from a low-grade astrocytoma. Despite their distinctive clinical courses, they arrive at an indistinguishable clinical and pathologic end point highlighted by widespread invasion and resistance to therapy and, as such, are managed clinically as if they are one disease entity. Because the life history of a cancer cell is often reflected in the pattern of genomic alterations, we sought to determine whether primary and secondary glioblastomas evolve through similar or different molecular pathogenetic routes. Clinically annotated primary and secondary glioblastoma samples were subjected to high-resolution copy number analysis using oligonucleotide-based array comparative genomic hybridization. Unsupervised classification using genomic nonnegative matrix factorization methods identified three distinct genomic subclasses. Whereas one corresponded to clinically defined primary glioblastomas, the remaining two stratified secondary glioblastoma into two genetically distinct cohorts. Thus, this global genomic analysis showed wide-scale differences between primary and secondary glioblastomas that were previously unappreciated, and has shown for the first time that secondary glioblastoma is heterogeneous in its molecular pathogenesis. Consistent with these findings, analysis of regional recurrent copy number alterations revealed many more events unique to these subclasses than shared. The pathobiological significance of these shared and subtype-specific copy number alterations is reinforced by their frequent occurrence, resident genes with clear links to cancer, recurrence in diverse cancer types, and apparent association with clinical outcome. We conclude that glioblastoma is composed of at least three distinct molecular subtypes, including novel subgroups of secondary glioblastoma, which may benefit from different therapeutic strategies.

A novel genome-wide screen that combines patient outcome analysis with array comparative genomic hybridization and mRNA expression profiling was developed to identify genes with copy number alterations, aberrant mRNA expression, and relevance to survival in glioblastoma. The method led to the discovery of physical gene clusters within the cancer genome with boundaries defined by physical proximity, correlated mRNA expression patterns, and survival relatedness. These boundaries delineate a novel genomic interval called the functional common region (FCR). Many FCRs contained genes of high biological relevance to cancer and were used to pinpoint functionally significant DNA alterations that were too small or infrequent to be reliably identified using standard algorithms. One such FCR contained the EphA2 receptor tyrosine kinase. Validation experiments showed that EphA2 mRNA overexpression correlated inversely with patient survival in a panel of 21 glioblastomas, and ligand-mediated EphA2 receptor activation increased glioblastoma proliferation and tumor growth via a mitogen-activated protein kinase-dependent pathway. This novel genome-wide approach greatly expanded the list of target genes in glioblastoma and represents a powerful new strategy to identify the upstream determinants of tumor phenotype in a range of human cancers.