bluuebunny/crossref_metadata_2025 huggingface dataset:
BIOM-15. SERUM AMYLOID-β42 AS A NONINVASIVE BIOMARKER FOR PROGNOSIS AND HISTOLOGIC FEATURES OF GLIOMA
Authors: Hwang Kihwan , Noh Minhee , Park Jay , Ji So Young , Han Jung Ho , Ahn Kwang Sung , Kim Chae-Yong
Published: November 2021

INTRODUCTION

Glioma is the most common primary tumor in central nervous system and is often refractory. Histopathologic examination is essential to establish initial diagnosis, and multiple imaging studies are conducted to assess for treatment response. However, those conventional approaches usually accompanies high risks and costs during treatment. The purpose of this study is to find a novel candidate of noninvasive biomarker for histologic prediction and prognostic assessment of glioma.

METHODS

Serum was prepared from blood samples collected preoperatively from 65 patients with WHO grade II–IV glioma from October 2004 to December 2017 in a single tertiary-level institution. Concentration of amyloid beta-42 (Aβ42) was measured by SMCxPRO (Merck) immunoassay. Clinical characteristics and histologic features of the patients including the molecular subtype were reviewed. Progression-free survival has been evaluated for the primary outcome.

RESULTS

The mean age of the patients was 53.7±12.2 years. 37 (56.9%) patients were males and 21 (32.3%) cases were primary tumors. In Kaplan-Meier survival analysis, higher serum Aβ42 (>5.7 pg/ml) group showed poor outcome with a statistical significance (p=0.014). In multivariate regression analysis, the concentration of serum Aβ42 showed a significant association with EGFR expression, or Ki-67 labeling index. The higher concentration of serum Aβ42 was associated with EGFR wild type (odds ratio 0.237, p=0.022), high cell proliferation (β=0.339, p=0.007) and poor outcome (hazard ratio 0.339, p=0.046).

CONCLUSION

Serum Aβ42 level is a possible good candidate of noninvasive biomarkers for prediction of histologic features and prognosis in glioma patients. Further studies with large cohorts might be mandatory for the clinical use of Aβ42.
BIOM-23. A PILOT STUDY OF WHOLE GENOME SEQUENCING (WGS) OF PLASMA CELL-FREE DNA (cfDNA) FOR ULTRASENSITIVE DETECTION OF TUMOR DNA IN PATIENTS WITH GLIOBLASTOMA (GBM)
Authors: Bagley Stephen J , Till Jacob , Abdalla Aseel , Nasrallah MacLean , Lauterman Tomer , Tavassoly Iman , Oklander Boris , Zviran Asaf , Carpenter Erica
Published: November 2021

BACKGROUND

Plasma circulating tumor DNA (ctDNA) is rarely detectable by traditional methods in patients with GBM. As a result, unlike in lung and other cancers, serial next generation sequencing of ctDNA for monitoring GBM tumor burden has been challenging. In light of the low tumor fraction (TF) of DNA fragments in GBM patient plasma and the urgent need to improve upon MRI for tracking GBM tumor burden, we conducted a pilot study in patients with newly diagnosed GBM using the C2 intelligence platform (C2i Genomics), which leverages genome-wide mutational integration for highly sensitive ctDNA detection.

METHODS

Plasma was collected pre- and post-operatively in patients with newly diagnosed GBM undergoing surgical resection/biopsy. cfDNA was extracted, quantified, and analyzed for fragment size. Genomic DNA (gDNA) was extracted from matched tumor tissue. Whole genome sequencing (WGS) was performed on both gDNA and cfDNA. A specific copy number alteration (CNA) compendium was created for each patient to generate a readout of TF (Zviran, Nat Medicine 2020). We assessed the association between TF at post-operative day 1 (a surrogate for residual disease) and OS, adjusting for other prognostic factors using Cox regression.

RESULTS

37 patients were enrolled. For samples with high tumor fraction (n=5), a statistically significant (p< 1e-4) correlation between CNA profiles of tumor tissue and plasma samples was observed. Post-operative TF above the median value was associated with inferior OS (median 7.7 vs. 19.3 months, p=0.019). This association persisted after adjusting for age, O6-methylguanine-DNA methyltransferase methylation status, extent of resection, and performance status (adjusted HR 2.5, 95% CI 1.1-5.6, p=0.03).

CONCLUSION

Genome-wide mutational integration enables ultra-sensitive detection of ctDNA in GBM patient plasma. Post-operative TF measured by the C2i test is independently associated with OS in newly diagnosed GBM, providing the foundation to evaluate this technology for personalized prognostication and disease monitoring.
BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA
Authors: Dmello Crismita , Sonabend Aarón , Arrieta Víctor , Zhang Daniel , Kanojia Deepak , Chen Li , Gould Andrew , Kane J Robert , Zhang Jiangshan , Kang Seong , Winter Jan , Horbinski Craig , Győrffy Balázs , Cordero Alex , Chang Catalina , Castro Brandyn , Hsu Patrick , Ahmed Atique , Lesniak Maciej , Stupp Roger , Sonabend Adam M
Published: November 2021

Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.
BIOM-41. LIVE-CELL IMAGING SHOWS UNEVEN SEGREGATION OF EXTRACHROMOSOMAL DNA ELEMENTS AND TRANSCRIPTIONALLY ACTIVE EXTRACHROMOSOMAL DNA CLUSTERS IN CANCER
Authors: Yi Eunhee , Gujar Amit , Guthrie Molly , Kim Hoon , Zhao Dacheng , Johnson Kevin , Amin Samirkumar , Das Sunit , Jillette Nathaniel , Clow patricia , Cheng Albert , Verhaak Roel
Published: November 2021

Oncogenic extrachromosomal DNA elements (ecDNAs) promote intratumoral heterogeneity, creating a barrier for successful cancer treatments. The underlying mechanisms are poorly understood and studies are hampered in part by a lack of adequate tools enabling studies of ecDNA behavior. Here, we show that single-cell ecDNA copy numbers greatly vary between tumor cells, both in vitro and in patient glioblastoma specimens, suggesting uneven ecDNA segregation during mitosis. We established a CRISPR-based approach which leverages unique ecDNA breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying this method during mitosis revealed disjointed ecDNA inheritance patterns, providing an explanation for rapid ecDNA accumulation in cancer. Post-mitosis, ecDNAs tended to cluster and clustered ecDNAs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and sheds new light on mechanisms through which ecDNAs contribute to oncogenesis.
BIOM-45. CHARACTERIZATION OF EVs RELEASED FROM 5-ALA DOSED GLIAL AND EXTRA-AXIAL TUMORS
Authors: Hsia Tiffaney , Yekula Anudeep , Carter Bob , Balaj Leonora
Published: November 2021

INTRODUCTION

The administration of 5-Aminolevunlinic acid (5-ALA) in the context of fluorescence-guided surgery is contingent on the highly selective accumulation of PpIX in tumor cells. PpIX is a precursor in the heme pathway that naturally exhibits fluorescent and phototoxic properties upon excitation. We have previously reported evidence of PpIX fluorescence in tumor-specific extracellular vesicles (EVs). Here, we explore the implications of exogenous 5-ALA on cellular function, EV characteristics, and gene expression.

METHODS

We have characterized a range of glioblastoma, meningioma, and healthy cell lines (Gli36 EGFRvIII, Gli36 EGFR WT, U87, CH-157, IOMM-Lee, and HBMVEC). At equal confluency, cells were dosed with 0.8 mM 5-ALA or mock dosed. Media was collected and processed to eliminate debris after 24 hours. Analysis of cells and EVs was conducted using Amnis ImageStream (IFC) and Nanoparticle Tracking Analysis. EV subpopulations were analyzed with IFC following antibody staining. RNA was extracted using Qiagen exoRNeasy and RNeasy. Libraries were prepared via Qiagen UPX 3’ Transcriptome kit and sequenced using Illumina MiSeq. Data analysis was carried out via GeneGlobe, CLC workbench, and MATLAB.

RESULTS

Following 5-ALA dosing, all tumor cells and their derived EVs exhibit significant levels of fluorescence. Analysis of EV subpopulations demonstrated a general decrease in tetraspanin-positive EVs following 5-ALA dosing. At 24 hours, Gli36 cell lines exhibited increased EV release post 5-ALA whereas U87 and meningioma lines resulted in a decreased EV release rate. This clustering is also reflected in EV size distributions and in cellular and EV differential gene expression analysis. Gene ontology analysis of common genes from mock and dosed EVs demonstrated high counts of genes controlling biological regulation as well as cellular and metabolic processes.

CONCLUSION

Collection and characterization of cancer specific EVs may be advantageous to liquid biopsy development.
STEM-03. Oct4/Sox2 DRIVE AN IMMUNOSUPPRESSIVE GSC PHENOTYPE BY INDUCING T-REG EFFECTOR GENES VIA TGFBR2 SIGNALING
Authors: Johnson Amanda , Korleski Jack , Laterra John , Lopez-Bertoni Hernando
Published: November 2021

Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3- cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in Jurkat cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P- GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies.
STEM-04. PD-1 INDEPENDENT OF PD-L1 LIGATION PROMOTES GLIOBLASTOMA GROWTH THROUGH THE NFkB PATHWAY
Authors: Mirzaei Reza , Yong Wee
Published: November 2021

Brain tumor-initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that a proportion of human and murine BTICs expressed programmed cell death protein (PD-1). Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation, and self-renewal of BTICs. Mechanistically, site-directed mutagenesis, RNA sequencing and pharmacological inhibitors implicated SHP-2-mediated activation of NFkB downstream of PD-1 in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require PD-L1 ligation; thus, the therapeutic blocking antibodies inhibiting PD-1/PD-L1 interaction which failed in glioblastoma trials could not overcome the growth advantage of PD-1 in BTICs. Finally, mice with intracranial Pdcd1 over- or underexpressing BTICs had shorter or longer survival, respectively, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a non-immune resistance mechanism of GBM patients to PD-1 or PD-L1 blocking therapies.
STEM-05. FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND UNRECOGNIZED PATHWAY DEPENDENCIES IN RECURRENT GLIOBLASTOMA
Authors: Chokshi Chirayu R , Tieu David , Brown Kevin R , Venugopal Chitra , Liu Lina , Kuhlmann Laura , Rossotti Martin A , Chan Katherine , Tong Amy H Y , Savage Neil , McKenna Dillon , Aghaei Nikoo , Subapanditha Minomi , Vaseem Shaikh Muhammad , Tatari Nazanin , Brakel Benjamin , Nachmani Omri , Ignatchenko Vladimir , Salamoun Joseph M , Wipf Peter , Sharlow Elizabeth , Provias John P , Lu Jian-Qiang , Murty Naresh K , Lazo John S , Kislinger Thomas , Henry Kevin A , Lu Yu , Moffat Jason , Singh Sheila K
Published: November 2021

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses support parallel tumor-intrinsic mechanisms of treatment resistance which rely on acquisition of immunosuppressive capacity, including a defective mismatch repair pathway, ablation of PTEN activity, and a novel combination of de novo mutations in SWI/SNF components. We map a multilayered genetic and functional response to resist chemoradiotherapy and drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel driver of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 repress axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and exploit a genetic dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We conclude that functional reprogramming drives tumorigenicity and present a dependence on a PTP4A2-ROBO1 signaling axis at GBM recurrence.
STEM-08. ST6Gal1-MEDIATED SILALYLATION IS CRITICAL FOR GLIOBLASTOMA GROWTH
Authors: GC Sajina , Tuy Kaysaw , Rickenbacker Lucas , Jones Brent , Chakraborty Asmi , Miller Ryan , Liu Xiaoguang , Mobley James , Bellis Susan , Hjelmeland Anita
Published: November 2021

Although altered cell surface glycosylation was one of the earliest modifications observed in neoplastic progression, this facet of cancer cell biology has received meager attention, particularly in brain tumors. Among the various glycosyltransferases present in human cells, golgi sialyltransferase ST6Gal1 [beta-galactoside alpha-2,6-sialyltransferase 1] adds sialic acid residues in α2-6 linkage to membrane-bound and secreted N-glycans. ST6Gal1 is known to be pro-tumorigenic in epithelial cancers where it can promote epithelial to mesenchymal transformation, tumor-initiating cell (TIC) phenotypes, and survival of cells exposed to stressors such as chemo- and radiotherapy, hypoxia, or serum starvation. However, roles for this potent TIC regulator have not been well explored in GBM as experiments in standard cell lines suggested ST6Gal1 was epigenetically silenced. To explore our hypothesis that ST6Gal1-mediated α2,6 sialylation is elevated in Brain Tumor Initiating Cells (BTICs) and promotes GBM growth, we utilized GBM patient-derived xenografts (PDXs). ST6Gal1 is expressed in GBM PDX tissue sections and elevated in stem-like BTICs in comparison to differentiated GBM cells or astrocytes. Knockdown of ST6Gal1 in BTICs decreased growth and neurosphere formation capacity in vitro, suggesting that ST6Gal1 regulates BTIC maintenance. Similarly, cells isolated directly from PDXs that were sorted for high and low expression of α2,6 sialylation showed that α2,6 sialylationhigh GBM PDX have elevated neurosphere formation capacity and growth. Further, immunocompromised mice injected with sorted α2,6 sialylationhigh PDX cells had significantly lower survival compared to mice injected with α2,6 sialylationlow cells. Using proteomic analysis of ST6Gal1 KD vs NT PDX, we identified novel regulators of cancer stem cell biology directly modulated by ST6Gal1. As we identified a small subset of IDHwt GBMs with ST6Gal1 and SOX2 amplification, we are generating a novel gliomagenesis model with conditional ST6Gal1 overexpression. Together, our data strongly implicates ST6Gal1 as a regulator of GBM BTIC maintenance and GBM growth.
STEM-09. DEFINING THE ROLE OF CD97 IN GLIOBLASTOMA STEM CELL SELF-RENEWAL
Authors: Ravn-Boess Niklas , Bhowmick Nainita , Hattori Takamitsu , Corrado Alexis , Koide Akiko , Karimkhan Afreen , Koide Shohei , Park Christopher , Placantonakis Dimitris
Published: November 2021

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Despite multimodal therapy, resistant GBM stem-like cells (GSCs) inevitably mediate disease recurrence. To identify novel vulnerabilities of GSCs, we performed an arrayed CRISPR/Cas9 screen against select adhesion G protein-coupled receptors (aGPCRs), many of which we found to be de novo expressed in GBM. Knockout of CD97 (ADGRE5), previously implicated in GBM cell migration, produced the most striking proliferative disadvantage in patient-derived GBM cultures (PDGC) among aGPCRs tested. We found high CD97 surface expression in all our PDGCs, while levels remained nearly undetectable in non-neoplastic brain cells, confirming that CD97 is de novo expressed in GBM. Upon shRNA-mediated knockdown of CD97 in PDGCs from all three TCGA transcriptional subtypes, we observed significantly reduced proliferation, as measured by Ki67 and Hoechst cell cycle analysis, and significantly diminished surface expression of CD133, a GSC marker. Notably, CD97 knockdown also significantly reduced tumorsphere initiation capacity in six PDGCs, as measured by extreme limiting dilution assays. These findings suggest that CD97 regulates GSC self-renewal in vitro. RNA-sequencing and GSEA pathway analysis from PDGCs following CD97 knockdown indicate an enrichment of aerobic respiratory gene sets, suggesting one of the major regulatory roles of CD97 is metabolic regulation. Indeed, metabolic assays show that CD97 knockdown alters oxygen consumption and glycolysis rates in PDGCs. Lastly, we have developed human synthetic antibodies to target CD97 in order to investigate its therapeutic potential. We have observed internalization of some of these antibodies, thus identifying candidates for the development of antibody-drug conjugates. In addition, other clones reduced GBM cell proliferation and elicited expression of various differentiation markers. Overall; our studies identify novel roles of CD97 in regulating the cellular hierarchy in GBM and tumor cell metabolism, and provide a strong scientific rationale for developing biologics to target CD97 in GBM.
STEM-14. THE WRAD COMPLEX REPRESENTS A THERAPEUTIC TARGET FOR CANCER STEM CELLS IN GLIOBLASTOMA
Authors: Mitchell Kelly , Alvarado Joseph , Goins Christopher , Martinez Steven , Macdonald Jonathan , Silver Daniel J , Roversi Gustavo , Kay Kristen , Schafer Rachel , Kashyap Anjali , Lauko Adam , Mulkearns-Hubert Erin E , Johnson Sadie , Rich Jeremy N , Stauffer Shaun , Hubert Christopher , Lathia Justin
Published: November 2021

Glioblastoma (GBM) progression and resistance to conventional therapies is driven in part by cells within the tumor with stem cell properties including quiescence, self-renewal and drug efflux potential. It is thought that eliminating these cancer stem cells (CSCs) is a key component to successful clinical management of GBM. However, currently, few known molecular mechanisms driving CSCs can be exploited for therapeutic development. Core transcription factors such as SOX2, OLIG2, OCT4 and NANOG maintain the CSC state in GBM. Our laboratory recently uncovered a self-renewal signaling axis involving RBBP5 that is necessary and sufficient for CSC maintenance through driving expression of these core stem cell maintenance transcription factors. RBBP5 is a component of the WRAD complex, which promotes Lys4 methylation of histone H3 to positively regulate transcription. We hypothesized that targeting RBBP5 could be a means to disrupt epigenetic programs that maintain CSCs in stemness transcriptional states. We found that genetic and pharmacologic inhibition of the WRAD complex reduced CSC growth, self-renewal and tumor initiation potential. WRAD inhibitors partially dissembled the WRAD complex and reduced H3K4 trimethylation both globally and at the promoters of key stem cell maintenance transcription factors. Using a CSC reporter system, we demonstrated that WRAD complex inhibition decreased growth of SOX2/OCT4 expressing CSCs in a concentration-dependent manner as quantified by live imaging. Overall, our studies assess the function of the WRAD complex and the effect of WRAD complex inhibitors in preclinical models and specifically on the stem cell state for the first time in GBM. Studying the functions of the WRAD complex in CSCs may improve understanding of GBM pathogenesis and elucidate how CSCs survive despite aggressive chemotherapy and radiation. Our ongoing studies aim to develop brain penetrant inhibitors targeting the WRAD complex as an anti-CSC strategy that could potentially synergize with standard of care treatments.
STEM-21. INHIBITING ADENINE SYNTHESIS ATTENUATES GLIOBLASTOMA CELL STEMNESS AND TEMOZOLOMIDE RESISTANCE
Authors: Singh Simranjit , Hansen Landon , Du Changzheng , Roso Kristen , Yang Rui , Greer Paula , He Yiping
Published: November 2021

Glioblastoma (GBM) is a lethal brain cancer that exhibits high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work has shown that homozygous MTAP deletion, a frequent genetic alteration in GBM, promotes the stemness of GBM cells. Here, exploiting the MTAP loss-conferred deficiency in adenine salvage, we demonstrated that subtle levels of adenine blockade via treatment with Alanosine, an inhibitor of de novo adenine synthesis, attenuate the stemness of MTAP-null GBM cells. Transcriptomic profiling performed on Alanosine-treated GBM cells revealed a reduction of mitochondrial DNA-encoded gene expression. Furthermore, Seahorse XF analysis showed that Alanosine treatment led to a reduction in mitochondrial respiration and eliminated GBM cells’ spare respiratory capacity, an important metabolic measure of cell fitness that is representative of their ability to respond to oxidative stress. Importantly, long term adenine shortage via treatment with low doses of Alanosine resulted in similarly compromised mitochondria functionality and attenuated GBM cells’ stemness. In further supporting this adenine blockade – compromised mitochondria – reduced stemness cascade, treatment with a mitochondrial respiration inhibitor attenuated the stemness of GBM cells, suggesting the importance of mitochondrial function in maintaining GBM stemness. Finally, in agreement with the diminished stemness and compromised mitochondrial function, we showed that Alanosine sensitized GBM cells to temozolomide (TMZ) in both in vitro cultures and in an orthotopic GBM model. Collectively, these results identify critical roles of adenine supply in maintaining stemness and mitochondrial function in GBM cells, suggest a targeted method of abrogating stemness and chemoresistance in GBMs, and support targeting adenine synthesis as a complementary approach for treating the half of GBMs harboring MTAP deletions.
STEM-23. LncRNA HOTAIR SPONGES miR301a-3p TO PROMOTE GLIOMA’S PROLIFERATION AND INVASION THROUGH THE ACTIVATION OF FosL1
Authors: King Pendelton , Guo Alyssa , Wan Jingwei , Liu Mingli
Published: November 2021

BACKGROUND

Non-coding RNAs, including LncRNAs, function in regulating glioblastomas’ numerous oncogenic phenotypes, such as invasiveness and stemness of glioblastoma stem cells. These LncRNAs contain long non-encoding RNA transcripts >200 nucleotides in length, many of which show cell type-specific expression. In this project, we investigated how TRPM7, a promoter for glioma’s proliferation and invasion, regulates LncRNA and contributes to glioma tumorigenesis.

METHODS

1) Total RNA, from either with A172 glioma cells or A172 glioma cells with TRPM7 knocked out (A172 KO), were extracted and then parallelly subjected to Human Cancer Pathway Finder, RT2 LncRNA PCR Array. 2) We then analyzed the prognostic role of LncRNAs using a publicly available bioinformatics data set (www.Oncolanc.org). 3) We examined the effects of TRPM7-regulated LncRNA, and its downstream molecules miR301a-3p and FosL1 oncogene, on the proliferation and invasion of glioma cells in A172 and PDX-L12 cells by either inhibition or activation of the above molecules.

RESULTS

1) Data analysis from RT2 data resulted in a list of 10 downregulated and 7 upregulated LncRNAs whose transcripts are statistically significant with fold changes greater than 2.0 by TRPM7 knockout. 2) The results showed that TRPM7 functions as a positive regulator of LncRNA, HOTAIR, which is an unfavorable prognostic factor of 7-year overall survival in glioma patients. 3) In addition, we found that LncRNA HOTAIR sponges miR-301a-3p to promote proliferation, invasion, and stemness of glioma through upregulating the oncogene FosL1. 4) Furthermore, we identified that FosL1 is a novel prognostic marker of glioma patients.

CONCLUSION

Our results demonstrated the role of TRPM7-regulated LncRNA HOTAIR as a miRNA sponge in glioma, which shed new light on LncRNA-directed therapeutics in glioma.
STEM-26. BLOOD-TUMOR BARRIER IS COMPOSED OF MECHANOSENSING TUMOR CELLS THAT MASK THERAPEUTIC VULNERABILITY
Authors: Momin Ali , Chen Xin , Wang Gousiyi , Wang Xian , Min Hyun-Kee , Dou Wenkun , Gong Zheyuan , Chan Jade , Chen Yuki , Veerasammy Kelly , Dong Weifan , Fan Jerry , Fekete Adam , Dou Zhengchao , Hu Malini , Pardis Parnian , Kumar Sachin , Juraschka Kyle , Donovan Laura , Dirks Peter , Taylor Michael D , Wang Lu-Yang , Hui Chi-chung , Abzalimov Rinat , He Ye , Sun Yu , Li Xuejun , Huang Xi
Published: November 2021

Two major obstacles in brain cancer treatment are the blood-tumor barrier (BTB), which restricts delivery of most therapeutic agents, and the quiescent brain tumor-initiating cells (BTICs), which evade cell cycle-targeting chemotherapy. Mechanosensation, the transduction of mechanical cues into cellular signaling, underlies physiological processes such as touch, pain, proprioception, hearing, respiration, epithelial homeostasis, and vascular and lymphatic development. We report that medulloblastoma (MB) BTICs are mechanosensing, a property conferred by force-activated ion channel Piezo2. In contrast to the prevailing view that astrocytes function as a physical barrier in BTB, BTICs project endfeet to ensheathe capillaries. MB develops a tissue stiffness gradient as a function of distance to capillaries. Piezo2 senses substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion at BTIC growth cones, which allow BTICs to mechanically interact with their substrate and sequester β-Catenin to prevent WNT/β-Catenin signaling in BTICs. Our single cell analysis uncovers a two-branched BTIC trajectory that progresses from a deep quiescent state to two cycling states. Tumor cell-specific Piezo2 knockout reverses the off-on WNT/β-Catenin signaling states in BTICs and endothelial cells, collapses the BTB, reduces quiescence depth of BTICs, and markedly enhances MB response to chemotherapy. Our study reveals that BTICs co-opt astrocytic mechanism to contribute to the BTB and provides the first evidence that BTB depends on mechanochemical signaling to mask tumor chemosensitivity. Targeting Piezo2 addresses BTB and BTIC properties that underlie therapy failures in brain cancer.
CBIO-01. INHIBITION OF EXTRACELLULAR CARBONIC ANHYDRASES INHIBITS GLIOBLASTOMA CELL INVASION
Authors: Proescholdt Martin , Qiu Zhenwei , Falter Johannes , Lohmeier Anette , Schmidt Nils-Ole
Published: November 2021

BACKGROUND

Malignant gliomas metabolize glucose preferably by glycolysis which is in accordance with the Warburg effect. This induces a high demand of glucose combined with a significant lactic acid load. The hypoxia-inducible carbonic anhydrase (CA) IX has been shown to moderate the extrusion of hydrogen ions into the extracellular space. Since the acidification of the extracellular environment contributes to host tissue invasion due to activation of proteolytic enzymes, we hypothesized that CA IX plays an important role in malignant glioma Recently, specific small molecule inhibitors of this enzyme have been developed and may provide an innovative strategy for anti – invasive treatment.

METHODS

Two established and 4 primary GBM cell lines (2 with mesenchymal and 2 with proneural transcriptional profile) were exposed to the CAIX inhibitor U104 under normoxic and hypoxic conditions. Cell toxicity was measured by ATP and crystal violet assay. For invasion assessment, a matrigel invasion chamber system with 8 µm pore size polycarbonate filter was used. CAIX expression was analyzed by quantitative RTPCR and Western Blot.

RESULTS

Hypoxia significantly induced CAIX expression in all cell lines. Invasiveness increased significantly under hypoxic conditions in the mesenchymal cells (p < 0.01). Regardless of oxygenation status, the mesenchymal group displayed significantly higher invasiveness compared to the proneural group (p = 0.006). Looking at all cell lines, invasion is significantly inhibited by U104, both under normoxic and hypoxic conditions (p < 0.01). However, while the mesenchymal group showed the highest susceptibility to CAIX inhibition followed by the proneurally differentiated group, the established cell lines were entirely refractory to CAIX inhibition.

CONCLUSION

Our data demonstrate that CAIX inhibition can effectively inhibit invasion in malignant glioma cells independent from oxygenation status, however the effects are significantly influenced by cell type specific biological features.
CBIO-03. A COMPREHENSIVE CHARACTERIZATION OF THE GBM LIPIDOME REVEALS A MOLECULARLY-DEFINED SUB-GROUP WITH HEIGHTENED SENSITIVITY TO FERROPTOSIS
Authors: Morrow Danielle , Minami Jenna , Bayley Nicholas , Williams Kevin , Prins Robert , Liau Linda , Cloughesy Timothy , Bensinger Steven , Nathanson David
Published: November 2021

Cancers, including the universally lethal glioblastoma (GBM), have reprogrammed lipid metabolism to fuel tumor growth. However, the molecular alterations responsible for aberrant lipid metabolism, and the potential for identifying new therapeutic opportunities are not fully understood. To systematically investigate the GBM lipidome, we performed integrated transcriptomic, genomic and shotgun lipidomic analysis of an extensive library of molecularly diverse patient-derived GBM tumors and model systems. Using this comprehensive approach, we discovered two GBM sub-groups defined by their combined molecular and lipidomic profile. Among the most significant differences between the two groups were lipid length and desaturation. As a consequence of this signature, a subset was more sensitive to lipid peroxidation and ferroptosis. Our findings suggest a novel association between specific molecular signatures of GBM, lipid metabolism and lipid peroxidation-induced cell death. This relationship may present a new therapeutic opportunity to target reprogrammed lipid metabolism in a molecularly-defined subset of GBMs.
CBIO-05. SOX2 CHROMATIN ARCHITECTURE AND ENHANCER ACTIVITY IN THE MAINTENANCE AND DEVELOPMENT OF NEURAL STEM CELLS
Authors: Bready Devin , Modrek Aram , Frenster Joshua , Skok Jane , Placantonakis Dimitris
Published: November 2021

Gain of function mutations in isocitrate dehydrogenase I (IDH1) result in the formation of the oncometabolite 2-hydroxyglutarate (2HG) in adult lower grade gliomas. To gain insight into mechanisms of gliomagenesis, our lab previously created a tractable human cellular model of low grade astrocytoma (LGA) using the putative cell-of-origin, human neural stem cells (NSCs), engineered to express mutant IDH1 and knockdown constructs against TP53 and ATRX, the two other genetic changes that accompany the IDH mutation in these tumors. We found that transcription factor (sex determining region Y)-box 2 SOX2, which is essential to NSC multipotency, the ability to differentiate to neuroglial lineages, behaves as a tumor suppressor during glioma initiation. In this context, we showed SOX2 is transcriptionally downregulated to impair NSC multipotency, thus locking NSCs in an undifferentiated state to initiate gliomagenesis. This downregulation occurs secondary to dynamic reorganization of the topologically associating domain (TAD) of SOX2 and the loss of contact with several genomic loci with histone modifications and chromatin accessibility suggestive of being enhancers. Here we show that those putative enhancers acquire enhancer-like features simultaneous to tje TAD organizing in a way that facilitates interaction with the SOX2 promoter during the process of pluripotent stem cell differentiation into neuroectodermal lineages, suggesting a developmental role. Preliminary data suggests that disruption of the SOX2 TAD by preventing binding of the genome organizer CTCF downregulates SOX2 expression in NSCs. Targeted silencing of several regions of a putative enhancer with CRISPRi also downregulates SOX2. In human embryonic stem cells (hESCs), interfering with these CTCF binding sites biases their differentiation away from the neuroectoderm. We are currently performing CRISPRi screen against all putative enhancer loci, teratoma formation assays on hESCs lacking relevant CTCF binding, and CRISPR mediated deletion of putative enhancers. Understanding this developmental process may reveal underlying vulnerabilities in LGA.
CBIO-06. CELL INTRINSIC HFE DRIVES SEX-SPECIFIC GLIOBLASTOMA GROWTH
Authors: Troike Katie M , Mulkearns-Hubert Erin E , Silver Daniel J , Connor James , Lathia Justin
Published: November 2021

Iron is an essential element required for a number of cellular processes and can contribute to malignant transformation and tumor expansion. In glioblastoma (GBM), tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The homeostatic iron regulatory (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by modulating iron uptake and release. HFE is upregulated in GBM tumors compared to non-tumor brain and expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. However, it is unclear how HFE impacts sex-specific GBM growth. To interrogate the underlying mechanism of HFE-mediated sex differences, we employed genetic loss and gain of function approaches using syngeneic mouse glioma models. We observed significant alterations in the expression of several iron-associated genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. We found that knockdown of Hfe decreased cell number and increased apoptosis in vitro and led to a significant impairment of tumor growth in vivo, with a more pronounced effect seen in female mice. Conversely, overexpression of Hfe increased cell number and significantly decreased survival only in female animals. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression in a sex-dependent manner. These data also suggest an unexplored link between cell intrinsic iron signaling and sex-specific microenvironmental and immune responses, which is the focus of ongoing studies.
CBIO-19. GENOME SHREDDING ENABLES CRISPR-MEDIATED GLIOBLASTOMA ONCOLYSIS
Authors: Fellmann Christof , Tan I-Li , Perez Alexendar , Lew Rachel , Zhu Karen , Berger Mitchel , Doudna Jennifer
Published: November 2021

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults 1. Despite multimodal treatment regimens including surgical resection, radio- and chemotherapy, the growth of residual tumor often results in therapy resistance and ultimately death. GBMs are highly diffuse and exhibit extensive intratumoral heterogeneity 2,3, confounding diagnostic efforts and presenting opportunities for therapy evasion. Therefore, innovative treatment paradigms that can efficiently eliminate GBM cells irrespective of their mutational and epigenetic profile are urgently needed. CRISPR technologies have revolutionized medicine by enabling targeted genome editing through RNA-guided introduction of DNA double-strand breaks 4,5. Here, we show that CRISPR-Cas9 mediated genome fragmentation through targeting of highly repetitive loci, termed “genome shredding”, enables rapid and robust elimination of GBM cells. We characterized genome shredding across mammalian and vertebrate cells, and identified optimal repetitive pan-vertebrate and species-specific loci. Genome shredding is equally effective in temozolomide (TMZ)-sensitive and -resistant GBM cells, and multi-cycle treatment regimens are feasible. Importantly, when deployed in intracerebral GBM xenografts through local delivery, CRISPR-Cas9 genome shredding efficiently eliminated all targeted cells. Together, genome shredding enables the rapid and efficient fragmentation of a target cell’s genome and subsequent DNA damage-induced cell death. This provides an innovative treatment paradigm that is independent of a tumor’s mutational and epigenetic profile and leverages CRISPR-Cas9 as a breakthrough therapeutic modality for GBM.
CBIO-23. ANTIAPOPTOTIC Bcl-xL RESTRICTS APOPTOSIS IN SHH MEDULLOBLASTOMA AND PROMOTES PROGRESSION
Authors: Cleveland Abigail , Veleta Katherine , Gershon Timothy
Published: November 2021

Medulloblastomas in most patients are distinctively sensitive to radiation therapy, but the mechanisms that mediate this sensitivity are unclear. Current treatments still fail 20%-60% of patients with SHH medulloblastoma and can leave survivors with long-term neurocognitive and social deficits. Understanding the mechanisms driving the typical radiation-sensitivity may identify less-toxic therapeutic strategies and provide insight into treatment failure. We previously showed that radiation sensitivity depends on the intrinsic apoptotic pathway, mediated by pro-apoptotic BAX. In cerebellar granule neuron progenitors (CGNPs), the cell of origin for SHH medulloblastoma, BAX activity is directly inhibited by anti-apoptotic BCL-xL; Bcl-xL-deleted CGNPs undergo spontaneous apoptosis. To test the therapeutic potential of disrupting BCL-xL in medulloblastoma, we conditionally deleted Bcl-xL in mice genetically engineered to develop SHH medulloblastoma. Here, I show that Bcl-xL deletion slows SHH medulloblastoma growth and prolongs survival of medulloblastoma-bearing mice. Bcl-xL-deleted tumors initially showed increased rates of spontaneous apoptosis, but this effect waned over time, suggesting the emergence of BCL-xL-independent survival mechanisms. We also noted increased microglial infiltration in Bcl-xL-deleted medulloblastomas. We hypothesize that IGF1 produced by microglia in the tumor microenvironment may be contributing to tumor resistance by upregulating translation of MCL-1, an anti-apoptotic BCL-xL homolog. IGF1 is known to upregulate translation through the mTOR pathway, while anti-apoptotic MCL-1 protein abundance is dependent upon translation regulation. Our on-going studies are testing the efficacy of pharmacologically targeting BCL-xL in mice with medulloblastoma, in combination with targeting IGF1 signaling using mTORC1 inhibitors.
CSIG-12. THE ROLE OF HYPOXIA SIGNALING AS AN ONCOGENIC EFFECTOR IN ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Johansson Elinn , Pietras Alexander
Published: November 2021

Diffuse intrinsic pontine gliomas (DIPG) are aggressive and highly treatment resistant pediatric tumors of the brainstem. The majority of the DIPG tumors (60-80%) carry somatic histone H3 mutations resulting in a global reduction of H3K27 trimethylation and subsequently a unique epigenetic landscape. Moreover, activating mutations in ACVR1 – the gene encoding for the BMP receptor ALK2 – were recently described in a large subset of DIPGs, suggestive of an oncogenic role of signaling from this receptor. Potential mechanisms of ACVR1-driven oncogenesis, however, remain poorly understood. Similar and overlapping ACVR1 mutations have been described in the rare disorder Fibrodysplasia Ossificans Progressiva (FOP), and evidence from FOP models have suggested several links between hypoxia signaling, BMP signaling, and FOP pathogenesis. We used cultured DIPG cells and adult glioma cells respectively to test the activity of the hypoxia pathway using a hypoxia-responsive elements (HRE)-luciferase reporter assay. Intriguingly, DIPG but not glioma cells overexpressing a mutant ACVR1 construct significantly elevated hypoxia signaling at hypoxia. BMP ligand stimulation resulted in a dose-dependent increase of hypoxia signaling in DIPG cells. By querying clinical gene expression data, we found elevated hypoxia signature scores in pediatric gliomas with mutant ACVR1 as compared to those with wildtype ACVR1. Finally, ACVR1 mutations frequently co-occur with Histone 3.3 and 3.1 mutations, and the hypoxia signature score was significantly higher than baseline only in tumors also harboring these Histone 3.3 or 3.1 mutations. Together, these data support the notion that hypoxia signaling is elevated downstream of ACVR1 specifically in pediatric gliomas and prompts for evaluation of hypoxia signaling as potential therapeutical target in DIPG.
CSIG-19. DISRUPTION OF DNA DAMAGE RESPONSE MODULATES THE EFFICACY OF LOCAL IMMUNOTHERAPIES IN EXPERIMENTAL GLIOMA
Authors: Koch Marilin , Zdioruk Mykola , Nowicki Michal , Schregel Katharina , Chiocca E Antonio , Tabatabai Ghazaleh , Lawler Sean
Published: November 2021

RATIONALE

Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR).

METHODS

We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF.

RESULTS

The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors.

CONCLUSION

DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.
CSIG-22. Cd95l/Cd95 GENE DELETION DECREASES MOUSE GLIOMA TUMORIGENESIS THROUGH THE INHIBITION OF NON-CANONICAL CD95L/CD95-MEDIATED CELL GROWTH AND IMMUNOSUPPRESSION
Authors: Quijano-Rubio Clara , Weller Michael
Published: November 2021

CD95 is a transmembrane receptor with potential to promote both cell death and growth. Initially described to trigger apoptosis upon ligand (CD95L) engagement, CD95 may also prompt cell proliferation, invasion and stemness. CD95 stimulation to induce cancer cell apoptosis has been proved clinically impracticable. However, in tumors expressing both CD95 and CD95L, strategically inhibiting CD95-CD95L interactions to simultaneously block cancer cell growth and apoptotic cell death in tumor microenvironment components, including CD95-expressing antitumor immune effector cells, may represent an alternative therapeutic strategy. Here we characterized the expression of CD95 and CD95L in murine glioma models in vitro and in vivo. To fully disrupt CD95-CD95L interactions, we deleted Cd95 or Cd95l by CRISPR-Cas9-mediated knockout (KO) and assessed the consequences on cell growth and tumorigenicity in immunocompetent and immunocompromised mice. CD95 expression was identified in selected murine glioma cell lines. In vitro, expression of the canonical, membrane-bound, form of CD95L was not detected but cell lines expressed a shorter non-canonical, soluble, Cd95l variant. Tumors generated upon implantation of the same cells in vivo expressed both Cd95l variants. Upon Cd95l KO, all investigated cell lines exhibited reduced growth in vitro. Cell growth reduction upon Cd95 KO in SMA-497 murine glioma cells was rescued upon Cd95 re-transfection, validating CD95 specificity of the phenotype. Cd95-overexpression in Cd95-expressing cells did not increase growth. In vivo, Cd95 or Cd95l KO cell implantation in syngeneic mice generated smaller tumors than wildtype cells, resulting in prolonged survival. While 40% Cd95l KO cell-implanted immunocompetent mice did not develop tumors, all immunodeficient mice did. Altogether, these data reveal a growth-promoting role of non-canonical CD95L-CD95 interactions in murine gliomas, which blockade through gene KO results in decreased tumorigenicity. Furthermore, our data suggest the contribution of CD95L-mediated immunosuppression to the reduction of Cd95l KO-associated tumorigenicity.