GBM Subtypes Research Articles

OTEH-9. scRNA sequencing of proneural GBM avatar model reveals acquisition of oncogenic transcriptional programming and infers a developmental path towards a genomically unstable state

Glioblastomas (GBMs) are the most common malignant primary brain tumors, and the paucity of novel treatments warrants an investigation of its origins and development into aggressive, lethal tumors. Koga & Chaim et al. have recently shown that human pluripotent stem cells (hiPSCs) with different combinations of driver mutations can be differentiated into neural progenitor cells (NPCs) and engrafted into mice to form high grade gliomas (iHGGs). In this work, scRNA seq analysis was used to investigate the development of TP53−/−;PDGFRAΔ8–9 iHGGs, an avatar model that has been shown to recapitulate the proneural subtype of GBM. After re-engrafting the primary avatar cultures (secondary tumor stage), the TP53−/−;PDGFRAΔ8–9 iHGGs developed diverse transcriptional programming and acquired a subpopulation of cells with high expression of known GBM oncogenes, such as MYC, CDK4, and PDGFRA. Notably, when all datasets were aggregated, this oncogene amplifying transcriptional program became the largest source of variation between all stages and replicates of the TP53−/−;PDGFRAΔ8–9 iHGGs. Indicated by a larger total copy number variation (CNV), this oncogene-amplifying program was associated with a genomically unstable developmental state. Trajectory inference could track the development of this population from the initial primary culture of TP53−/−;PDGFRAΔ8–9 iHGG. Differential gene expression analysis identified distinct divergences in clonal evolution––e.g., high expression of the S100 protein family in one cluster––following the acquisition of this genomically unstable state. Lastly, genomic PCR was used to ascertain whether these changes in transcriptional programming were reflected in changes in DNA copy number and identified DNA amplifications of MYC and CDK4. Our scRNA seq analysis of the GBM avatar model platform provides novel insight into how oncogenic states in GBM develop from a small number of driver mutations.

Abstract SY07-02: Gaining biologic insights into glioblastoma using proteomics

Abstract Glioblastoma (GBM) is the most common type of primary malignant tumor in adults and contributes disproportionately to cancer morbidity and mortality. Understanding its molecular pathogenesis is crucial for improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomics data on 99-treatment naïve GBMs provided insights to GBM biology. Multiple key findings emerged that were not known from traditional genomic analyses: (1) Analysis of protein phosphorylation identified key signaling intermediates in the RTK/RAS path-way common to multiple RTK genomic alterations (PTPN11 and PLCG1), potentially offering common therapeutic targets for different oncogenic drivers in GBM. (2) Phosphoproteomics also identified potential druggable targets based on kinase-substrate pathway analysis, as well as novel phosphoprotein targets associated with the regulation of telomere length by ATRX in IDH mutants. (3) TP53 protein abundance in GBM is regulated by protein/phosphoprotein effectors (4) Four immune GBM subtypes exist, characterized by distinct immune cell population differences – Immune High and Immune Low phenotypes in GBM were driven by tumor-associated macrophage markers, and associated with distinct epigenetic modifications and histone acetylation patterns. (5) The mesenchymal subtype displays EMT signatures specific in tumor cells, distinct from infiltrating immune cells. (6) Histone H2B acetylation and immune-low GBM was driven largely by BRDs, CREBBP, and EP300. (7) Identification of key metabolic changes in IDH mutants facilitating the accumulation of onco-metabolite 2-HG were also associated with lipidomic changes. This work identifies additional therapeutic channels for GBM and novel information useful for more accurate stratification patients for effective treatment. Citation Format: Jill S. Barnholtz-Sloan. Gaining biologic insights into glioblastoma using proteomics [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 SY07-02.

Differential Predictors and Clinical Implications Associated With Long-Term Survivors in IDH Wildtype and Mutant Glioblastoma.

BackgroundGlioblastoma (GBM) is the most aggressive intracranial tumor which can be divided into two subtypes based on status of isocitrate dehydrogenase (IDH). A small fraction of patients after receiving standard treatment can be long-term survivors (LTS). This study was designed to disclose the predictors and clinical implications associated with LTS in IDH wildtype and mutant GBM.MethodsPatients who survived beyond five years after diagnosis of GBM were defined as LTS, while those with a survival less than one year were defined as short-term survivors (STS). A total of 211 patients with diagnosis of GBM in Beijing Tiantan Hospital from January 2007 to January 2015 were enrolled, including 44 (20.9%) LTS and 167 (79.1%) STS. The clinical, radiological and molecular features between groups were systematically compared.ResultsCompared with STS, LTS were a subgroup of patients with a younger age at diagnosis (P=0.006), a higher KPS score (P=0.011), higher rates of cystic change (P=0.037), O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (P=0.007), and IDH mutation (P=0.049), and more likely to have undergone gross total resection (P<0.001). Survival analysis demonstrated that LTS with wildtype IDH conferred a longer progression-free survival (66.0 vs. 27.0 months, P=0.04), but a shorter post-progression survival (46.5 months vs. not reached, P=0.0001) than those of LTS with mutant IDH. LTS with mutant IDH showed a trend towards increased survival after receiving re-operation (P=0.155) and reirradiation (P=0.127), while this clinical benefit disappeared in the subset of LTS with wildtype IDH (P>0.05).ConclusionThe prognostic value and therapeutic implications associated with LTS in GBM population significantly differed on the basis of IDH status. Our findings provide a new approach for physicians to better understand the two subtypes of GBM, which may assist in making more tailored treatment decisions for patients.

Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion

SummaryGlioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

Abstract PO052: Uncovering the evolution of Glioblastoma proteome landscape from primary to the recurrent stage for development of novel diagnostic and predictive biomarkers

Abstract Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. A wealth of literature describes the biology of primary GBM (p-GBM), but we currently lack an understanding of how GBM evolves through therapy to become a very different tumor at recurrence, which may explain why therapies against p-GBM fail to work in recurrent GBM (r-GBM). Therefore, to understand the evolution of r-GBM, we aimed to characterize patient-matched p-GBM and r-GBM proteome and identify potential therapeutic targets for r-GBM. We collected one of the world’s largest patient-matched p-GBM and r-GBM samples from the Hamilton Health Sciences for gene expression profiling, proteomic analyses and tissue microarray (TMA) construction. Nano-String analysis was performed for GBM subtype identification. Furthermore, patient demographics was generated for survival analysis. The top potential therapeutic targets for r-GBM were identified by proteomic analysis and were validated on TMA using immunohistochemistry. The essentiality of each protein in r-GBM were assessed using CRISPR KO studies and the top hit were selected for pre-clinical testing. 6798 proteins were detected by shotgun, label-free proteomic analyses. Differential expression analysis on the surface proteins revealed a distinct set of proteins overexpressed in r-GBM among which 7 proteins were selected as top potential therapeutic targets for r-GBM. Besides, the patients were grouped based on survival rate and the differential expression analysis revealed significantly enriched proteins and pathways in short-term survivors which cause aggressive phenotypes in GBM. In addition, consensus clustering identified five protein clusters which show distinction between primary vs recurrent tumors. Our data also strongly supports a preponderance of immune regulatory/suppressive genes as important drivers of r-GBM. This study resulted in identification of diagnostic and predictive biomarkers which is extremely complementary and instructive for the development of new poly-therapeutic paradigms for GBM patients at the recurrent level and will lead to improvement of patient’s survival. Citation Format: Nazanin Tatari, Shahbaz Khan, Julie Livingstone, Chitra Venugopal, Jennifer Chan, Cynthia Hawkins, John Provias, Jian-Qiang Lu, Kjetil Ask, Thomas Kislinger, Sheila Singh. Uncovering the evolution of Glioblastoma proteome landscape from primary to the recurrent stage for development of novel diagnostic and predictive biomarkers [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO052.

Genomic alterations predictive of response to radiosurgery in recurrent IDH-WT glioblastoma.

Despite aggressive treatment, glioblastoma invariably recurs. The optimal treatment for recurrent glioblastoma (rGBM) is not well defined. Stereotactic radiosurgery (SRS) for rGBM has demonstrated favorable outcomes for selected patients; however, its efficacy in molecular GBM subtypes is unknown. We sought to identify genetic alterations that predict response/outcomes from SRS in rGBM-IDH-wild-type (IDH-WT). rGBM-IDH-WT patients undergoing SRS at first recurrence and tested by next-generation sequencing (NGS) were reviewed (2009-2018). Demographic, clinical, and molecular characteristics were evaluated. NGS interrogating 205-genes was performed. Primary outcome was survival from GK-SRS assessed by Kaplan-Meier method and multivariable Cox proportional-hazards. Sixty-three lesions (43-patients) were treated at 1st recurrence. Median age was 61-years. All patients were treated with resection and chemoradiotherapy. Median time from diagnosis to 1st recurrence was 8.7-months. Median cumulative volume was 2.895cm3 and SRS median marginal dose was 18Gy (median isodose-54%). Bevacizumab was administered in 81.4% patients. PFS from SRS was 12.9-months. Survival from SRS was 18.2-months. PTEN-mutant patients had a longer PFS (p = 0.049) and survival from SRS (p = 0.013) in multivariable analysis. Although no statistically significant PTEN-mutants patients had higher frequency of radiation necrosis (21.4% vs. 3.4%) and lower in-field recurrence (28.6% vs. 37.9%) compared to PTEN-WT patients. SRS is a safe and effective treatment option for selected rGBM-IDH-WT patients following first recurrence. rGBM-IDH-WT harboring PTEN-mutation have improved survival with salvage SRS compared to PTEN-WT patients. PTEN may be used as a molecular biomarker to identify a subset of rGBM patients who may benefit the most from SRS.

The Multifaceted Glioblastoma: From Genomic Alterations to Metabolic Adaptations.

Glioblastoma multiforme (GBM) develops on glial cells and is the most common as well as the deadliest form of brain cancer. As in other cancers, distinct combinations of genetic alterations in GBM subtypes induce a diversity of metabolic phenotypes, which explains the variability of GBM sensitivity to current therapies targeting its reprogrammed metabolism. Therefore, it is becoming imperative for cancer researchers to account for the temporal and spatial heterogeneity within this cancer type before making generalized conclusions about a particular treatment's efficacy. Standard therapies for GBM have shown little success as the disease is almost always lethal; however, researchers are making progress and learning how to combine therapeutic strategies most effectively. GBMs can be classified initially into two subsets consisting of primary and secondary GBMs, and this categorization stems from cancer development. GBM is the highest grade of gliomas, which includes glioma I (low proliferative potential), glioma II (low proliferative potential with some capacity for infiltration and recurrence), glioma III (evidence of malignancy), and glioma IV (GBM) (malignant with features of necrosis and microvascular proliferation). Secondary GBM develops from a low-grade glioma to an advanced-stage cancer, while primary GBM provides no signs of progression and is identified as an advanced-stage glioma from the onset. The differences in prognosis and histology correlated with each classification are generally negligible, but the demographics of individuals affected and the accompanying genetic/metabolic properties show distinct differentiation [3].

Multiple positron emission tomography tracers for use in the classification of gliomas according to the 2016 World Health Organization criteria.

BackgroundThe molecular diagnosis of gliomas such as isocitrate dehydrogenase (IDH) status (wild-type [wt] or mutation [mut]) is especially important in the 2016 World Health Organization (WHO) classification. Positron emission tomography (PET) has afforded molecular and metabolic diagnostic imaging. The present study aimed to define the interrelationship between the 2016 WHO classification of gliomas and the integrated data from PET images using multiple tracers, including 18F-fluorodeoxyglucose (18F-FDG), 11C-methionine (11C-MET), 18F-fluorothymidine (18F-FLT), and 18F-fluoromisonidazole (18F-FMISO).MethodsThis retrospective, single-center study comprised 113 patients with newly diagnosed glioma based on the 2016 WHO criteria. Patients were divided into 4 glioma subtypes (Mut, Codel, Wt, and glioblastoma multiforme [GBM]). Tumor standardized uptake value (SUV) divided by mean normal cortical SUV (tumor–normal tissue ratio [TNR]) was calculated for 18F-FDG, 11C-MET, and 18F-FLT. Tumor–blood SUV ratio (TBR) was calculated for 18F-FMISO. To assess the diagnostic accuracy of PET tracers in distinguishing glioma subtypes, a comparative analysis of TNRs and TBR as well as the metabolic tumor volume (MTV) were calculated by Scheffe's multiple comparison procedure for each PET tracer following the Kruskal–Wallis test.ResultsThe differences in mean 18F-FLT TNR and 18F-FMISO TBR were significant between GBM and other glioma subtypes (P < .001). Regarding the comparison between Gd-T1WI volumes and 18F-FLT MTVs or 18F-FMISO MTVs, we identified significant differences between Wt and Mut or Codel (P < .01).ConclusionCombined administration of 4 PET tracers might aid in the preoperative differential diagnosis of gliomas according to the 2016 WHO criteria.

PATH-29. COMPARATIVE ANALYSIS OF DNA METHYLATION PROFILES ASSOCIATED WITH IDH-WILDTYPE GLIOMA AND GLIONEURONAL TUMOR SUBTYPES

Abstract DNA Methylation profiles are highly robust and reproducible as a classification tool. Less is understood regarding the methylation differences that exist among gliomas and glioneuronal tumors. To address this, we analyzed differentially methylated probes (DMPs) of gliomas and glioneuronal tumors compared to normal brain white matter controls. After filtering (Δbeta &amp;gt;0.3, logFC &amp;gt;±1) of significant probes we observed that low grade glioma/glioneuronal tumors (LGGs) had significantly fewer DMPs (Hyper/Hypo) as compared to GBMs. For example, posterior fossa pilocytic astrocytomas (PA’s) showed 2861 DMPs (1916 hypo/945 hyper) versus 9653 for GBM-RTKI ((6563/3090) respectively, while tumors such as PXA and anaplastic PA showed intermediate changes between LGG’s and GBMs. Hypomethylated and hypermethylated probes were analyzed for gene ontology and KEGG pathway enrichment, with LGG subtypes showing hypomethylated probes/genes associated with cell adhesion, blood vessel development and viral infection (P-value = 10-7). In contrast, hypomethylated probes in GBM subtypes were enriched for plasma membrane and cell periphery gene ontologies (P-value = 10-52). With respect to hypermethylated probes, LGG subtypes showed enrichment for myelination, glial cell differentiation and sphingolipid metabolism (P-value = 10-5) while DNA-binding transcription factor activity was seen in GBM subtypes (P-value=10-35). Examples of the most significantly hypermethylated genes in GBM included the transcription factors, GATA3 and PAX9. Intermediate-grade gliomas such as anaplastic PA and PXA showed enrichment of hypermethylated genes similar to GBM, but of lower significance (P-values = 10-6 and 10-4). Overall, understanding of cancer-associated DNA methylation changes in glioma subtypes suggests a hierarchy of biological changes that may underlie the pathogenesis of these tumors and interestingly, highlight tumor types such as PXA and anaplastic PA as having intermediate methylation changes, between benign LGG and GBM. Hypermethylation of transcription-factor genes will be investigated in GBM and compared with changes in gene expression to understand possible roles in the pathogenesis.

BIOM-16. IMMUNOMIC ANALYSIS OF GLIOBLASTOMA (GBM) USING GENE EXPRESSION PROFILING

Abstract BACKGROUND Despite the success of immunotherapy across the spectrum of human cancer, a successful strategy has not emerged for GBM. While PD-L1 IHC and TMB have demonstrated some utility as predictors of immunotherapy benefit, responsiveness is complexly determined by factors affecting T cell trafficking, antigen presentation, other immune checkpoints, and mediators of immune exhaustion. Thus, we set out to to characterize mediators of immune resistance and their diversity in a population of GBM patients utilizing quantitative gene expression. METHODS A set of 54 immunotherapy and checkpoint relevant genes and seven genes related to immune failure were selected from the literature. RNA gene counts for TCGA glioblastoma multiforme samples (N=163) were downloaded from https://portal.gdc.cancer.gov/. Annotation on subtypes and PFS values were obtained from PMID: 24120142. Gene expression normalization as FPKM, hierarchical clustering and box-plots were performed using R-3.6.0. Statistical differences of gene expression between subtypes were quantified using a TurkeyHSD test. RESULTS A heatmap with hierarchical clustering for immune related genes for the TCGA GBM cohort was generated including colored annotation for the subtype and progression free survival. The graph shows a rough separation into two groups, where one group of the genes is tentatively associated with mesenchymal subtype and shorter survival and showing higher expression for most immune evasion genes. However, a heterogeneity of immune evasion signatures was identified within and across subtypes. Transcripts related to antigen presentation, EZH2, and LDHA varied significantly between GBM subtypes (p &amp;lt; 0.05). CONCLUSION Gene expression analysis has utility to identify specific mediators of immune evasion and to inform the selection of combination therapies for discrete subsets of patients. A Bayesian approach to patient selection for specific immunotherapy strategies may enhance the likelihood of successful implementation of immunotherapy in the clinic.

TMOD-11. APPLICATION OF A NOVEL TRANSGENIC AND SYNGENEIC MOUSE MODELS OF HUMAN CLASSICAL GLIOBLASTOMA FOR DRUG SCREENING AND PRE-CLINICAL TRIALS

Abstract Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with 1 year median survival. The classical subtype of GBM occurs in about 10% of GBM cases and has the worst clinical outcome. The most frequent genetic alteration associated with classical GBM is amplification in the epidermal growth factor receptor (EGFR) and homozygous deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A). We generated a transgenic mouse harboring EGFRvIII, a constitutively active mutant of EGFR, knock in and homozygous deletion in p19 ARF, an alternative spliced form of the CDKN2a gene, restricted to glial cells. Transgenic mice develop tumors as early as 1 month and 73% of mice die by 6 months of age. MRIs reveal that mice develop infiltrative tumor cells throughout the brain and craniospinal junction with some infiltration within the leptomeninges that can result in hydrocephalus. Histology shows that 72% of tumors are high grade gliomas and some tumors demonstrate classical histological features of human GBM such as pseudopallisading necrosis, hyper vascularity and cellular atypia. We generated primary cell lines from transgenic mice that retained EGFRVIII expression and P19 ARF deletion and formed allografts in syngeneic mouse brains. Epigenetic modifications are considered a key mechanism in GBM development. We have therefore evaluated the efficacy of a panel of 42 compounds that target various epigenetic proteins including the readers, writers, and editors of several histone marks available from the Structural Genomics Consortium in primary cell lines derived from our mouse model. The primary cell lines were exposed to epigenetic drugs showing different responses to the same epigenetic modifiers. Key candidate drugs will further be tested pre-clinically in our syngeneic mouse model. In summary, we developed a novel transgenic and syngeneic mouse model for human classical GBM that can be used for drug screening and pre-clinical trials.

TAMI-56. HYPOXIA-DRIVEN NECROSIS DRIVES DYNAMIC TUMOR PROGRESSION IN GLIOBLASTOMA

Abstract The onset of necrosis correlates with aggressive malignant progression in solid tumors. Whether necrosis arises from or promotes accelerated tumor proliferation and progression remains unknown, primarily due to inadequate models systems that fail to induce or capture these dynamic changes as they develop. In glioblastoma (GBM; WHO grade IV), the most malignant primary brain tumor, vascular pathology and central necrosis precedes rapid, radial expansion resulting in patient mortality. While genetic alterations in GBM have been highly characterized, biological adaptations arising from sustained hypoxia/anoxia require further mechanistic investigation. This sustained nutrient deprivation leads to necrosis which dramatically changes the tumor microenvironment (TME). To reveal the dynamic temporal and spatial changes promoting expansive progression, we are generating mouse models that more appropriately capture events found in human gliomas, accounting for unique microenvironmental stressors often lacking in GBM animal models, specifically central necrosis. Combining hypoxia-induced focal necrosis within high grade gliomas with intravital microscopy captures TME restructuring to understand its impact on glioma progression. Our studies use both genetically characterized patient-derived orthotopic GBM xenografts, alongside an immunocompetent RCAS/tv-a model, to determine how antagonizing these processes impacts disease progression and outcomes across multiple GBM subtypes. Complementary to this, we are investigating how increasing hypoxia and necrosis-driven signaling events promotes tumor-associated macrophages (TAMs) influx and reprogramming during glioma progression. Our preliminary data indicate substantial differences pre- and post-necrosis regarding TAM enrichment, immunosuppressive phenotypic conversion and their biological impact, however these mechanisms have not yet been elucidated. Our models capture glioma growth dynamics, reactive neuroinflammation and therapeutic resistance, facilitating innovative pre-clinical interventions to improve patient outcome.

Secretory Pathway Kinase FAM20C, a Marker for Glioma Invasion and Malignancy, Predicts Poor Prognosis of Glioma.

PurposeGlioblastoma (GBM) is the most lethal primary cancer in adult central nervous system, and new strategies are desperately needed. The secretory pathway kinase or kinase-like proteins (SPKKPs) have been shown to mediate multiple physiological functions by phosphorylating extracellular proteins and proteoglycans. However, their roles in cancers, especially GBM, remain poorly defined.MethodsThe least absolute shrinkage and selection operator (LASSO) regression was employed for establishing the SPKKPs signature for IDH wild type (wt) GBM prognosis. Integrative analyses with multiple datasets were employed to identify the core member of this gene family in glioma. The receiver operator characteristic (ROC) curves and immunohistochemistry were further used for evaluating its association with progressive malignancy in glioma and GBM patients’ survival, respectively. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to interpret its functions in GBM, which were further verified in vitro.ResultsA SPKKPs classifier was constructed with 3 genes of this family. This signature could effectively distinguish IDH wt GBM survival. Family with sequence similarity 20 C (FAM20C) was further identified as the core member of this family in glioma. Elevated FAM20C expression was not only closely correlated with glioma malignancy progression and the mesenchymal subtype of GBM but also indicated unfavorable survival of GBM patients. FAM20C was also found to be associated with the disrupted immune response in GBM microenvironment and was required for the migration of glioma and immune cells.ConclusionThese data indicate that the potential of FAM20C serving as a predictive molecule and a therapeutic target for GBM.

A simplified integrated molecular and immunohistochemistry-based algorithm allows high accuracy prediction of glioblastoma transcriptional subtypes

AbstractGlioblastomas (GBM) can be classified into three major transcriptional subgroups (proneural, mesenchymal, classical), underlying different molecular alterations, prognosis, and response to therapy. However, transcriptional analysis is not routinely feasible and assessment of a simplified method for glioblastoma subclassification is required. We propose an integrated molecular and immunohistochemical approach aimed at identifying GBM subtypes in routine paraffin-embedded material. RNA-sequencing analysis was performed on representative samples (n = 51) by means of a “glioblastoma transcriptional subtypes (GliTS) redux” custom gene signature including a restricted number (n = 90) of upregulated genes validated on the TCGA dataset. With this dataset, immunohistochemical profiles, based on expression of a restricted panel of gene classifiers, were integrated by a machine-learning approach to generate a GliTS based on protein quantification that allowed an efficient GliTS assignment when applied to an extended cohort (n = 197). GliTS redux maintained high levels of correspondence with the original GliTS classification using the TCGA dataset. The machine-learning approach designed an immunohistochemical (IHC)-based classification, whose concordance was 79.5% with the transcriptional- based classification, and reached 90% for the mesenchymal subgroup. Distribution and survival of GliTS were in line with reported data, with the mesenchymal subgroup given the worst prognosis. Notably, the algorithm allowed the identification of cases with comparable probability to be assigned to different GliTS, thus falling within overlapping regions and reflecting an extreme heterogeneous phenotype that mirrors the underlying genetic and biological tumor heterogeneity. Indeed, while mesenchymal and classical subgroups were well segregated, the proneural types frequently showed a mixed proneural/classical phenotype, predicted as proneural by the algorithm, but with comparable probability of being assigned to the classical subtype. These cases, characterized by concomitant high expression of EGFR and proneural biomarkers, showed lower survival. Collectively, these data indicate that a restricted panel of highly sensitive immunohistochemical markers can efficiently predict GliTS with high accuracy and significant association with different clinical outcomes.The authors developed a novel simplified assay for glioblastoma transcriptional classification on formalin-fixed-paraffin-embedded tissue samples. On such dataset, immunohistochemical profiles, based on expression of a restricted panel of gene classifiers, were integrated by machine learning approach to generate a glioblastoma transcriptional signature based on protein quantification that allowed to efficiently assign transcriptional subgroups to an extended cohort. Correlations with both histopathological features and clinical outcome have been also performed.

Abstract 1917: The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma

Abstract Glioblastoma (GBM) is an incurable form of brain cancer with a median survival of ~15 months. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP) represents a significant clinical discovery as these patients have an enhanced survival, with a median survival of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2) which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases. This mutation occurs early in gliomagenesis and further results in aberrant DNA methylation and widespread transcriptional repression. Histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain (MBD) proteins, so we hypothesized that IDH mutant GBM exhibits enhanced reliance on HDAC activity, which is functionally significant to their cell proliferation and survival. Using a panel of 6 patient-derived cell lines grown in defined growth media, we show that G-CIMP is significantly more sensitive in vitro to the clinically approved pan histone deacetylase (HDAC) inhibitor panobinostat, with IDH mutant GBM cells exhibiting ~4 and ~10 fold lower IC/EC50 values in flow cytometric cell viability and apoptosis assays compared to IDH wild type (WT) GBM cells, respectively. For IDH mutant GBM, the average IC50 value was 13.5 nM for cell viability assays and the average EC50 value was 4.8 nM for apoptosis assays. Induction of cleaved caspase 3, a marker of apoptosis, was observed only in IDH mutant GBM cells when exposed to 10 nM panobinostat over 5 days. Analysis of proliferation via BrdU incorporation assays show that treatment with 10 nM panobinastat for 48 hours results in a 90% reduction in proliferation of IDH mutant GBM compared to only 25% reduction in IDH WT GBM cells. Molecular analysis via western blot shows that various acetylated chromatin marks, i.e. H3K14/18/27Ac, are preferentially upregulated in our IDH mutant GBM cell lines when exposed to 10 nM panobinostat for 48 hours. This was also supported by ELISA data showing that IDH mutant GBM cells have ~2 fold increase in histone H3 acetylation compared to IDH WT cells in response to 10 nM and 50 nM panobinostat. These data led us to perform a HDAC activity assay with nuclear extracts from our IDH WT and mutant GBM cells, but this assay showed no significant difference in basal HDAC activity. Overall, our results support a hypothesis where IDH mutant GBMs are more sensitive to panobinostat via preferential uptake of the drug. These data ultimately suggest that G-CIMP tumor cells display a profound sensitivity to the cytotoxic and antiproliferative effects mediated by exposure to the HDAC inhibitor panobinostat. These studies provide a strong foundation for future preclinical work evaluating the use of HDAC inhibitors as a personalized cancer therapy to treat G-CIMP. Citation Format: Thomas K. Sears, Kevin D. Woolard. The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1917.

Abstract 3780: Cerebrospinal fluid-responsive factor SERPINA3 induces an increase in the malignancy of glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with a median survival of 14 months despite multimodal treatment. Patients with GBM tumors exhibit a post-resection recurrence rate of almost 100%. The tumor recurrence and treatment resistance properties of GBM reside in a stem-like cell population called Brain Tumor Initiating Cells (BTIC). Among primary GBMs, periventricular tumors exhibit worse survival than tumors distal to the lateral ventricles (LV), the cause for this worse outcome is not known. A potential explanation is the proximity of these tumors to the cerebrospinal fluid (CSF) and the neurogenic niche in the sub-ventricular zone (SVZ). Chemotactic signals present in the cerebrospinal fluid (CSF) regulate neural progenitor cells migratory response and differentiation profile. Our preliminary data show that cancer-derived CSF induces an increase in the migration and proliferation of GBM cells when compared to non-cancer CSF. We evaluated the transcriptome of human GBM cells in response to cancer and non-cancer CSF and identified alpha-1-antichymotrypsin (SERPINA3) as one of the genes that show higher over expression upon cancer CSF-stimulation. SERPINA3 is a circulating anti-protease that is produced and released in different tissues such as liver, prostate, lungs, endometrium and brain that is over-expressed in multiple cancers. We hypothesize that the overexpression of SERPINA3 in response to CSF is a contributing factor to the increase malignancy of periventricular GBM tumors. We tested this hypothesis in silico, in vitro and in vivo. Using TCGA and Rembrandt databases we observed a higher expression of SERPINA3 in brain tumor tissue compared to controls with no differences among GBM subtypes. In GBM patients, expression of SERPINA3 negatively affects survival expectancy. In vitro, Using intraoperative samples, we confirmed that SERPINA3 is overexpressed in brain cancer tissue by western-blot and qRT-PCR. Interestingly, we observed a higher nuclear localization of SERPINA3 in cancer tissue and cells. To determine if SERPINA3 has an effect on the malignancy of GBM cells, we silenced its expression using shRNA. SERPINA3 silencing induced a decrease in cell proliferation (p&amp;lt;0.05), as well as migration (p&amp;lt;0.05), and invasion (p&amp;lt;0.05). Remarkably CD133 expression and stem cell markers were decreased when SERPINA3 is silenced. In contrast, the addition of recombinant SERPINA3 induced an increase in cell migration (p&amp;lt;0.05) of GBM cells. Moreover, cancer CSF is no longer able to induce an increase in GBM cell migration in SERPINA3-KD cells (p&amp;lt;0.05). In vivo, mice orthotopically-injected with SERPINA3-KD cells showed a prolonged overall survival (p&amp;lt;0.05). In summary, our results support the hypothesis that SERPINA3 plays a key role in GBM malignancy in response to CSF and its inhibition results in a better outcome using GBM preclinical models. Targeting the CSF-GBM interaction is a potential therapeutic approach that remains to be explored. Citation Format: Montserrat A. Lara Velazquez, Natanael Zarco, Anna Carrano, Jordan Phillips, Paula Schiapparelli, Emily S. Norton, Stephanie Jeanneret, Teresita Corona, Jose Segovia, Kaisorn Chaichana, Yan Asmann, Alfredo Quinones Hinojosa, Hugo Guerrero-Cazares. Cerebrospinal fluid-responsive factor SERPINA3 induces an increase in the malignancy of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3780.

Abstract 1113: Evaluation of novel therapeutics using models from the Mayo Clinic GBM patient-derived xenograft (PDX) collection

Abstract The Mayo Clinic collection of patient-derived xenograft (PDX) models has been developed from the direct implantation of fresh glioblastoma samples into immunodeficient mice. Over 95 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated clear infiltration of tumor cells into the brain parenchymal in 90% of PDXs with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed that our collection of PDXs captures the genetic heterogeneity of GBM, with the majority of known alterations represented at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations were the most frequent overall (86%) followed by homozygous deletion of CDKN2A (70%). Multiple alterations were identified in the p53 pathway, including TP53 mutations (36%) and MDM2/4 (10%/2%) amplification. PI3-kinase pathway alterations mainly involved PTEN (48%) while MAP kinase pathway alterations were present in a subset of PDXs (NF1 alterations 17%, BRAF 4%). IDH mutations were found in only two models. Gene expression analysis revealed that our PDX collection includes all GBM subtypes with classical being the most represented (46% vs mesenchymal 31% and proneural 23%). MGMT promoter methylation was also observed in 45% of PDXs. The high fidelity of PDX models to their human counterpart makes these models ideal for testing standard of care and novel targeted therapies. To assess if PDXs are predictive of patient treatment response, 37 orthotopic PDX lines were treated with radiation (RT), temozolomide (TMZ), or concurrent RT/TMZ. Across all PDXs, RT and TMZ demonstrated median survival extension of 90 and 46 days relative to vehicle. Concurrent RT/TMZ further extended survival by 146 days. MGMT promoter methylation was strongly associated with response to TMZ-based strategies but not RT alone.This observation suggests that our PDX collection possesses clinically relevant predictive value. To date, we have characterized treatment responses to over 135 regimens including both GBM standards of care and targeted strategies in our PDX models. Recent lab efforts strive to define the effect of blood brain barrier disruption on drug delivery. With over 20 years of experience, our group has extensive expertise in the development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Since 2016, we have shared over 5200 individual samples (tumor tissue, cells, DNA/RNA, protein extracts, tissue microarray slides) in both internal and external collaborators. Our models have been utilized in over 41 NIH funded grants, including 33 funded RO1s, as well as by the pharmaceutical community with 50 working relationships to date (20 contracts, 30 collaborations). Finally, our PDX collection has been used in over 120 peer-reviewed published manuscripts. Citation Format: Danielle Burgenske, Ann C. Mladek, Rachael A. Vaubel, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Evaluation of novel therapeutics using models from the Mayo Clinic GBM patient-derived xenograft (PDX) collection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1113.

Glutamine uptake and utilization of human mesenchymal glioblastoma in orthotopic mouse model

BackgroundGlioblastoma (GBM) are highly heterogeneous on the cellular and molecular basis. It has been proposed that glutamine metabolism of primary cells established from human tumors discriminates aggressive mesenchymal GBM subtype to other subtypes.MethodsTo study glutamine metabolism in vivo, we used a human orthotopic mouse model for GBM. Tumors evolving from the implanted primary GBM cells expressing different molecular signatures were analyzed using mass spectrometry for their metabolite pools and enrichment in carbon 13 (13C) after 13C-glutamine infusion.ResultsOur results showed that mesenchymal GBM tumors displayed increased glutamine uptake and utilization compared to both control brain tissue and other GBM subtypes. Furthermore, both glutamine synthetase and transglutaminase-2 were expressed accordingly to GBM metabolic phenotypes.ConclusionThus, our results outline the specific enhanced glutamine flux in vivo of the aggressive mesenchymal GBM subtype.

Abstract B17: Hyperactivating the Hippo pathway effector TAZ distorts the immune microenvironment in promoting the mesenchymal transformation in glioblastoma

Abstract Glioblastoma (GBM), the deadliest and most common primary brain malignancy in adults, is highly heterogeneous with variable prognoses and treatment responses. The mesenchymal subtype of GBM is associated with the worst prognosis and highest treatment resistance. It is still not clear how GBM mesenchymal differentiation is achieved. Hyperactivity of transcriptional coactivator with PDZ-binding motif (TAZ) was linked to the GBM mesenchymal transformation, although its role in this process remains unclear. Here, by using orthotopic xenograft mouse GBM models, we found that hyperactive TAZ in tumor cells promotes myeloid cells to infiltrate into tumors. Such enhanced myeloid cell infiltration is associated with the mesenchymal transformation and tumor progression. We demonstrated that these TAZ-induced tumorigenic properties rely on its interaction with the transcription factor, TEA domain family member (TEAD). Such interaction induces the transcription of chemokines that are able to recruit the myeloid cells. Specific depletion of the infiltrated myeloid cells is able to slow down the tumor progression. Our studies suggested that recruiting myeloid cells by hyperactive TAZ contributes the mesenchymal transformation and tumor progression in glioblastoma. Citation Format: Patricia Yee, Yiju Wei, Zhijun Liu, Wei Li. Hyperactivating the Hippo pathway effector TAZ distorts the immune microenvironment in promoting the mesenchymal transformation in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B17.

B3GNT5 is a novel marker correlated with stem-like phenotype and poor clinical outcome in human gliomas.

AimsGlioblastoma multiforme (GBM) is the most lethal tumor with a median patient survival of 14 to 15 months. Glioma stem cells (GSCs) play a critical role in tumor initiation and therapeutic resistance in GBM. B3GNT5 has been suggested as the key glycosyltransferase in the biosynthesis of the (neo‐) lacto series of glycosphingolipid. In this study, we evaluated the B3GNT5 expression in GSCs as well as the correlation with clinical data in GBM.MethodsThe mRNA levels of B3GNT5 in normal astrocytes, four glioma cell lines, and four GSCs were evaluated using real‐time PCR. Small interference RNAs (siRNAs) were used to inhibit B3GNT5 expression and analyze its ability to form neurospheres. Statistical analyses were conducted to determine the association with B3GNT5 expression and tumor grade and GBM subtypes as well as patient survival using public datasets.Results B3GNT5 expression was significantly elevated in GSCs compared with normal astrocytes, glioma cell lines, and their matched differentiated tumor cells. Knockdown of B3GNT5 in GSCs decreased the neurosphere formation. Patients with high B3GNT5 expression had a short overall survival. B3GNT5 is correlated with classical and mesenchymal GBM subtypes.ConclusionThe findings suggest the central role of B3GNT5 in regulating malignancy of GBM.