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PDF - 1816K, Supplementary Figure 1: CGH probe for Cre-mediated Pten deletion provides a surrogate of tumor DNA content for mouse tumors. Supplementary Figure 2: The genomic architecture of mouse tumor gene deletions suggests independent selection pressure for genes within the same deletion in human tumors. Supplementary Figure 3: Mouse and Human Proneural GBM share a common regulatory network. Supplementary Figure 4: *PTEN/p53 tumors rapidly progress and acquire the histological features of GBM. Supplementary Figure 5: *Pten tumors acquire and express mutations in p53. Supplementary Figure 6: *p53 tumors develop the histological features of GBM, and have an intermediate survival between *PTEN and *PTEN/p53 tumors.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression. Cancer Res; 74(5); 1440–51. ©2014 AACR.

XLSX - 1231K, Supplementary Table 1: List of genes deleted in 75 to 100% of the end-stage *PTEN tumors Supplementary Table 2: List of genes showing copy number gains in *PTEN end-stage and *PTEN/p53 mouse tumors. Supplementary Table 3: Genes that were deleted in 75-100% of *PTEN end-stage mouse tumors and at least 10% of human tumors within one of the four GBM subtypes mapped to their chromosomal location in the human genome. Supplementary Table 4: List of gene deletions that were specific for one of the four GBM subtypes. Supplementary Table 5: Correlation of proneural-specific genetic alterations and deletions identified by cross-species comparison. Supplementary Table 6: Spearman correlation for Verhaak gene sets show highest correlation for human proneural GBM subtype for 21 dpi *PTEN tumors, end-stage *PTEN tumors and *p53 end-stage tumors. Supplementary Table 7: MR identified from NB versus *PTEN 21 dpi and NB versus *PTEN end-stage mouse MARINas, and human NB versus proneural GBM (TCGA) MARINa.

PDF - 106K, Contains the legends for Supplementary Figures 1-6 and Supplementary Tables 1-7.

Memory difficulties are commonly associated with temporal lobe epilepsy (TLE) and cause significant disability. This article reviews the role of altered hippocampal theta oscillations and theta-gamma coupling as potential causes of memory disturbance in temporal lobe epilepsy, dissecting the potential mechanisms underlying these changes in large-scale neuronal synchronization. We discuss development of treatments for cognitive dysfunction directed at restoring theta rhythmicity and future directions for research.

The phytocannabinoid Δ(9)-tetrahydrocannabinol (THC) was isolated and synthesized in the 1960s. Since then, two synthetic cannabinoids (SCBs) targeting the cannabinoid 1 (CB1R) and 2 (CB2R) receptors were approved for medical use based on clinical safety and efficacy data: dronabinol (synthetic THC) and nabilone (synthetic THC analog). To probe the function of the endocannabinoid system further, hundreds of investigational compounds were developed; in particular, agonists with (1) greater CB1/2R affinity relative to THC and (2) full CB1/2R agonist activity. This pharmacological profile may pose greater risks for misuse and adverse effects relative to THC, and these SCBs proliferated in retail markets as legal alternatives to cannabis (e.g., novel psychoactive substances [NPS], “Spice,” “K2”). These SCBs were largely outlawed in the U.S., but blanket policies that placed all SCB chemicals into restrictive control categories impeded research progress into novel mechanisms for SCB therapeutic development. There is a concerted effort to develop new, therapeutically useful SCBs that target novel pharmacological mechanisms. This review highlights the potential therapeutic efficacy and safety considerations for unique SCBs, including CB1R partial and full agonists, peripherally-restricted CB1R agonists, selective CB2R agonists, selective CB1R antagonists/inverse agonists, CB1R allosteric modulators, endocannabinoid-degrading enzyme inhibitors, and cannabidiol. We propose promising directions for SCB research that may optimize therapeutic efficacy and diminish potential for adverse events, for example, peripherally-restricted CB1R antagonists/inverse agonists and biased CB1/2R agonists. Together, these strategies could lead to the discovery of new, therapeutically useful SCBs with reduced negative public health impact.

Traditionally, the World Health Organization classification of gliomas is based on tumor histology. Recently, major efforts in characterizing the global expression profiles and genetic alterations in glioblastoma have led to significantly improved understanding of this disease.1–6 Of all these efforts, it is evident that one of the most robust and identifiable gene expression patterns in gliomas is the proneural (PN) subtype.7,8 Differences in glioblastoma molecular phenotypes can have differences in clinical response to therapies. The PN subgroup in particular has been found to be associated with younger patient age at diagnosis and a phenotype that is unresponsive to aggressive traditional therapies that do reduce mortality for the other subgroups.9 Therefore, there is a pressing need to identify proper therapeutic interventions that can benefit PN glioblastoma patients. Our group previously described a murine glioma model that is phenotypically classifiable with human PN glioblastoma based on its gene expression profile.10 In this model, PN murine tumors are generated in vivo by retrovirally induced overexpression of platelet derived growth factor (PDGF) and deletion of phosphatase and tensin homolog (PTEN) with or without additional p53 deletion in glial progenitor cells using the Cre-lox system in transgenic mouse strains. More recently, we showed that cell lines derived from these murine glioma models retain their PN phenotype in vitro and can be utilized for in vitro and in vivo experimentation, providing a reasonable PN glioma model.11 We looked for potential therapeutic targets among PN-specific genes, hoping to find genes that are overexpressed in this group of patients. Topoisomerase II (TOP2) was previously identified as a PN-specific gene based on its high expression levels.9 Thus, in this current study we explored the therapeutic effects of TOP2 inhibition as a therapeutic strategy in a murine PN glioma model. Poisons that act on TOP2, such as etoposide, have been used in combination therapies for the treatment of malignant gliomas. Unfortunately these trials have shown limited antitumoral activity and poor response rates for chemotherapy regimens involving systemic delivery of etoposide.12–14 It is likely that factors such as poor penetration across the blood–brain barrier and systemic toxicities limit the therapeutic efficacy of etoposide following systemic delivery. Moreover, as seen in expression data of The Cancer Genome Atlas (TCGA; http://tcga-data.nci.nih.gov/tcga/), TOP2 expression is variable across glioblastoma samples, suggesting that clearly defined inclusion criteria restricted to those patients with high expression of this enzyme might lead to better therapeutic results of targeting TOP2. In this study, we explore the efficacy of etoposide in a preclinical model of PN glioma. To maximize the efficacy of this agent and overcome the challenging pharmacokinetics of systemic administration for brain tumors, we tested the use of convection-enhanced delivery (CED) for this agent. CED involves the use of catheters surgically implanted into the tumor and surrounding brain parenchyma, allowing for a continuous delivery of chemotherapy directly into the tumor through positive pressure microperfusion.15 This delivery method provides an advantage over systemic administration in bypassing the blood–brain barrier and reducing toxicities to other organs. In the context of this background, here we present preclinical data suggestive of the efficacy of etoposide treatment for the PN subgroup of glioblastoma.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression. Cancer Res; 74(5); 1440–51. ©2014 AACR.

PTEN Variations The product of the tumor suppressor gene phosphate and tensin homolog on chromosome ten ( PTEN) is a lipid and protein phosphatase that regulates important cellular processes, including growth, survival, and metabolism (see the Perspective by Leslie and Brunton ). Though PTEN is best known for effects on the phosphatidylnositol 3-kinase (PI3K) signaling pathway, the PTEN protein is also found in the nucleus. Bassi et al. (p. 395 ) found that PTEN's presence in the nucleus was regulated in response to covalent modification of the protein by SUMOylation and phosphorylation. Cells lacking nuclear PTEN showed increased sensitivity to DNA damage and underwent cell death if the PI3K pathway was also inhibited. Hopkins et al. (p. 399 , published online 6 June) discovered an alternative translation start site in human PTEN messenger RNA that allowed expression of a protein, PTEN-Long, with about 170 extra amino acids. The unusual enzyme was released from cells and then taken up into other cells. In a mouse tumor model, uptake of the enzyme inhibited the PI3K pathway and inhibited tumor growth.

Topoisomerase II (TOP2) is highly expressed on proneural gliomas, but its specific function on these tumors is unknown. We found that TOP2 is expressed in gliomas from early stages, increases during progression, and is elevated in radiographically localized non-enhancing regions of human glioblastoma, an area that is challenging to target systemically, and a common source of recurrence. TOP2A is highly expressed by infiltrating tumor cells, in the tumor center and at the edges, and low on surrounding brain. TOP2 enzymatic activity was detected on proneural glioma cell lines. TOP2 poison etoposide and 3 TOP2 inhibitors showed significant cytotoxicity on a proneural glioma cells (IC50 0.3-4 mM). Moreover, intracranial etoposide convection-enhanced delivery (CED) (80 mM) was tolerated and led to a significant survival benefit in mouse proneural gliomas. In contrast, etoposide CED (4 mM) showed transient decrease in tumor growth but failed to prolong survival, consistent with ineffective tumoral concentrations in trials of systemic etoposide for recurrent gliomas. TOP2 transcriptional modulation on proneural gliomas was suggested as the activity of many key transcription factors correlates with TOP2 levels across glioblastoma patients. TOP2 pharmacological targeting is a promising treatment target for some gliomas. Refining patient selection and drug delivery is key for optimizing this therapy.

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation.

BACKGROUND: Two of the defining features of glioblastoma are their ability to invade brain and to proliferate. While both processes are stimulated by the multiple signaling cascades that are activated in GBM, the redundancy present in these pathways has limited the clinical efficacy of kinase inhibitors. An alternative would be to directly target the cellular machinery that drives dispersion and proliferation. This machinery includes “molecular motors”—a superfamily of enzymes that generate the forces needed for a wide variety of cellular physiologies. In this study, we have examined two members from this family that can be targeted with clinically available drugs—cytoplasmic myosin II, which drives cellular motility, and Eg5, which drives the separation of the chromosomes during mitosis. METHODS: Rodent proneural glioblastomas were generated by intracranial injection of PDGF-expressing bi-cistronic retroviruses, and tumor cell invasion in vitro and ex vivo was examined as described (Ivkovic et al., Mol. Biol. Cell, 23, 533-42; Isermann et al., Curr. Protoc. Cell Biol., 2012). Patient-derived human glioma stem cells (GSCs) were isolated and maintained as described (Cheng et al., Cell, 153, 139-52). RESULTS: Myosin II: Myosin II isoforms are upregulated in all four subtypes of GBM, and expression correlates inversely with survival. We find that PDGF abolishes the anti-invasive effect of the EGFR inhibitior erlotinib on EGF-stimulated tumor dispersion, and, likewise, EGF abolishes the corresponding effect of the PDGFRα inhibitor imatinib on PDGF-stimulated tumor migration. However, blebbistatin, an allosteric inhibitor of myosin II, effectively blocks glioma dispersion even with simultaneous activation of both pathways. Furthermore, the rho kinase inhibitor fasudil, which inhibits myosin II and is used clinically for the treatment of pulmonary hypertension, also blocks PDGF-stimulated tumor invasion. Eg5: We find that Eg5 is consistently overexpressed in GSCs, compared to non-GSCs and that GSCs exhibit a greater sensitivity to ispinesib, a clinically available Eg5 inhibitor, compared to non-GSCs. Eg5 inhibition compromises the tumor initiation capability of GSCs as well as tumor progression in an orthotopic xenograft model. CONCLUSIONS: Prior efforts to block glioma invasion and proliferation have been hampered both by therapy-resistant GSCs and the redundancy present in pro-migratory signaling cascades. Our results indicate that molecular motors represent a set of targets whose inhibition blocks glioma dispersion and proliferation regardless of the activity of upstream growth and dispersion-promoting pathways and they imply that further pre-clinical and clinical development of molecular motor inhibitors in malignant glioma is warranted. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Glioblastoma (GBM) remains prognostically dismal, with care centered on resection, motivating research into novel therapies. Although inducing anti-tumor immunity remains an attractive target for therapeutic and preventative intervention, the interplay between evolving dysregulation of the glioma microenvironment and T cell inefficacy remains poorly understood. In our murine model of proneural glioma, retroviral delivery of PDGF and cre-mediated knockout of PTEN in glial progenitors of adult C57BL/6 gives rise to slow-growing tumors, which were harvested at early- mid- and late-stage progression timepoints following induction, along with peripheral blood. From human patients, tissue from low- and high-grade glioma resections and corresponding peripheral lymphocytes were cryofrozen during surgery at New York Presbyterian-CUMC. For both species, we employed a commercially available primer set (iRepertoire) for nested PCR of the complementarity-determining region 3 (CDR3) of the TCR-alpha and TCR-beta chains from the T cell RNA, followed by next-generation sequencing on an Illumina MiSeq. We developed a computational pipeline for mapping TCR cassettes, in silico translation, pairwise analysis of tissue/periphery per subject, and error analysis. In the murine model, we observe that at late-stage, the intratumoral TCR repertoire diverges significantly from the peripheral, including dramatic expansion of single tumor-associated CDR3s, while the peripheral repertoire itself diverges from those of healthy mice. In both human patients and mice, we observed tumor-associated CDR3s, disproportionately abundant in tumor tissue compared to the corresponding peripheral blood, at both the amino acid and nucleotide level. In human samples we observed tumor-specific TCR expansions that were associated with particular functional subsets (CD8+, CD4+, Treg, NKT). Sequence-level study of the TCR repertoire promises new insight into the scope of glioma immunosuppression, especially systemic effects which remain elusive and the origins of intratumoral suppressive populations, and holds the potential for immunotherapeutic interventions, non-invasive diagnostics, and direct assessment of global responses to immunotherapy.