bluuebunny/crossref_metadata_2025 huggingface dataset:
CNSC-31. CRISPR FUNCTIONAL SCREEN IDENTIFIES A NOVEL LONG NONCODING RNA MODULATING GLIOBLASTOMA INVASION
Authors: Tsung Kathleen , Han Jane , Liu Kristie , Loh Eddie , Attenello Frank
Published: November 2022

INTRODUCTION

Glioblastoma(GBM) invasion studies traditionally target coding genes. Long noncoding RNAs (lncRNAs), are transcripts without coding potential, with limited data regarding function. We leveraged CRISPR-interference, repressing lncRNA transcription via Cas9-KRAB, to evaluate invasive function of GBM-associated lncRNAs in a CRISPRi functional screen.

METHODS

2,307 candidate lncRNAs were assembled from GBM differential gene expression. Guide RNAs targeting candidates were lentivirally incorporated into GBM screening populations. Screen populations were evaluated via Matrigel invasion, comparing sgRNA populations before/after a 24-hour invasion period. ASO treatment evaluated pharmacologic targeting of candidate lncRNAs. Top screening candidates were evaluated in a patient derived glioma stem cell (GSC) line, USC02, as well as U87 and U251 commercial lines. Differential gene expression was assessed in multiple cell lines following lncRNA knockdown via RNA-seq followed by GSEA and pathway analysis was evaluated to assess downstream mechanistic candidates affected by lncRNA KD.

RESULTS

Forty-eight lncRNAs were significantly associated with GBM invasion, with lncRNA repression associated with decreased invasion of 31-85%(p< 0.01). Individual validation of a candidate lncRNA KD, LH02236, confirmed 85% decrease in tumor cell invasion versus control. Evaluation of gene expression across normal cortex and tumor samples from GSEA/TCGA datasets revealed LH02236 is significantly more expressed in GBM versus low grade glioma, and in low grade glioma versus normal cortex. Expression of LH02236 was significantly associated with patient survival (p< 0.0001). Gene expression analysis of LH02236 knockdown revealed Sox10 to be highly correlated with lncRNA expression across multiple cell lines.

CONCLUSION

Large scale CRISPRi screening identified LH02236, a previously unannotated lncRNA, as essential to invasion in patient lines. Gene expression is significantly associated with tumor grade and patient survival. Analysis of knockdown across multiple cell lines suggests Sox 10, commonly associated with GSC function, is modulated by this novel lncRNA.
TMIC-85. MICROGLIA REPLACEMENT CHANGES THE TRANSCRIPTIONAL PROFILE OF TUMOR ASSOCIATED MYELOID CELLS IN MURINE MODELS OF BRAIN MALIGNANCIES
Authors: Mader Marius , Rodrigues Adrian , Chernikova Sophia , Wong Zheng Hao Samuel , Wang Yuelong , Petritsch Claudia , Wernig Marius , Gephart Melanie Hayden
Published: November 2022

Tumor associated microglia and macrophages (TAMs) represent a main cell type of brain malignancies and demonstrate complex interactions with cancerous cells and the tumor microenvironment. These interactions have important implications for the progression and treatment of malignant central nervous system (CNS) tumors. In a non-oncological context, it has been shown that endogenous microglia can be replaced by peripheral hematopoietic cells by genetic or pharmacological intervention. A highly efficient replacement protocol (Shibuya et al, Sci Transl Med 2022, PMID:35294256) utilizing myeloablative bone marrow transplantation (BMT) followed by microglial depletion via pharmacological inhibition of the Colony-stimulating factor-1 receptor (CSF-1R) leads to almost complete repopulation of the CNS myeloid niche by circulation-derived myeloid cells (CDMCs). We investigated if this approach could be utilized to integrate CDMCs into brain tumors and study associated changes in the TAM signature. Microglia replacement in immune-competent mice was followed by intracranial transplantation of syngeneic cell lines of either high-grade glioma (GL261) or breast cancer metastasis (E0771). Lineage tracing proved high intra- and peri-tumoral chimerism of BMT-graft derived cells among CNS myeloid cells in both models two weeks after tumor cell injection. Tumor associated CDMCs (TA-CDMCs) showed a similarly activated morphology as naïve TAMs. We used single cell RNA sequencing of CD45-positive cells to further profile the transcriptomic identity of glioma TA-CDMCs. Pathway enrichment analysis revealed an upregulation of immune response pathways in TA-CDMCs compared to regular TAMs, including the overexpression of genes relevant for the regulation of T cell activation, suggesting possible modification of the glioma immune-environment. Strategies to manipulate the myeloid niche have the potential to provide further understanding of the pathobiology of TAMs and to build the basis for novel cell therapeutic approaches in the neuro-oncological domain.
MODL-02. GLIOMA ORGANOID MODEL PHENOTYPICALLY RECAPITULATES KEY ASPECTS OF MALIGNANT TRANSFORMATION IN GLIOBLASTOMA
Authors: Zhao Yi , Kant Shiva , Kesarwani Pravin , Hubert Christopher , Nakano Ichiro , Fullmer Joseph , Chinnaiyan Prakash
Published: November 2022

Glioblastoma (GBM) displays extensive intra-tumoral heterogeneity, serving as a major obstacle for effective treatment. The identification of molecular subtypes offered promise for personalized treatment regimens in GBM, however, it has seen been determined these subtypes are reflective of intra- rather than inter-tumoral heterogeneity, with proneural (PN) and mesenchymal (MES) subtypes corresponding to the infiltrative edge and peri-necrotic core of a tumor, respectively. Further, plasticity between subtypes has been identified, with PN-to-MES transition being described as a mode of resistance. Our laboratory utilizes patient-derived, subtype-specific GBM neurospheres as a model to understand GBM heterogeneity and have previously demonstrated that these models faithfully recapitulate human disease, with PN-neurospheres expressing Olig2, having a metabolic phenotype consistent with low-grade glioma, and an indolent growth pattern in vivo when compared to aggressive, CD44 expressing MES-neurospheres. In parallel, our group demonstrated 3D-organoid models of GBM recapitulate the tumor ecology of this malignancy ex vivo. Therefore, we sought to determine if this model could be utilized to provide a window into the plasticity of GBM molecular subtypes. Consistent with their phenotype when grown as neurospheres, MES-organoids had a homogeneous distribution of proliferating cells and retained CD44 expression. In contrast, cellular heterogeneity emerged in PN organoids that could be morphologically divided into an outer, cell-dense rim and an inner core with a lower cell density. The outer rim, recapitulating a perivascular niche, contained proliferating cells, and the inner core contained necrotic cells, mimicking the hypoxic microenvironment of GBM. Intriguingly, PN organoids retained Olig2 expression in the edge, however, actively transitioned into a MES-like state in the core, expressing CD44 and metabolic reprogramming consistent with malignant transformation. Collectively, these finding support that microenvironment contributes towards PN-MES transition and PN organoid models may serve as a tool to provide a window into specific molecular and metabolic factors contributing towards transformation.
MODL-13. Sensitivity of Tumor Treating Fields (TTFields) treatment planning to healthy tissue segmentation inaccuracies
Authors: Ze’evi Oshrit , Marciano Tal , Naveh Ariel
Published: November 2022

TTFields are FDA approved for treating patients with GBM. TTFields utilize low intensity electric fields with intermediate frequency (100-500 kHz) that disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields have been shown to disrupt multiple phases of the cell cycle including metaphase, anaphase, and telophase, resulting in cellular apoptosis. TTFields are administered through two pairs of capacitive arrays placed on the patient’s scalp. As TTFields are a dose dependent therapy, optimization of array layout for maximizing field intensity may improve survival rates of patients. Thus, Novocure is developing a treatment planning system, in which MR images are used to create a patient-specific model, which in turn is used for simulating electric fields distribution. However, the segmentation process of the MRIs is prone to errors that may influence the choice of array layout, and consequently provide lower TTFields dose than possible. To test the sensitivity of layout chosen to such inaccuracies, we placed different array layouts on the highly detailed MIDA head model and simulated the resulting LMiPD from each configuration. We then simplified the model in several phases and compared the resulting array layout ranking between the full model and each simplified model. Our results show that though the calculated LMiPD is very sensitive to the quality of the model, the ranking pattern between the detailed and simplified models is similar. Some alterations do occur, but this mostly happens when the LMiPD values between switched layouts are very close, rendering these alterations clinically insignificant. Based on these results, we conclude that the ranking order of array layouts by LMiPD dose does not significantly change between detailed and simplified head models even though the LMiPD dose itself is sensitive to the quality of the head model.
MODL-24. ESTABLISHING A CLINICALLY RELEVANT CT AND ASSOCIATED RADIOMICS PIPELINE FOR INTRACRANIAL RODENT TUMOUR MODELS
Authors: Connor Kate , Conroy Emer , White Kieron , Shiels Liam , Keek Simon , Ibrahim Abdalla , Gallagher William , Clerkin James , Sweeney Kieron , O'Brien David , Cryan Jane , Heffernan Josephine , Brett Francesca , Lambin Philippe , Woodruff Henry , Byrne Annette
Published: November 2022

While magnetic resonance imaging (MRI) is the predominant imaging modality for glioblastoma (GBM), pre-clinical MRI scanner availability is limited. As pre-clinical CT is more widely available and cost-effective, this study aimed to 1) establish preclinical-GBM CT and CT-radiomic workflows, 2) identify whether CE-CT could detect murine orthotopic GBM tumours on two CT instruments [TRIUMPHX-O-CT; IVISSPECTRUM-CT], 3) assess whether CT-radiomic features could distinguish tumour from normal tissue, and support earlier detection of tumours, 4) verify translation of pre-clinical CT-radiomic pipelines to, and assess pre-clinical CT-features in, clinical CE-CT scans.U87R-Luc2(n=25) and NFpp10a-Luc2(n=10) orthotopic GBM models were established and tumours monitored via bioluminescence imaging (BLI). Concurrently, mice underwent CE-CT (IV-iodine/300mg/mL/50kV-scan). Extracted radiomic features (PyRadiomics) underwent dimensionality reduction (Spearman correlation; >0.85). Remaining features were analysed (Recursive feature elimination (RFE)/RepeatedCV/randomforest) in normal and tumour tissue and across timepoints (TRIUMPHX-O-CT-Wk3vsWk6,Wk6vsWk9/12; IVISSPECTRUM-CT-Wk6vsWk9/12).CE-CT and radiomic pipelines were successfully established for orthotopic GBM models, using both CT-systems. On visual assessment of images, BLI was significantly more sensitive, with tumours detectable at Wk1 (BLI) vs Wk9 (CE-CT). However, RFE analysis identified CT-radiomic features (first_order&glcm) which differentiated tumour from normal tissue (TRIUMPHX-O-CT). A subsequent feature set (first_order,glcm,gldm&glzm) were identified (TRIUMPHX-O-CT/IVISSPECTRUM-CT), detecting tumours earlier (Wk3&Wk6) than possible by visual assessment of CTs. Preclinical radiomic methods were successfully applied to exploratory clinical CE-CT scans(n=10). Here, several preclinical CT-features (e.g. Zone_Entropy) showed increased intensity in tumour regions. Overall experimental BLI is the most sensitive method for pre-clinical intracranial tumour detection. However, analysis of clinically relevant CT-radiomic features may facilitate tumour identification and earlier tumour detection (Wk3/Wk6-TRIUMPHX-O-CT/Wk6-IVISSPECTRUM-CT) than possible by visual assessment of CT (Wk9). Clinically relevant CT-derived radiomic features may therefore support intracranial rodent tumour assessment. Importantly, preclinical radiomic methods successfully translate to clinical CT-radiomic analysis. Parallel trends in tumour-specific feature intensities across pre-clinical and clinical scans suggest species conservation of features.
MODL-28. DEVELOPING A STRATEGY FOR MODELING HIGH-GRADE GLIOMA IN GӦTTINGEN MINIPIGS
Authors: Tora Muhibullah S , Lei Kecheng , Nagarajan Purva P , Bray David P , Rindler Rima S , Neill Stewart G , Zhang Michelle , Texakalidis Pavlos , Krasnopeyev Andrey , Gergye Crystal , James Raphael , Oshinski John N , Federici Thais , Bruce Jeffrey N , Canoll Peter , Boulis Nicholas M
Published: November 2022

BACKGROUND

The current literature does not describe a reproducible large animal model of intracranial high-grade glioma (HGG). Prior work has demonstrated the feasibility of inducing HGG de-novo in rodents by targeting specific oncogenic pathways. Here we report our approach to the production of supratentorial HGG in a series of minipigs through lentiviral gene transfer and subsequent initial characterization of a porcine glioma cell line.

METHODS

Four minipigs received injections into the subcortical white matter using a combination of lentiviral vectors expressing platelet-derived growth factor beta (PDGF-B), HRAS, and shRNA-p53. Animals underwent behavioral monitoring through porcine neurobehavioral scoring (PNS) and veterinary monitoring. Magnetic resonance imaging (MRI) was conducted at endpoint prior to necropsy. Post-mortem tissue biopsies underwent tissue culture and neuropathologic evaluation with hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescent staining. Data are presented using appropriate statistical tests where relevant and descriptive statistics.

RESULTS

Two pigs received 50ul injections and reached endpoint by the end of post-operative week 1 and 2. Two pigs received 25 ul injections and were asymptomatic until a pre-determined endpoint of 4 weeks. MRI scans at endpoint demonstrated contrast enhancing, mass forming lesions at the site of injection with evidence of hemorrhage and perilesional edema, consistent with high-grade glioma. On H&E staining high-grade glioma growth was identified in 100% of animals. We observed immunopositivity for tumor markers GFAP, OLIG2, NG2, SOX2, and PDGFRA, as well as redox markers, and microenvironmental features consistent with high-grade glioma. Porcine glioma cell cultures were found to have significantly greater proliferative rate compared to control, and demonstrated GFAP, OLIG2, PDGFRA, and CD68 immunopositivity.

CONCLUSIONS

Lentiviral gene transfer represents a feasible strategy for glioma modeling in the Gӧttingen minipig. With our described methodology, we present a realistic strategy for reproducible modeling of intracranial glioma as a platform for preclinical neurosurgical development programs.
MODL-29. IN SILICO MODELING TO PREDICT ONCOGENICITY AND POTENTIAL TARGETABILITY OF NOVEL FGFR VARIANTS IN PEDIATRIC LOW GRADE GLIOMA
Authors: Laxer Ben , Nobre Liana , Tabori Uri , Vadivel Arun Anguraj , Levine Adrian , Johnson Monique , Ryall Scott , Ku Michelle , Milos Scott , Hawkins Cynthia
Published: November 2022

Pediatric Low grade gliomas (pLGGs) are largely driven by alterations in the RAS-MAPK pathway. The third most commonly mutated gene in pLGGs is the FGFR1 gene, having alterations including fusions, duplications or missense mutations. Accurate identification of these alterations has become increasingly important as FGFR inhibitors enter the therapeutic space. Here we describe a case of a pLGG patient with an FGFR1 W289_insW290 found through NGS in the tumor at diagnosis, and progression, without additional alterations. This extracellular, third-Ig domain mutation has not been reported. Transcriptional and proteomic profiling of the tumor showed low MAPK and high PIK-AKT pathway activation when compared to other gliomas. Literature review identified disorders of bone formation with similar mutations in the homologous FGFR2 gene. One such mutation, FGFR2 W290G favors inter-receptor disulfide bond formation over the normal intra-receptor disulfide bond. To determine if this insertion is a possible driver mutation in our pLGG case, Alphafold2 protein simulation software was used to make in silico models; quality metrics from both Alphafold2 and VADAR1.8 were used. Structure simulations of the FGFR1 insertion mutant (pLDDT = 81.8) and the mutant FGFR2 W290G (pLDDT = 83.4), were generated and compared to a protein data base, experimentally determined structure of wildtype FGFR1. When monomers of all three structures were aligned using PyMol, the root mean square deviations were < 1.5 indicating the structures are correct or the same. Based on these results we concluded that, like the known FGFR2 mutation, this insertion is predicted to cause hyperactivity by inducing prolonged dimerization due to an aberrant inter-receptor disulfide bond but any structural or functional differences only occur when receptors are dimerized. We are currently generating isogenic cell line models to test this hypothesis and treat these cell lines with inhibitors to determine which treatments might work best for our patient.
MODL-33. AN ANIMAL MODEL OF GLI2-AMPLIFIED MEDULLOBLASTOMA
Authors: Murad Najiba , Liu Xiao , Tao Ran , Pei Yanxin
Published: November 2022

Medulloblastoma (MB) is the most common malignant brain tumor affecting children. Among the known subtypes of MB, GLI2-amplified SHH-MB associated with P53 mutations has the worst prognosis, and a 5-year survival rate of less than 30%. Moreover, GLI2-amplified MBs are non-responsive to SMO inhibitors, the only targeted treatment option available for SHH-MB. To address this treatment gap, there is an urgent need to identify novel potential therapeutic targets, an effort that requires better understanding of key cellular and molecular mechanisms underlying GLI2-amplified MB. To that end, we generated a mouse model of GLI2-amplified MB and identified GL12 as the critical driver of tumorigenesis, and granule cell progenitors (GCPs) as the cells of tumor origin. We further found that GLI2 specifically drives embryonic, but not neonatal, Math1+ GCPs to form SHH-MB tumors. An scRNA-seq analysis revealed MAPK pathway activation specifically in embryonic Math1+ GCPs. The MAPK pathway was activated in mouse and human GLI2-driven MB tumors, and a MEK/ERK inhibitor significantly inhibited GLI2-driven MB in vivo. Based on these findings, we hypothesize that GLI2-driven MB originates from a specific population of Math1+ GCPs during a discrete temporal interval in cerebellar development, and that targeting MAPK/MEK/ERK pathway components represents a novel approach to treatment of GLI2-amplified MB.
MODL-36. EXPRESSION OF YAP1-MAML2 AND CONSTITUTIVELY ACTIVE YAP1 DRIVE THE FORMATION OF MENINGIOMA-LIKE TUMORS IN MICE THAT RESEMBLE NF2-MUTANT MENINGIOMAS
Authors: Szulzewsky Frank , Arora Sonali , Arakaki Aleena , Sievers Philipp , Bonnin Damian Almiron , Paddison Patrick , Sahm Felix , Cimino Patrick , Gujral Taranjit , Holland Eric
Published: November 2022

YAP1 is a transcriptional co-activator and oncogene under the control of the Hippo Signaling Pathway. Functional inactivation of Hippo Pathway tumor suppressors, including NF2, are frequent events in human cancers. Meningiomas are the most common primary brain tumors, and a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 gene copy, implicating oncogenic YAP activity in the pathobiology of almost half of these tumors. An alternate type of activating YAP1 mutation are YAP1 gene fusions that have been identified in several cancer subtypes. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2-wild type meningiomas. Here, we show that the expression profile of human YAP1-MAML2-positive pediatric meningiomas resembles that of the common NF2-mutant meningiomas based on global and YAP-related gene expression signatures. We then use the RCAS/tv-a system for postnatal gene transfer and show that the intracranial expression of YAP1-MAML2 in neonatal mice results in the formation of meningioma-like tumors that exert a similar gene expression pattern as seen in human YAP1 fusion-positive and NF2-mutant meningiomas and regulate classical YAP1 target genes. We demonstrate that YAP1-MAML2 exerts oncogenic YAP activity that is resistant to inhibitory Hippo pathway signaling and relies on the interaction with TEAD transcription factors. Pharmacological disruption of this interaction is sufficient to inhibit the viability of YAP1-MAML2-expressing mouse tumors ex vivo. Finally, we show that constitutively active YAP1 (S127/397A-YAP1) is also sufficient to cause the formation of similar meningioma-like tumors suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a sufficient oncogenic driver in YAP1-MAML2 fusion-positive meningiomas, which mimic NF2-mutant meningiomas, and highlight TEAD-dependent YAP activity as a potential therapeutic target in these tumors.
CNSC-38. EGFR AMPLIFICATION IS A PUTATIVE RESISTANCE MECHANISM FOR NSCLC-LM PATIENTS WITH TKI THERAPY AND IS ASSOCIATED WITH POOR OUTCOME
Authors: Yang Hainan , Wen Lei , Zhou Caicun
Published: November 2022

BACKGROUND

Leptomeningeal metastases (LM) have become increasingly common in NSCLC patients who harbor epidermal growth factor receptor (EGFR) mutation treated with EGFR-TKI and are correlated with inferior prognosis. Evidence in prior research demonstrated that EGFR amplification was more likely presented in advanced clinical stages and was associated with worse survival. However, whether the EGFR amplification is a prognostic marker in NSCLC-LM is still inconclusive.

METHODS

This study enrolled patients diagnosed with NSCLC-LM from June 2019 to September 2021, who had received previous EGFR-TKI at Guangdong Sanjiu Brain Hospital. Cerebrospinal fluid (CSF) samples were collected and subjected to targeted next-generation sequencing (NGS) of 168 cancer-related genes. Clinical characteristics and overall survival (OS) were compared in patients with and without EGFR amplification.

RESULTS

This study enrolled 53 NSCLC-LM patients, all of whom had EGFR mutations. TP53 and EGFR amplification are the two most frequent mutations in the study cohort, presenting 72% (38 of 53) and 40% (21 of 53), respectively. The rate of EGFR amplification was much higher at the time of leptomeningeal progression than at initial diagnosis (p< 0.01). Karnoskfy performance status was poorer (p=0.021), and CSF pressure was higher (p=0.0067) in patients with than without EGFR amplification. A multivariable Cox proportional hazard regression model showed that EGFR amplification was an independent prognostic factor for poorer OS (8.3 vs 15 months; p=0.017). Median OS was shorter in NSCLC-LM patients with mutated than wild-type TP53, but the differences was not statistically significant (10 vs 17.3 months, p=0.184).

CONCLUSIONS

EGFR gene amplification could be a potential resistance mechanism to EGFR-TKI failure in NSCLC-LM and is associated with inferior clinical outcomes.
DNAR-15. POLYMORPHISM AT P21 CODON 31 PROVIDED A PREDICTION FOR BEVACIZUMAB THERAPY IN GLIOBLASTOMA MULTIFORME
Authors: Cheng Wen-Yu , Shen Chiung-Chyi , Chiao Ming-Tsang
Published: November 2022

Glioblastoma (GBM) is a common, malignant brain tumor. While it rarely metastasizes to distant organs, the diffuse invasion of glioma cells within the brain makes it difficult to resect. P21 (Waf-1) is a cyclin-dependent kinase inhibitor that plays an essential role in cell growth arrest, terminal differentiation, and apoptosis. Thus, the existence of natural variants of p21 could be linked to specific cancer. The purpose of this study was to identify the polymorphism variants of p21 codon 31 (Ser31Arg) and to explore its role in the therapy of glioblastoma multiforme. A total of unrelated 99 GBM subjects, were enrolled in the study, and genomic DNA was obtained from each tumor sample for genotyping. Genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism method (PCR-RFLP analysis. Odds ratios (Ors) and 95% confidence intervals (CIs) were estimated by unconditional logistic regression analysis. We found that among these GBM patients, the distribution of codon 31 polymorphisms was 20.2% for Serine homozygotes (Ser/Ser), 32.3% for arginine homozygotes (Arg/Arg), and 47.5% for Serine/arginine heterozygotes (Ser/Arg). Although the polymorphisms of p21 codon 31 were not directly associated with the overall survival of GBM, both the Arg/Arg and Arg/Ser genotypes were associated with significant benefits in terms of overall survival in patients treated with CCRT plus bevacizumab compared to patients treated with CCRT alone. Our findings suggest that the p21 Ser31Arg polymorphism may play an important role in the pathogenesis and initiation of GBM. Moreover, the polymorphism seems to contribute to a significant prediction at early diagnosis for bevacizumab therapy.
SURG-42. MOLECULAR PROFILE CONSENSUS CLUSTERING DEFINES CLINICALLY DISTINCT GROUPS IN IDH WILD-TYPE GLIOBLASTOMA
Authors: Gersey Zachary , Mallela Arka , Zhang Xiaoran , Pease Matthew , Abdelhakiem Mohamed , Pearce Thomas , Lieberman Frank , Mantica Megan , Phillips Richard , Bagley Stephen , Nasrallah MacLean , Binder Zev , Brem Steven , Chen Isaac , O’Rourke Donald M , Zinn Pascal , Siddiqui Zaid , Amankulor Nduka
Published: November 2022

OBJECTIVES

Although the impact of surgery in glioblastoma (GBM) is well-established, there is insufficient data to determine how surgical resection affects overall survival (OS). The impact of other GBM genomic alterations on EOR-induced survival remains poorly understood. Here, we leverage a large cohort of GBM patients with genomic data to establish the impact of genomic alterations on resection-dependent overall survival in GBM.

METHODS

537 IDH WT GBMs from UPMC were analyzed using Glioseq next generation sequencing panel. Using molecular profiles derived from Glioseq data, semi-supervised Monte Carlo reference-based consensus clustering was used to define 5 distinct groups. Cox Proportional Hazards models were used to identify predictors of OS within molecular groups.

RESULTS

Clustering of 537 IDH wild-type GBM identified 5 distinct groups: 1: TERT mutated/MGMT methylated/EGFR mutated or gain; 2: TERT mutated /MGMT methylated/p53 mutated; 3: TERT WT; 4: TERT mutated/MGMT methylated/CDKN2A loss; 5: TERT mutated/MGMT unmethylated. Thresholds in postoperative residual contrast enhancing volume (rCEV) and residual non-contrast enhancing volume (rNCEV) were identified which marked significant differences in median overall survival (MOS) between molecular groups in rCEV -Group 1: 11.7cc, Group 2: 14.4cc, Group 3: 1.1cc, Group 4: 0.1cc, and Group 5 1.1cc. Sex, age, KPS >70, temozolomide, and radiation along with thresholded rCEV and rNCEV were used in multivariate Cox analyses. Groups demonstrated distinct predictors of OS: radiation only in Group 1 tumors, low rCEV in Group 2 tumors, younger age, temodar, low CEV, and KPS >70 in Group 3 tumors, female sex, younger age, radiation, KPS >70 low rCEV in Group 4 tumors, and KPS >70, temodar, and low rCEV in Group 5 tumors.

CONCLUSIONS

Clustering results suggest that there are clinically distinct molecular groups of IDH wild-type glioblastomas. Age, sex, KPS >70, temozolomide treatment, radiation therapy, and EOR have varying affects on these molecular groups.
STEM-03. MOLECULAR PROFILING OF PRIMARY VERSUS RECURRENT GLIOBLASTOMA BRAIN TUMOR STEM CELLS UNCOVERS SIGNALING MECHANISMS THAT PROMOTE THE AGGRESSIVENESS OF RECURRENT GLIOBLASTOMA
Authors: Heemskerk Kyle , Hao Xiaoguang , Cseh Orsolya , Luchman H Artee , Weiss Samuel
Published: November 2022

Glioblastoma (GBM) is a devastating brain cancer with a median overall survival of a mere 12-15 months. Patients receive the standard of care treatment, comprised of maximum surgical resection, ionizing radiation, and temozolomide chemotherapy, however the tumor inevitably recurs. Recurrent GBM is more invasive, difficult to resect, and treatment resistant. GBM brain tumor stem cells (BTSCs), a sub-population of stem-like tumor cells with the ability to self-renew and differentiate into a heterogeneous tumor, are thought to be at the root of recurrent disease. BTSCs isolated from primary and recurrent tumors from the same patient are rare due to fewer resections of recurrent tumors and reduced quality of recurrent tumor samples used to initiate cell lines. As a result, the process of tumor recurrence is vastly understudied in BTSCs. To further understand the process of recurrence and elucidate potential mechanisms of invasion and treatment resistance, we explored global transcriptomics in 40 primary versus 17 recurrent BTSC cultures. Additionally, we profiled changes at the chromatin level in a subset of primary versus recurrent BTSCs using ATAC sequencing. Several pathways were found to be upregulated in recurrent GBM BTSCs, including innate immune signaling at the mRNA and chromatin level, which may mediate the aggressive nature of recurrent BTSCs. Moreover, these signaling changes may be unique to the stem cell population within the tumor, as they are not observed when profiling primary versus recurrent bulk tumors. We are currently targeting these pathways genetically and pharmacologically in primary and recurrent GBM BTSCs established from the same patient and have uncovered mechanisms for treatment resistance, invasion, and recurrence. Our work investigating global signaling changes in primary versus recurrent BTSCs holds promise for uncovering new therapeutic targets or biomarkers for treatment of recurrent GBM.
STEM-08. MODELING AND UNDERSTANDING GLIOBLASTOMA EDGE CELLS
Authors: Maturi Naga Prathyusha , Neves Ines , Dang Yonglong , Yildirim Irem , Latini Francesco , Bergström Tobias , Sundström Anders , Xing Pengwei , Jarvius Malin , Larsson Rolf , Fryknäs Mårten , Ryttlefors Mats , Chen Xingqi , Swartling Fredrik , Uhrbom Lene
Published: November 2022

Glioblastoma is a devastating disease with an overall median survival of 8 months from diagnosis. The majority of patients die of a tumor relapse in close proximity of the resected primary tumor. Glioblastoma is intensely researched but most studies have been performed on tissues and cultures derived from the bulk tumor while it is the remaining edge cells that cause lethality. Investigations of edge cells are rare and few experimental models exist. Here we have established and analyzed a series of matched cell cultures derived from the tumor bulk and outer edge of six IDH wildtype glioblastoma patients with the purpose to understand glioblastoma edge cell biology. Tumor samples were resected guided by 5-ALA fluorescence using neuro-navigation and stringent procedures to not contaminate edge samples with bulk tumor cells. First bulk tumor samples were resected from 5-ALA fluorescent tissue. After removal of all fluorescent areas and careful irrigation of the cavity the edge sample was resected 1-2 cm outside of the fluorescent border in a non-eloquent area. Following dissociation the samples were used in sphere assays and for explantation. There was a significant difference in self-renewal across all patients between matched bulk and edge cultures, in line with results from sphere assays on acute cells, suggesting maintenance of glioblastoma cell properties in established cultures. Invasion analysis showed a reverse significant difference between matched bulk and edge cultures strengthening a general functional distinction between tumor bulk and edge cells across patients. To investigate the molecular basis of our findings we performed whole exome sequencing (WES) and combined single cell RNA- and ATAC-sequencing (10X Multiome). Analyses are ongoing but WES data does not support genetic causes for their differences while the 10X Multiome data indicate that epigenetic regulation may underlie the different properties of bulk and edge glioblastoma cells.
STEM-16. MACROH2A2 REPRESSES AN EPIGENETIC SELF-RENEWAL NETWORK IN GLIOBLASTOMA STEM CELLS
Authors: Nikolic Ana , Bobyn Anna , Maule Francesca , Ellestad Katrina , Paik Seungil , Marhon Sajid , Mehdipour Parinaz , Lun Xuequng , Johnston Michael , Gafiuk Christopher , Zemp Franz , Shen Yaoqing , Ninkovic Nicoletta , Labit Elodie , Berger N Daniel , Brownsey Duncan , Kelly John , Biernaskie Jeff , Dirks Peter , Derksen Darren , Jones Steven J M , Senger Donna L , Chan Jennifer , Mahoney Douglas J , de Carvalho Daniel , Gallo Marco
Published: November 2022

Glioblastoma is a highly malignant tumour driven by a subset of self-renewing cells termed glioblastoma stem cells. This self-renewal phenotype is largely epigenetically driven, however, the exact epigenetic mechanisms underpinning it are poorly understood. We found that the histone variant macroH2A2 is repressed in a subset of glioblastoma tumours, and that this repression is associated with poorer survival. We interrogate the function of macroH2A2 using in vitro and in vivo patient-derived models. We show that macroH2A2 antagonizes self-renewal and expression of NPC and OPC-like markers, and rewires accessible chromatin by maintaining accessibility at a subset of enhancer elements. We identify small molecules that can upregulate macroH2A2 expression, and find that treatment with a small molecule reduces self-renewal, induces interferon sensitive genes, and a viral mimicry response, effects which are abolished by macroH2A2 knockdown. In summary, we identify macroH2A2 as a repressor of self-renewal in glioblastoma and suggest it may be a novel biomarker and potential marker of therapeutic susceptibility.
STEM-18. EPIGENETIC AND MOLECULAR COORDINATION BETWEEN HDAC2 AND SMAD3-SKI IS REQUIRED FOR GROWTH AND STEM CELL CHARACTERISTICS OF BRAIN TUMOUR STEM CELLS
Authors: Bahia Ravinder , Hao Xiaoguang , Hassam Rozina , Cseh Orsolya , Bozek Danielle , Luchman H Artee , Weiss Samuel
Published: November 2022

Brain tumour stem cell population in glioblastoma (GBM) display key cancer stem cell characteristics of high self-renewal and drug resistance that are maintained by the coordinated functions of epigenetic and molecular regulators. Yet, specific epigenetic mechanisms that, in collaboration with relevant molecular pathways, help maintain a stem-like state in BTSCs remain poorly understood. Here, we identify HDAC2 as a foremost epigenetic regulator in BTSCs that specifically utilizes the transforming growth factor-β (TGF-β) pathway related proteins, SMAD3-SKI, for remodelling BTSC chromatin accessibility and transcriptional programs to facilitate their stemness and tumorigenic potentials. Our initial drug screening revealed that selective inhibition of HDAC1 and 2 with romidepsin was effective in targeting BTSC viability, cell proliferation and self-renewal in vitro. Using CRISPR-cas9 knockout and shRNA knockdown strategies, we further demonstrated that loss of HDAC2 disrupts an epigenetic and molecular coordination between HDAC2 and SMAD-SKI proteins, which negatively impacts BTSC survival, cell proliferation and self-renewal in vitro and improves median survival in orthotopic xenograft mouse models. Loss of HDAC2 showed reduction in the protein abundance of transcriptional regulator, SMAD3 and negative regulator protein, SKI. However, overexpression of SMAD3 in HDAC2 deficient BTSCs could partially rescues their cell functional deficits. These findings suggest that context-specific epigenetic regulations by HDAC2 and its interaction with the critical transcriptional regulators, SMAD3-SKI, maintains the stemness and growth characteristics of BTSCs. Further HDAC2 overexpression increases cell proliferation and self-renewal abilities in normal neural stem cells (NSCs). These findings thus support the role of HDAC2 as a key epigenetic determinant of stemness in normal NSCs and of cancer stem cell characteristics and tumorigenic potential in BTSCs. Collectively, our data raises the potential that disruption of the coordinated mechanisms regulated by HDAC2-SMAD3-SKI axis may be an effective therapeutic approach for targeting GBM BTSCs.
STEM-21. REPURPOSING CLEMASTINE TO SUPPRESS GLIOBLASTOMA STEM CELLS
Authors: Sun Michael , Yang Rui , Liu Heng , Wang Wenzhe , Song Xiao , Hu Bo , Reynolds Nathan , Roso Kristen , Chen Lee , Greer Paula , Cheng Shi-Yuan , Ashley David , Pirozzi Christopher , He Yiping
Published: November 2022

Glioblastoma stem cells (GSCs), also known as brain tumor initiating cells (BTICs), drive tumor progression, heterogeneity, and resistance to treatments, posing formidable challenges to advancing effective treatments against glioblastoma (GBM). We postulated that inducing BTIC differentiation can serve as a solution to diminishing their stem-like features. Here, we report that clemastine, an over-the-counter oral medication for allergy relief, attenuates stemness and proliferation of BTICs. These effects of clemastine were accompanied by altered transcriptional programs suggestive of a shift from maintaining stem cell identity to differentiation, resonating with the ability of clemastine to promote oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes. Genetic perturbation and small-molecule inhibition of putative pharmacological targets of clemastine revealed that Emopamil-binding protein (EBP), an enzyme in the sterol biosynthesis pathway, played a pivotal role in mediating the differentiating and anti-tumor effects of clemastine. Notably, loss-of-function assays showed that EBP expression was indispensable for BTIC propagation. In contrast, overexpression of EBP stimulated BTIC proliferation, thus uncovering a previously unknown role of sterol metabolism in BTIC maintenance. Finally, we demonstrated that a mouse neural stem cell-derived glioma model was similarly susceptible to clemastine treatment. Taken together, our study identifies pathways essential for the perpetuation of stemness in GBM, and implicates a non-oncology drug with a well-established safety profile that can be repurposed to mitigate the stem-like properties of GBM.
STEM-23. SLOW CYCLING CELL RNA BASED T CELL THERAPY TO PREVENT RECURRENCE IN GBM
Authors: Dajac Mariana , Conforti Cole , Tian Guimei , Moore Ginger , Flores Catherine , Huang Jianping , Mitchell Duane , Deleyrolle Loic
Published: November 2022

Glioblastoma (GBM), the most common primary malignancy of the central nervous system (CNS), is almost universally fatal due to inevitable recurrence despite aggressive therapeutics. Considering the infiltrative nature of GBM, targeting residual cells after surgical tumor resection is challenging. Development of effective novel adjuvant therapies against drivers of disease recurrence is of imperative priority. Anticancer treatments most effectively eliminate rapidly dividing cells but spare dormant or infrequently dividing populations. Multiple studies have reported in numerous cancers the existence of slow-cycling cells (SCCs) that are refractory to therapies. Our laboratory identified SCCs in high-grade glioma, which represents an enriched reservoir of highly infiltrative and treatment-resistant cancer propagating cells known to drive recurrence. An effective approach to specifically eliminate this cell population is urgently required to limit recurrence and improve survival. Importantly, these cells reliably defined a cellular niche characterized by definite potentially immunogenic neoantigens, which can serve as immune targets. Our ultimate goal is to leverage the power of adoptive cellular therapy (ACT) to recognize GBM SCC-derived antigens to achieve robust tumor control through targeting of these critical drivers of recurrence. Our data revealed that dendritic cells electroporated with slow-cycling cell RNA successfully primed T cells to recognize and target glioma treatment resistant cells. These experiments demonstrated that slow cycling cell-specific T cells (SCC-T cells) exhibit enhanced activation when in presence of treatment-resistant tumor cells as shown by increased CD8 differentiation, amplified effector and memory differentiation compared to total tumor RNA-specific T cell and control groups. Additionally, SCC-T cells presented the highest anti-tumor activity, as demonstrated by the greatest decrease of tumor cell proliferation and increase of T cell apoptosis. Together these results demonstrate the efficacy and superiority of SCC-based immunotherapy platform to target treatment-resistant glioblastoma cells.
CCRG-02. THE DIFFUSE MIDLINE GLIOMA MUTATIONS, H3.3 K27M AND G34R/V, INHIBIT MITOTIC PHOSPHORYLATION OF SERINE 31, LEADING TO TUMOR FORMATION THROUGH INCREASED CHROMOSOMAL INSTABILITY
Authors: Day Charles , Grigore Florina , Langfald Alyssa , Hakkim Faruck , Daniels David , Vaughan Kevin , Robinson James , Hincliffe Edward
Published: November 2022

Diffuse midline gliomas (DMG) are high-grade pediatric gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. In an aberrant division, chromosome missegregation triggers S31 phosphorylation along chromosome arms in anaphase leading to p53-induced cell cycle arrest. Here we show in vitro and in vivo, that H3.3 K27M, G34R, and G34V significantly suppress S31 phosphorylation by Chk1 (the mitotic S31 kinase). In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used genome editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of DMG. Expression of PDGFβ linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDGFβ-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single DMG-associated point mutation in H3.3 can generate chromosomal instability and suggests that chromosomal instability is a major contributing factor to DMG K27-altered and DMG G34-altered tumor formation.
CSIG-02. FOSL1 PROMOTES GLIOMA STEM CELLS TRAITS THROUGH INTERACTING WITH ITS BINDING PROTEIN PHOSPHORYLATED STAT3
Authors: Go Shanchun , King Pendelton , Liu Mingli
Published: November 2022

BACKGROUND

Glioma stem cells (GSCs) have key roles in tumorigenesis, progression, and resistance to conventional treatments for glioblastoma. We reported that TRPM7 channels, which fused with C-terminal kinase, endow glioma cells with stem cell-like capacities of self-renewal. Here, we investigated the role of TRPM7 downstream target gene FOSL1’s post transcription in glioma stemness.

METHODS

1) The immunohistochemical staining for FOSL1 was performed using the rabbit polyclonal anti-FOSL1 antibody in glioma tissue arrays from 76 Chinese patients and 10 normal individuals. The staining intensity of cells in TMA was evaluated in three different fields (≥100 cells/field). The semi-quantitative HSCORE was calculated for FOSL1. 2) Upon suppression of FOSL1 by siRNA FOSL1, GSC markers, CD133 expression was evaluated by flow cytometry using PE anti-human CD133 antibody, while ALDH1 enzymatic activity was assessed using an Aldefluor kit, in A172 and U87MG glioma cell lines and patient-derived xenoline PDX-L14 of proneural subtype. 3) To assay FOSL1 gene promoter activity, the 5’-flanking region of the human FOSL1 gene (1201bp) was inserted into the firefly luciferase reporter vector pGL3-Basic between MluI and BglII sites. For mutant construct, STAT3 binding sites were mutated using Q5 site-directed mutagenesis kit.

RESULTS

1) Quantification of the IHC results showed that FOSL1’s positive nuclear staining was significantly higher in GBM (n=13, p=0.0005), grade III (n=12, p=0.0116), and grade II astrocytoma (n=51, p=0.0485) compared to that of normal brain tissue (n=10). 2) GSC markers CD133 and ALDH1 were positively associated with FOSL1 in glioma. 3) Phosphorylated STAT3 directly binds to the FOSL1 promoter and the activation levels of STAT3 have a positive effect on the transcriptional activity of FOSL1.

CONCLUSION

Our results demonstrated the role of TRPM7-regulated STAT3 activation directly regulated FOSL1 gene transcription, which shed new light on TRPM7/FOSL1-directed therapeutics in glioma. (This study was supported by NIH NIGMS GM121230)
CSIG-13. CLINICAL IMPACT OF CDKN2A/B DELETIONS IN IDH-MUTANT ASTROCYTOMAS
Authors: Yuile Alexander , Satgunaseelan Laveniya , Alexander Kimberley L , Thavaneswaran Subotheni , Krasovitsky Michael , Buckland Michael , Lee Maggie , Wei Grace , Kastelan Marina , Wong Mark , Wilson Isabella , Bayly Angela , Varikat Winny , Sim Hao-Wen , Kong Ben , Lwin Zarnie , Turner Cassie , Back Michael , Miller Samuel , Lee Adrian , Wheeler Helen
Published: November 2022

BACKGROUND

IDH-mutant astrocytomas with CDKN2A/B homozygous deletions (HD) are now categorized as grade 4 astrocytomas. It is unclear how these HD astrocytomas should be treated. We aimed to compare 3-year outcomes for HD versus heterozygous deleted (HeD) IDH-mutant astrocytomas, describe the prognostic impact of deletions relative to histologic grade and describe differences in outcomes based on treatment regimen.

METHODS

Molecular and clinical data concerning IDH-mutant glioma patients +/-CDKN2A/B deletions were retrieved across four neuro-oncology centers and two tumor banks from 01/2016-07/2021.

RESULTS

48 patients with HD or HeD were identified. Median follow-up time was 4.0 years, median age 33 (range 20-74) years. Sixteen astrocytomas were histologic grade 4, remainder were grade 2 (n= 9) and grade 3 (n= 23). HD were detected in 16 patients. Survival data were unavailable in n= 1 HD and n= 2 HeD patients. 3-year OS for CDKN2A/B HD and HeD groups were 47% versus 67% (log-rank p= 0.004). For grades 2, 3 and 4, 3-year OS for HD were 100% (n= 1), 57% (n= 7) and 29% (n= 7) respectively, 3-year OS for HeD were 100% (n= 7), 56% (n= 16) and 57% (n= 7) respectively. In HD patients, six were treated with concurrent temozolomide-radiotherapy (3-year OS 50%) and five with radiotherapy and sequential temozolomide only (3-year OS 80%, log-rank p= 0.38). Treatment data were unavailable in two patients and three were treated with “other” regimens. Sequential regimens were preferentially used in histologic grade 2 and 3 cases.

CONCLUSION

Preliminary data are in keeping with current literature and are one of the first to describe outcomes based on clinical regimen. There was insufficient evidence of OS differences between concurrent and sequential therapy, though conclusions are limited by sample size and data maturity. Additional follow-up data are required to further define optimal treatment for these patients.