bluuebunny/crossref_metadata_2025_split huggingface dataset:
STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA
Authors: Lauko Adam , Turaga Soumya M , Volovetz Josephine , Bayik Defne , Bao Shideng , Yu Jennifer , Lathia Justin 
Published: November 2021

Abstract
Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.
STEM-20. THE ROLE OF HUMAN BETA DEFENSINS IN THE CLONOGENICITY OF GLIOBLASTOMA MULTIFORME
Authors: Harris Peggy , Kerstetter-Fogle Amber , Sloan Anthony , Raghavan Alankrita , Hoffman Harry , Barnholtz-Sloan Jill , Couce Marta , Jin Ge , Weinberg Aaron , Sloan Andrew 
Published: November 2021

Abstract

INTRODUCTION
Glioblastoma (GBM) is the most common primary malignant brain tumor with a median overall survival of 12-15months. GBM aggressiveness and poor response to treatment are often attributed to a small population of stem-like cells referred to as glioma stem cells (GSCs). Human Beta-Defensins (HBDs), a family of small molecules initially thought to function as anti-microbials, have been implicated in various cancers with functions that are cancer type specific, including proinflammatory and immunosuppressive roles. GOAL: This study aimed to elucidate HBDs expression in GSCs and differentiated gioma cells (DGSs).

METHODS
We identified HBD2 and HBD3 in glioma and GSCs and DGSs by immunofluorescence (IF) and immunohistochemistry (IHC). We also assessed the role HBD2/3 play in GBM oncogenesis by investigating their effects on the self-renewal capacity of GSCs.

RESULTS
HBD2 and HBD3 are found in both bulk tumor and GSCs by IHC and IF. Expression of HBD2 and HBD3 mRNA levels are elevated in GBM patient tissue in comparison to lower grade gliomas by 16.2 & 1.9 fold (respectively; p &lt; 0.0001). Additionally, transcriptional expression of HBD2 and HBD3 are increased by 0.68 and 1.15 fold respectively in GSCs versus autologous DGCs (p &lt; 0.05; p&lt; 0.005 respectively), suggesting a role for HBD 2/3 in oncogenesis. Interestingly however, HBD2 and HBD3 pretreatment of GSC cell lines showed decreased self-renewal capacity by 34.2 and 66.4% (p &lt; 0.001), determined by the reduced number of large neurospheres ( &gt;250mm).

CONCLUSIONS
Our results demonstrate the presence of HBD2/3 in GBM samples by IHC and IF with increased HBD2/3 mRNA expression in GBM samples. Interestingly DGCs contain less HBD2/3 than their GCS counterparts, and clonogenicity assays demonstrate a decrease in oncogenesis, suggesting that HBD’s role in proliferation and clonogenicity of DGCs and GSCs may be context dependent, leading to additional questions and future studies.
STEM-22. TARGETING REPLICATION STRESS RESPONSE FOR GLIOMA STEM CELL SPECIFIC CYTOTOXICITY
Authors: Clough Emily , Ballinger Tracy , Semple Colin , Strathdee Karen , carruthers ross 
Published: November 2021

Abstract

BACKGROUND
Evidence suggests treatment resistant glioma stem cells (GSCs) drive glioblastoma (GBM) recurrence. Current treatments fail to eradicate GSC and novel GSC targeting therapies are a priority. GSC exhibit elevated DNA replication stress (RS) versus non GSC tumour cells driving constitutive DNA damage response (DDR) activation and efficient DNA repair. We previously demonstrated that targeting RS response with combined ATR and PARP inhibition (CAiPi) (VE821 and Olaparib) provides potent GSC specific cytotoxicity. In this study we investigated the underlying DDR phenotype which determines this vulnerability.

RESULTS
Paired GSC enriched (‘GSC’) and GSC depleted, differentiated (‘bulk’) populations were cultured from GBM specimens in neurobasal media with growth factors or serum containing media respectively. GSC exhibited reduced survival following exposure to CAiPi versus bulk. CAiPi significantly increased 53BP1 G1 phase nuclear bodies (53BP1NBs) in GSC, which are known to shield under-replicated DNA in actively transcribed genes. Mapping the genomic distribution of endogenously occurring replication dependent DNA double strand breaks via Breaks Ligation In Situ Sequencing (BLISS), revealed reduced intragenic DSB in long actively transcribed genes in GSC versus bulk at baseline, suggesting a reliance upon transcription coupled DSB repair in GSC. RNA seq demonstrated CAiPi-induced transcriptomic alterations in GSC including replication regulation and initiation. DNA fibre assay showed that CAiPi increased GSC new origin firing which correlated with PARP trapping. GSCs were rescued from CAiPi by roscovitine induced inhibition of excess origin firing. CAiPi is potently radiosensitizing by clonogenic assay and we demonstrated murine blood brain barrier penetration of CAiPi utilising VE822 and pamiparib in vivo.

CONCLUSION
Dysregulation of origin firing by CAiPi exposes a GSC specific vulnerability which results in DNA under-replication and abrogation of proficient DNA repair seen at long actively transcribed genes and has potential to be clinically translated as a GSC specific cytotoxic therapy.
CBIO-17. A NOVEL ROLE FOR ATR KINASE DETERMINES GLIOBLASTOMA TUMOUR CELL INVASION
Authors: carruthers ross , Derby Sarah , Strathdee Karen , Chalmers Anthony , Norman Jim , Birch Joanna 
Published: November 2021

Abstract
BACKGROUND: Widespread contamination of the brain with malignant cells is a predominant feature of glioblastoma (GBM) and fatal brainstem infiltration is frequently observed at autopsy. Whilst radiotherapy improves survival, irradiation increases GBM cell invasion, resulting in sublethal dose to cells migrating outside the irradiated volume. Tumour cell invasion should be a therapeutic priority if survival is to be improved. The responsible molecular mechanisms are key to improving outcomes but remain enigmatic. Ataxia telangiectasia and rad3-related (ATR) is a DNA damage response (DDR) kinase involved in DNA replication stress (RS) response and is an established therapeutic target for GBM. In this study we demonstrate a novel role for ATR kinase in facilitating malignant cell invasion. METHODS AND RESULTS: Invading margins of human GBM samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated a reduction in cell velocity following ATR inhibition (ATRi; VE822) or ATR siRNA, and a retraction defect was evident in vitro. Extensive cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures on electron microscopy which could engulf high molecular weight dextran, suggesting blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed integrin sequestration within macropinosomes and reduced integrin internalisation respectively. Interrogation of a published ‘ATR interactome’ revealed ATR targets with functions in endocytic vesicle trafficking. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further study demonstrated reduced invading tumour cells following ATRi in intracranial xenografts. CONCLUSION: We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin internalisation. ATRi results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.
CBIO-27. INTEGRATED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA
Authors: Fernandez Elizabeth , Mia Wilson , Bayley Nicholas , Zhu Henan , Tse Christopher , Liau Linda , Cloughesy Timothy , Nathanson David 
Published: November 2021

Abstract
Molecular profiling frequently fails to predict therapeutic outcomes in cancer. This is in part due to the myriad of genetic alterations comprising a tumor which enable rapid rewiring of cancer signaling pathways. Functional profiling ascertains signaling dynamics and has emerged as an alternative method to predict drug responses. It is unknown whether incorporating molecular with functional profiling offers superior insight into specific tumor dependencies and consequently therapeutic susceptibilities. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that treatment with standard of care (e.g., temozolomide or radiation) rewired the apoptotic machinery in a p53 dependent manner, eliciting an exclusive survival dependency on BCL-xL in a genetically defined subset of GBM tumors. However, functional BH3 profiling of ‘apoptotic priming’ was required to predict response to combined IR/TMZ and BCL-xL inhibition in p53 WT GBM tumors. Accordingly, a composite of genetic and functional profiling predicted the response of patient derived GBM models to IR/TMZ with a novel GBM specific BCLxL antagonist. Collectively, these studies identify the mechanisms of intrinsic apoptosis resistance in GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.
CSIG-04. UNCOUPLING OF ETS1 FROM MAPK PATHWAY SIGNALS AS RESISTANCE MECHANISM TOWARDS BRAF INHIBITORS IN BRAF-MUTATED CHILDHOOD GLIOMA
Authors: Gabler Lisa , Lötsch-Gojo Daniela , Kirchhofer Dominik , Laemmerer Anna , Mayr Lisa , Pirker Christine , Jaunecker Carola , Kastler Lucia , Azizi Amedeo A , Dorfer Christian , Czech Thomas , Haberler Christine , Peyrl Andreas , Slavc Irene , Spiegl-Kreinecker Sabine , Roessler Karl , Gojo Johannes , Berger Walter 
Published: November 2021

Abstract

BACKGROUND
High-grade gliomas are among the most aggressive brain tumors across all age groups. BRAF is within the most frequently altered genes in pediatric glioma, sometimes connected with telomerase reverse transcriptase (TERT) promoter mutations, predicting a particularly aggressive course of disease. Precision medicine approaches targeting the MAPK pathway have shown promising results in patients with BRAF-mutated glioma. Although acquired insensitivity to BRAF inhibitors appears as major issue for therapy failure, underlying molecular mechanisms are still poorly understood.

METHODS
Cell models from an anaplastic pleomorphic xanthoastrocytoma with BRAF V600E and TERT promoter mutations and the recurrent tumor, operated following MAPK pathway-targeting therapy, were established. Furthermore, a dabrafenib-resistant subline of the recurrent tumor was generated. The patient-derived cell models were genetically characterized using array-based genomic hybridization (aCGH). Sensitivity of the cells towards different MAPK pathway inhibitors was tested. Basal expression and activation of MAPK pathway and downstream signals were analyzed by qRT-PCR and Western blots.

RESULTS
Screening a panel of both primary and immortalized glioma cell models with different BRAF and TERT promoter status revealed significantly induced MAPK pathway activation and enhanced TERT levels in BRAF V600E and TERT promoter double-mutant gliomas. Furthermore, cells with both mutations were hyper-sensitive towards BRAF-targeting agents and BRAF inhibition resulted in reduced TERT levels. ETS1 expression was strongly increased in the recurrent tumor and identified as important player in telomerase re-activation. aCGH revealed gains of chromosomal regions encoding for different ETS-factors in the dabrafenib-resistant subline. Western blot analyses suggested a BRAF/ERK-independent survival mechanism in the dabrafenib-resistant subline. Accordingly, dabrafenib insensitivity triggered cross-resistance towards the MEK inhibitor trametinib. Uncoupled from the MAPK pathway, ETS1 expression was further upregulated in the dabrafenib-resistant subline. CONSLUSION: Taken together, our data demonstrate that MAPK-independent ETS transcription factor upregulation is a central mechanism of BRAF inhibitor therapy failure in BRAF-mutated pediatric glioma patients.
CSIG-13. A DYNAMIC CAUSAL MODEL OF GLIOBLASTOMA STEM CELL SIGNALING PREDICTS EFFECTS OF KINASE INHIBITORS
Authors: Holzapple Emilee , Miskov-Zivanov Natasa , Cochran Brent 
Published: November 2021

Abstract
Glioblastomas and glioblastoma stem cells are heterogeneous with respect to mutations, gene expression, and response to drugs. To make predictive responses of individual GBM stem cell lines to kinase inhibitors, we have constructed a causal model of glioblastoma stem cell signaling. The core model was built starting from pathways identified from TCGA mutation data with the addition of the Jak/STAT, Hedgehog, and Notch pathways. Elements and relations between them were validated and extended using the PCNet interaction database and the INDRA database which includes machine read extractions from the biomedical literature. The result is a high confidence executable model consisting of 209 elements (proteins, genes, RNAs) and 370 regulatory logic rules between the elements. Stochastic simulations of the model provide dynamic (quantile) changes in time and responses to perturbations. The output simulates activity of individual nodes as well as cell cycle progression, apoptosis, and differentiation. To simulate the responses of individual cell lines to kinase inhibitors, the model was initialized using DNA sequencing data, RNA-seq, and reverse phase protein array (RPPA) data from each cell line. Comparing the results of the simulations to the drug responses of 11 different kinase targets in 3 cell lines, the model was 88% accurate in predicting effects on growth and survival. The model was further tested by comparing the effects of Mek inhibition of each of the cell lines in the model to the results observed in the RPPA data which overlap by 127 elements. In this case, there was less than 65% concordance between the model and the data for individual nodes. Discrepancies between the model predictions and the data are being investigated to determine whether the model logic or extent needs to be revised to improve the model. This modeling approach is a step toward developing algorithms for personalized therapeutics for GBM.
CSIG-18. HYPERMETHYLATION OF SLIT-ROBO PATHWAY GENES RESULTS IN INACTIVATION IN GLIOMA PROGRESSION
Authors: Kerstetter-Fogle Amber , Harris Peggy , Hoffman Harry , Sloan Anthony , Elder Theresa , Couce Marta , Barnholtz-Sloan Jill , Sloan Andrew 
Published: November 2021

Abstract

INTRODUCTION
Glioblastomas (GBM) are the most common and malignant primary brain tumors. Their rapid growth and invasion into neuronal parenchyma is devastating, with limited treatment options. Genomic alterations have been extensively studied in gliomas tumors, including aberrations of the Slit-Robo pathway genes. The Slit family of secreted proteins modulates migration of somatic cells during development and mediate their effect by binding to its receptor Roundabout (Robo). Genes in the Slit-Robo pathways have been shown to be inactivated by promoter hypermethylation in a number of human cancers. We hypothesize that Slit-Robo signaling pathways are modulated via promotor methylation, controlling GBM malignancy and regulating the invasive capacity of glioma stem cells (GSCs).

METHODS
We characterized expression of mRNA and protein via real-time PCR and Western blots in primary GBM tissue. We then assessed whether epigenetic alterations correlate with Slit-Robo expression by conducting in vitro demethylation assays in patient derived GSCs followed by real-time PCR. We conducted invasion assays to elucidate the role of Slit in GSC invasion.

RESULTS
The Cancer Genome Atlas indicates a negative correlation between Robo2 expression and glioma patient survival (17.1 months versus 37.4 months; p &lt; 1.4E-4). We discovered differential expression of the Slit (1-3) and Robo (2, 3) genes and protein of up to 8-fold between grades 3 and 4 astrocytomas (8.34 versus 4.73 density). Treating patient derived glioma stem cells with 5-aza-2-deoxycytidine results in induction in Slit-Robo gene expression ranging from 5 – 40-fold (p &lt; 0.01). Addition of Slit 2 and 3 in an invasion assay induced migration of GSCs in a differential and concentration dependent manner of 10 - 25% (p &lt; 0.05).

CONCLUSIONS
Our results suggest that promoter methylation and gene expression of the Slit-Robo genes correlates with protein expression, tumor invasiveness, and prognosis and a potential therapeutic target.
CSIG-29. TRIBBLES-1 (TRIB1) INCREASES TUMOR FORMATION IN AN ORTHOTOPIC MOUSE MODELS BY PROMOTING SURVIVAL OF GBM CELLS AND TREATMENT RESISTANCE
Authors: Singh Karnika , Han Chunhua , Fleming Jessica , McElroy Joseph , Kumar Ashok , Sebastian Ebin , Bell Erica , Robe Pierre , Haque Jahar , Chakravarti Arnab 
Published: November 2021

Abstract

INTRODUCTION
Glioblastoma (GBM) is the most aggressive CNS tumor with an average survival of about 15 months after diagnosis. The current gold standard therapy comprises radiation therapy (RT) and concurrent and adjuvant temozolamide (TMZ). Due to poor prognosis, novel therapeutic targets need to be identified for drug development. To this end, we identified TRIB1 through correlative studies using patient derived methylation data from an institutional cohort as a novel therapeutic target. TRIB1 is a Ser/Thr pseudokinase that functions as a scaffold for the degradation of its substrates and activates Akt and MEK oncogenic pathways. In this study, we show that TRIB1 promoted GBM progression by upregulating survival pathways and reducing RT/TMZ-induced cell death.

MATERIALS AND METHODS
In vitro functional validation was performed by overexpression and knockdown approaches. GBM patient derived (PDX) cell lines overexpressing the TRIB1 transgene were used to create an orthotopic tumor model for in vivo studies. Mice were monitored for changes in tumor volume and overall survival. Stable cell lines were generated by puromycin selection. Western blotting was utilized to detect protein levels.

RESULTS
Mice inoculated with PDX cells overexpressing TRIB1 transgene had increased tumor volume and worse overall survival compared to the empty vector control. We also observed that TRIB1 overexpression caused decreased apoptosis of PDX cell lines after RT/TMZ treatment. Additionally, an increase in the phosphorylation of ERK and Akt was also noted after TRIB1 overexpression. Consistent with these observations, TRIB1 knockdown sensitized the cells towards radiation and caused decreased Akt phosphorylation/activation as well.

CONCLUSION
TRIB1 overexpression decreases overall survival of xenograft bearing mice and promotes GBM cell survival by upregulating survival signaling pathways. This compromises the effects of RT/TMZ therapy, which is reversed after TRIB1 knockdown. Targeting of TRIB1 may reduce oncogenic signaling in GBM cells and therefore would sensitize them towards RT/TMZ therapy.
CSIG-30. TARGETING TELOMERASE VIA MEK INHIBITION IN AGGRESSIVE TERT PROMOTER MUTATED BRAIN TUMORS
Authors: Lötsch-Gojo Daniela , Gabler Lisa , Jaunecker Carola , Fürnweger Kerstin , Bruckner Katharina , Mayr Lisa , Madlener Sibylle , Kirchhofer Dominik , Spiegl-Kreinecker Sabine , Roessler Karl , Berger Walter , Gojo Johannes 
Published: November 2021

Abstract
Activating point mutations within the TERT promoter (C228T or C250T) account for the most frequent alteration in aggressive brain tumors. Presence of these alterations results in the generation of binding sites for E-twenty-six (ETS) transcription factors accompanied by enhanced TERT expression. Accordingly, TERT promoter mutations foster cellular immortalization and subsequently tumor aggressiveness. Due to the limitation of treatment options in aggressive brain tumors, including glioblastoma and medulloblastoma, new therapeutic targets need to be discovered. As we previously described a strong interaction of oncogenic MEK/ETS signaling and TERT promoter mutations, we hypothesize that inhibition of these factors halters cell immortalization in TERT-driven brain tumors. Our study included three TERT promoter wild-type (TERTwt), six mutated (TERTmut) glioblastoma and three TERTmut medulloblastoma cell models and tested the effect of MEK inhibitors (U0126 and trametinib) and the ETS inhibitor YK-4-279 on cell viability and clone formation. Cellular senescence upon treatment was evaluated by beta-galactosidase assays. Impact on TERT mRNA expression and TERT promoter activity were analyzed by quantitative real-time PCR and luciferase reporter assays, respectively. Furthermore, the effects on MAPK- and PI3K pathway activation were evaluated by Western blot. Amongst the investigated inhibitors, tumor cells harboring C228T mutation were distinctly more sensitive against trametinib as compared to TERTwt and C250T TERTmut cells. Similar effects were observed on clonogenicity upon long-term exposure to this inhibitor. Regarding MAPK signaling activation, trametinib treatment completely blocked ERK phosphorylation in every cell model, while activation of ETS1 was more effectively reduced in C228T TERTmutcells. Accordingly, exposure to trametinib reduced TERT expression and promoter activity accompanied by induction of cellular senescence in cells with C228T mutation. Impact of trametinib is currently investigated in preclinical experiments using TERTmut brain tumor models. Summarizing, MEK inhibition represents a novel strategy to overcome cell immortalization especially in C228T TERTmut brain tumors.