bluuebunny/crossref_metadata_2025_split huggingface dataset:
STEM-11. HYDROGEN SULFIDE FUNCTIONS AS A TUMOR SUPPRESSION IN GLIOBLASTOMA
Authors: Silver Daniel J , Roversi Gustavo , Bithi Nazmin , Wang Sabrina , Troike Katie M , Neumann Chase K , Ahuja Grace , Reizes Ofer , Brown J Mark , Hine Christopher , Lathia Justin 
Published: November 2021

Abstract
Glioblastoma (GBM) cancer stem cells (CSCs) respond to a variety of stimuli within their immediate surroundings. However, little is known about the lifestyle factors that alter CSC enrichment and function within the tumor microenvironment (TME). To examine the consequences of diet-induced obesity on the progression of GBM, we interrogated tumor growth using patient-derived and syngeneic GBM models implanted into the brains of mice fed either an obesogenic high-fat diet (HFD) or a low-fat, control diet. HFD consumption resulted in an accelerated disease trajectory, presenting significantly shortened overall survival. HFD reshaped the TME altering the lipid landscape, enhancing the CSC phenotype, stimulating tumor cell proliferation, and protecting from necrotic cell death. Similar results were not observed in metabolically obese, leptin-deficient (ob/ob) mice. We simultaneously identified a potent suppression of the gasotransmitter, hydrogen sulfide (H2S). H2S functions principally through protein S-sulfhydration and regulates multiple programs including bioenergetics, metabolism, and immune response. Inhibition of H2S increased tumor cell proliferation and chemotherapy resistance, whereas treatment with H2S donors reduced tumor cell fitness in vitro and attenuated GBM growth in vivo. Exogenous treatment with H2S donors also rescued the lipid-mediated increase in tumor cell proliferation. As H2S exerts its action though protein S-sulfhydration, we confirmed that HFD-fed mice, which experienced decreased H2S synthesis, presented a severely depleted S-sulfhydrated protein landscape. Loss of this post-translational modification was confirmed in GBM patient tissues compared to age- and sex-matched controls. Taken together, our findings provide evidence that H2S functions as a tumor suppressor in GBM. Our observations highlight a new mechanism for tumor growth dynamics that can be leveraged for new therapeutic strategies focused on boosting H2S. Finally, our findings indicate that lifestyle factors can have pleiotropic effects on GBM progression through concomitant mechanisms involving tumor metabolism, modifications to the TME, and regulation over the CSC phenotype.
STEM-19. NON-REDUNDANT, ISOFORM-SPECIFIC ROLES OF HDAC1 IN THE REGULATION OF THE GLIOMA STEM CELL PHENOTYPE
Authors: Cascio Costanza Lo , McNamara James , Melendez Ernesto Luna , Lewis Erika , Dufault Matthew , Sanai Nader , Plaisier Christopher , Mehta Shwetal 
Published: November 2021

Abstract
Glioblastoma (GBM) is characterized by an aberrant yet druggable epigenetic landscape. One major family of epigenetic regulators, the Histone Deacetylases (HDACs), are considered promising therapeutic targets for GBM due to their repressive influences on transcription. Although HDACs share redundant functions and common substrates, the unique isoform-specific roles of different HDACs in GBM remain unclear. There is a temporal and cell-type specific requirement of HDAC1 and 2 during normal brain development, with HDAC2 being indispensable in neural stem cells. Here, we specifically investigated the functional importance of HDAC1 in glioma stem cells, an HDAC isoform whose expression increases with brain tumor grade and is correlated with decreased survival. Using cell-based and biochemical assays, transcriptomic analyses and patient-derived xenograft models, we report that knockdown of HDAC1 alone has profound effects on the glioma stem cell (GSC) phenotype and survival in a p53-dependent manner. HDAC1 is the essential class I deacetylase in glioma stem cells, and its loss is not compensated for by its paralogue HDAC2 or other HDACs. Loss of HDAC1 expression significantly suppresses viability of GSCs harboring functional p53, and that HDAC2 expression is completely dispensable in GSCs. In addition, HDAC1 silencing but not HDAC2, stabilizes and acetylates p53 in GSCs, resulting in upregulation of key p53 target genes and induction of programmed cell death. Furthermore, ablation of HDAC1 function alone results in histone hyperacetylation and a collapse of the stemness state in GSCs. We demonstrate significant suppression in tumor growth upon targeting of HDAC1 and identify compensatory pathways that provide insights into combination therapies for GBM. Our study highlights the importance of HDAC1 in GBM and the need to develop isoform-specific HDAC inhibitor drugs.
STEM-24. INTERFERON SIGNALING REGULATES THE PROLIFERATION AND MESENCHYMAL PHENOTYPE OF GLIOBLASTOMA STEM CELLS
Authors: Khan Sabbir , Mahalingam Rajasekaran , Sen Shayak , Gandy Kaitlin , Alfaro-Munoz Kristin , Majd Nazanin , Balasubramaniyan Veerakumar , DeGroot John 
Published: November 2021

Abstract
Interferon (IFN) signaling contributes to stemness, cell proliferation, cell death, and cytokine signaling in cancer and immune cells; however, the role of IFN signaling in glioblastoma (GBM) and GBM stem-like cells (GSCs) is unclear. This study aimed to investigate the cancer cell-intrinsic IFN signaling in tumorigenesis and malignant phenotype of GBM. We characterized cell-intrinsic IFN signaling in The Cancer Genome Atlas, patient-derived cohorts of GSCs, and published single-cell RNA sequencing datasets by in-silico analyses. The in-silico findings were further validated by evaluating the cytokine secretion and using pharmacological activators and blockers of IFN/transducer and activator of transcription 1 (STAT1) signaling. We found that GSCs and GBM tumors exhibited differential cell-intrinsic IFN signaling, and high IFN/STAT1 signaling is associated with mesenchymal phenotype and poor survival outcomes. Ruxolitinib, a pharmacological inhibitor of IFN/STAT1, abolished the IFN/STAT1 signaling in GSCs with intrinsically high IFN signaling. IFN-γ treatment for 1 week promotes the mesenchymal phenotype in GSCs with low IFN signature. In addition, chronic inhibition of IFN/STAT1 signaling with ruxolitinib decreased cell proliferation and mesenchymal signatures (CD44, YKL40, and TIMP1) in GSCs with intrinsically active IFN/STAT1 signaling. Publicly available human glioma single-cell RNA-seq (scRNA-seq) datasets analyses showed that both tumor and nontumor cells expressed IFN signaling genes, and the mesenchymal signature was highly expressed in the same cluster where IFN signaling genes were upregulated. We demonstrated that cell-intrinsic IFN signaling in GSCs and GBM tumors is associated with mesenchymal signatures and cell proliferation. Our study provides evidence for the possibility of targeting IFN signaling in a specific group of GBM patients.
CBIO-02. IN VITRO TUMOR TREATING FIELDS (TTFields) REDUCE PROLIFERATION AND ALTER MLH1 EXPRESSION IN TEMOZOLOMIDE RESISTANT (TMZR) PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS
Authors: Michelhaugh Sharon , Degen Katherine , Walker Blake , Mittal Sandeep 
Published: November 2021

Abstract

BACKGROUND
TTFields therapy is approved as a monotherapy for the treatment of recurrent GBM, tumors that often have acquired resistance to TMZ. To examine TTFields efficacy in the context of TMZR in vitro, TTFields were applied to TMZR GBM cells to assess proliferation, clonogenicity, and changes in expression of O6-methylguanine-DNA methyltransferase (MGMT) that confers TMZ resistance and DNA Mismatch Repair Protein Mlh1 (MLH1) that may confer TMZ sensitivity.

METHODS
15-037 GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter. Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. U-251 MG cells and 15-037 cells were incubated with 100 µM TMZ for three 5-day cycles to establish TMZR. TMZR cells were plated on coverslips (1×104cells/coverslip) and incubated overnight prior to TTFields application. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 14 days. Then coverslips (n=3/group) were harvested, counted, and replated for clonogenic assay at 1000 cells/35mm2 well. Clonogenic assay plates were stained with crystal violet, imaged, and colonies counted. Additional coverslips (n=3/group) were harvested and MGMT and MLH1 expression were assessed by qRT-PCR with the delta-delta Ct method using GAPDH as housekeeping control. Cell counts and colony counts were compared with two-tailed t-test with significance at p&lt; 0.05.

RESULTS
In U-251 MG and 15-037 TMZR cells, TTFields application significantly reduced both cell counts and clonogenic assay colony counts compared to untreated controls (U-251 MG: 45.4% and 26.5%; 15-037: 96.2% and 60.9%, respectively, p&lt; 0.05). In both TMZR cell lines, MGMT expression was unchanged by TTFields, but MLH1 expression increased 2.2 fold in U-251 MG cells and 7.1 fold in 15-037 cells after TTFields application.

CONCLUSIONS
Not only are TMZR GBM cells sensitive to TTFields in vitro, but TTFields increased expression of MLH1 which may be able to reduce resistance to TMZ.
CBIO-07. ST-401: A BRAIN-PENETRANT MICROTUBULE-TARGETING AGENT THAT KILLS CANCER CELLS IN INTERPHASE
Authors: Ruiz Juan Jesus Vicente , Wordeman Linda G , Stella Nephi 
Published: November 2021

Abstract
Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers, and evidence suggests that glioblastoma is sensitive to disruptions in microtubule (MT) functions; but most MTAs do not cross the blood brain barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioblastoma in culture at nanomolar concentrations (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165). Of note, ST-401 binds to the colchicine site, inhibits tubulin assembly and reversibly reduces MT dynamics in cells with a milder activity compared to the reference MTA, nocodazole. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 demonstrated significant antitumor and radiosensitizing activities. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity 1) did not correlate with any of the compounds tested thus far and 2) only weakly correlated with MTAs that act through alternate mechanisms [Taxol and Vinblastine]. Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function that is different from MTAs tested thus far on the NCI 60 cancer cell panel. As initial step to discover this novel mechanism of action, we studied the anti-tumor activity of ST-401 and nocodazole in HCT116 cells in culture. As expected, low concentrations (30 and 100 nM) of nocodazole killed HCT116 cells in mitosis and increased p-P53; by contrast these low concentrations of ST-401 killed HCT116 cells in interphase and did not increase p-P53. Our study shows that the brain-penetrant MTA, ST-401, kills cancers in interphase, a response that contrasts with the activity of the reference MTA nocodazole. ST-401 represents a promising chemical scaffold to develop brain-penetrant therapeutics for the treatment of patients diagnosed with glioblastoma.
CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS
Authors: Kitange Gaspar , Vaubel Rachael , Sarkaria Jann 
Published: November 2021

Abstract
Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.
CBIO-14. SINGLE CELL RNA SEQUENCING ANALYSIS OF HUMAN GLIOBLASTOMA STEM-LIKE CELL CULTURES AND XENOGRAFT TUMORS
Authors: Arora Sonali , Mihalas Anca , Bassett John , Patel Anoop , Paddison Patrick 
Published: November 2021

Abstract
Single cell RNA-seq (scRNA-seq) studies for glioma have yielded critical insight into intratumoral heterogeneity and developmental gene expression patterns for primary gliomas. One key conclusion from these studies is that each tumor represents a complex, yet maligned, neuro-developmental ecosystem, harboring diverse cell types, which presumably contribute to tumor growth and homeostasis in specific ways (e.g., vascular mimicry, immune evasion, recreating NSC niches, neural injury responses, etc.). Here, to better understand experimental models of human glioblastoma (GB), we performed single cell RNA-seq analysis of human GB stem-like cells (GSCs) of distinct tumor subtypes (mesenchymal and proneural) during their in vitro culture in serum-free conditions and also during tumor formation in immunocompromised mice. This analysis revealed surprising differences between in vitro and in vivo grown GSCs. Among our results, we find that in vivo mesenchymal GSCs are capable of transitioning to proneural-like states, while proneural GSCs are capable of transitioning to mesenchymal states. We characterize cycling cells based on expression of and G2/M and S phase makers, estimate RNA velocity, and examine different developmental trajectories arising in vitro and in vivo. We also compare and discuss different analysis pipelines for scRNA-seq data.
CBIO-15. LOSS OF WILLIAMS SYNDROME TRANSCRIPTION FACTOR (WSTF) LEADS TO IMPROVED TEMOZOLOMIDE SENSITIVITY IN HUMAN GLIOBLASTOMA CELLS IRRESPECTIVE OF MGMT EXPRESSION
Authors: Sarnow Katharina , Schwab Stephanie , Rio Oline , Mukherjee Joydeep , Bjerkvig Rolf , Johannessen Tor-Christian 
Published: November 2021

Abstract

BACKGROUND
The prognosis for glioblastoma multiforme (GBM) patients is poor with a median survival of approximately 15 months. The DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) counteracts the effects of temozolomide (TMZ) chemotherapy and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its functions in the DDR in GBMs, is so far unknown. In this study, we investigated whether a loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment.

METHODS
We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA.

RESULTS
WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p&lt; 0.0001, U-251: Clone 7/35.1/70.2 vs control: p&lt; 0.0001/p= 0.0107/p= 0.0119).

CONCLUSION
WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.
CBIO-16. DOSE INTENSIFIED TEMOZOLOMIDE IMPROVES INFILTRATION OF CD70 CAR T CELLS INTO THE GBM TUMOR MICROENVIRONMENT
Authors: Karachi Aida , Huang Jianping , Tao Haipeng , Dastmalchi Farhad , Jin Linchun , Mitchell Duane , Rahman Maryam 
Published: November 2021

Abstract
CD70 CAR T cells developed by our group have anti-tumor efficacy in syngeneic murine GBM model and are currently being developed for first-in-human testing. The objective of this study was to evaluate the impact of temozolomide (TMZ) in various dosing strategies on the expansion, persistence and function of the CD70 CAR T cells after infusion. C57BL/6 mice underwent intracranial implantation of KR-158 overexpressing CD70 tumor cells. Tumor bearing animals were treated with standard dose (SD) (50 mg/kg x 5 days), metronomic dose (MD) (25mg/kg x 10 days), or dose intensified (DI) (75mg/kg x 5 days) of TMZ, followed by 10×106 CAR T cells infusion. Peripheral blood was collected to monitor the persistence of CAR T cells in the systemic blood circulation. Six weeks post treatments, spleens and tumors were collected and CAR T cells abundance and function were measured. TMZ preconditioning resulted in the expected lymphopenia in animals by generation of dose dependent lymphopenia. Circulating CD70 CAR T cells peaked in the systemic blood 2 weeks after infusion (3-fold, p&lt; 0.0001). Markers of T cell exhaustion including PD-1 and TOX expression on CAR T cells were not different between the three TMZ treatment groups compared to control. Six weeks post treatment, CD70 CAR T cells were found to be highly infiltrated within the tumor microenvironment in the DI TMZ group compared to the other groups (4-fold,p &lt; 0.0001). Tumor infiltrating CD70 CAR T cells in the DI TMZ group did not have an increase in PD-1 and TOX expression which was seen in the MD and SD TMZ groups. DI TMZ preconditioning results in greater CD70 CAR T cell trafficking to the tumor without T cell exhaustion compared to lower doses of TMZ. The affects of TMZ on the immune microenvironment to enhance CAR T cell regimens warrants further study.
CSIG-05. DISSOCIATION OF THE INTRAMOLECULARLY CLEAVED N- AND C-TERMINAL FRAGMENTS OF ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 (ADGRD1) INCREASES ITS CANONICAL SIGNALING IN GLIOBLASTOMA
Authors: Frenster Joshua , Stephan Gabriele , Ravn-Boess Niklas , Bready Devin , Wilcox Jordan , Liebscher Ines , Schöneberg Torsten , Placantonakis Dimitris 
Published: November 2021

Abstract
We previously demonstrated that GPR133 (ADGRD1), an adhesion GPCR that signals via cytosolic cAMP increase, is de novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells. Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic xenograft initiation in vivo. GPR133’s requirement for GBM growth and its absence in non-malignant brain suggest its therapeutic potential, yet its mechanisms of action and activation remained unclear. Here, we demonstrate in patient-derived GBM cultures and HEK293T cells that GPR133 gets intramolecularly cleaved into N-terminal and C-terminal fragments (NTF and CTF) right after synthesis in the endoplasmic reticulum. The resulting NTF and CTF remain non-covalently bound to each other, until the mature receptor reaches the plasma membrane, where we observe dissociation of the extracellular NTF from the transmembrane-spanning CTF. While cleavage is not required for correct subcellular trafficking, the cleaved wild-type GPR133 generates significantly higher cytosolic cAMP levels than an uncleavable point mutant GPR133 (H543R), suggesting that cleavage and dissociation are involved in receptor activation. To test this hypothesis in a more controllable proxy system, we generated a fusion of the CTF of GPR133 and the N-terminus of human protease-activated receptor 1 (hPAR1). Indeed, acute thrombin-induced cleavage and shedding of the hPAR1 NTF increases intracellular cAMP levels generated by the GPR133 CTF. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. To test whether extracellular binding proteins could influence NTF shedding and/or GPR133 signaling activation, we conducted ligand discovery screens and indeed found a new GPR133 binding protein in GBM cells, which is capable of influencing receptor signaling. Together, these findings provide critical insights into GPR133’s mechanism of activation, that will guide future approaches of therapeutic targeting of GPR133 in GBM.
CSIG-06. ELUCIDATING THE ROLE OF CO-OCCURRING MUTATIONS IN FGFR1-DRIVEN PEDIATRIC LOW-GRADE GLIOMA
Authors: Morin Eric , DiGiacomo Jeromy , Malinowski Seth , Alexandrescu Sanda , Jones David , Ligon Keith , Bandopadhayay Pratiti 
Published: November 2021

Abstract
Pediatric low-grade gliomas (pLGGs) have excellent survival, however, with current standard of care, most patients suffer lifelong severe sequalae. pLGGs are almost exclusively driven by single activating mutations in the mitogen-activated protein kinase (MAPK) pathway. As targeted molecular therapy clinical trials focusing on BRAF-altered pLGGs are showing promising results in early clinical trials, similar efforts are underway for FGFR1-altered tumors. In our cohort of FGFR1-4 altered gliomas, we observe frequent occurrence of a second alteration associated with activation of the MAPK or mammalian target of rapamycin (mTOR) pathways in pLGGs. Most commonly in the phosphatase NF1, PIK3CA, PIK3R1 PTPN11 or within the FGFR1 gene itself. However, the impact of second co-occurring mutations on therapeutic response and resistance has not been explored. To address this, we established mouse neural stem cell models over-expressing hot-spot mutated FGFR1 alone or in combination with a second alteration. Immunoblotting revealed that the addition of a second alteration attenuated phosphorylation of ERK, AKT and S6 and influenced cell proliferation both in normal growth conditions and in absence of growth factor. Treatment with an FGFR inhibitor (Infigratinib) showed reduced drug response in double mutant cells compared to hot-spot mutated FGFR1 alone. This was associated with less reduction of phosphorylation of ERK and S6 in the double mutant cells upon treatment. In conclusion, the presence of a second alteration influences proliferation and drug response in models of FGFR1-mutated pLGG, potentially by modulating MAPK and mTOR signaling.