bluuebunny/crossref_metadata_2025_split huggingface dataset:
DDDR-08. ARF4-MEDIATED RETROGRADE TRAFFICKING AS A MECHANISM OF CHEMORESISTANCE IN GLIOBLASTOMA
Authors: Budhiraja Shreya , Baisiwala Shivani , Perrault Ella , Cho Sia , Chen Li , Dmello Crismita , Park Cheol , Sonabend Adam M , Ahmed Atique 
Published: November 2022

Abstract
Glioblastoma (GBM) remains a deadly disease with an extremely poor prognosis, owing largely to its high rate of resistance to conventional temozolomide (TMZ) chemotherapy. To identify the unknown drivers of this chemoresistance, we performed a genome-wide CRISPR knockout sensitivity screen. Results showed significant enrichment of ~200 novel genes, including ARF4, a previously unstudied gene involved in retrograde trafficking—a well-regulated process by which cargo is transported internally from endosomes to ultimately the nucleus. Initial investigation showed that ARF4-knockdowns resulted in significantly heightened susceptibility to TMZ in multiple GBM patient-derived xenograft lines and extended survival compared to the controls (p&lt; 0.01) in vivo in both primary and recurrent lines. Live-cell imaging further revealed that ARF4-knockdowns significantly inhibited retrograde trafficking, while ARF4-overexpressions resulted in an untenable increase in retrograde trafficking (p &lt; 0.001) in vitro. Enhanced trafficking was also observed in TMZ-treated cells (p &lt; 0.001), suggesting that ARF4 may drive dysregulation of retrograde trafficking to promote chemoresistance.We then performed an unbiased proteomics screen in control and ARF4-knockdown GBM cells with and without therapy to identify which proteins were being uniquely transported to the nucleus during therapy as a result of ARF4-mediated retrograde trafficking. Results showed the greatest enrichment of EGFR signaling, as validated by an increase in EGFR trafficking and nuclear EGFR expression in ARF4-overexpression and TMZ-treated conditions and a decrease of this phenomenon in ARF4-knockdown conditions.We also show that DNA-PK, a DNA repair protein that is transcriptionally activated by nuclear EGFR, is similarly downregulated in ARF4-knockdown conditions and elevated in ARF4-overexpression and TMZ-treated conditions. Treatment with DNA-PK-inhibitor, KU57788, resulted in extended survival compared to the controls (p &lt; 0.01) in vivo in a recurrent line, indicating potential clinical benefit from targeting this pathway. Overall, we present a novel understanding of how ARF4 acts during therapy to increase nuclear localization of chemoresistance-promoting proteins.
DDDR-15. SPIDER VENOM MOLECULES IMPAIR HUMAN GLIOMA CELLS MIGRATION: POTENTIAL APPLICATION IN CANCER THERAPY
Authors: Barreto Natalia , da Silva Monize Valeria Ramos , da Rocha-E-Silva Thomaz Augusto Alves , Sutti Rafael , Sciani Juliana Mozer , Veiga Jose Carlos Esteves , Verinaud Liana , Araujo Joao Luiz Vitorino , Raposo Catarina 
Published: November 2022

Abstract
Gliomas correspond to approximately 80% of malignant neoplasms of the central nervous system. Due to its aggressiveness, there is a growing need to seek new strategies to control the disease. The components of spider venoms are stable peptides with high specificity against biological molecular targets of therapeutic relevance. It has already been demonstrated by our group that two subfractions (SFs) isolated from the Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) can delay the migration of glioblastoma (GB) well-established cell lineage in invasions assays. Therefore, the present study aimed to evaluate the responsiveness of samples collected from Brazilian patients with a presumptive diagnosis of glioma (high and low grades), to PnV SFs, in order to contribute to the development of new drugs. After patient consent, 13 samples were collected for cultivation and attempt to reach the tenth passage, to ensure the establishment of the lineage. Four lineages were established: J01 and N07 – glioblastoma (high grade); C03 and L09 – astrocytoma and oligodendroglioma (low grade) respectively. Cells were incubated for 48 hours with SF1 and SF11, during the scratch wound-healing assay. The results demonstrated that the SFs, mainly SF11 (which proved to be a pure peptide with 1603,88 Da, sequenced as PyrKKDKKDKFY–GLM–NH2), harmed the migration of all these samples when compared to the control group. In addition, initial data with J01 lineage demonstrated that migration was also affected during the transwell invasion assay; as well as ROCK protein, involved in control of cell migration, was decreased (demonstrated by western blot) by both SFs, suggesting that ROCK is involved in the mechanism of the components. Ongoing tests continue to establish other samples collected so that the tests described above will also be carried out. These results will contribute to the development of new therapies against these tumors. Support: FAPESP #2019/10003-3.
DDDR-17. CHEMOTHERAPEUTIC STRESS INDUCES NONCANONICAL HIF1Α SIGNALING UNDER NORMOXIA IN GLIOBLASTOMA DRIVING THERAPEUTIC RESISTANCE
Authors: Ventarapragada Divya , Ahmed Atique , Perrault Ella , Park Cheol 
Published: November 2022

Abstract
Glioblastoma (GBM) is an aggressive brain tumor with no cure. Standard treatment includes adjuvant, concomitant temozolomide (TMZ) and radiation, but most patients do not survive more than 15 months. Tumor adaptations to temozolomide (TMZ) contribute to glioblastoma (GBM) chemoresistance and recurrence, and hypoxia inducible factor subunit alpha (HIF1α) and downstream effectors are implicated in attenuated therapeutic responses. We have previously shown that TMZ-dependent hijacking of HIF1α signaling under normoxia is independent from physiological hypoxia in the tumor core. Our results further showed that TMZ promotes HIF1α accumulation enhancing stem-like characteristics of GBM cells driving therapy-resistant populations involved in recurrence. However, noncanonical HIF1α signaling mechanisms are not well understood preventing the success of HIF1α inhibitors in GBM thus far. Here, we investigate how TMZ induces HIF1α in an oxygen-independent manner by characterizing the HIF1a binding profile post-therapy. We cultured patient-derived xenograft (PDX) models of GBM in vitro and found HIF1α expression and hypoxia response element (HRE) activity peak 96h post-therapy using western blot and luciferase assays. Our lab also performed a whole-genome CRISPR-Cas9 knockout screen in GBM cells over 28 days to determine genetic drivers of GBM aggression. HIF1α was found to be particularly enriched in this screen, further supporting the role of chronic hypoxia signaling for GBM proliferation (p-value &lt; 0.05). We show TMZ therapy prevents proteasome-dependent degradation through reduced binding of PHD2 and VHL allowing nuclear translocation of HIF1a through western blot and immunocytochemistry. Thus, we performed immunoprecipitation of HIF1α 96h post-therapy for proteomic characterization to establish how TMZ uniquely regulates HIF1α stabilization in the absence of oxygen tension, and these results will be presented. We will subsequently characterize the regulatory role of TMZ-dependent post-translational modifications to HIF1α. Elucidating the molecular mechanisms of therapy-mediated HIF1α regulation can produce better therapeutic targets and increase TMZ efficacy.
DDDR-24. INTEGRATED ANALYSIS OF SINGLE CELL CHROMATIN ACCESSIBILITY AND RNA EXPRESSION IDENTIFIED COMMON VULNERABILITY DESPITE GLIOBLASTOMA HETEROGENEITY
Authors: Raviram Ramya , Raman Anugraha , Preissl Sebastian , Wu Shaoping , Koga Tomoyuki , Zhu Chenxu , Luebeck Jens , Van Deynze Kinsey , Han Jee Yun , Hou Xioameng , Ye Zhen , Mischel Anna , Li Yang Eric , Fang Rongxin , Baback Tomas , Mugford Joshua , Han Claudia , Glass Christopher , Barr Cathy , Mischel Paul , Bafna Vineet , Escoubet Laura , Ren Bing , Chen Clark 
Published: November 2022

Abstract

INTRODUCTION
In 2021, the World Health Organization (WHO) reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH) wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intra-tumoral heterogeneity is a key contributor to therapeutic failure.

METHODS
we applied integrated genome-wide chromatin accessibility (snATACseq) and transcription (snRNAseq) profiles to clinical specimens derived IDHwt glioblastomas and G4 IDHm) astrocytomas, with goal of therapeutic target discovery.

RESULTS
The integrated analysis achieved resolution of intra-tumoral heterogeneity not previously possible, providing a molecular landscape of extensive regional and cellular variability. snATACseq delineated focal amplification down to an ~40 KB resolution. The snRNA analysis elucidated distinct cell types and cell states (neural progenitor/oligodendrocyte cell-like or astrocyte/mesenchymal cell-like) that were superimposable onto the snATACseq landscape. Paired-seq (parallel snATACseq and snRNAseq using the same clinical sample) provided high resolution delineation of extrachromosomal circular DNA (ecDNA), harboring oncogenes including CCND1 and EGFR. Importantly, the copy number of ecDNA genes correlated closely with the level of RNA expression. Integrated analysis across all specimens profiled suggests that IDHm grade 4 astrocytoma and IDHwt glioblastoma cells shared a common chromatin structure defined by open regions enriched for Nuclear Factor 1 transcription factors (NFIA and NFIB). Silencing of NF1A or NF1B suppressed in vitro and in vivo growth of patient-derived IDHwt glioblastomas and G4 IDHm astrocytoma models that mimic distinct glioblastoma cell states.

CONCLUSION
Our findings suggest despite distinct genotypes and cell states, glablastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intra-tumoral heterogeneity.
DDDR-26. IDENTIFICATION OF OLIG2 AS A PREDICTOR OF OSIMERTINIB’S EFFICACY AGAINST EGFRVIII+ GLIOBLASTOMA
Authors: Adams Jackson , Kwatra Gita , Bordeaux Zachary , Kwatra Madan 
Published: November 2022

Abstract

BACKGROUND
Molecular characterization of glioblastomas by The Cancer Genome Atlas revealed molecular abnormalities in epidermal growth factor receptor (EGFR) in over 50% of glioblastomas. These included gene amplification, a large deletion in ligand binding domain (EGFRvIII), kinase duplications and gene fusions. Our laboratory has been focusing on targeting EGFRvIII in glioblastomas and we noted EGFRvIII heterogeneity. We found that growth inhibition of EGFRvIII+ glioblastoma stem cell lines D317 and D10-0171 by osimertinib was different, with D317 being more sensitive. Molecular analyses revealed that D10-0171 has 35-fold higher levels of the transcription factor OLIG2. Here we hypothesize that increased OLIG2 expression confers osimertinib resistance in EGFRvIII+ GBM.

METHODS
An OLIG2 knock out GSC, D10-0171 (OLIG2-KO) was generated using CRISPR. Efficacy of osimertinib in growth inhibition of D10-0171 (WT) and OLIG2-KO was examined in a subcutaneous xenograft model. Once tumors became visible, mice were treated daily with either 200 μl vehicle or 25 mg/kg osimertinib by oral gavage. RNA-seq and reverse phase protein array (RPPA) analyses were then performed on both GSC’s.

RESULTS
Assessment of tumor growth of both GSC’s demonstrated that OLIG2-KO cells formed tumors at a significantly lower rate than WT with a rate-based (T/C) ratio of 0.0077. Administration of osimertinib led to tumor growth inhibition in both GSCs but had a greater effect in OLIG2KO than WT, with rate-based T/C ratios of 0.099 and 0.1669, respectively. Transcriptomic analysis of OLIG2KO revealed downregulation of several pathways when compared to WT, including MYC targets V1 and V2, oxidative phosphorylation, and fatty acid metabolism, while proteomic analysis revealed downregulation of several growth-promoting pathways including ErbB and MAPK signaling.

CONCLUSION
Lower levels of OLIG2 increase osimertinib sensitivity of EGFRvIII+ glioblastoma. We propose a schema for selecting EGFRvIII+ patients who will respond to Osimertinib.
DDDR-30. UNRAVELING THE NOVEL ANTI-TUMOR MECHANISM OF ST-401, A BRAIN PENETRANT MICROTUBULE TARGETING AGENT
Authors: Khan Kainat , Vicente Juan , McFaline-Figueroa José , Wordeman Linda , Stella Nephi 
Published: November 2022

Abstract
Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers. While evidence suggests that glioblastoma (GBM) is sensitive to disruptions in microtubule (MT) functions; most MTAs do not reach the brain. To address this limitation, we developed a brain-penetrant MTA, ST-401, that kills patient-derived GBM in culture at nanomolar concentrations, binds to colchicine sites through a unique interaction mode, gently and reversibly inhibits MT assembly and kills cancer cells through a novel anti-tumor mechanism as determined by analysis of its activity in the NCI 60 cancer cell panel compared to the Approved Oncology Agents library (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165).We used flow cytometry to confirm that nocodazole (10-100 nM) treatment of HCT116 cells triggered apoptosis, necrosis, and accumulation of autophagosomes, as well as increase in p-P53. By sharp contrast, ST-401 did not trigger these responses. Live cell imaging showed that NOC preferentially kills HCT116 cells in mitosis whereas ST-401 preferentially kills these cells in interphase. ScRNAseq analysis of NOC and ST-401 treatment of HCT-116 cells showed that ST-401 down-regulates MYC mRNA expression, suggesting that ST-401 might affect MYC function. MYC protein levels are regulated by multiple mechanisms, including phosphorylation at residue Thr58 that promotes its ubiquitination and proteasome degradation. We discovered that ST-401 increases MYC phosphorylation on Thr58 and down-regulates MYC protein expression in both HCT116 and the GBM cell line, U251.Our result suggests that the novel anti-tumor mechanism of ST-401 might involve cell death in interphase and reduced MYC expression. Funded by NIH (CA244213 to NS and NS106924 to NS).
DDDR-31. THE THERAPEUTIC POTENTIAL OF THE LONG NON-CODING RNA LNC-HLX-2-7 IN GROUP 3 MEDULLOBLASTOMAS IN CHILDREN
Authors: Katsushima Keisuke , Yuan Menglang , Lee Bongyong , Stapleton Stacie , Jallo George , Seal Sudipta , Eberhart Charles , Perera Ranjan 
Published: November 2022

Abstract
Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB formation and tumor progression. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 MBs. In this study, we report that lnc-HLX-2-7 RNA specifically accumulates in the HLX (host gene of lnc-HLX-2-7) promoter region and activates HLX expression by recruiting multiple factors including enhancer elements. RNA sequencing and chromatin immunoprecipitation revealed that HLX directly binds to the promoters of several tumor-promoting genes, including MYC, and activates their expression. Furthermore, intravenous treatment with antisense oligonucleotides targeting lnc-HLX-2-7 coated with cerium-oxide nanoparticle (CNP-lnc-HLX-2-7) reduced tumor growth (40-50%) in intracranial MB xenograft mouse model (n = 10, p &lt; 0.01, t-test). We found that the combinatorial therapy of CNP-lnc-HLX-2-7 and cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared to CNP-lnc-HLX-2-7 monotherapy (n = 10, p &lt; 0.01, t-test) only. We report here the importance of the lnc-HLX-2-7-HLX-MYC axis in regulating group 3 MB progression and provide a strong rationale for using lnc-HLX-2-7 as a specific and potent therapeutic target for the group 3 MBs in children.
DDDR-35. TARGETING GBM INVASION BY INHIBITING KCNA1 WITH 4-AMINOPYRIDINE: AN FDA APPROVED DRUG THAT EASILY PASS THROUGH THE BBB
Authors: Xiao Sophie , Li Lin , Yang Deying , Zhang Huiyuan , Huang Yulun , Teo Wan-Yee , Patel Akash , Du Yuchen , Li Xiao-Nan 
Published: November 2022

Abstract
Diffuse invasion is a hall mark of glioblastoma (GB) and one of the primary causes of poor clinical outcomes in GBM patients. Tumor cells migrate deep into the normal brain tissues are frequently protected by the BBB, making them particularly difficult to treat. New therapeutic targets are needed. Our previous studies using spatially dissected and functionally validated matching pairs of invasive and tumor core GBM cells identified KCNA1 as a shared gene that is selectively over-expressed in the invasive GBM cells in 6 patient derived orthotopic xenograft (PDOX) mouse models of pediatric GBM (Huang YL et al, Adv Science 2021). A subsequent analysis of adult GBM RNAseq data from IVY Atlas revealed a significantly elevated expression of KCNA1 (4.9 fold) in the invasive edges of patient GBM tumors. It is also one of the 11 core molecules identified in GEO and TCGA databases through an integrated bioinformatic analysis (Yang J, Front Onc 2021). To determine the anti-invasive activities of targeting KCNA1, we treated three highly invasive adult GBM PDOX models with 4-aminopyridine (4-AP), an old lipid soluble drug that easily penetrate the BBB, at 5 mg/kg, i.p., 5 days/week for 8 weeks acing alone or in combination with fractionated radiation (at 2 Gy/day x days). As single agent, 4-AP significantly extended median animal survival times in 1/3 GBM models (69 to 77 days, P = 0.033). Combination with XRT did not significantly improve the animal survival times in the three models. Systematic analysis of GBM invasion in mouse brains of the 3 PDOX models before, during and after 4-AP treatment revealed remarkable inhibition of tumor invasion. Our data highlighted the role of KCNA1 in promoting GBM invasion and support the fine tuning of 4-AP dose, schedule, and length of treatment to serve as a novel component of anti-GBM invasion therapies.
DDDR-37. GB5121 IS A NOVEL, IRREVERSIBLE, COVALENT BTK INHIBITOR WITH HIGH SELECTIVITY AND CNS-PENETRANCE FOR TREATMENT OF CNS MALIGNANCIES
Authors: Yusuf Isharat , Guimond David , Naiman Zachary , Schiff Theodore , Clemons Bryan , Paulson Bryanna , Hou Kay , Meadows Kristen Taylor , Rose Mark , Carter Laura 
Published: November 2022

Abstract
Inhibitors of Bruton’s tyrosine kinase (BTK) are approved treatments for several B cell lymphomas. However, they are characterized by modest selectivity and/or limited central nervous system (CNS) exposure which is important in instances of CNS disease. GB5121 is an orally available, selective, irreversible, covalent small molecule BTK inhibitor that was designed to address these limitations. Here, GB5121 was profiled for selectivity, potency, inactivation kinetics and CNS penetrance in comparison to ibrutinib. GB5121 pharmacodynamic properties were evaluated using both cell-free enzymatic and cell-based functional assays, showing nM potency for BTK inhibition and rapid BTK inactivation kinetics (Kinact/Ki) in both peripheral and CNS tissue, a critical parameter for irreversible inhibitors. In a kinome scan, GB5121 exhibited high kinase selectivity against 349 kinases with only TEC/TXK demonstrating &gt; 50% inhibition at 1 µM; GB5121 did not inhibit phosphorylation of EGFR in a cell-based assay. When compared with other BTK inhibitors, GB5121 showed superior CNS target occupancy using a probe-based ELISA measuring free BTK in the brain of mice receiving 3 daily oral doses. GB5121 also demonstrated significantly higher brain to plasma ratio in mice with an intact blood brain barrier. In non-human primates, a 1:1 brain to plasma concentration ratio was demonstrated for GB5121 for up to 8 hours with both oral (30 mg/kg) and IV (2 mg/kg) doses. Together, these features differentiate GB5121 from both FDA-approved BTK inhibitors, as well as those currently under clinical investigation. Our data supports the use of GB5121 in clinical trials where BTK is a known driver of malignancies, including CNS lymphoma. This research has been previously presented at the AACR Annual Meeting 2022.
DDDR-38. A LENTIVIRAL CRISPR SCREEN FOR EPIGENETIC MODULATORS OF CAR T CELL-TARGETED ANTIGENS IN GLIOBLASTOMA CELL LINES
Authors: Jordan Walter T , Ayad Nagi 
Published: November 2022

Abstract
Glioblastoma is the most common malignant brain tumor. Unfortunately, outcomes for this cancer are poor. Even with standard of care treatment, the median survival time is only 15 months and the disease is almost universally lethal. Therefore, more effective therapies are urgently needed to treat this condition. One promising treatment is chimeric antigen receptor (CAR) T cell therapy. CAR T cells are white blood cells that are engineered to kill cancer cells expressing certain antigens; however, the potential of this treatment has yet to be fully realized and further work is required for it to be more effective. One strategy for augmenting CAR T cell therapy would be to increase the expression of the targeted antigens on cancer cells. This could be done using small molecule inhibitors of epigenetic pathways that regulate expression of these antigens. Therefore, the goal of this study was to screen for epigenetic genes that regulate expression of antigens currently being targeted in CAR T cell clinical trials for glioblastoma. Using a CRISPR/Cas9 lentiviral vector library, we knocked out various genes encoding epigenetic proteins in human glioblastoma cell lines. Flow cytometry was used to sort for cells with the highest levels of target surface antigen expression. Genomic DNA was isolated from the cells with the highest surface expression of the antigens of interest. The isolated DNA was sequenced to identify the genetic manipulations responsible for increased antigen expression. Future studies will use small molecule inhibitors of the identified epigenetic modulators to augment CAR T cell therapy in mice.
DDDR-40. OVERCOMING DRUG RESISTANCE BY TARGETING AUTOPHAGY REGULATED BY A LONG INTERGENIC NONCODING RNA
Authors: Zhang Alexander , Sheng Zhi , Pridham Kevin 
Published: November 2022

Abstract

BACKGROUND
Autophagy is a self-eating process that supports cancer cell survival. Autophagy activation during drug treatment often prolongs cancerous cell survival, causing drug resistance, as seen in glioblastoma multiforme (GBM) to temozolomide (TMZ). Thus, blocking autophagy is an appealing approach to overcoming cancer drug resistance. Long noncoding RNAs (LincRNAs) have emerged as potential targets. LincRNA LINC00467 (renamed ARLINC) was recently identified as a potential autophagy inhibitor in cancer cells. However, ARLINC’s mechanism of inhibition and whether its modulation can suppress autophagy in cancer remains unknown.

OBJECTIVE
Research Question: Can blocking autophagy restore the sensitivity of cancer cells to chemotherapy? Hypotheses: 1) ARLINC regulates chemotherapy drug sensitivity by inducing autophagy. 2) Overexpression of ARLINC can increase chemotherapy drug sensitivity, as measured by cell viability and autophagy assays

METHODS
Aim 1) Endogenous ARLINC levels in GBM cell lines will be measured using qRT-PCR. Aim 2) Cell viability of GBM upregulated with pBabe-ARLINC (experiment) and pBabe (control) then exposed to increasing concentrations of TMZ will be analyzed using MTS assay. Student’s T-test will be used to determine significance. Aim 3) Autophagy flux will be measured in GBM lines that are transiently transfected with ARLINC compared to the empty vector via Cyto-ID assay.

RESULTS
Aim 1: Compared to Normal Human Astrocytes (NHA), U87MG had higher levels, LN18 and SF295 and lower, and LN229 had around the same levels of ARLINC. Aim 2: MTS cell viability assays in SF295 upregulated with ARLINC showed no significant decrease in TMZ sensitivity. Aim 3: Cyto-ID in U87MG and LN229 upregulated with ARLINC showed no significant decrease in autophagy flux.

CONCLUSIONS/FUTURE WORK
Results show no effect of ARLINC upregulation on cell viability and autophagy flux. Future work involves knockdown experiments to decrease intracellular levels of ARLINC and determine the effects of upregulation of autophagy on sensitization.