Primary GBM Tumors Research Articles

Abstract 1073: PRMT5 inhibition synergizes with a natural small molecule compound to kill MTAP-deleted cells and suppress tumor growth

Abstract Homozygous deletion of MTAP occurs in about 15% of all human cancers, such as glioblastoma, pancreatic cancer, mesothelioma, urothelial bladder carcinoma, and lung squamous cell carcinoma. PRMT5 inhibitors show activity against MTAP-deleted cancer cells in culture and xenografts with a mechanism that relies on the significant elevation of the MTAP substrate, methylthioadenosine (MTA). Previously, we have shown that unlike cells in culture, MTA levels in MTAP-deleted primary human GBM tumors are not significantly higher than in MTAP-intact tumors. Therefore, combining the PRMT5 inhibitor with another drug may be required to increase the therapeutic window and clinical efficacy of a PRMT5 inhibitor in MTAP-deleted patients. Here, we identified a natural small molecular chemical compound with a good safety profile that synergizes with a PRMT5-MTA complex inhibitor, which boosts the efficacy of MTAP-deleted selective cell killing in the presence of MTA sequestering cells. This combination therapy significantly increases the potency of PRMT5 inhibitor treatment in MTAP-deleted cells across various tumor cell lines and lowers the IC50 of PRMT5 inhibitor treatment. In vivo, PRMT5 inhibitor combination treatment leads to smaller tumor volumes in MTAP-deleted CDX tumors (U87, glioma cell line) compared to PRMT5 inhibitor monotherapy. In summary, our proposed combination therapy of PRMT5 inhibition with a natural compound may increase the therapeutic window and clinical efficacy of PRMT5 inhibitors leading to better treatment options for patients harboring MTAP-deleted cancer. Citation Format: Yasaman Barekatain, Kyle LaBella, Hikaru Sugimoto, Kristen Harris, Sunada Khadka, Florian Muller, Raghu Kalluri. PRMT5 inhibition synergizes with a natural small molecule compound to kill MTAP-deleted cells and suppress tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1073.

TMIC-12. COMPLEMENT SIGNALING IN HYPOXIC NICHES OF THE GLIOMA MICROENVIRONMENT

Abstract Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.

BSCI-23 ANEUPLOIDY PROFILING IN GLIOBLASTOMA IDENTIFIES MECHANISMS OF DISEASE PROGRESSION AND TREATMENT VULNERABILITIES

Glioblastoma (GBM) is the most common and malignant adult brain tumor. Despite years of research, few advancements have been made in its management. One challenging area of glioblastoma research is patient stratification in clinical trials based on genomic features. Although several regions of aneuploidy have been known to drive disease progression in GBM, the degree of aneuploidy across the genome varies widely and the significance of regions of aneuploidy has not been assessed. Using whole genome sequencing profiles for matched tumor and non-tumor samples, we were able to accurately determine the degree of aneuploidy and loss of heterozygosity for a set of primary GBM tumors. Next, using machine learning techniques, distinct patterns of aneuploidy and loss of heterozygosity emerged among a set of GBM tumors, allowing us to define distinct aneuploidy subgroups. Interestingly, these aneuploidy subgroups showed distinctly different rates of patient survival, suggesting that regions of aneuploidy may be driving disease progression. Differing rates of various GBM genomic subtypes including IDH mutation, EGFR mutation, MGMT methylation, and tumor subtypes was also seen among the aneuploidy subgroups. We were able to derive a gene expression signature for each of these aneuploidy subgroups and revealed distinct pathways that were driving tumor growth. Furthermore, using a perturbagen-response dataset we were able to predict compounds to distinctly target each subgroup. Collectively, this suggests that aneuploidy profiling provides important clues to varying mechanisms of disease progression and is a promising approach for targeted therapy in a patient-specific manner.

IM-2 Possibility of immunotherapy for the glioblastoma patients with O6-methyl-guanine DNA methyltransferase (MGMT) expression or promoter unmethylated

Background: MGMT is a DNA repair protein that removes the cytotoxic O6-methylguanine (O6MG) DNA lesions generated by TMZ; thereby, MGMT expression is mechanistically linked to TMZ resistance. However, thus far, there is no effective treatment for these patients with MGMT promoter unmethylated. Therefore, a new treatment for GBM patients with MGMT expression is urgently needed. To this end, we examined the tumor microenvironment in GBM with or without MGMT expression. Methods: Based on The Cancer Genome Atlas (TCGA) primary GBM cohort, the tumor-infiltrating lymphocytes (TILs) expression level was calculated using the CIBERSORTx algorithms and the single-sample Gene Set Enrichment Analysis (ssGSEA) method. Furthermore, the differential expression gene analysis was conducted and pathway analysis was performed using Ingenuity Pathway Analysis (IPA). The results were validated using the GBM cohort from the Chinese Glioma Genome Atlas (CGGA) database. In addition, TILs were isolated from 13 surgically removed primary GBM tumors in our institution. Their responses to autologous tumors were evaluated by IFNγ ELISA. Results: T cells CD8 score by CIBERSORTx was significantly higher in the MGMT-high tumor. Similarly, ssGSEA scores for activated CD8 T cell, Macrophage, activated B cell, and Type 1 T helper cell were significantly higher in the MGMT-high tumor. Conversely, T cells CD4 naive was significantly higher in the MGMT-low tumor. Consistently, tumor-reactive TILs were detected in the MGMT-high tumor. Pathway analysis showed that Rictor was highly enriched in the MGMT-high tumor and Rictor inhibited lymphocyte activation. Coclusion: In this study, we demonstrated that macrophage was highly activated in the MGMT-high tumors. Thus, CSF-1R inhibitor can be combined with immunotherapy in these MGMT-high tumors to enhance anti-tumor immune responses. In addition, TMZ + mTOR2 inhibitors + PD-1 inhibitors may be effective against the MGMT-L group.

BIOM-20. TUMOR-INTRINSIC AND PERIPHERAL FEATURES ASSOCIATE WITH SURVIVAL AFTER POLIO VIROTHERAPY IN RECURRENT GBM

Abstract BACKGROUND PVSRIPO is a live-attenuated recombinant rhino:poliovirus that mediates antitumor efficacy by engaging antitumor immunity. A subset (~20%) of patients with recurrent GBM (rGBM) survive >24 months after therapy. We previously reported that low tumor mutation burden (TMB) is associated with longer survival after PVSRIPO and immune checkpoint blockade therapy in rGBM, and that low TMB associates with higher inflammatory gene expression signatures in rGBM tumors. METHODS Clinical features were tested for association with survival after PVSRIPO therapy. Whole exome sequencing and RNA-sequencing of tumors were used to correlate mutational landscape, tumor mutation burden (TMB), and gene expression signatures of patient tumors with survival. An in vitro assay that measures inflammatory responses of patient PBMCs to PVSRIPO was performed. An independent cohort of paired primary and recurrent GBM tumors was used to assess longitudinal changes in TMB and gene expression signatures after standard of care treatment. RESULTS A short time to recurrence and low TMB associated with longer survival after PVSRIPO therapy; these features were not prognostic for longer survival in immunotherapy naïve rGBM cohorts. Unexpectedly, higher pre-treatment polio neutralizing antibody titers were also associated with longer survival after PVSRIPO therapy in two independent clinical cohorts. PBMCs from patients surviving longer after PVSRIPO therapy mounted higher TNF, but lower IFN-a responses after in vitro challenge with PVSRIPO. In analysis of paired primary vs recurrent GBM tumors, we discovered that patients with low TMB upon recurrence were more likely to experience increased tumor inflammation and suppression of overall TMB. Low TMB in rGBM tumors was also associated with neoantigen depletion. Collectively, these observations imply that patients with low TMB and/or shorter duration of standard of care therapy may have intact immune surveillance, and that pre-treatment immunological status may dictate survival response to polio virotherapy.

Abstract 2203: Identifying the transcriptomic signatures of mutational heterogeneity in GBM using single cell genomics

Abstract Glioblastoma (GBM) is the most prevalent and lethal primary brain tumor, which carries a poor prognosis due to a high level of therapeutic resistance and rate of tumor recurrence. Underlying this aggressive behavior is extraordinary genomic and cellular heterogeneity, conferring numerous avenues for treatment escape and refractory growth. This work leverages single cell genomic analyses to link somatic mutations to transcriptomic signatures within the native tumor tissue. We implemented a three-stage pipeline to perform the following tasks: variant calling, machine learning (ML) classification, and biological pathway analysis. Somatic variants were called from smart-seq based scRNA-seq data and filtered based on predicted lethality, reference in cancer mutation databases e.g. COSMIC, and population prevalence. In the second stage, the genotyped cells and normalized gene expression profiles were pre-processed using differential gene expression, and input into ML classifiers trained on the task of predicting mutation status. The utilized ML models included a Support Vector Machine and Ridge Classifier and were selected based on performance and interpretability. The efficacy of the models was evaluated using the AUC-ROC metric across multiple dataset partitions. The output of this stage was a set of “important genes” identified using the learned ML model weights. In the third stage, the sets of identified genes were subsampled and analyzed using the g:Profiler API to identify significant biological pathways. The analysis was performed on 2 datasets containing 677 samples from 5 patients and 1091 samples from 4 patients. The EGFRvIII mutation was used to validate the performance of the methodology due to its prevalence in primary GBM tumors. The mutation was detected in 17 single-cell samples. The ML models both produced a mean AUC-ROC of 0.93 [0.92, 094], and the classifiers identified NPM1, TCEA1, PPIA, SUMO2, among other genes, to be of significance. The individual genes have functions involving cell cycle regulation and protein folding, while the identified biological pathways associated with gene subsets were linked to ribonuclease activity and transcription factor post-transcriptional modifications. The ML component was applied to an additional dataset containing cell samples from 4 patients and correctly identified the 2 patients positive for EGFRvIII. The larger analysis identified point mutations in EGFR, TP53, and ATRX, and the classifiers exhibited AUC-ROC metrics ranging from 0.94 [0.93, 0.94] to 0.99 [0.99, 1.0]. Overall, we developed and employed a pipeline to link somatic variants with their gene expression signatures at the single cell level, identified biological pathways for further examination and established a ML classifier that can be applied to high-throughput scRNA-seq methods that lack the transcript coverage of the variant loci. Citation Format: Christopher Streiffer, Daniel Zhang, Ryan Salinas, Steven Brem, Donald O'Rourke, Zev Binder, Han-Chiao Isaac Chen, Hongjun Song. Identifying the transcriptomic signatures of mutational heterogeneity in GBM using single cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2203.

Identification of a Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance primarily contribute to an inevitable tumor recurrence and dismal patient outcome in glioblastoma. Although GBM stem cells (GSCs) are shown to play key roles in these processes, the molecular pathways driving the GSC phenotype (GBM-stemness) remain poorly defined. A global miRNA expression analysis was performed using FFPE GBM tumor samples, and we found that low expression of miR-146a correlated with poor clinical outcome and tumor recurrence. Based on this finding, the hypothesis is that miR-146a functions as a tumor suppressor and inhibits stemness in GBM. Expression analysis was performed using FFPE GBM tumor samples (n = 268) followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a-POU3F2/SMARCA5 pathway in GBM tumorigenesis and therapeutic response. MicroRNA-146a was one of the top miRNAs that correlated with better prognosis in GBM patients by UVA analyses (p = 9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p<0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p = 0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p<0.01) and prolonged survival (p = 0.001) in vivo. In addition, miR-146a was downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly reduced stemness. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Moreover, overexpression of miR-146a significantly inhibited NF-κB pathways through targeting POU3F2 and SMARCA5 in GBM. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. Fundings: R01CA188228 (to A.C., R.B., K.L., and J.S.B-S.), and the T&P Bohnenn Fund for Neuro-Oncology Research (to P.A.R.). R01CA169368, R01CA11522358, R01CA1145128, R01CA108633, 1RC2CA148190, and U10CA180850-01; A Brain Tumor Funders Collaborative Grant; Ohio State University Comprehensive Cancer Center Award (all to A.C.).

COMP-16. COMPREHENSIVE TRANSCRIPTOMIC ANALYSIS OF SINGLE CELLS FROM RECURRENT AND PRIMARY GLIOBLASTOMA TO PREDICT CELL-TYPE SPECIFIC THERAPEUTICS

Abstract Glioblastoma (GBM) remains the most common adult brain tumor, with poor survival expectations, and no new therapeutic modalities approved in the last decade. Our laboratories have recently demonstrated that the integration of a transcriptional disease signature obtained from The Cancer Genome Atlas’ GBM dataset with transcriptional cell drug-response signatures in the LINCS L1000 dataset yields possible combinatorial therapeutics. Considering the extreme intra-tumor heterogeneity associated with the disease, we hypothesize that the utilization of single-cell RNA-sequencing (scRNA-seq) of patient tumors will further strengthen our predictive model by providing insight on the unique transcriptomes of the cellular niches present within these tumors, and into the transcriptional dynamics of these same cellular niches. By sequencing single-cell transcriptomes from recurrent GBM tumors resected from patients at the University of Miami, and integrating our datasets with previously published scRNA-seq data from primary GBM tumors, we are able to gain additional insight into the differences between these clinical distinctions. We have analyzed the differential expression of kinases both across and within distinct cell populations of primary and recurrent GBM tumors. This transcriptional map of kinase expression represents the heterogeneity of potential targets within individual tumors and between recurrent and primary GBM. Additionally, by generating disease signatures unique to each cellular population, and integrating these with transcriptional drug-response signatures from LINCS, we are able to predict compounds to target specific cell populations within GMB tumors. Additional computational techniques such as RNA velocity analysis and cell cycle scoring elucidate temporal insights to further prioritize these cell-type specific therapeutics, and reveal the intra-cellular dynamics present within these tumors. Collectively, our studies suggest that we have developed a novel omics pipeline based on the single cell RNA-sequencing of individual GBM cells that addresses intra-tumor heterogeneity, and may lead to novel therapeutic combinations for the treatment of this incurable disease.

CSIG-30. miR-146a INHIBITS TUMORIGENESIS AND INDUCES TEMOZOLOMIDE SENSITIVITY VIA AFFECTING CANCER STEM CELL PROPERTIES IN GBM

Abstract BACKGROUND Glioblastomas (GBMs) are the most aggressive primary brain tumors, with an average survival time of less than 15 months. miRNAs are emerging as promising and novel biomarkers in GBM. The aims of this study are: 1) to investigate novel miRNAs biomarkers that affect tumorigenesis and therapeutic sensitivity, and 2) to study the underlying molecular mechanisms in GBM. METHODS Nanostring v3 was performed followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a in GBM tumorigenesis and therapeutic response and the molecular mechanisms were investigated. RESULTS UVA analyses demonstrated that miR-146a is one of the top miRNAs that correlated with better prognosis in GBM patients (p=9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p&lt; 0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p=0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p&lt; 0.01) and prolonged survival (p=0.001) in vivo. In addition, miR-146a is downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly affected glioma cancer stem cell self-renewal. We also found that overexpression of miR-146a significantly inhibited the NF-κB, AKT, and ERK pathways. CONCLUSION Our data suggest, for the first time, that miR-146a predicts favorable prognosis for GBM patients and sensitizes primary GBM cells to TMZ treatment in vitro and in vivo through regulating glioma stem cells. Importantly, miR-146a may prove to be a master switch shutting off AKT, NF-κB, as well as other pathways and may overcome redundancies among these pathways leading to resistance. FUNDING: Bohnenn Fund (to PR), R01CA108633, R01CA169368, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC).

Abstract 4742: The influence of cellular developmental state on response to therapeutics in glioblastoma

Abstract Glioblastoma (GBM) is an aggressive tumor treated with ionizing radiation (IR) and temozolomide (TMZ). Here we investigate how developmental state of glioblastoma cells: cancer stem cells (CSC) or differentiated cells, influences cellular response to treatment. Tissue from untreated primary GBM tumors representing diverse genotype were dissociated and cultured in conditions selective for CSCs, which were subsequently differentiated into an astrocytic phenotype by culturing in media supplemented with 2% FBS. Stemness index was evaluated using a machine learning predictive algorithm. Sensitivity of both isogenic CSC and serum differentiated cell (SDC) populations to TMZ and IR was determined by non-linear fitting of dose-response curves, using CellTiterGlo to measure cell viability (n=5) . CSCs and SDCs were then subjected to sub-lethal TMZ, IR (4 Gy), or control treatments, in triplicate. Cells were harvested, and DNA was isolated and Illumina 450k DNA methylation array data was analyzed by using the Methylumi package in R software. RNA was isolated for sequencing, and differentially expressed genes were determined by NOISeq R package. Our results show that GBM CSCs are more vulnerable to both TMZ and IR treatment than the isogenic SDCs. Transcriptome profiling show that IR leads to increase in p53 mediated expression of pro-apoptotic genes such as MDM2, PUMA and BCL2 binding protein relative to control in CSCs. In differentiated cells, IR lead to an increase in WNT signaling, DNA-repair activity and histone modification /remodeling proteins. Enrichment analysis using Fisher’s exact test identified significant enrichment in lipid, fatty acid and metabolic processes as well as receptor tyrosine signaling. Globally, CSCs exhibited a decrease in protein coding genes and increase in regulatory RNA expression. DNA methylation show a global increase in CpG promoter methylation in differentiated cells relative to isogenic stem-like counterparts. We recovered significant differentially methylated probes between treatment groups (Wilcoxon signed rank test) as well as a global increase in CpG promoter methylation in IR treated cells relative to control. These findings underscore the challenge of treating a highly heterogeneous tumor and challenge a strategy of inducing differentiation to increase GBM sensitivity to treatment. Citation Format: Oluwademilade Nuga, Ana deCarvalho, Douglas Ruden, Stephen Brown, Indrani Datta, Tathianne Malta. The influence of cellular developmental state on response to therapeutics in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4742.

Abstract 4222: Targeting HMGA2 suppresses GBM stemness, invasion and tumorigenicity

Abstract Glioblastoma Multiforme (GBM) causes the majority of brain tumor-related deaths in the United States. The high invasiveness of GBM prevents complete surgical resection, allowing tumor recurrence. The lack of curative treatment highlights a dire need to develop specific targeted therapeutics. Identifying and targeting novel molecular markers in glioma stem-like cells (GSCs) thought to be critical for tumor invasion and recurrence could lead to development of better therapies. HMGA2, a non-histone transcriptional modulator, is a regulator of normal and cancer stem cells. The significance of HMGA2 as a potential therapeutic target in GBM has not been investigated. We found increased HMGA2 expression in a subset of primary human GBM tumors, in multiple patient-derived GBM neurosphere and adherent cell lines, and in the mesenchymal subgroup of GBM in The Cancer Genome Atlas (TCGA) database. Lentiviral short hairpin RNA (shRNA)-lowered HMGA2 protein levels and significantly reduced invasion and clonogenicity of GBM cell lines in transwell invasion and soft agar colony formation assays. Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited growth of the U-87 MG GBM cell line (P&amp;lt;0.01) in MTS assays. Mice bearing orthotopic GBM neurosphere xenograft lines transduced with HMGA2 shRNA lived longer (median survival = 108 days) compared to control shRNA (median survival = 67.5 days) (P&amp;lt;0.0001 by log-rank analysis). Conversely, lentiviral-mediated overexpression of HMGA2 in GBM cell lines increased invasion, clonogenicity and resistance to radiation in vitro (P&amp;lt;0.01). Overexpression of HMGA2 also promoted GBM tumor formation in vivo in orthotopic xenografts. To elucidate the molecular mechanism by which HMGA2 promotes invasion and stemness in GBM, we performed mRNA expression profiling on GBM neurosphere lines lentivirally transduced to express HMGA2 or empty vector control. We found increased CD44 expression in HMGA2-overexpressing GBM neurosphere lines compared to control, and confirmed this result by western blotting. Together, our results suggest an oncogenic role of HMGA2 in promoting GBM tumorigenicity by increasing invasiveness and a stem cell-like state, and show that HMGA2 is a potential therapeutic target in GBM. Citation Format: Harpreet Kaur, Marianne Hütt-Cabezas, Isabella Taylor, Laura Asnaghi, Fausto Rodriguez, Brent A. Orr, Charles G. Eberhart, Eric H. Raabe. Targeting HMGA2 suppresses GBM stemness, invasion and tumorigenicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4222. doi:10.1158/1538-7445.AM2015-4222

Abstract 2318: Genetic and functional diversity of propagating cells in glioblastoma

Abstract Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure.This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo. Citation Format: Sara Grazia Maria Piccirillo, Sue Colman, Nicola E. Potter, Frederik W. van Delft, Suzanne Lillis, Maria-Jose Carnicer, Lyndal Kearney, Colin Watts, Mel Greaves. Genetic and functional diversity of propagating cells in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2015-2318

Genetic and Functional Diversity of Propagating Cells in Glioblastoma

SummaryGlioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure. This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo.

IT-06 * PERSONALIZED IMMUNOTHERAPY FOR THE TREATMENT OF GLIOBLASTOMA

BACKGROUND: Immunotherapy harnesses the immune system to specifically attack malignant cells thereby offering a targeted treatment option without systemic toxicity for patients with GBM. However, immunotherapy remains limited by the relative dearth of antigen specific tumor targets. Since GBMs have been shown to express cytomegalovirus (CMV) antigens, we can use these viral epitopes as a novel, effective, and specific targets in conjunction with other tumor associated antigens (TAAs) for effective glioma therapy. We hypothesize that elucidating a more full antigenic profile of CMV and other TAAs for use in a multi-component immunotherapeutic approach will provide robust immunologic responses against GBM. METHODS: We designed a multiplex RT-qPCR SYBR array (Bio-Rad) containing 27 CMV genes that were shown to be associated with immune reactivity. Agilent 4x44k custom gene expression arrays were designed and developed to screen GBM patient tumor samples for the presence of human TAA genes. RT-qPCR products were verified via Sanger sequencing and select TAA microarray genes were validated via RT-qPCR. Targets identified within GBM specimens were evaluated for immunogenicity in vitro using RNA-pulsed dendritic cells. RESULTS/CONCLUSIONS: As expected, there was significant heterogeneity in the expression of CMV and TAA antigens within primary GBM tumors. The capacity to effectively target GBM tumors using autologous T cells stimulated by antigen pulsed DCs was demonstrated in vitro. In summary, given differences in antigenic profiles of GBM patients, a personalized targeted approach will allow us to select the appropriate RNA transcripts for use in a multi-component dendritic cell based therapy. The enrichment for personalized tumor antigens using transcript specific autologous RNA amplification may further refine immunologic targeting and we have demonstrated the feasibility and immunologic potency of this novel therapeutic approach.

NI-58 * LOCATION OF GLIOBLASTOMA INTERSECTIING WHITE MATTER TRACTS PREDICT PATIENT PROGNOSIS AND RESPONSE TO BEVACIZUMAB PRIOR TO THERAPY

INTRODUCTION: Brain tumors are known to invade healthy brain along white matter tracts. We therefore hypothesized that specific white matter tracts would be associated with poorer prognosis based on their connection to primitive brain regions necessary for essential life functions. We also hypothesized that tumors in these regions treated with bevacizumab would alter patient survival. METHODS: 109 subjects with primary GBM tumors were retrospectively included in this study. Voxel-wise survival analyses were performed to test for spatial and treatment dependence of GBM survival prognosis. An intersection analysis was first performed between an enhancing tumor mask and template-space diffusion tensor imaging (DTI) derived tractography which highlighted voxels with tumor-intersecting white matter tracts. Maps of voxels with these tracts were made for each patient. A voxel-wise Kaplan-Meier test was performed comparing the survival times of patients with and without a tumor-intersecting white matter tract in that voxel. The same analysis was implemented to compare survival of participants with and without bevacizumab. This highlighted regions where survival was effected due to anti-VEGF treatment. A Monte Carlo simulation was performed to validate the observed survival differences induced by bevacizumab considering inconsistencies in treatment regimens seen in the patient population. RESULTS: Glioblastoma patients with tumors intersecting the right anterior thalamic radiation, right inferior fronto-occipital fasciculus, bilateral corticospinal tracts, and corpus callosum have decreased survival compared to those with tumors intersecting elsewhere. Patients treated with bevacizumab and who also had tumors intersecting the corpus callosum, corticospinal tract, or the thalamic radiations were found to be at a survival advantage when compared to those who did not receive bevacizumab. CONCLUSIONS: Patient prognosis is associated with the location of tumor intersecting WM tracts. A survival advantage was found in tumors intersecting specific WM tracts treated with bevacizumab.

Abstract 3031: HMGA2 promotes invasion and stemness in glioblastoma

Abstract Glioblastoma Multiforme (GBM) causes the majority of brain tumor-related deaths in the United States. The high invasiveness of GBM prevents complete surgical resection, allowing tumor recurrence. The lack of curative treatment highlights a dire need to develop specific targeted therapeutics. Identifying and targeting novel molecular markers in glioma stem-like cells (GSCs), one of the major causes of tumor invasion and recurrence, could lead to development of better therapies. HMGA2, a non-histone transcriptional modulator, is a regulator of normal and cancer stem cells. The significance of HMGA2 as a potential therapeutic target in GBM has not been investigated. To elucidate the role of HMGA2 in GBM, we have used a combination of in vitro and in vivo techniques. We found increased HMGA2 expression in the mesenchymal subgroup of GBM tumors in the The Cancer Genome Atlas (TCGA) database and in a subset of primary human GBM tumors. HMGA2 is expressed in multiple patient-derived GBM neurosphere lines with increased expression in CD133+ neurospheres compared to CD133- neurospheres. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 significantly inhibited invasion of GBM neurosphere lines (040821, 62% reduction with HMGA2 shRNA vs. control shRNA, P&amp;lt;0.001) in transwell invasion assays. Conversely, lentiviral-mediated overexpression of HMGA2 in GBM neurosphere lines increased invasion (GBM1 and 040821, 100-130% increase with HMGA2 overexpression vs. control vector, P&amp;lt;0.001) in transwell invasion assays. In addition, overexpression of HMGA2 increased clonogenicity of GBM neurosphere lines in vitro in soft agar and methylcellulose colony formation assays (40-60% increase with HMGA2 overexpression vs. control vector, P&amp;lt;0.001). Importantly, log-rank analysis of Kaplan-Meier plots showed increased survival of mice injected with GBM neurosphere lines transduced with HMGA2 shRNA compared to control shRNA (P&amp;lt;0.05) in an in vivo, orthotopic, intracranial mouse xenograft model. To elucidate the molecular mechanism by which HMGA2 promotes invasion and stemness in GBM, we performed mRNA expression profiling on GBM neurosphere lines lentivirally transduced to express HMGA2 or empty vector control. We found increased CD44 expression in HMGA2-overexpressing GBM neurosphere lines compared to control. Together, our results suggest an oncogenic role of HMGA2 in promoting GBM tumorigenicity by increasing invasiveness and a stem cell-like state, thereby suggesting that HMGA2 is a potential therapeutic target in GBM. Citation Format: Harpreet Kaur, Marianne Hütt, Xing-gang Mao, Brent A. Orr, Charles G. Eberhart, Eric H. Raabe. HMGA2 promotes invasion and stemness in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3031. doi:10.1158/1538-7445.AM2014-3031

Abstract 1379: Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells

Abstract Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2014-1379

Abstract 4102: FAT1: A novel regulator of cancer and inflammation.

Abstract FAT1, a member of cadherin superfamily is a large trans-membrane protein with 34 cadherin repeats. FAT1 was originally identified as a tumor suppressor gene in Drosophila but its role in human cancers is just beginning to unfold. Considering its tumor suppressive role in Drosophila, few studies have reported similar function of FAT1 in human cancers but on the contrary several studies have also demonstrated oncogenic role of FAT1. In this study we identified oncogenic role of FAT1 in human glioma where knockdown of FAT1 was found to inhibit migration and invasion of glial cells. Further investigation into molecular mechanism of FAT1 function revealed direct link to AP-1 mediated transcription via Programmed Cell Death 4 (PDCD4). Knockdown of FAT1 was found to inhibit AP-1 mediated transcription and expression of downstream target genes. AP-1 is a known mediator of cancer and inflammation and accordingly we found decreased expression of several pro-inflammatory markers like COX-2, cytokines like IL-6, IL-1β and chemokines like IL-8, MCP-1 and MIP-2 after FAT1 knockdown. Interestingly, the expression of anti-inflammatory cytokine IL-10 was found to be increased after FAT1 knockdown. Moreover FAT1 expression was also found to positively correalate with COX-2 and IL-6 expression in primary GBM tumors. Thus, in this study FAT1 was found to act as an oncogene as well as to orchestrate the inflammatory milieu of the glioma cells by regulating AP-1 mediated transcription via PDCD4. Citation Format: Bhawana Dikshit, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1: A novel regulator of cancer and inflammation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2013-4102

Abstract 4637: Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme.

Abstract Glioblastoma Multiforme (GBM) is associated with a dismal prognosis despite intensive multimodal therapy, highlighting the need for novel therapeutic approaches. Chromatin remodeling complexes and associated co-repressors such as histone deacetylases (HDAC), DNA methyltransferases (DNMT) and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor and regulatory gene expression and may contribute to glial cell transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. We have previously identified PRMT5 over-expression in GBM cell lines, primary GBM tumors, and GBM that spontaneously develop in a pre-clinical mouse model. The degree of PRMT5 over-expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p&amp;lt;0.0001), PRMT5 works concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. PRMT5 silending in GBM cell lines leads to growth arrest, apoptosis and loss of HDAC2, DNMT3a and PRMT5 co-repressor complex recruitment at chemokine gene promoters. These findings provide evidence that PRMT5 is a central repressive element required for maintenance of target gene silencing. Thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for GBM. A rational design of small molecule compounds to inhibit PRMT5 activity led us to construct an in silico model of the human PRMT5 catalytic domain based on available homologous crystal structures. We screened a library of 10,000 compounds and eight small molecules were identified based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays showed that a lead compound (BLL1) was capable of selectively inhibiting PRMT5 and not PRMT1 or PRMT7 activity (p&amp;lt;0.0001). BLL1 interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in GBM cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 showed a dose-dependent response of inhibition of cellular proliferation in GBM cell lines. Combination treatment of GBM cells with subtoxic doses of BLL1, hypomethylating agent 5-azacitidine, and HDAC inhibitor TSA showed synergistic induction of cell death, loss of the epigenetic mark S2Me-H4R3, and de-repression of the immune modulating chemokine CXCL10. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for BLL1. We have successfully developed a first in class drug to selectively target dysregulated PRMT5 enzymatic activity in GBM. We are currently developing drugs with improved selectivity and potency. Citation Format: Fengting Yan, Kate Gordon, Kiran Mahasenan, Mark Lustberg, Lapo Alinari, Christian T. Earl, Balveen Kaur, Chenglong Li, Robert A. Baiocchi. Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4637. doi:10.1158/1538-7445.AM2013-4637

Abstract 2896: Genomic determinants and signature of MET-targeted therapy in glioblastoma.

Abstract Glioblastoma (GBM) is one of the most devastating cancers, because the intrinsic capability of single tumor cells to invade normal brain impedes surgical eradication, predictably resulting in early local recurrence and death. The goal of this study is to develop therapeutic strategies for treating GBM patients by identifying and targeting the molecular mechanisms of GBM invasiveness through inhibiting MET signaling pathway. Overexpression of hepatocyte growth factor (HGF) and its receptor MET are associated with poor prognosis of GBM. Genomic analysis of primary GBM tumors indicates that MET is overexpressed in tumors of the mesenchymal phenotype, which is more invasive and results in short patient survival. More recently, MET activation was found to contribute to recurrence in GBM patients after bevacizumab treatment, further highlighting the utility of blocking the MET pathway in this disease. With MET inhibitors’ entering clinical trials, there is increased need to find the molecular determinants of MET sensitivity. Because GBM is a heterogeneous disease in which drug response in the individual patient can be regulated by different mechanisms, approaches for understanding and predicting treatment outcomes based on the molecular features of individual tumor will be of a high value. Based on our previous finding that HGF-autocrine activation predicts sensitivity to MET inhibition, we have developed a two-step methodology using microarray technology and bioinformatics analysis to identify and test genomic determinants in order to effectively deploy MET-targeted therapy in GBM patients. By analyzing the data sets from The Cancer Genome Atlas Network (TCGA) and those from GBM xenograft models sensitive and insensitive to MET inhibitors, a 25-gene signature highly associated with HGF-autocrine activation is identified. When performing validation analysis, the signature scored 40 human GBM xenograft models by HGF expression level, providing a good rationale for further testing its predictive value in identifying tumors sensitive to MET inhibitors. We conclude that HGF-autocrine activation can result in oncogene addiction to MET signaling in GBM. Via a unique panel of signature genes we may be able to identify a subset of patients vulnerable to anti-MET drugs. Citation Format: Qian Xie, Jennifer Johnson, Maria Libera Ascierto, Liang Kang, Robert Bradley, Sandeep Mittal, Kyle Furge, Michael Briggs, Kirk Tanner, Michael E. Berens, Francesco M. Marincola, George F. Vande Woude. Genomic determinants and signature of MET-targeted therapy in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2013-2896