bluuebunny/crossref_metadata_2025 huggingface dataset:
CNSC-19. HYPOXIA-INDUCED NEURONAL ACTIVITY IN GLIOMA PATIENTS POLARIZES MICROGLIA BY POTENTIATING RNA M6A DEMETHYLATION
Authors: Guo Xiaofan , Wei Qiu , Li Gang
Published: November 2023

Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. In this study, by applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. Here, we demonstrate that hypoxia induces glioma stem cells (GSCs) to produce glutamate, which activates local neurons. The hypoxia-induced neuronal activity promotes GBM progression by facilitating microglial M2 polarization. We found that when microglia were co-cultured with activated neurons, the level of RNA m6A methylation in microglia was impaired, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted regional microglial M2 polarization while cortical neuronal inhibition decreased regional microglial M2 polarization in GBM xenografts. Together, these findings indicate that hypoxic GSC-induced neuronal activation promotes GBM progression by polarizing microglia via decreasing their m6A methylation levels.
CNSC-21. INVESTIGATING THE EVOLUTION OF NEURON-GLIOMA CIRCUIT DYNAMICS USING AN IN VIVO IMAGING METHOD
Authors: Shamardani Kiarash , Keough Michael , Monje Michelle
Published: November 2023

Pediatric high-grade gliomas (pHGG) are aggressive primary brain neoplasms with a dismal prognosis, making them the leading cause of brain tumor-related deaths in children. pHGGs occur in specific anatomical locations at specific ages underscoring their origin in neurodevelopment and the critical importance of the brain tumor microenvironment. Activity-regulated mechanisms are major regulators of neural development and plasticity. Many pHGGs originate from oligodendroglial precursor cells (OPC) and, like OPCs, neuronal activity promotes pHGG proliferation. Recent work has demonstrated that pHGG cells form calcium-permeable AMPAR-mediated synapses with neurons analogous to the axo-glial synapses that form between neurons and OPCs. Neuronal activity drives pHGG growth through secretion of activity-regulated mitogens and through electrochemical communication with pHGG cells that integrate into neural circuits. In turn, pHGG increases neuronal excitability and remodels functional neural circuits. The interconnected network of glioma cells and neurons is fundamental to pHGG progression. However, how these neuron-glioma networks evolve over time remains to be fully understood. We hypothesize that as gliomas progress, the neuron-glioma malignant circuitry evolves chiefly through synaptogenesis to promote activity that fosters glioma progression. Thus, we developed a two-color in vivo imaging method to study neuron-glioma cell interactions over time in freely behaving mice enabling us to record the frequency, pattern, and synchronicity of calcium transients in neurons, glioma cells, and their coactivity giving us a comprehensive view of the changes in neuron-glioma circuit dynamics over time. We have observed increasing neuronal activity throughout the disease, consistent with increasing neural hyperexcitability over time, and have observed that different types of gliomas exhibit unique patterns of calcium transients (rise time, peak amplitude, decay time, and half-width). Our imaging paradigm offers a unique ability to study neuron-glioma interactions at the systems level and will allow us to study how pharmacological inhibitors and neuronal experience shape neuron-glioma malignant circuit dynamics.
CNSC-23. ROLE OF GLIAL PH-DEPENDENT METABOLIC REGULATION IN GLIOMA PROGRESSION
Authors: Ye Qi , Oh Heavin , Choy Tiffany , Madamba Christine , Serin-Harmanci Akdes , Rao Ganesh , Lee Hyun Kyoung
Published: November 2023

Malignant glioma is a devastating brain cancer with barely any improvement in prognosis over decades. One key mechanism for glioma invasiveness is the dynamic metabolic communication between glioma and surrounding cells shaping the tumor microenvironment. While extracellular acidosis is a pathological hallmark of glioma, pH-dependent mechanisms underlying tumor metabolic reprogramming remain poorly defined. Our study focuses on a glial-enriched sodium-bicarbonate cotransporter, Slc4a4, which was previously identified as intracellular and extracellular pH regulator. While Slc4a4-mediated pH regulation is important for physiological brain function and responses after injury, its role in glioma progression is largely undefined. To begin addressing this question, we analyzed The Cancer Genome Atlas (TCGA) database, finding high Slc4a4 expression correlates with prolonged survival in glioma patients. Corroboratively, Slc4a4 gain-of-function (GOF) drastically decreases tumorigenesis and angiogenesis in patient-derived-xenograft and CRISPR-mediated de novo mouse glioma models. These results are further complemented by loss-of-function (LOF) studies using glial-specific Slc4a4 knockout mice, suggesting an inhibitory role of Slc4a4 in glioma growth and associated angiogenesis. A combination of unbiased metabolomic and cancer-targeted proteomic profiling of Slc4a4 GOF/LOF mouse glioma reveals an inverse correlation between Slc4a4 and a key lipid metabolism protein, FABP5 (Fatty Acid Binding Protein 5), which is coupled with dysregulated lipid metabolism. Moreover, analysis of TCGA database shows inverse expression and survival correlation of Slc4a4 and FABP5 in glioma patients. Further functional study shows that overexpression of FABP5 reverses Slc4a4’s inhibitory effects on glioma growth in immunocompetent mouse glioma model, supporting a functional antagonism between Slc4a4 and FABP5. Together, our study indicates that Slc4a4 plays a protective role in glioma progression by regulating metabolic activities, providing critical insights into pH-dependent metabolic reprogramming in glioma.
CNSC-27. LOSS OF CAPICUA IMPAIRS OLIGODENDROGENESIS IN THE DEVELOPING MOUSE BRAIN
Authors: Pyczek Joanna , Hall Courtney F , Johnston Michael , Singhal Divya , Gallo Marco , Morrissy Sorana , Chan Jennifer
Published: November 2023

INTRODUCTION

70% of all oligodendrogliomas (ODG) harbor hemizygous loss-of-function Capicua (Cic) mutations. Cic is a transcriptional repressor downstream of MAPK signaling. Loss of Cic in neural precursors has been linked to increased proliferation and developmental bias towards differentiation-incompetent oligodendrocyte precursor cells (OPCs). This study aims to elucidate the consequences of OPC-specific Cic loss for oligodendroglia development and assess its contribution to ODG formation.

METHODS

Pdgfra-CreERT2;Cicfl/+;Rosa26EYFP (heterozygote control, Cic-HET) and Pdgfra-CreERT2;Cicfl/fl;Rosa26EYFP (homozygous knockout, Cic-KO) mice were used to induce Cic deletion and EYFP expression in OPCs during the postnatal wave of oligodendrocyte production (P3+P5). Double labeling with BrdU at P6 and EdU 6 hours prior to experimental endpoint was used to birthdate and assess active proliferation, respectively. Cic knockout was confirmed on the genomic level by qPCR. Formalin-fixed mouse brains were analyzed at 7-, 14- and 28-days post injection (dpi) for expression of proliferation and oligodendrocyte stage-specific markers by multiplex immunofluorescent staining. FACS-sorting was used to enrich for recombined cells based on EYFP expression. The 10x Genomics platform was used to analyze the single cell transcriptome (scRNAseq) of Cic-KO vs Cic-HET brains at designated time points.

RESULTS

7dpi Cic-KO brains displayed an increased percentage of Pdgfra+ OPCs (25.76% KO vs. 15.95% HET) and a decreased percentage of CC1+ mature oligodendrocytes (27.69% KO vs. 33.93% HET) compared to heterozygote control. Cic-KO OPCs showed an increased proliferation rate as demonstrated by higher frequency of Ki67+ (3.16% HET vs 7.26%KO) and EdU+ (22.47% HET vs 44.44% KO) cells. scRNAseq revealed upregulation of genes involved in “nervous system development” and downregulation of “myelination” program upon Cic knockout.

CONCLUSION

We showed that OPC-specific Cic knockout results in increased cellular proliferation and maturation defects in the developing mouse brain. Cic loss may contribute to ODG formation by promoting developmental programs and inhibiting oligodendrocyte maturation in postnatal OPCs.
CNSC-30. CD4 T CELLS MEDIATED UPREGULATION OF CCL4 CHEMOKINE DRIVES GLIOBLASTOMA TUMOR MICROENVIRONMENT REPROGRAMMING FOLLOWING TREATMENT WITH A NOVEL MRNA VACCINE
Authors: Chauhan Anjali , Ebrahim Ghaidaa , Dastmalchi Farhad , McGrath Kyle , Thomas Nagheme , Takacs Gregory Pal , Harrison Jeffrey , Frain Matthew , Mendez-Gomez Hector , Sayour Elias , Subramaniam Vignesh , Angelini Thomas , Von Roemeling Christina , Mitchell Duane , Rahman Maryam
Published: November 2023

INTRODUCTION

Intra-tumoral immunosuppression is a major cause of treatment failure for patients with glioblastoma (GBM). Our group has developed a proprietary vaccine that combines mRNA encapsulated in lipid nanoparticles with PEG hydrogel and a chemokine (CXCL9). This hydrogel-chemokine-mRNA (HCM) vaccine is delivered subcutaneously (SQ) and results in significant anti-tumor efficacy in syngeneic murine glioma models. The objective of this study was to evaluate the effects of the vaccine on the tumor microenvironment (TME).

METHODS

C57BL/6 mice underwent intracranial implantation of KR158-luc tumor cells and underwent SQ injection of the HCM vaccine using total tumor mRNA. Tumors were collected for analysis. Q-PCR and flow cytometry were done to evaluate the differential gene expression and CCR5 expression (receptor for CCL4) in the TME.

RESULTS

CCL4 was found to be upregulated in tumor-bearing animals treated with HCM vaccine using PCR. In a separate experiment, CCL4 blockade was given prior to HCM vaccination and this resulted in abrogation of the survival benefit of the vaccine. Next, flow cytometry was performed and microglia in the TME had the highest expression of the CCR5 receptor. CD4 T cells in the TME had significantly increased CCL4 expression after treatment with HCM vaccine.

CONCLUSION

The HCM vaccine results in significant anti-tumor efficacy in murine syngeneic murine glioma which is dependent on upregulation of CCL4 in the TME driven by CD4 T cells. The CCL4 exerts its effect through microglia expressing CCR5 in the TME. Further analysis of the effects of vaccination on microglia phenotype and function are underway.
CNSC-35. EVALUATING THE THERAPEUTIC POTENTIAL OF NOVEL NTRK AGONISTS AND ANTAGONISTS IN HIGH-RISK NEUROBLASTOMA
Authors: Johnson David , Schlichthaerle Thomas , Pattwell Siobhan , Baker David
Published: November 2023

Neuroblastoma is the most common extra-cranial tumor diagnosed in children within the first two years of their lives. Although low-risk and intermediate -risk neuroblastoma patients have a survival rate of 90-95%, high-risk neuroblastoma patients have a survival rate of approximately 50%. To improve the survival rate in high-risk patients, our group focuses on tropomyosin receptor kinases (TRKs), which are family of tyrosine kinases that have been heavily implicated in crucial developmental processes, such as differentiation, that are typically hijacked in cancer. Using de novo agonists specifically designed against three different neurotrophin receptors (TrkA, TrkB, TrkC), our group is investigating the fundamental biological role of TRK receptors in mediating tumorigenesis in neuroblastoma. Preliminary results show that our TrkA agonist not only increase both ERK and AKT activity but also causes a significant decrease in TrkA expression in several neuroblastoma cell lines which we hypothesize is due to constitutive activation of TrkA, leading to its internalization and degradation. Phenotypically, we observe that neuroblastoma cell line SK-N-SY5Y, which is typically used in differentiation studies, lose their neuroblast-like phenotype when treated with TrkA agonist, and begin to exhibit neurite outgrowth which may be indicative of differentiation-associated pathways being activated due to TrkA activation. Using a combination of de novo protein design, immunoblotting, RNA-sequencing, and in vivo studies to characterize the effect(s) TRK agonists have on neuroblastoma, we hope to assess whether targeting and modifying the activity of TrkA, and other Trk receptors such as TrkB and TrkC, holds new therapeutic promise for patients with high-risk neuroblastoma.
CNSC-36. INDOLENT INTRAVASCULAR LARGE B-CELL LYMPHOMA (IVLBCL) ORIGINALLY PRESENTING AS A STROKE MIMIC
Authors: Cabrera Nynoshka Bassatt , Philip Karan , Nasany Ruham Alshiekh
Published: November 2023

BACKGROUND

IVBCL is a rare subtype of extranodal diffuse large B-cell lymphoma, characterized by the growth of neoplastic cells in blood vessels with no other apparent extravascular mass, making diagnosis a challenge. It usually has an aggressive and fast-paced course; however, there is limited data regarding incidence due to its rarity.

CASE

A 73 year-old woman presented with progressive gait worsening a year prior to presentation to our institution. Her symptoms then developed to include left-sided hemiparesis with associated headaches, warranting a local emergency department visit. Brain magnetic resonance imaging (MRI) without contrast showed a Flair hyperintense lesion in the right MCA territory with restricted diffusion, assumed to be an ischemic stroke, and she was started on stroke prevention measures. The patient continued to decline, becoming wheelchair-bound and developing short-term memory loss, chronic headaches, and pseudobulbar affect. Repeat brain MRI without contrast five months later showed progression of the Flair hyperintensity to the left hemisphere with restricted diffusion. Brain MRI with contrast followed, revealing subtle enhancement. Vessel imaging was unremarkable, and cerebrospinal fluid (CSF) analysis was normal with negative cytology and flow cytometry, yet elevated beta-2-microglobulin at 3.25 mcg/mL. A year after symptoms onset, the patient presented to our Neuro-Oncology clinic. Repeat Brain MRI with contrast and spectroscopy showed progression on the left-sided lesion with persistent restriction diffusion and enhancement, prompting a biopsy. Pathology confirmed large B cell lymphoma. PET-CT body scan showed no evidence of systematic disease. The patient was treated with Rituximab, Methotrexate, Vincristine, and Procarbazine (RMVP) with complete response demonstrated by complete resolution of the diffusion restriction and symptoms improvement.

CONCLUSIONS

Although IVLBCL is known for being a fast progressive disease, we present a case of indolent course delaying diagnosis. Unexplained progressive neurological symptoms with restricted diffusion on MRI should prompt consideration for this diagnosis.
CNSC-41. DOES ISCHEMIC STROKE DRIVE BRAIN TUMOR PROGRESSION?
Authors: Ye Qi , Madamba Christine , Khatri Latha , Yang Yuhui , Rao Ganesh , Lee Hyun Kyoung
Published: November 2023

Glioblastoma multiforme is the most common and lethal primary brain tumor, with a 5-year survival rate of less than 5%. Its complex pathophysiology, highlighted by massive inflammatory responses, enhances tumor progression and impedes therapeutic interventions, leading to its dismal prognosis. Inflammatory responses are also a cardinal feature of brain injury and remarkably, clinical evidence from large-scale patient cohort studies indicates a link between glioma and cerebral ischemic stroke, where approximately 10% of ischemic stroke patients develop glioma. This observation suggests that brain injury may be a risk factor for the development of brain tumors; however, whether and how stroke occurrence promotes brain tumor progression is unknown. To address this knowledge gap, we employed a photothrombotic stroke mouse model in conjunction with established glioma models. In these models, we observed a significant increase in tumor cell proliferation and infiltration towards the infarct region and blood-brain barrier disruption. Intriguingly, spatial transcriptomic analysis in stroke-glioma models highlighted altered pH regulation in the microenvironment at the leading edge of tumor cells near stroke lesions. Further analysis revealed that the astrocytic pH regulator, Slc4a4, is significantly downregulated in the infiltrating tumor cells near infarct lesions, and that Slc4a4 inhibits tumor progression in our glioma models. Based on patient cohort studies that link glioma occurrence with cerebral ischemia and our new mouse model results, our data demonstrates that exposure to cortical ischemia-induced-hypoxia during tumorigenesis, promotes malignant glioma progression by altering the brain microenvironment via pH modulation. These conceptually innovative studies will integrate our new stroke-glioma models and advanced methodologies to investigate the stroke-glioma interaction via hypoxia and pH modulation, potentially identifying new therapy targets.
STEM-02. SYNGN: A NOVEL EPIGENETIC PLATFORM FOR PATIENT-SPECIFIC DRUG-MATCHING IN GLIOBLASTOMA
Authors: Vinel Claire , Boot James , Zabet Radu , Brandner Sebastian , Marino Silvia
Published: November 2023

The alteration of the glioblastoma genome by epigenetic mechanisms that share functions with normal developmental processes, such as self-renewal and fate specification of NSC, is a key piece of evidence that links brain cancer pathogenesis with dysregulated stem cell functions. A patient-specific comparison of glioblastoma cells with NSC, a putative cell of origin of at least a proportion of these tumours, is not feasible as patient-matched endogenous NSC are not surgically accessible and all epigenetic studies in glioblastoma have so far compared epigenetic changes of different tumours with each other or to comparators obtained from foetal brains or an unrelated donor. We reasoned that availability of syngeneic GSC and NSC pairs would allow to identify crucial epigenetic differences on a patient-specific basis and would provide essential therapeutic contrast to define disease-and patient-intrinsic biomarkers of drug response that are less confounded by germline variation. We have derived GSC from IDH-wildtype glioblastoma and harnessed state-of-the-art stem cell technologies to generate patient-matched fibroblast-derived EPSC, which were induced to NSC (iNSC). We demonstrate that integrated analysis of the transcriptome and DNA methylome of GSC/iNSC pairs identifies druggable target genes (PTGER4, ALDH3B1, NTRK2) in a proportion of patients and we validate this patient-specific prediction of drug response at pre-clinical level in 3D synGLICO and in in vivo models. Integration of small RNA regulation to the pipeline allows refined in silico prediction of drug responses on a patient-specific basis. Most recently we have added assessment of chromatin remodelling (H3K4me3, H3K27ac, H3K3me3, H3K27me3 and ATACSeq) to the pipeline and shown that redistribution of selected histone marks as well as shifts in chromatin states across the genome identifies known and novel druggable regulatory mechanisms in GSC, which are specific of the neoplastic context. In conclusion, SYNGN is an epigenetic platform which can identify patient-specific druggable targets in glioblastoma.
STEM-05. HYPOXIA AND ACIDOSIS PROMOTE YAP/TAZ ACTIVATION, POTENTIALLY SUPPORTING GLIOMA STEM CELL MAINTENANCE IN THE PERI-NECROTIC ZONES
Authors: Shih Ling-Kai , Li Jiabo , Carneiro-Lobo Tatiana , Olson Cheryl , Brat Daniel
Published: November 2023

Glioblastoma (GBM) is a highly malignant brain tumor characterized by rapid growth and poor clinical outcomes. The development of necrosis is associated with massive tumor microenvironment (TME) reshaping and rapid progression characterized by accumulation of tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) in peri-necrotic zones. Using Ivy-GAP and single-cell RNA seq data, we found that many canonical stem-cell associated transcription factors (SOX2, OLIG2, FOXM1) were not upregulated in the hypoxic peri-necrotic zone, yet Hippo transcriptional activation was consistently noted under these conditions. We hypothesized that Hippo activation promotes GSC stemness and may be due to direct effects of hypoxic; the acidic conditions associated with necrosis; or by cytokines secreted by adjacent TAMs. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to 1% hypoxia and found that nuclear expression of YAP1 and TAZ, as well as Hippo pathway readout CYR61, were upregulated compared to normoxia (21%) at 48 hrs, indicating Hippo activation. Exposing cells to an acidic environment (pH 6.3) also caused Hippo transcriptional activation independent of hypoxia. Using single-cell RNA-seq data, we uncovered potential hypoxia-regulated upstream genes that could be responsible for nuclear YAP/TAZ transportation under hypoxia, including Zyxin, WBP2, and PP2A. We also found that culturing GBM cells with media conditioned by human primary macrophages activated Hippo transcription. Using human cytokine arrays, we identified cytokines that showed increased levels in TAM-conditioned media, including EGF, CXCL8, TGF-β, and CXCL5. Treating GBM cells with either hypoxia or a pharmacological activator of YAP/TAZ led to the upregulation of proteins in the inhibitor of differentiation (ID) family (ID1, ID2), which could represent a downstream mechanism related to stem-like behavior following Hippo activation. Our data support the conclusion that hypoxia and TAM cytokines directly stimulate YAP/TAZ activation to potentially promote GSC maintenance in the GBM peri-necrotic niche.
STEM-06. OSMR INTERACTOME MAPPING REVEALS THE SIGNIFICANCE OF A CHLORIDE CHANNEL IN CYTOKINE REGULATION AND BRAIN TUMOUR STEM CELL SELF-RENEWAL
Authors: Randhawa Kamaldeep , Raco Laura , Snider Jamie , Stagljar Igor , Jahani-Asl Arezu
Published: November 2023

Oncostatin M Receptor (OSMR) is a key regulator of brain tumour stem cell (BTSC) self-renewal and glioblastoma (GB) tumourigenesis. This receptor is found in a complex with Epidermal Growth Factor Receptor variant III (EGFRvIII) and orchestrates a positive feedback loop with STAT3. OSMR also regulates oxidative phosphorylation and participates in promoting the transition of cells towards mesenchymal-like glioma states. How OSMR drives GB tumorigenesis via multiple mechanisms remains largely unclear. Here, we map OSMR interactome in different GB subtypes using the Mammalian Membrane Two Hybrid High Throughput Screen (MaMTH-HTS). Analysis of these data followed by functional validation has uncovered distinct OSMR interactome in a context dependent manner. Follow up investigations, however, have led to the discovery of the Chloride Intracellular Channel 1 (CLIC1), essential in the regulation of OSMR cytokine signaling and GB progression independently of the genetic profile of the GB tumour. CLIC1 forms a complex with OSMR to promote BTSC self-renewal and knockout of CLIC1 or pharmacological inhibitors to CLIC1 impairs OSM mediated STAT3 phosphorylation. Our work suggests that CLIC1 and OSMR form a complex in BTSCs and functionally interact to activate STAT3.
STEM-10. SOX2 POSITIVE, PRESUMED TUMOR INVASION MEASURED WELL BEYOND CONTRAST ENHANCEMENT AND FLAIR HYPERINTENSITY IN BOTH TREATED AND UNTREATED GLIOBLASTOMA PATIENTS ASSESSED AT AUTOPSY
Authors: Stebbins Margaret , Bobholz Samuel , Duenweg Savannah , Lowman Allison , Flatley Michael , Nath Biprojit , Kyereme Fitzgerald , Winiarz Aleksandra , Connelly Jennifer , Coss Dylan , Krucoff Max , Banerjee Anjishnu , LaViolette Peter
Published: November 2023

In the treatment of glioblastoma (GBM), MRI is used to determine location and extent of cancer. It is poorly understood how far tumor invasion may exist outside of contrast enhancement (CE) and FLAIR hyperintensity (FL). This study identifies presumed non-enhancing tumor invasion using SOX2 staining on tissue collected at autopsy, which may highlight invading glioma cells. Three patients with primary GBM were included for this study. Patient 1 (P1) with an overall survival (OS) of 148 days received no treatment following sub-total resection. Patient 2 (P2) (OS = 1601) and patient 3 (P3) (OS = 629 days) underwent the clinical standard of care treatment including chemo-radiation and bevacizumab at recurrence. At autopsy, brains were sliced axially to match patients’ last MRI scan. Samples were obtained including regions suspicious of tumor and regions appearing normal. SOX2 positive cells were identified visually on digitized autopsy histology. Samples were visually matched to the patient’s last T1C and FLAIR MRI acquisition prior to death. Approximate distance from CE and FL from SOX2 positive cells was measured digitally, limited to the same MRI slice (2D assessment). For P1 who received no treatment, SOX2 positive cells were identified 3.9cm from CE and 4.1cm from FL within the same hemisphere as the primary tumor, and 2.1cm and 2.3cm within the opposite hemisphere, respectively. For P2 and (P3), SOX2 positive cells were identified approximately 4.4cm (5.2 cm) from CE and 4.0cm (2.0cm) from FL within the same hemisphere as primary tumor, and 4.7cm (4.4cm) from CE and 4.1 cm (4.7cm) from FL within the opposite hemisphere. This study uses SOX2 to identify presumed GBM cells well beyond clinically identified regions of tumor, on T1C and FLAIR imaging. Additional research is necessary to determine SOX2 sensitivity and specificity for identifying GBM cells outside regions with readily identifiable GBM characteristics.
STEM-12. TARGETING GLIOBLASTOMA WITH A BRAIN PENETRANT DRUG THAT IMPAIRS BRAIN TUMOUR STEM CELLS
Authors: Jahani-Asl Arezu
Published: November 2023

Brain tumour stem cells (BTSCs) are a population of self-renewing malignant stem cells that play an important role in glioblastoma tumour hierarchy and contribute to tumour growth, therapeutic resistance, and tumour relapse. Thus, targeting of BTSCs within the bulk of tumours represents a crucial therapeutic strategy. Here, we report a potent brain penetrant drug (AJ5939) that impairs BTSCs and impedes glioblastoma progression. We show that AJ5939 inhibits the self-renewal and growth of BTSCs harbouring a diverse range of oncogenic mutations without affecting non-oncogenic neural stem cells. Global gene expression profiling revealed significant alterations in BTSC transcriptome and attenuation of the expression of a large panel of genes involved in cell cycle progression, stemness, and DNA repair mechanisms. Mechanistically, we show that AJ5939 impairs Notch signalling pathway via directly targeting this pathway and altering the stemness signature. In combination with ionizing radiation (IR), however, AJ5939 suppresses the DNA repair mechanisms and facilitate the response of the BTSC to ionizing radiation induced cell death. Finally, we present data that AJ5939 treatment of brain tumours in preclinical models significantly prolongs lifespan.
STEM-15. STAT5-OLIG2 TRANSCRIPTIONAL COMPLEX MEDIATES GLIOMA STEM CELL SELF RENEWAL AND INVASION
Authors: Miller Aunay , Cascio Costanza Lo , Blomquist Mylan , McNamara James , Orndorff Keely , Dufault Matthew , Sereduk Christopher , Loftus Joseph , Mehta Shwetal , Tran Nhan
Published: November 2023

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion.
STEM-20. REGULATION OF GLIOMA STEM CELL MAINTENANCE AND THERAPEUTIC RESISTANCE BY EGFR MISSENSE MUTATIONS
Authors: Weaver Sidney , Miller Aunay , Blomquist Mylan , McNamara James , Orndorff Keely , Kloss Jean , Sereduk Christopher , Loftus Joseph , Mehta Shwetal , Tran Nhan
Published: November 2023

Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM. EGFRvIII (deletion of exons 2-7) is observed in GBM (20-25%) resulting in a constitutively activated EGFR receptor. In addition, extracellular domain missense point mutations, (e.g. G598V, A289V, R108K) are observed in GBM (14.4%). These missense mutations in the EGFR extracellular domain also induces a highly active receptor by enhancing ligand binding. Earlier reports have shown that these EGFR missense point mutations have exhibited therapeutic resistance; however, the molecular mechanism of these missense point mutations in GBM remains unknown. Previous studies in our lab showed that EGFRvIII promotes GSC maintenance and therapeutic resistance via oligodendrocyte transcription factor 2 (OLIG2) signaling. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Increased OLIG2 expression promotes invasion, growth, proliferation, and survival. Here we report an increase in OLIG2 protein expression in GSCs harboring EGFR-G598V or EGFR-A289V mutations, suggesting these mutations may support stemness properties in GBM tumors. GSC expressing these point mutations display increased self-renewal, growth, and survival. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. Hence, we hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients.
CCRG-04. AUTOMATED REAL TIME TRACKING OF GLIOBLASTOMA CELL CYCLE TRANSITIONS IN ASSEMBLOIDS LINKS G2 CELLS TO INFILTRATION
Authors: Akhunbay-Fudge Christopher Yusuf , Critchley Kevin , Mathew Ryan , Wurdak Heiko
Published: November 2023

Glioblastoma (GB) is the most aggressive primary brain tumour in adults associated with survival rates below 5% despite treatment. Tumour repopulating residual cells in the surgical cavity resist treatment due to their molecular heterogeneity. Single-cell transcriptomic studies have linked GB heterogeneity to the cell cycle; however, it remains unclear to what extent cell cycle states affect the infiltrative capacity of GB. The “go or grow” paradigm predicts that migration and proliferation are separate spatiotemporal events. We used a brain tumour-cerebral organoid (assembloid) approach to test the migratory potential of GB cell cycle phases during assembloid infiltration. Human induced pluripotent cells were differentiated into cerebral organoids that self-assemble with spheroids of patient-derived GB cells, expressing a fluorescent cell cycle reporter (FUCCI). Utilising live-cell confocal imaging, the process of GB assembloid infiltration was captured. Resultant videos were analysed with automated image analysis capable of tracking cell cycle transitions and cellular migration in confocal microscopy planes, enabling the longitudinal quantification of GB migration and invasion. Our analysis revealed a subset of highly infiltrative GB cells, containing a significant number of cycling G2 phase cells that demonstrated a ~1.4-fold increase in directional infiltrative capacity compared to their non cycling G1 phase counterparts. To isolate and characterize the G2 migratory GB cell state, we developed a phenotypic separation assay using microenvironmental cues. Specifically, we found that these cues markedly affect GB migration routes enabling the isolation of the highly migratory GB cells for further investigation. Our results question the “go or grow” hypothesis at the level of GB cellular subpopulations. We hypothesise that the observed G2 migratory capacity of GB cells could be critical to tumour recurrence following surgery. Further research is underway to define the underlying mechanisms of G2 GB cell migration.
CCRG-05. A UNIFYING AND SELF-REINFORCING MECHANISM OF TUMOR IMMORTALITY IN TERT PROMOTER MUTANT CANCERS
Authors: Stevers Nicholas , Barger Carter , Yuan Jimmy , Kim Somang , Hong Chibo , Lenzo Olivia , McKinney Andrew , Wu Samuel , Lee Yu-Jin , Collins Sara , Batsios Georgios , Kwok Darwin , Suwala Abigail , Appin Christina , Viswanath Pavithra , Kasahara Noriyuki , Gordan John , Molinaro Annette , Song Jun , Costello Joseph
Published: November 2023

Telomerase Reverse Transcriptase promoter mutations enable tumor cell immortality in millions of cancer patients annually. While prior therapies targeting telomerase lacked tumor selectivity and were poorly tolerated, TERT promoter mutations and their regulation present a unique opportunity for tumor specific reversal of cellular immortality. The two hotspot mutations, G228A and G250A, generate identical de novo E26 transformation specific (ETS) transcription factor binding sites. However, the 28 ETS factors share a similar binding site preference, raising the question of whether all ETS factors can reactivate TERT. In glioblastoma and a few other cancers, we have shown the de novo ETS site along with a nearby native ETS site (ETS-195/200) recruit one ETS factor, the GA-binding protein (GABP) tetramer, to reactivate TERT. This GABP-mediated mechanism may be restricted to specific cancer types and the two hotspot mutations or could be widely relevant to any cancer with a de novo ETS TERT promoter mutation. We have found that the GABP tetramer activates 6 distinct mutant TERT promoters across 16 different cancer types. However, TERT expression is maintained in most tetramer depleted tumor cells as the tetramer is serially replaced by GABP dimers and weakly by a paralogous tetramer complex that increase following disengagement of GABP tetramer-mediated negative feedback loops. Elimination of both the tetramer and dimer reinstates epigenetic repression of TERT, shortens telomeres, and prevents cancer cell division. However, some knockout cancer cells avoid this fate through weak activation of TERT by the paralogous GABP tetramer complex. The compensatory maintenance of TERT expression can be overcome with a dominant negative GABPB1, leading to TERT silencing, evidenced by gene expression and non-invasive metabolic imaging correlates, and telomere shortening in TERT promoter mutant glioblastoma. We are currently investigating the delivery of this dominant negative to tumor cells via clinically approved retroviral replicating vectors.
CCRG-07. GOLD NANOPARTICLES DEMONSTRATE FAVORABLE EFFECTS ON THE BEHAVIOR OF MESENCHYMAL STEM CELLS, THEREBY AUGMENTING THEIR OVERALL BIOLOGICAL PERFORMANCE
Authors: Shen Chiung-Chyi , Lin Ruei-Hong , Yang Yi-Chin , Hung Huey-Shan
Published: November 2023

Gold nanoparticles (AuNPs) have been extensively studied for their interaction with cells, particularly mesenchymal stem cells (MSCs), and their potential to influence various cellular behaviors such as proliferation and differentiation. In this research, AuNP solutions at concentrations ranging from 1.25 to 10 ppm were prepared to investigate their biocompatibility. The presence of AuNPs was confirmed using ultraviolet-visible spectroscopy, while Dynamic Light Scattering assay characterized their size and scanning electron microscopy revealed their shape. MSCs were treated with different concentrations of AuNPs to assess biocompatibility. The results indicated that 1.25 and 2.5 ppm concentrations of AuNPs significantly enhanced MSC proliferation, reduced reactive oxygen species generation, and attenuated platelet/monocyte activation. Furthermore, the presence of filopodia and lamellipodia suggested improved adhesion ability of MSCs when incubated with 1.25 and 2.5 ppm AuNPs. The expression of matrix metalloproteinase (MMP-2/9) was also upregulated under these concentrations, indicating enhanced cell migration ability. Lower levels of apoptosis were observed in MSCs treated with 1.25 and 2.5 ppm AuNPs compared to higher concentrations, as assessed by Annexin-V/PI double staining and Fluorescence Activated Cell Sorting (FACS). Western blotting analysis showed increased expression of anti-apoptotic proteins Cyclin-D1 and Bcl-2. Additionally, real-time PCR analysis demonstrated reduced expression of inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6, and IL-8) in MSCs treated with 1.25 and 2.5 ppm AuNPs. Animal model tests revealed that these concentrations of AuNPs exhibited better biocompatibility, including anti-inflammatory effects and endothelialization. Overall, 1.25 and 2.5 ppm AuNP solutions were shown to enhance the biological functions of MSCs, suggesting their potential as biocompatible nanomedicine for regenerative research.
CSIG-01. DEFINING THE INVOLVEMENT OF THE PERIVASCULAR NICHE IN BRAIN TUMOR METASTASES
Authors: Fong Vernon , Abeysundara Namal , Livingston Bryn , Richman Cory , Erickson Anders , Taylor Michael
Published: November 2023

Medulloblastomas (MB) are the most prevalent malignant pediatric brain tumors, originating in either the cerebellum or the dorsal brainstem. They are classified into four distinct subgroups (WNT, SHH, Group 3, and Group 4), characterized by unique gene expression profiles, metastatic patterns, and recurrence rates. Metastatic progression of MB is frequently associated with poor prognosis, affecting approximately 30% of patients at diagnosis. Despite considerable research on primary tumors, there are currently no approved specific treatments for MB leptomeningeal metastases. Thus, gaining further insights into metastatic MB and developing targeted therapies are imperative. The leptomeningeal niche, where metastatic MB tumor cells reside, comprises a complex network of blood vessels supported by perivascular cells, including pericytes, smooth muscle cells, and fibroblasts. However, the specific adhesion molecules and signaling pathways within the leptomeningeal niche that contribute to tumor cell colonization remain elusive. In this study, we hypothesize that the perivascular niche plays a critical role in the colonization and survival of metastatic cells within the leptomeninges. To investigate this, we employed an innovative sLP-mCherry niche labeling system in conjunction with single-cell RNA sequencing to spatially identify and examine the perivascular cells neighboring metastatic MB tumor cells. Through our analysis, we identified upregulated genes associated with cholesterol export in tumor-associated perivascular cells, suggesting their potential involvement in supporting metastatic cell colonization. To further explore this mechanism, we utilized CRISPR and lentiviral overexpression constructs to modulate cholesterol uptake in MB cell lines, observing subsequent changes in metastatic burden. In summary, our study elucidates the importance of the perivascular niche in metastatic medulloblastomas and provides novel insights into the molecular mechanisms that drive tumor cell colonization within the leptomeninges. These investigations into the lipid transport mechanism in the vascular microenvironment opens doors for the development of innovative treatments targeting MB metastases.
CSIG-02. SHH PATHWAY ACTIVATION DRIVES NEURAL CREST CELL SIGNATURE IN A SUBGROUP OF MPNSTS
Authors: Suppiah Suganth , Liu Jeffrey , Li Mira , Heidari Mohsen , Yakubov Rebeca , Bunda Severa , Zadeh Gelareh
Published: November 2023

INTRODUCTION

Neurofibromatosis Type 1 is hereditary tumor predisposition syndrome that results in the development of innumerable nerve sheath tumors that fall within the spectrum of benign, premalignant and malignant lesions. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs.

METHODS

We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, a SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment.

RESULTS

We observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6 . We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines.

CONCLUSION

SHH pathway activation drives dedifferentiation into a neural crest cell-like state in a subset of MPNSTs. We confirm that inhibiting SHH pathway activity in a subset of MPNSTS prevent growth and malignant progression, providing a rational for future clinical trials.
CSIG-05. A FIRST COMPREHENSIVE ANALYSIS OF TRANSCRIBED ULTRA CONSERVED REGIONS UNCOVERS IMPORTANT REGULATORY FUNCTIONS OF NOVEL NON-CODING TRANSCRIPTS IN GLIOMAS
Authors: Gibert Myron , Saha Shekhar , Zhang Ying , Marcinkiewicz Pawel Pawel , Chagari Bilhan , Roig-Laboy Christian , Sarkar Aditya , Dube Collin , Hudson Kadie , Sun Yunan , Kefas Benjamin , Abounader Roger
Published: November 2023

Most glioma research has focused on protein-coding genes and much less on non-coding RNAs (ncRNAs). Transcribed Ultra-Conserved Regions (TUCRs) represent a severely understudied class of ncRNAs that are highly conserved across multiple species, including 100% conservation in mouse and rat genomes. These 481 transcripts are highly resistant to variation and are commonly deregulated in cancer, which suggests regulatory and functional importance when considered along with their high degree of conservation. We performed the first-ever analysis of TUCRs in glioblastoma (GBM) and low-grade gliomas (LGG). We first annotated all TUCRs in gliomas and described their genomic locations. We then showed that numerous TUCRs are highly expressed and deregulated in gliomas, and that this deregulation is associated with clinical outcomes. We then used a WGCNA pipeline to predict the functions for all TUCRs in gliomas by compiling gene ontology (GO) terms for coregulated genes. These data were used to identify several TUCRs that are potentially relevant to glioma initiation and progression. Of these, we investigated the most highly upregulated intergenic TUCR, uc.110, in vitro and in vivo in GBM. Knockdown of uc.110 in vitro reduced GBM cell accumulation and invasion and knockdown in vivo inhibited GBM xenograft growth and increased animal survival. We then performed mechanistic analyses of uc.110 and found that it regulates the expression of a Wnt pathway member, membrane frizzled-related protein (MFRP). We discovered that uc.110 sponges the tumor suppressor microRNA miR-544, which targets and inhibits MFRP, thus increasing the bioavailability of MFRP in GBM. This study is the first of its kind in gliomas. It shows important regulatory roles for TUCRs in gliomas and outlines novel methods for studying TUCRs in any disease context.
CSIG-08. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS TO CONTROL HIF1Α TRANSCRIPTION IN GLIOBLASTOMA
Authors: Zhang Ying , Yuan Ying , Grello Cassandra , Reon Brian , Gibert Myron , Dube Collin , Yang Yanzhi , Hudson Kadie , Kefas Benjamin , Dutta Anindya , Abounader Roger
Published: November 2023

P53 is mutated in a large subset of glioblastoma (GBM). Most hot-spot p53 mutants are gain-of-function (with only partially understood mechanisms). To investigate the role of gain-of-function mutant p53 (GOF-Mut-p53), we performed ChIP-seq analysis of GOF-Mut-p53 R273H, the most common p53 mutation in GBM. We identified and verified numerous GOF-Mut-p53 targets that differed from those of wildtype p53. Among the 2,830 targets of GOF-Mut-p53 were 535 long non-coding RNAs (lncRNAs), the majority of which we also found enriched in Mut-p53 human tumors and deregulated in the TCGA database. LncRNAs play important regulatory roles in cancer. One of the most highly enriched lncRNAs was LINC00643. We first performed ChIP confirmation of GOF-Mut-p53 binding to the promoter of LINC00643. We then showed that GOF-Mut-p53 inhibits LINC00643 expression. Furthermore, we found that LINC00643 is deregulated in human GBM and LGG and correlated with patient survival. Overexpression of LINC00643 led to a significant inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. Co-expression of human LINC00643 or its mouse homologue with PDGF and Cre in RCAS transgenic mice (N/tv-a; Ink4a-arf-/-; Pten-fl/fl) reduced tumor growth and improved animal survival. These data show that GOF-Mut-p53 exerts oncogenic effects in GBM by downregulating LINC00643. To elucidate the mechanisms of action of LINC00643, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to the HIF1α enhancer region. Overexpression of LINC00643 and knockdown of GOF-Mut-p53 in GBM cells under hypoxia growth conditions reduced HIF1α mRNA and protein expression. LINC00643 also regulated the expression of numerous hypoxia-regulating genes downstream of HIF1α. Our study shows for the first time that GOF-Mut-p53 transcriptionally regulates a subset of lncRNAs and that GOF-Mut-p53-regulated LINC00643 regulates hypoxia in GBM.