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Supplementary Figure 1 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure Legends 1-4 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 2 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 3 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 4 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

SummaryTo understand the relationships between the non-GCIMP glioblastoma (GBM) subgroups, we performed mathematical modeling to predict the temporal sequence of driver events during tumorigenesis. The most common order of evolutionary events is 1) chromosome (chr) 7 gain and chr10 loss, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes. We then developed a computational methodology to identify drivers of broad copy number changes, identifying PDGFA (chr7) and PTEN (chr10) as driving initial nondisjunction events. These predictions were validated using mouse modeling, showing that PDGFA is sufficient to induce proneural-like gliomas and that additional NF1 loss converts proneural to the mesenchymal subtype. Our findings suggest that most non-GCIMP mesenchymal GBMs arise as, and evolve from, a proneural-like precursor.

Glioblastoma is the most common adult primary brain tumor and has a dismal median survival. Radiation is a mainstay of treatment and significantly improves survival, yet recurrence is nearly inevitable. Better understanding the radiation response of glioblastoma will help improve strategies to treat this devastating disease. Here, we present a comprehensive study of the in vivo radiation response of glioma cells in a mouse model of proneural glioblastoma. These tumors are a heterogeneous mix of cell types with differing radiation sensitivities. To explicitly study the gene expression changes comprising the radiation response of the Olig2(+) tumor bulk cells, we used translating ribosome affinity purification (TRAP) from Olig2-TRAP transgenic mice. Comparing both ribosome-associated and total pools of mRNA isolated from Olig2(+) cells indicated that the in vivo gene expression response to radiation occurs primarily at the total transcript level. Genes related to apoptosis and cell growth were significantly altered. p53 and E2F were implicated as major regulators of the radiation response, with p53 activity needed for the largest gene expression changes after radiation. Additionally, radiation induced a marked shift away from a proneural expression pattern toward a mesenchymal one. This shift occurs in Olig2(+) cells within hours and in multiple genetic backgrounds. Targets for Stat3 and CEBPB, which have been suggested to be master regulators of a mesenchymal shift, were also up-regulated by radiation. These data provide a systematic description of the events following radiation and may be of use in identifying biological processes that promote glioma radioresistance.

Brainstem gliomas (BSG) are a rare group of central nervous system tumors that arise mostly in children and usually portend a particularly poor prognosis. We report the development of a genetically engineered mouse model of BSG using the RCAS/tv-a system and its implementation in preclinical trials. Using immunohistochemistry, we found that platelet-derived growth factor (PDGF) receptor α is overexpressed in 67% of pediatric BSGs. Based on this observation, we induced low-grade BSGs by overexpressing PDGF-B in the posterior fossa of neonatal nestin tv-a mice. To generate high-grade BSGs, we overexpressed PDGF-B in combination with Ink4a-ARF loss, given that this locus is commonly lost in high-grade pediatric BSGs. We show that the likely cells of origin for these mouse BSGs exist on the floor of the fourth ventricle and cerebral aqueduct. Irradiation of these high-grade BSGs shows that although single doses of 2, 6, and 10 Gy significantly increased the percent of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)–positive nuclei, only 6 and 10 Gy significantly induce cell cycle arrest. Perifosine, an inhibitor of AKT signaling, significantly induced TUNEL-positive nuclei in this high-grade BSG model, but in combination with 10 Gy, it did not significantly increase the percent of TUNEL-positive nuclei relative to 10 Gy alone at 6, 24, and 72 hours. Survival analysis showed that a single dose of 10 Gy significantly prolonged survival by 27% (P = 0.0002) but perifosine did not (P = 0.92). Perifosine + 10 Gy did not result in a significantly increased survival relative to 10 Gy alone (P = 0.23). This PDGF-induced BSG model can serve as a preclinical tool for the testing of novel agents. Cancer Res; 70(6); 2548–57

Platelet-derived growth factor receptor alpha–positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. Human glioblastoma multiforme tumors often contain rapidly proliferating oligodendrocyte lineage transcription factor 2 (Olig2)-positive cells that resemble OPCs. In this study, we sought to identify candidate pathways that promote OPC differentiation or quiescence rather than proliferation. Gene expression profiling conducted in both normal murine OPCs and highly proliferative Olig2-positive glioma cells identified all the transcripts associated with the highly proliferative state of these cells and showed that among the various cell types found within the brain, Olig2-positive tumor cells are most similar to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have similar expression profiles in human tumors and were significantly enriched in genomic deletions, suggesting an antiproliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings show that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies. Cancer Res; 72(18); 4856–68. ©2012 AACR.

Malignant gliomas remain the most devastating childhood and adult tumors of the central nervous system. Although adult and pediatric gliomas are histologically indistinguishable, they differ in location, behavior, and molecular characteristics. This implies that the molecular pathways and pathophysiology of malignant gliomagenesis in these two populations are distinct. Such differences between adult and pediatric gliomas may predict different therapeutic responses. Therefore, accurate genetically engineered models of adult and pediatric gliomas may help understand the biology of these tumors and evaluate therapeutic agents in preclinical studies. It has been proposed that gliomas arise from the subventricular zone in mice during development. Here, we demonstrate that, in adult mice, gliomas may arise not only when injected in the subventricular zone but also when injected in the cortex and cerebellum. Our work demonstrates a versatile and highly reproducible adult mouse model of glioma, which can be easily incorporated into preclinical studies.

Post-transcriptional regulation of gene expression contributes to the protein output of a cell, however, methods for measuring translational regulation in complex in vivo systems are lacking. Here, we describe a sensitive method for measuring translational regulation in defined cell populations from heterogeneous tissue in vivo. We adapted the translating ribosome affinity purification (TRAP) methodology to measure the relative occupancy of individual mRNA transcripts in translating ribosomes in the Olig2-positive tumor cell population in a genetically engineered mouse model (GEM) of glioma. Global measurement of paired ribosome-bound and total cellular mRNA populations from tumor cells in vivo identified a broad distribution of relative ribosome occupancies amongst mRNA species that was highly reproducible across biological samples. Comparison of the translation state of glioma cells to non-transformed oligodendrocyte progenitor cells in normal brain identified global alteration of translation in tumor, and specifically of genes involved in cell division and synthetic metabolism. Furthermore, investigation of alteration in steady state translational efficiencies upon loss of PTEN, one of the most frequently mutated and deleted tumor suppressors in glioma, identified differential translation of proteins involved in cellular respiration, canonically regulated by PI3K/Akt signaling, and cellular glycosylation profiles, deregulation of which is known to be associated with tumor progression. Application of the translation efficiency profiling method described here to other biological contexts and conditions would extend our knowledge of the scope and impact of this important mode of gene regulation in complex in vivo systems.

Integration of expression, copy number, methylation, and regulatory sequence information identifies miRNAs and transcription factors that drive the global expression changes associated with different glioblastoma subtypes., The proneural and mesenchymal transcriptomic subtypes of glioblastoma are associated with distinct regulatory programs. REST, miR-124 and miR-132 are potential drivers of expression changes in proneural glioblastoma, and the inferred extent of dysregulation of miR-132 correlates with survival in the proneural subtype. The expression changes in proneural glioblastoma associated with key regulators in the regression model are consistent with in vivo expression changes in mouse PDGF-driven tumors. Transfection of miR-124 and miR-132 in proneural neurospheres induces expression changes that are concordant with proneural tumor-versus-normal expression changes., Large-scale cancer genomics projects are profiling hundreds of tumors at multiple molecular layers, including copy number, mRNA and miRNA expression, but the mechanistic relationships between these layers are often excluded from computational models. We developed a supervised learning framework for integrating molecular profiles with regulatory sequence information to reveal regulatory programs in cancer, including miRNA-mediated regulation. We applied our approach to 320 glioblastoma profiles and identified key miRNAs and transcription factors as common or subtype-specific drivers of expression changes. We confirmed that predicted gene expression signatures for proneural subtype regulators were consistent with in vivo expression changes in a PDGF-driven mouse model. We tested two predicted proneural drivers, miR-124 and miR-132, both underexpressed in proneural tumors, by overexpression in neurospheres and observed a partial reversal of corresponding tumor expression changes. Computationally dissecting the role of miRNAs in cancer may ultimately lead to small RNA therapeutics tailored to subtype or individual.

Background The tumor microenvironment contains normal, non-neoplastic cells that may contribute to tumor growth and maintenance. Within PDGF-driven murine gliomas, tumor-associated astrocytes (TAAs) are a large component of the tumor microenvironment. The function of non-neoplastic astrocytes in the glioma microenvironment has not been fully elucidated; moreover, the differences between these astrocytes and normal astrocytes are unknown. We therefore sought to identify genes and pathways that are increased in TAAs relative to normal astrocytes and also to determine whether expression of these genes correlates with glioma behavior. Methodology/Principal Findings We compared the gene expression profiles of TAAs to normal astrocytes and found the Antigen Presentation Pathway to be significantly increased in TAAs. We then identified a gene signature for glioblastoma (GBM) TAAs and validated the expression of some of those genes within the tumor. We also show that TAAs are derived from the non-tumor, stromal environment, in contrast to the Olig2+ tumor cells that constitute the neoplastic elements in our model. Finally, we validate this GBM TAA signature in patients and show that a TAA-derived gene signature predicts survival specifically in the human proneural subtype of glioma. Conclusions/Significance Our data identifies unique gene expression patterns between populations of TAAs and suggests potential roles for stromal astrocytes within the glioma microenvironment. We show that certain stromal astrocytes in the tumor microenvironment express a GBM-specific gene signature and that the majority of these stromal astrocyte genes can predict survival in the human disease.

BackgroundGliomas are thought to form by clonal expansion from a single cell-of-origin, and progression-associated mutations to occur in its progeny cells. Glioma progression is associated with elevated growth factor signaling and loss of function of tumor suppressors Ink4a, Arf and Pten. Yet, gliomas are cellularly heterogeneous; they recruit and trap normal cells during infiltration.Methodology/principal findingsWe performed lineage tracing in a retrovirally mediated, molecularly and histologically accurate mouse model of hPDGFb-driven gliomagenesis. We were able to distinguish cells in the tumor that were derived from the cell-of-origin from those that were not. Phenotypic, tumorigenic and expression analyses were performed on both populations of these cells. Here we show that during progression of hPDGFb-induced murine gliomas, tumor suppressor loss can expand the recruited cell population not derived from the cell-of-origin within glioma microenvironment to dominate regions of the tumor, with essentially no contribution from the progeny of glioma cell-of-origin. Moreover, the recruited cells can give rise to gliomas upon transplantation and passaging, acquire polysomal expression profiles and genetic aberrations typically present in glioma cells rather than normal progenitors, aid progeny cells in glioma initiation upon transplantation, and become independent of PDGFR signaling.Conclusions/significanceThese results indicate that non-cell-of-origin derived cells within glioma environment in the mouse can be corrupted to become bona fide tumor, and deviate from the generally established view of gliomagenesis.

SummaryMaintenance of genomic integrity is essential for adult tissue homeostasis and defects in the DNA-damage response (DDR) machinery are linked to numerous pathologies including cancer. Here, we present evidence that the DDR exerts tumor suppressor activity in gliomas. We show that genes encoding components of the DDR pathway are frequently altered in human gliomas and that loss of elements of the ATM/Chk2/p53 cascade accelerates tumor formation in a glioma mouse model. We demonstrate that Chk2 is required for glioma response to ionizing radiation in vivo and is necessary for DNA-damage checkpoints in the neuronal stem cell compartment. Finally, we observed that the DDR is constitutively activated in a subset of human GBMs, and such activation correlates with regions of hypoxia.