OTEH-11. Single Cell RNA Sequencing to identify cellular heterogeneity with in Pituitary Adenomas

Abstract Pituitary adenomas (PA) are one of the most common primary brain tumors and comprise approximately 15% of brain neoplasms. Most PA are histologically benign, but can cause significant morbidity. Previous studies utilized whole genome and exome sequencing to identify a few somatic variants, but no recurrent mutations were observed. Further studies are warranted to identify driver mutations occurring at low frequencies. We used single-cell RNA sequencing (10X Genomics) to investigate cellular heterogeneity in 12 non-functioning pituitary adenomas. Our analysis identified discrete clusters of cells associated with specific functional pathways. One of these clusters corresponded to cells expressing genes related to metabolic pathways, primarily lipid metabolism. Another cluster consistent amongst the three patients comprised cells involved in antigen presentation and processing. In addition, the copy number variation analysis highlighted distinct chromosomal alterations within our samples. Interestingly, we were able to identify clonal variations within each tumor based on chromosomal aberrations. For example, in our first patient we observed a gain of chromosome 19 and loss of chromosome 2. Our analysis showed three different clonal populations within this tumor. All three populations harbored the loss of chromosome 2, one population exhibited gain of chromosome 19, while a third population exhibited loss of chromosome 19. These early results indicate the loss of chromosome 2 as an early event in tumorigenesis and gain/loss of chromosome 19 as late events. We are currently in a process of identifying somatic variations within these tumors by variant calling. Currently we are expanding our analysis to 20 non-functional PA. Mapping the single-cell gene expression profiles with mutational phylogeny will reveal the differences in clonal evolution within the tumor subtypes. This study will help us define the molecular fingerprint of pituitary adenomas and provide insights which could be utilized in the clinic for better management of these tumors.

The great phenotypic heterogeneity of glioblastoma (GBM) -both inter and intratumorally -has hindered therapeutic efforts. While genome-based molecular subtyping has revealed that GBMs may be parsed into several molecularly distinct categories, this insight has not translated to a significant extension of patient survival. We hypothesize that, rather than gene expression as a whole, analysis of targetable pathways could yield important insights into the development of novel classification schemes and, most importantly, to targeted therapeutics. Here, we interrogated tumor samples using a pathway-based approach to resolve tumoral heterogeneity. The Cancer Genome Atlas samples were clustered using gene set enrichment analysis and the resulting 3 clusters were informative of patient survival and only modestly overlapped with prior molecular classification. We validated our approach by generating gene lists from common elements found in the top contributing genesets for a particular cluster and testing the top targets in appropriate gliomasphere patient-derived lines. Samples enriched for cell cycle related genesets showed a decrease in sphere formation capacity, proliferation and in vivo tumor growth when E2F1, our top target, was silenced. Consistent with our theory, E2F1 knockdown had little or no effect on the growth of the non-enriched lines, despite their ability to proliferate in vitro and in vivo. We similarly analyzed single cell RNAseq datasets and correlated cell cycle and stemness signatures with the gene lists we generated, concluding that cells with stem cell signatures were depleted of E2F1 and its downstream targets. Finally, we confirmed a connection between E2F1 and cellular inhibitor of PP2A (CIP2A) in a cluster of samples. Loss of function studies reveal a diminished capacity for DNA damage regulation in E2F1 activated samples. Our studies relate inter-and intratumoral heterogeneity to critical cellular pathways dysregulated in GBM, with the ultimate goal of establishing a pipeline for patient-and tumor-specific precision medicine. 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. Gliomas are the most common malignant brain tumor, have an aggressive behavior, and invariably relapse and progress. Despite the recent advancements, little is known about the role of the epigenome in glioma disease progression and recurrence. To investigate the molecular dynamics over time and in response to therapeutic pressures, the Glioma Longitudinal AnalySiS (GLASS) Consortium, a multinational collaboration, is investigating epigenome-wide molecular data from primary and recurrent matched pairs, including IDH mutant (IDHmut) and IDH wildtype (IDHwt) gliomas. We have compiled a total of 357 samples comprising 143 primary-recurrent pairs profiled by DNA methylation, of which 157 samples have genomic data (WXS/WGS) and 120 have transcriptomic data (RNAseq). IDHwt gliomas have a distinct epigenetic evolution compared to IDHmut after treatment. IDHwt gliomas are more epigenetically stable over time, while IDHmut gliomas display a loss of DNA methylation throughout disease progression. Next, we investigated the molecular drivers of longitudinal gliomas by integration of DNA methylation and gene expression data. We identified epigenetic activation of cell cycle pathways in recurrent IDHmut compared to initial tumors. Transcription factors musculin, ZNF367, and ZNF682 are enriched among recurrent IDHmut gliomas and potentially regulate IDHmut recurrence and/or progression. We next used a DNA methylation-based deconvolution approach to estimate the tumor microenvironment (TME) composition. We found that the TME among IDHmut subtypes (Codel, GCIMP-high, and GCIMP-low) presented less immune infiltration than IDHwt (Classic-like, Mesenchymal-like, and PA-like). Post-treatment, we found a decrease of CD4+T and an increase of CD8+T cells in IDHmut. In conclusion, IDHmut gliomas present a more unstable epigenome, while the epigenome of IDHwt gliomas seems relatively preserved after treatment. We identified potential master regulators of cell cycle deregulation of IDHmut recurrence. Finally, the TME differs across IDHmut and IDHwt gliomas and the cell composition changes over time.

OTEH-11. SINGLE CELL RNA SEQUENCING TO IDENTIFY CELLULAR HETEROGENEITY WITH IN PITUITARY ADENOMAS
Saket Jain, Husam Babikir, karin Shamardani, Aaron Diaz, Manish Aghi; University of California San Francisco, San Francisco, CA, USA Pituitary adenomas (PA) are one of the most common primary brain tumors and comprise approximately 15% of brain neoplasms. Most PA are Abstracts ii13 NEURO-ONCOLOGY ADVANCES • JULY 2021 histologically benign, but can cause significant morbidity. Previous studies utilized whole genome and exome sequencing to identify a few somatic variants, but no recurrent mutations were observed. Further studies are warranted to identify driver mutations occurring at low frequencies. We used single-cell RNA sequencing (10X Genomics) to investigate cellular heterogeneity in 12 non-functioning pituitary adenomas. Our analysis identified discrete clusters of cells associated with specific functional pathways. One of these clusters corresponded to cells expressing genes related to metabolic pathways, primarily lipid metabolism. Another cluster consistent amongst the three patients comprised cells involved in antigen presentation and processing. In addition, the copy number variation analysis highlighted distinct chromosomal alterations within our samples. Interestingly, we were able to identify clonal variations within each tumor based on chromosomal aberrations. For example, in our first patient we observed a gain of chromosome 19 and loss of chromosome 2. Our analysis showed three different clonal populations within this tumor. All three populations harbored the loss of chromosome 2, one population exhibited gain of chromosome 19, while a third population exhibited loss of chromosome 19. These early results indicate the loss of chromosome 2 as an early event in tumorigenesis and gain/loss of chromosome 19 as late events. We are currently in a process of identifying somatic variations within these tumors by variant calling. Currently we are expanding our analysis to 20 non-functional PA. Mapping the single-cell gene expression profiles with mutational phylogeny will reveal the differences in clonal evolution within the tumor subtypes. This study will help us define the molecular fingerprint of pituitary adenomas and provide insights which could be utilized in the clinic for better management of these tumors.

OTEH-12. ASSESSING ADAPTIVE RESPONSES TO LOSS OF EXTRACHROMOSOMAL DNA AMPLIFICATION
Artem Berezovsky 1,2 , Indrani Datta 1 , Ruicong She 1 , Laura Hasselbach 1 , Laila Poisson 1 , Ana C. deCarvalho 1,2 ; 1 Henry Ford Health System, Detroit, MI, USA. 2 Wayne State University, Detroit, MI, USA BACKGROUND: Oncogene activation through somatic gene amplification happens frequently in GBM, with over 70% of these tumors presenting amplification of at least one putative driver gene, oftentimes in small extrachromosomal circular DNA segments composed of chromatin (ecDNA). A molecularly diverse and representative panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX), which retain the original genomic abnormalities and ecDNA amplifications, was employed to assess adaptive response to the absence of ecDNA amplification. METHODS: We have isolated ecDNA negative cell populations from two patient-derived models. HF3035 harbors a MET amplification and HF3253 harbors a PDGFRA constitutively active genomic rearrangement and extrachromosomal amplification. We conducted paired, whole RNA-sequencing on 20 HF3253 populations (ecDNA+/-: 6 clones from 3 biological replicate PDXs and 4 clones from 4 in vitro technical replicates) and 12 HF3035 population (ecDNA+/-: 6 clones from 3 biological replicate PDXs). RESULTS: Nonparametric differentially expressed gene (DEG) analysis using NOISeqBio (R/Bioconductor), identified 564 differentially expressed genes (482 upregulated in ecDNA(-)) employing a stringent false discovery rate of 0.05. Genes significantly associated with PDGF stimulation, central carbon metabolism, and H3K27me3 were downregulated in ecDNA(-), while genes significantly associated with astrocytic processes, neuronal differentiation, and EGFR signaling were upregulated in ecDNA(-) (EnrichR). We employed an additive linear model with PDX serving as a blocking factor to compare ecDNA+ and ecDNA-populations in both models (R/edgeR). 2071 genes were upregulated in ecDNA+ PDX specimens and 2365 genes were downregulated. Specifically, E2F targets were highly enriched in ecDNA+ populations, in addition to mRNA pre-processing. ecDNA loss primarily targeted glycogen metabolism, NTRK signaling, and inositol phosphate catabolism. CONCLUSIONS: We have identified PDX-specific and non-specific features to an adaptive response to the loss of ecDNA amplification. Notably, a signature adaptation is an upregulation of seemingly redundant receptor tyrosine kinases.

OTME-1. TAMEP ARE BRAIN TUMOR PARENCHYMAL CELLS CONTROLLING NEOPLASTIC ANGIOGENESIS AND PROGRESSION
Roland Kälin 1 , Linzhi Cai 1 , Yuping Li 1 , Louisa von Baumgarten 1 , Christian Schulz 1 , Ines Hellmann 2 , Rainer Glass 1 ; 1 University Clinics Munich, Munich, Bavaria, Germany. 2 Ludwig Maximilians University, Munich, Munich, Bavaria, Germany Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote spe-cific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression. Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most common primary brain tumor in adults. GBM has the poorest prognosis and no efficient cure to date due to diffusive growth into the surrounding brain, preventing complete surgical resection and leading to inevitable tumor relapse. Tumor microenvironment (TME) of GBM contains brain-residing microglia and bone-marrow derived macrophages (collectively known as glioma-associated microglia/macrophages, GAMs) that constitute up to 30% of the tumor mass and promote tumor invasion. Hypoxia (a shortage of oxygen) is a key factor in tumor progression of GBM as it can globally and rapidly alter the gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can enhance the protumorigenic function of GAMs, e.g. by inducing expression of cytokines and cell surface receptors both in GAMs and glioma cells, but little is known about chromatin alterations of GBM under hypoxia. Since regulation of expression of such molecules could depend on the epigenetic alterations, we hypothesize that hypoxia may potently alter the chromatin accessibility and functions of GAMs and glioma cells. We determine the genome-wide changes in chromatin accessibility in GAMs and glioma cells in response to hypoxic stress using single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing), which allows simultaneous genome-wide assessment of chromatin accessibility and expression of intracellular protein markers in single cells, allowing faithful selection of hypoxic and non-hypoxic cells. Secondly, we are employing an oxygen-dependent co-culture model in vitro to study the mechanisms of chromatin alterations in GAMs and glioma cells under controlled hypoxic conditions and test how these changes depend on the glioma -GAMs cross-communication. In summary, we characterize the interactions between innate immune cells and glioma cells by looking at their chromatin alterations under hypoxia.

OTME-3. DISSECTION OF THE ROLE OF STROMAL MICROENVIRONMENT AND TUMOR-TME CROSSTALK IN PEDIATRIC BRAIN CANCER
Aniello Federico 1,2 , Marcel Kool 1,2 ; 1 Hopp-Children's Cancer Center (KiTZ), Heidelberg, Germany. 2 Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany Brain tumors are the deadliest malignancies that occur during childhood and strong efforts are required to develop innovative therapeutic strategies. The intrinsic capacity of malignant cells to organize, shape and exploit the surrounding environment where they develop (tumor microenvironment, TME), has not been fully elucidated for pediatric brain cancers yet. Here, we exploited a multi-omic approach to define the TME cell populations and their contributions in the most common pediatric brain tumor entities, such as medulloblastomas and ependymomas. Analysis of single-cell RNA sequencing data of human tumors resulted in the identification of heterogeneous populations of non-malignant cells present in the TME. In particular, re-clustering and marker-based cell type assignment strategies allowed to define a broad range of immune and stromal subclasses showing distinctive expression signatures reflecting variegated functional roles. By cross-matching the tumor data with normal brain expression atlases, we could further refine the annotation of the newly identified stromal functional subpopulations and define the "tumor-associated" marker signatures of genes exclusively enriched in stromal cells within the TME, linked to immune activation, cell