bluuebunny/crossref_metadata_2025_split huggingface dataset:
EXTH-29. INHIBITION OF LYSINE-SPECIFIC HISTONE DEMETHYLASE 1A (KDM1A/LSD1) SENSITIZES GLIOBLASTOMA CELLS TO CHEMO AND RADIATION THERAPY
Authors: Alejo Salvador , Palacios Bridgitte , Venkata Prabhakar Pitta , Chen Yihong , He Yi , Viswanadhapalli Suryavathi , Pratap Uday , Zheng Siyuan , Brenner Andrew , Vadlamudi Ratna , Sareddy Gangadhara 
Published: December 2020

Abstract

BACKGROUND
Despite treatment advances, glioblastoma (GBM) is the deadliest tumor of the central nervous system. Patients face a dismal 15-month median survival and average 5-year survival rate of 13%. Current standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, GBM patients often relapse and succumb to their disease. Recently, we have shown that lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM. In this study, we tested the hypothesis that KDM1A is essential for DNA damage response (DDR), inhibiting KDM1A thereby sensitizes GBM to either TMZ or radiation therapy.

METHODS
KDM1A knockout (KDM1A-KO) and knockdown (KDM1A-KD) cells were generated using CRISPR/Cas9 and KDM1A specific shRNA, respectively. Cell viability, clonogenic survival, and apoptotic assays were used to study the effect of KDM1A-KO, -KD, or pharmacological KDM1A inhibitors (NCD-38 and NCL-1) on radiation and TMZ sensitization. Mechanistic studies were conducted using RNA-seq, RT-qPCR, and western blot analysis. Furthermore, in vivo efficacy of KDM1A inhibitor and TMZ was established using orthotopic models of GBM.

RESULTS
Cell viability, survival, and apoptotic assays demonstrated that knockout/knockdown or inhibition of KDM1A sensitized GBM cells to both TMZ or radiation therapy. KDM1A inhibitors used in combination with radiation or TMZ significantly increased apoptosis in GBM cells. RNA-seq analysis and signaling studies showed attenuation of DDR pathways in KDM1A-KD cells. Radiation or TMZ treatment enhanced DNA damage in KDM1A-KD cells compared to controls. Importantly, combination of KDM1A inhibitor and TMZ significantly reduced in vivo tumor progression and improved overall survival in orthotopic GBM murine models.

CONCLUSIONS
Our results provide strong evidence that KDM1A inhibition sensitizes GBM to TMZ or radiation therapy via modulation of DDR. This suggests combination of KDM1A inhibitors with TMZ or radiation therapy could serve as a novel treatment for GBM patients.
EXTH-31. INCREASING TUMOR TREATING FIELDS (TTFIELDS) EFFICACY BY TARGETING THE G2 CELL CYCLE CHECKPOINT WITH WEE1 OR CHK1 INHIBITORS IN GLIOBLASTOMA CELL LINES
Authors: Slangen Paul L G , de Gooijer Mark C , van Geldorp Mariska , van Tellingen Olaf , Borst Gerben R 
Published: December 2020

Abstract
Tumor Treating Fields (TTFields) are a novel, noninvasive FDA-approved treatment modality for glioblastoma (GBM) that utilizes alternating electric fields. While the mechanism of action was at first exclusively attributed to the effects of TTFields on cells during mitosis, additional effects during interphase have recently come to light. The aim of our research is to elucidate the effect of TTFields on the cell cycle to advance the understanding of TTFields and to find novel targets for increasing its efficacy. We studied the effect of TTFields on cell cycle distribution by (propidium iodide and phospho-Histone H3 labeling) flow cytometry using double-thymidine block (DTB) synchronized cell populations. Following the release of the DTB, TTFields treatment caused GBM cells to accumulate in G2, which was prevented by concomitant exposure to a Wee1 inhibitor. Next, we compared the efficacy of TTFields with or without Wee1 or Chk1 inhibitors in multiple GBM cell lines (A172, SNB-19, and U251) by colony formation assays and observed a strong and synergistic decrease in colony formation potential in all cell lines. To investigate the underlying mechanism of G2 arrest, we quantified the amount of DNA damage, but found no difference in either γH2AX foci or comet tail moments between control and TTFields treated cells. To follow up on this surprising observation, we are using live cell imaging (inovitro Live™) of PIP-FUCCI-transduced cells. This tool will allow us to track individual cells, to evaluate the time spent in each phase of the cell cycle and to determine the ultimate cell fate in control and treatment conditions. Our finding that Wee1 or Chk1 inhibition dramatically boosts the efficacy of TTFields may have great implications for treatment of patients with TTFields. The in vivo validation of our in vitro findings will be performed in tumor-bearing mice by using the inovivo™ system.
EXTH-34. PHASE I TRIAL OF CONVECTION-ENHANCED DELIVERY OF IL13RA2 AND EPHA2 RECEPTOR TARGETED CYTOTOXINS IN DOGS WITH SPONTANEOUS INTRACRANIAL GLIOMAS
Authors: Rossmeisl John , Herpai Denise , D’Agostino Ralph , Rossmeisl John , Tatter Stephen , Dickinson Peter , Debinski Waldemar 
Published: December 2020

Abstract
The interleukin 13 receptor alpha 2 (IL13RA2) and ephrin type-A receptor 2 (EPHA2) receptors are attractive therapeutic targets, being expressed in ~90% of canine and human gliomas, and absent in normal brain. Clinical trials using an earlier generation IL-13 based cytotoxin showed encouraging clinical effects in human glioma, but met with technical barriers associated with the convection enhanced delivery (CED) method. In this study, IL-13 mutant and Ephrin-A1 (EFNA1)-based bacterial cytotoxins targeted to IL13RA2 and EPHA2 receptors, respectively, were administered locoregionally by CED to dogs with intracranial gliomas to evaluate their safety and preliminary efficacy. In this Phase I, 3 + 3 dose escalation trial, cytotoxins were infused by CED in 17 dogs with gliomas expressing IL13RA2 or EPHA2 receptors. CED was performed using a shape-fitting therapeutic planning algorithm, reflux-preventing catheters, and real-time intraoperative MRI-monitoring. The primary end point was to determine the maximum tolerated dose of the cytotoxic cocktail in dogs with gliomas. Consistent intratumoral delivery of the cytotoxic cocktail was achieved, with a median target coverage of 70% (range, 40–94%). Cytotoxins were well tolerated over a dose range of 0.012–1.278 mg/mL delivered to the target volume (median, 0.099 mg/mL), with no dose limiting toxicities observed. Objective tumor responses, up to 94% tumor volume reduction, were observed in 50% (8/16) of dogs, including at least one dog in each dosing cohort &gt;0.05 mg/mL. This study provides pre-clinical data fundamental to the translation of this multi-receptor targeted therapeutic approach to the human clinic.
EXTH-37. TARGETING EPIGENETIC VULNERABILITIES IDENTIFIED FROM A CRISPR SCREEN IN H3.3K27M DIPG
Authors: Panditharatna Eshini , Dharia Neekesh , Li Deyao , Beck Alexander , Shaw McKenzie , Jiang Li , Trissal Maria , Liu Ilon , Lareau Caleb , Anastas Jamie , Quezada Michael , Hack Olivia , Mire Hafsa , Jerome William , Kugener Guillaume , Root David , Vazquez Francisca , Dai Lingling , Wang Tingjian , Mathewson Nathan , Shi Yang , Stegmaier Kimberly , Monje Michelle , Golub Todd , Qi Jun , Filbin Mariella 
Published: December 2020

Abstract
Children diagnosed with diffuse intrinsic pontine glioma (DIPG), a type of high grade glioma in the brainstem, currently have a dismal 5-year overall survival of only 2%. The majority of DIPG patients harbor a K27M mutation in histone 3.3 encoding genes (H3.3K27M). To understand if the aberrant epigenetic landscape induced by H3.3K27M provides an opportunity for novel targeted therapies, we conducted the first CRISPR/Cas9 screen using a focused library of 1,350 epigenetic regulatory and cancer related genes in six H3.3K27M DIPG patient-derived primary neurosphere cell lines. We identified gene dependencies in chromatin regulators, polycomb repressive complexes 1 and 2 (PRC1 and PRC2), histone demethylases, acetyltransferases and deacetylators as novel tumor cell dependencies in DIPG. We hypothesized that targeting dysregulated functions of chromatin regulators by genetically deleting and chemically targeting these epigenetically induced vulnerabilities, we could ameliorate, or even reverse the downstream oncogenic effects of the aberrant epigenetic landscape of DIPG. In our secondary CRISPR nanoscreen, we first used six single guide RNAs (sgRNA) to knockout each gene using CRISPR/Cas9 ribonucleoprotein nucleofections, followed by use of three best sgRNAs combined with homology directed repair templates. Compared to lentiviral delivery, nucleofection is a rapid method, with reduced off-target toxicity, suitable for single gene knockouts in DIPG neurospheres. Secondary CRISPR validations confirmed dependencies in BMI1, CBX4, KDM1A, EZH2, EED, SUZ12, HDAC2, and EP300. Next, we conducted a chemical screen using 20 inhibitors and degraders to target the aberrant activity of HDAC, KDM1A, P300/CBP, PRC1 and PRC2. We identified eight chemical compounds that were effective in H3.3K27M DIPG neurosphere cell lines at low drug concentrations. Among these, an inhibitor and degrader targeting P300/CBP activity indicates a novel strategy of epigenetic therapy in DIPG. Through our combinatorial testing, we will identify a synergistic combination of epigenetic therapy for treating children diagnosed with H3.3K27M DIPG.
EXTH-39. HEXON SWAPPING MITIGATES ANTI-VIRAL IMMUNE RESPONSE DURING BRAIN TUMOR VIROTHERAPY
Authors: Ho Shin Dong , Nguyen Teresa , Jiang Hong , Sohoni Sagar , Gupta Sumit , Ossimetha Ashley , Alonso Marta M , Gomez-Manzano Candelaria , Fueyo Juan 
Published: December 2020

Abstract
Cancer virotherapy is a paradigm-shifting treatment modality based on the capabilities of virus-mediated oncolysis to elicit an anti-tumor immune response. Phase 1 and 2 clinical trials have demonstrated the safety and efficacy of our oncolytic adenovirus DNX-2401 (Delta-24-RGD) for patients with recurrent malignant gliomas. While a subset of the patients showed significant benefits, our goal is to improve the response rate. Clearance of the therapeutic virus by dominant anti-viral immune responses may contribute to the observed limits of the virotherapy. Adenovirus serotype 5 that provides the backbone of most oncolytic adenoviruses is highly prevalent in the human population and neutralizing antibodies against the capsid protein hexon may inhibit viral infection and replication. In this study, we showed using immunofluorescence that in mice bearing orthotopic syngeneic glioblastoma GSC005 treated with Delta-24-RGD, IgG antibodies crossed the disrupted blood-brain barrier and infiltrated the brain tumor parenchyma to colocalize with the viral hexon, suggesting that the systemic immune response may eradicate the virus within the infected tumor. To overcome this obstacle, we generated a chimeric virus called Delta-24-RGD-H43m with hexon hypervariable regions replaced with those from a lesser prevalent serotype 43 to avoid recognition by antibodies generated against serotype 5. The molecular swapping of the hexon did not significantly interfere with virion assembly nor attenuate its anti-glioma effect. Thus, the two viruses showed comparable efficacy in vitro (P= 0.568) and in vivo for animals without prior virus exposures (P= 0.228). Importantly, Delta-24-RGD-H43m evaded neutralizing antibodies generated against Delta-24-RGD and maintained its oncolytic ability (P&lt; 0.0001). We conclude that hexon swapping strategies may improve virotherapy by alleviating the dominant immune response against the virus. Despite limited understanding of the interaction between oncolytic viruses and the host immune system, further research on strategies to circumvent virus-specific immune responses should aid the development of enhanced, glioma-targeted virotherapies.
EXTH-40. THERAPEUTICALLY TARGETING DE NOVO PURINE BIOSYNTHESIS IN DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Mersich Ian , Dasgupta Biplab 
Published: December 2020

Abstract
Diffuse intrinsic pontine glioma (DIPG) is an incurable brainstem malignancy in children with median survival less than 1 year and 5-year overall survival only 2 percent. Little progress has been made in treating this deadly disease due to its inoperable location and treatments aimed at targets defined in adult gliomas. Despite recent advances in genetic characterization of DIPGs there are still no targeted therapies that significantly improve overall survival. We recently generated a metabolic profile for DIPG elucidating an upregulation in purine metabolism, specifically in de novo purine biosynthesis (DNPB). Normally nucleotide salvage maintains cellular purine levels by recycling degraded bases, however DNPB is needed when purine levels are depleted. Purine metabolism provides the basic components of nucleotides needed for tumor proliferation and thus considered a high-priority target in cancer treatment. DNPB is a sequential ten step enzymatic process resulting in the production of inosine monophosphate. The last step in DNPB is carried out by the bifunctional enzyme ATIC which is upregulated in DIPG cell lines, and in patient tumors. Our preliminary data demonstrates DIPG cell lines are sensitive to pharmacological inhibition and genetic ablation of multiple enzymes in the DNPB pathway. Strikingly, cell viability could be rescued by purine supplementation when inhibiting this pathway except when ATIC is inhibited indicating the mechanism of cell death for ATIC inhibition is independent of purine nucleotide levels. Furthermore, there is a therapeutic window for targeting ATIC in DIPG cell lines relative to normal neural stem cells and normal human astrocytes. Metabolic flux experiments have demonstrated DNPB is upregulated in DIPG cell lines and the reason these cells are more sensitive to ATIC inhibition is likely related to the rapid accumulation of a cytotoxic metabolite upstream of ATIC. In vivo studies are currently underway in pre-clinical mouse models for DIPG.
EXTH-43. GENERATION OF A B-CELL-BASED VACCINE FOR THE TREATMENT OF GLIOBLASTOMA
Authors: Lee Chang Catalina , Miska Jason , Hou David , Rashidi Aida , Zhang Peng , Burga Rachel , Torres Ignacio Jusue , Xiao Ting , Arrieta Victor , Zhang Daniel , Lopez-Rosas Aurora , Han Yu , Sonabend Adam , Horbinski Craig , Stupp Roger , Balyasnikova Irina , Lesniak Maciej 
Published: December 2020

Abstract
Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNg stimulation. BVaxmigrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunologic memory that prevented the growth of new tumors upon subsequent re-injection in cured mice. GBM patient-derived BVax were successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. In addition to the role in activating CD8+ T cells, BVax produce tumor-specific antibodies able to control tumor growth via antibody-mediated cell cytotoxicity. In conclusion, BVax tackles GBM immunosurveillance escape by using both cellular (CD8+ T-cell activation) and humoral (anti-tumor antibody production) immunity. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.
EXTH-44. DEVELOPMENT OF RETROVIRAL REPLICATING VECTORS ENCODING SEQUENCE-OPTIMIZED NITROREDUCTASE FOR PRODRUG ACTIVATOR GENE THERAPY IN HUMAN GLIOMA MODELS
Authors: Collins Sara , Inagaki Akihito , Tai Chien-Kuo , Logg Christopher R , Searle Peter , Tangney Mark , Kasahara Noriyuki 
Published: December 2020

Abstract
Early-phase clinical trials of Toca 511, a retroviral replicating vector (RRV) expressing an optimized form of yeast cytosine deaminase for prodrug activator gene therapy, showed highly promising evidence of therapeutic benefit and increased survival in recurrent high-grade glioma, but an international Phase 3 trial failed to meet primary and secondary endpoints. Nonetheless, promising results observed within predetermined subgroups, combined with the highly favorable safety profile of this approach, make it worthwhile to consider alternative prodrug-activating enzymes in order to increase therapeutic efficacy. In this study, we employed RRV to deliver modified versions of another prodrug activator gene, E.coli nitroreductase (NTR), which activates the prodrug CB1954 to generate a potent bifunctional alkylating agent. We constructed RRV encoding two different isoforms of wild-type E.coli NTR genes (NfsA, NfsB) as well as variants of both isoforms extensively modified to optimize human codon usage and vector stability. Further efforts to improve the potency of these prodrug activator enzymes included the construction of additional new vectors incorporating point mutations at the active site which have previously been demonstrated to improve prodrug conversion efficiency. NTR transgene insertion did not affect RRV replication, which resulted in increasing NTR expression over time in human glioma cells in culture. Sequence optimization resulted in enhanced genomic stability of the vectors upon serial passage and also in significantly reduced cell viability in vitro on treatment of fully transduced U87 and U87-EGFRviii glioma cells with CB1954 prodrug. In orthotopic U87-EGFRviii intracerebral tumor models, stereotactic intratumoral injection of sequence-optimized RRV-NTR and repeated cycles of prodrug treatment resulted in rapid and significant reduction of tumor burden as monitored by bioluminescence imaging, leading to prolonged survival benefit. These data indicate that improved therapeutic efficacy can be achieved with optimized NTR prodrug activator gene therapy delivered by RRV in experimental glioma models.
EXTH-45. DELTA-24-RGDOX ACTIVATION OF THE IDO-KYN-AHR CASCADE IN GLIOBLASTOMA: OLD TARGETS FOR A NEW THERAPY
Authors: Nguyen Teresa , Shin Dong Ho , Jiang Hong , Wainwright Derek , Sohoni Sagar , Gupta Sumit , Ossimetha Ashley , Lang Frederick , Alonso Marta M , Fueyo Juan , Gomez-Manzano Candelaria 
Published: December 2020

Abstract
Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.
EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Carvalho Diana , Richardson Peter , Olaciregui Nagore Gene , Stankunaite Reda , Lavarino Cinzia Emilia , Molinari Valeria , Corley Elizabeth , Ruddle Ruth , Donovan Adam , Pal Akos , Raynaud Florence I , Overington John P , Phelan Anne , Sheppard Dave , Mackinnon Andrew , Hubank Michael , Cruz Ofelia , Madrid Andres Morales La , Mueller Sabine , Carcaboso Angel Montero , Carceller Fernando , Jones Chris 
Published: December 2020

Abstract
Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.