bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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< 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

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

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

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

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

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.
EXTH-49. BXQ-350 TARGETS TO THE LYSOSOME AND KILLS GLIOBLASTOMA (GBM) CELLS VIA ACTIVATION OF APOPTOTIC CASPASES IN VITRO
Authors: Wilkins Nikhil , Stephens Timothy , Felix Laura
Published: December 2020

BACKGROUND

Apoptosis is a programmed cell death mechanism where cells respond to internal or external stimuli by initiating a cascade of events and enzymes leading to cell death. One of the hallmarks of cancer is the ability to resist apoptotic stimuli. Removing resistances to apoptosis can result in the death of these tumor cells.

METHOD

The GBM cell line Gli36ΔEGFR was used to determine Caspase activity and BXQ-350 cytotoxicity. Cells were treated with 9uM to 30uM BXQ-350 in triplicate and incubated for 24 hours at 37oC. Promega’s Caspase-Glo 9 or Caspase-Glo 3/7 reagent was added to each well of a plate and was incubated at room temperature in the dark for 3 hours then luminescence was read. The parallel cytotoxic assay was run under the same conditions except Roche’s MTT labeling reagent was added to each well after 24 hours and the plate was incubated at 370C for 4 hours. Solubilization solution was added to each well, the plate was incubated overnight then absorbance was read. The GBM cell line U87 MG was used to determine lysosomal targeting by treating with 10uM BXQ-350 and incubated at 37oC overnight. They were stained with anti-SapC (RFP) and anti-LAMP1 (GFP) antibodies and images were taken.

RESULT

BXQ-350 mediated cell death is correlated with a rise in Caspase 3, Caspase 7 and Caspase 9 activity. The caspase activity levels did not rise until after BXQ-350 passed its IC50 and stayed elevated. Caspases 3/7 levels showed higher activity compared to untreated than Caspase 9. BXQ-350 was seen to colocalize to LAMP1, a lysosomal membrane protein.

CONCLUSION

BXQ-350 tracks to the lysosomal membrane where it initiates a cascade of enzymes necessary to cause apoptosis. Caspases 3/7 are the effector caspases that complete the apoptotic process removing a major barrier to fight cancer.
EXTH-56. DISRUPTION OF FUNCTIONAL NETWORKS IN GLIOBLASTOMA TO OVERCOME THERAPY RESISTANCE
Authors: Joseph Kevin , Vollmer Lea , Schneider Matthias , Beck Juergen , Hofmann Ulrich , Schnell Oliver , Heiland Dieter
Published: December 2020

Neurobeachin plays a crucial role when it comes to the formation, maturation, and differentiation of functional synapses, within the central nervous system (CNS). Glioblastoma has cellular compartments that have been shown to form functional synapses, integrating themselves to existing neuronal networks. We present evidence that GBM cells form electrophysiologically active networks, mediated by both electrical and chemical synapses during their growth. These networks carry information about changes in the tumor microenvironment, similar to those observed in neuronal networks. We present data about signature signaling patterns, dependent on stimuli, leading to transcriptional changes. These patterns are conducted through the network by means of electrical/chemical synapses, mediated by Neurobeachin. When the tumor network was acutely treated with a signaling inhibitor, Meclofenamate, functional synapses were altered, leading to an abolishment of electrical signaling, resulting in loss of biological organization. These results were validated in both extracellular electrophysiological recordings and Ca2+ imaging experiments. The formation of these functional networks plays a vital role in the resistance to alkylating agents or harsh metabolic imbalances, which was completely lost when the network was disrupted. Acute downregulation of Neurobeachin expression causes electrophysiological disruption of the GBM network, leading to loss of connectivity, terminally resulting in cell apoptosis/necrosis, transcriptionally validated by means of RNA-seq. This combined therapeutic approach could lead to potential alternative therapy, where the basic biological functionality of the GBM is targeted.
EXTH-58. ONCOSCILLATORS: INDUCTION OF THE MITOCHONDRIAL PERMEABILITY TRANSITION IN CANCER CELLS
Authors: Sharpe Martyn , Baskin David , Helekar Santosh
Published: December 2020

Magnetic fields in the mT range influence spin state pairing in redox-active radical pairs generating spin-forbidden quantum states which are kinetically inert. Studies examining the effects of static magnetic fields on mitochondrial electron transfer kinetics have demonstrated only modest effects. When neodymium super-magnets are securely attached to and precision-balanced on the shafts of electronically-controlled motors it is possible to generate rotating magnetic fields of desirable strengths and frequencies. Unlike a static magnetic field or an alternating field in a static electromagnetic coil, oscillating magnetic field (OMF) produced by rotating lines of force of a spinning permanent magnet can dynamically couple the electron spins of radical pairs within proteins whose orientations are ‘fixed’. The frequencies of rotation of magnets can also be tuned to appropriate electron cycling resonances within the proteins. Using OMF of appropriate field strength, frequency and on/off acceleration/deceleration profiles we can completely arrest electron transport in isolated respiring rat liver mitochondria. Parallel to this inhibition of electron flux, we also independently observe an increase in superoxide and hydrogen peroxide. Under certain OMF exposure regimes, we observe membrane permeability transition in these mitochondria when using succinate as substrate, and show that the mitochondrial membrane permeability transition effect can be blocked by bongkrekic acid. We have examined the effect of OMF on oxygen consumption in cultured primary cancer cells with a rotating magnet (oncoscillator) that is an integral component of a new anti-cancer Oncomagnetic device. We observe three main effects in addition to the inhibition of respiratory flux in cancer cells – damage to the respiratory complex, uncoupling and generation of superoxide/hydrogen peroxide. OMF generated by oncoscillators can induce mitochondrial permeability transition in primary cultured malignant meningioma, diffuse intrinsic pontine glioma and GBM cells. Parallel experiments with normal human astrocytes show only minor changes in cellular/mitochondrial function under these conditions.
EXTH-63. IN VITRO DRUG SCREENING BASED ON IN SILICO DATA IDENTIFIES NEW THERAPEUTIC AGENTS FOR AGGRESSIVE MENINGIOMA
Authors: Jungwirth Gerhard , Cao Junguo , Yu Tao , Warta Rolf , Unterberg Andreas , Herold-Mende Christel
Published: December 2020

The mainstay of treatment for progressive or recurrent meningiomas is surgery and/or radiotherapy. Patients with refractory cancer might benefit from systemic treatment options. However, to date, no effective chemotherapy is available for these patients. For this reason, novel inhibitors for the treatment of aggressive meningiomas are urgently needed. Therefore, we used our previously published Affymetrix microarray dataset (GSE74385) consisting of 62 meningiomas enriched with 28 WHO°III MGMs to screen substantially expressed genes that can be targeted by available inhibitors. For each targeted gene, three compounds were selected based on their clinical development. This filter process resulted in 107 drugs targeting 57 different genes. Most compounds were oncology-related (n = 94, 88%) with the remaining compounds being non-oncology agents (n = 13, 12%). Thereafter, a 2-stage screening strategy was employed. First, drugs were screened at a single dose (2.5 µM) in two malignant meningioma cell lines (NCH93 and IOMM-Lee) with CellTiter-Glo (Promega). Only drugs resulting in a cell viability of 50% or less of either cell line were considered for further validation. Remaining drug candidates (n = 33) exclusively belonged to the oncology-related group, consisting of 4 FDA-approved antineoplastic drugs (12%). The other drugs are currently tested in clinical trials (24% in phase III, 39% in phase II, and 24% in phase I). Drug candidates were further analyzed in a six-point dose-response scheme ranging from 0.1 nM to 10 µM in three meningioma cell lines (Ben-Men-1, NCH93, IOMM-Lee). The top 5 drugs were selected based on the lowest mean of z-transformed area under the curve. Resulting drugs and corresponding targets are: OTSSP167 (MELK), Panobinostat (HDAC), Picropodophyllin (IGF-1R), KPT-9274 (PAK4 and NAMPT), and BI-2536 (PLK1). Taken together, our drug screening approach utilized in silico data to identify potential inhibitors for the treatment of aggressive meningiomas warranting further preclinical investigation.
EXTH-66. NEO100 TRANSIENTLY OPENS UP THE BLOOD BRAIN BARRIER VIA TIGHT JUNCTION INHIBITION
Authors: Chen Thomas , Wang Weijun , Ramos Nagore Marin , Schonthal Axel
Published: December 2020

The blood brain barrier (BBB) prevents effective entry of nearly all therapeutics to the central nervous system (CNS), preventing effective treatment of brain-related malignancies. Intracarotid mannitol injection has been the main technique to transiently open up the BBB, with its attendant variability and complications. A more direct and better tolerated method is needed to open up the BBB. We present our discovery that intraarterial (IA) injection of NEO100, a cGMP-quality form of perillyl alcohol (POH), transiently opens up the BBB in a safe and reversible manner. We used in-vitro models of MDCK1 and patient derived brain endothelial cell (BEC) + astrocyte barriers to determine that NEO100 increased FITC-antibody diffusion across the in-vitro BBB model and decreased trans-epithelial/endothelial electrical resistance (TEER). NEO100 effects on transcellular and paracellular pathways were studied using western blot, flow cytometry, HPLC, fluorescent probes, microarray analysis, and transmission electron microscopy. In-vivo studies were performed using ultrasound-guided intracardiac administration of NEO100 in mice with subsequent intravenous delivery of non-BBB permeable therapeutic agents. We determined that NEO100 transiently disrupts the transcellular pathway by permeabilizing BEC membranes, and the paracellular pathway via delocalization of tight junction proteins. In vivo IA NEO100 administration caused an effective dose- and time-dependent BBB permeabilization, which was reversible and well tolerated by the mice. This was evidenced by the spreading of Evans blue dye, and of therapeutics with different molecular weights, ie methotrexate, anti-PD-1 antibody, and CAR-T cells in the brain. Our results demonstrate that IA NEO100 is able to open the BBB in a controlled and reversible manner, allowing it to facilitate drug delivery to the CNS.
EXTH-74. IND-ENABLING CHARACTERIZATION OF DUAL DRD2- AND ClpP-TARGETING AGENT ONC206 AS THE NEXT IMIPRIDONE FOR CLINICAL NEURO-ONCOLOGY
Authors: Prabhu Varun , Morrow Sara , Kawakibi Abed Rahman , Jitkova Yulia , Jung Jinkyu , Madhukar Neel , Garnett Mathew , McDermott Ultan , Benes Cyril , Wechsler-Reya Robert , Elemento Olivier , DeMorrow Sharon , Schimmer Aaron , Stogniew Martin , Theeler Brett , Gilbert Mark , Allen Joshua
Published: December 2020

ONC201 is the first bitopic antagonist of dopamine receptor D2 (DRD2) and allosteric mitochondrial protease ClpP agonist, that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201, is also a bitopic DRD2 antagonist that exhibits enhanced non-competitive effects, nanomolar potency, and disruption of DRD2 homodimers. In these studies, a FITC-casein degradation assay revealed that ONC206 also acts as an agonist of human ClpP and has a 3-fold improved potency. GEPIA database analysis showed ClpP mRNA was overexpressed in glioblastoma cells relative to normal cells. Broad nanomolar efficacy of ONC206 (GI50 < 78-889nM, 72h) was observed in >1,000 GDSC cancer cell lines with the highest sensitivity in cell lines exhibiting high ClpP and/or DRD2+/DRD5- RNA expression signatures. Among solid tumors, nervous system related cell lines were particularly sensitive. ONC206 reduced the viability of normal human fibroblasts at higher doses (GI50 > 5µM), suggesting a wide therapeutic window. Antitumor efficacy without body weight loss was observed with 50 mg/kg weekly oral ONC206 in a subcutaneous xenograft model of DRD2-overexpressing, dopamine-secreting tumor cells. Oral ONC206 at 50mg/kg exhibited a ~12 µM plasma Cmax and ~6 hours terminal half-life in Sprague-Dawley rats. Additionally, 5–10 fold higher ONC206 concentrations were observed in adrenal gland, bile duct, brain and bone marrow relative to plasma. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs revealed no dose-limiting toxicities. Mild and reversible body weight changes were observed at the highest evaluated dose in both species. The no-observed-adverse-effect level was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats that exceed efficacious doses. A 50 mg starting dose of ONC206 was selected for the first-in-human open label, dose escalation, and food effect Phase I study in biomarker-enriched adult recurrent primary CNS tumors.
EXTH-75. IN VITRO AND IN VIVO EFFICACY OF COMBINATORIAL INHIBITION OF LSD1 AND HDACS IN PATIENT DERIVED GLIOBLASTOMA STEM CELL MODELS
Authors: Gangadharan Achintyan , Grasse Leslie , Espejo Alexsandra , Ezhilarasan Ravesanker , Collier Tiara , Henry Verlene , Gumin Joy , Sulman Erik , Lang Frederick , Chandra Joya
Published: December 2020

Inhibitors of histone deacetylases (HDACi) have been tested in glioblastoma (GBM), however, single agent clinical efficacy has not been proven, prompting study of combinatorial approaches. A rational HDACi combination strategy is with inhibition of LSD1, lysine specific demethylase 1, a histone demethylase known to exist in complex with HDAC1/2. We previously showed in vivo efficacy of combining a brain permeant LSD1 inhibitor, tranylcypromine (TCP) together with vorinostat. More selective inhibitors of LSD1 have been developed and were tested in the current study together with the HDACi, vorinostat or panobinostat in a panel of patient derived glioblastoma stem cell (GSC) lines that have been characterized as radio-resistant or radiosensitive. To test in vitro sensitivity, dose response experiments were conducted in nine GSC cell lines using three LSD1 inhibitors or two HDACi as single agents or in combination. Two non-tumor lines were also tested to assess selective cytotoxicity for brain tumor lines. Cell viability and clonogenicity assays were performed. Several radio-resistant GSC lines showed sensitivity to single agent LSD1 inhibitors, whereas some radio-sensitive GSCs did not, indicating distinct molecular mechanisms of response. Intracranial orthotopic xenograft models were used to test in vivo efficacy using the radioresistant GSC20 line. Interestingly, LSD1 inhibition alone promoted tumor burden reduction and better survival rates than combination treatment. RNA-Seq guided biomarkers of sensitivity to the LSD1/HDAC inhibitor combination were assessed in vitro and in vivo. Both in vivo and in vitro data show that GSC20 treated with GSK LSD1 inhibitor had higher levels of HKDC1 gene with in vivo data also showing elevated levels of FTH1. In vivo mouse data for vorinostat and combination treatment groups showed that only the FTH1 gene level was elevated, providing a potential explanation for low in vivo efficacy. Collectively, our data suggests that LSD1 inhibition represents a viable strategy in GBM.
EXTH-76. THE INOVIVO SYSTEM: A NOVEL PRECLINICAL TOOL FOR IN VIVO DELIVERY OF TUMOR TREATING FIELDS (TTFIELDS)
Authors: Davidi Shiri , Blat Roni , Shteingauz Anna , Porat Yaara , Giladi Moshe , Weinberg Uri , Palti Yoram
Published: December 2020

Tumor Treating Fields (TTFields) are an antineoplastic treatment modality targeting dividing cancer cells, approved by the FDA for treatment of glioblastoma and malignant pleural mesothelioma. TTFields are delivered to the patient continuously, using a portable signal generator and 2 pairs of transducer arrays attached to the skin. While in vitro research tools for TTFields have been available, animal experiments were so far limited due to the lack of a dedicated TTFields in vivo delivery system. The main challenges associated with TTFields application to mice are the absolute requirement for adequate and continuous contact between array electrodes and animal skin throughout treatments; and the need to deliver the electric fields through multiple wired conductors connected to electrodes, which limit animal movement thus imposing stress. This work reports on development of an in vivo system to facilitate continuous delivery of TTFields to mice bearing cancer tumors in the torso or flank that addresses the aforementioned challenges. Three major developments were introduced to the system: 1) transducer array electrodes composed of delicate, flexible inner layer to improve skin adherence and a breathable, durable, outer layer to secure electrodes to the skin; 2) a novel electric swivel to prevent cable coiling; and 3) a cage constructed to house 2 mice separately while still allowing socializing. During a 1-week study, the new inovivo system demonstrated improvement compared to previous systems in the following areas: increased animal treatment compliance, less weight loss, and fewer number of array replacements. In conclusion, the new inovivo system is a comprehensive tool for continuous, 2 directional TTFields delivery to tumors in the torso or flank of mice for conducting TTFields experiments with minimal animal stress, thus allowing further elucidation of the effects of TTFields on the whole animal.
IMMU-02. THE SURVIVAL OUTCOMES ASSOCIATED WITH IMMUNE CHECKPOINT INHIBITORS FOR NON-SMALL CELL LUNG CARCINOMA PATIENTS WITH BRAIN METASTASES IN THE UNITED STATES
Authors: Lamba Nayan , Smith Timothy , Iorgulescu Bryan
Published: December 2020

BACKGROUND

Management of advanced NSCLC has been transformed by PD-1/PD-L1 immune checkpoint inhibitors (ICI), with FDA approvals in 2015 (second-line) and 2016 (first-line). Because patients with brain metastases (BMs) were disproportionately excluded from the pioneering trials, herein we evaluated the overall survival (OS) associated with ICI in NSCLC BMs nationally.

METHODS

Patients newly-diagnosed with stage 4 NSCLC, including BMs, from 2010–2016 were identified from the National Cancer Database (comprising >70% of all newly-diagnosed cancers in the U.S.) Post-approval, median time from diagnosis to ICI was 58days, and this timepoint was selected for all landmark survival analyses (logrank test and multivariable Cox regression) and for multivariable logistic regression to identify predictors of ICI utilization.

RESULTS

50,858 patients presented with advanced NSCLC that involved the brain: representing 27.6% of all newly-diagnosed stage 4 cases. Following initial FDA approvals in 2015, ICI rates for BM patients rose from 7.2% in 2015 to 12.7% in 2016. OS for NSCLC BMs for patients diagnosed post-approval (median 6.3 months, 95%CI: 6.0–6.6) was substantially better than those diagnosed pre-approval (median 5.5 months, 95%CI: 5.4–5.7, p< 0.001) and, in fact, than those diagnosed in 2014 (median 5.9 months, 95%CI: 5.6–6.1, p=0.002). Among patients diagnosed post-approval, ICI receipt demonstrated substantially improved OS in landmark survival analyses (median 13.8 months, 95%CI: 12.2–15.1; vs. 8.5 months, 95%CI: 8.3–8.9, p< 0.001); benefits which persisted in multivariable landmark survival analyses (HR 0.83, 95%CI: 0.71–0.96, p=0.02), independent of patient characteristics, other therapies, and extracranial disease. For patients diagnosed post-approval, who reached the landmark timepoint, ICI receipt was independent of patient demographics, socioeconomic status, and hospital type—with the exception of Medicaid-insured patients, who were less likely than privately-insured patients to receive ICI (OR 0.77, 95%CI: 0.60–0.97, p=0.03).

CONCLUSIONS

Nationally, the dramatic OS benefits of ICIs for advanced NSCLC were also demonstrated for patients with BMs.
IMMU-07. TUMOR STROMA–TARGETING ANTIBODY-CYTOKINE CONJUGATES TO CONVERT THE IMMUNOLOGICALLY COLD GLIOMA MICROENVIRONMENT INTO A HOT ONE
Authors: Weiss Tobias , Puca Emanuele , Silginer Manuela , Hemmerle Teresa , Pazahr Shila , Bink Andrea , Weller Michael , Neri Dario , Roth Patrick
Published: December 2020

Glioblastoma is an immunological “desert”. The administration of pro-inflammatory cytokines could shift the balance between tumor-associated immune suppression and anti-tumor immunity but the systemic administration of therapeutically active doses of pro-inflammatory cytokines is hampered by toxic side effects. We investigated different antibody-cytokine fusion products that enable a targeted delivery of interleukin (IL-) 2, IL-12 or tumor-necrosis factor (TNF)α to the tumor site upon systemic administration by binding to a tumor-specific epitope of fibronectin. We confirmed the target antigen expression in ex vivo sections of orthotopic syngeneic glioma mouse models and human glioblastoma samples and characterized the distribution of these antibody-conjugates in glioma-bearing mice upon systemic administration using in vivo imaging. Subsequently, we demonstrated potent anti-tumor activity of these antibody-fusion proteins in fully immunocompetent orthotopic mouse glioma models and characterized their mode of action. We also translated this immunotherapeutic strategy to treat patients with recurrent glioblastoma systemically with an antibody-fusion protein that enables the targeted delivery of TNFα to the tumor site. This was well tolerated, led to a treatment-associated tumor necrosis and increased the number of tumor-infiltrating T cells. This work builds a basis for future studies with antibody-cytokine fusion proteins as a promising treatment strategy for central nervous system tumors.
IMMU-10. GENOMIC DIFFERENCES UNDERLIE MYELOID-DERIVED SUPPRESSOR CELL SEXUAL DIMORPHISM IN GLIOBLASTOMA
Authors: Bayik Defne , Zhou Yadi , Lo Alice , Park Chihyun , Hong Changjin , Vail Daniel , Watson Dionysios , Roversi Gustavo , Lauko Adam , Silver Daniel , Alban Tyler , Otvos Balint , Grabowski Matthew , Sorensen Mia , Sims Peter , Kristensen Bjarne , Horbinski Craig , Vogelbaum Michael , Hwang Tae Hyun , Khalil Ahmad , Iavarone Antonio , Ahluwalia Manmeet , Cheng Feixiong , Lathia Justin
Published: December 2020

A potently immunosuppressive tumor microenvironment facilitates progression of glioblastoma (GBM). We previously demonstrated that myeloid-derived suppressor cell (MDSC) subsets promote tumorigenesis in a sex-specific manner, contributing to sexual dimorphism in GBM incidence and prognosis. Our findings indicated that proliferating monocytic MDSCs (mMDSCs) accumulate in tumors of male mice and patients, while female tumor-bearing mice had an increase in circulating granulocytic MDSC (gMDSC) frequency, and a high gMDSC gene signature correlated with worse outcome of female patients. However, the mechanisms underlying sexual dimorphism of MDSC heterogeneity remain understudied and can provide insights for improved immunotherapy response. Using syngeneic mouse glioma models and sequencing approaches, we show that expression of Y-chromosome-linked genes correlates with upregulation of multiple RNA transcription-related pathways specifically in male mMDSCs. Consistently, adoptive transfer of male mMDSCs but not gMDSCs worsened GBM outcome in male recipients, while the transfer of sex-matched mMDSCs did not impact survival of female mice. In contrast to this cell-intrinsic regulatory pathway, sex steroids had no impact on MDSC profile, as castration or ovariectomy failed to alter MDSC subset accumulation patterns in GBM-bearing mice. Correspondingly, IL-1β, which we had identified as a female-specific drug target, was highly expressed in female but not male gMDSCs. Single-cell sequencing revealed that circulating but not tumor-infiltrating gMDSCs were the primary source of IL-1β and that its neutralization provided a female-specific survival advantage by reducing circulating gMDSCs. This was accompanied by declines in tumor infiltration of microglia, microglia activation status and tumor cell proliferation. In vitro, IL-1β inhibition reduced viability and expression of activation markers by primary microglia. These findings highlight a peripheral gMDSC-microglia communication axis mediated by IL-1β signaling in females with GBM and indicate that expression differences in MDSC subsets represent opportunities for improved immunotherapy efficacy while accounting for sex as a biological variable.
IMMU-13. EFFICACY OF CXCR6 BLOCKADE AS A POTENTIATOR OF ANTI-PD-1 THERAPY FOR THE TREATMENT OF GLIOBLASTOMA
Authors: Pant Ayush , Saleh Laura , Patel Kisha , Medikonda Ravi , Lim Michael
Published: December 2020

Glioblastoma (GBM) is an aggressive primary tumor of the brain with a dismal prognosis for patients. Despite the standard of care treatment, median survival is 12–15 months. The blockade of inhibitory checkpoints such as PD-1 and CTLA-4 has become the mainstay immunotherapy to treat solid tumors but it lacks efficacy in treating GBM patients. Emergence of alternative checkpoints on T cells as a mechanism of acquired resistance is considered one of the major hurdles for the success of anti-PD-1 therapy in GBM. Using an orthotopic mouse model of GBM, we have seen that cytotoxic T cells infiltrating the tumor show a preponderance of the chemokine receptor CXCR6 on exhausted cells. Furthermore, ablating CXCR6 along with anti-PD-1 therapy greatly improved anti-tumor immune response. Whereas PBS treated and CXCR6 KO mice had no long-term survivors 40 days post-tumor implantation, 90% of anti-PD-1 treated CXCR6 KO mice were long-term survivors, compared with 12% among anti-PD-1 treated wildtype mice. This supports our hypothesis that blockade of CXCR6 licenses anti-PD-1 blockade by alleviating acquired resistance to anti-PD-1 therapy. We have observed CXCR6 expression on exhausted T cells of GBM patients, making it a promising target for dual therapy with anti-PD-1 in clinical trials.
IMMU-16. TWO DISTINCT SUBSETS OF NATURAL KILLER CELLS ARE ENRICHED IN THE TUMOR MICROENVIRONMENT AND CORRELATE WITH SURVIVAL OUTCOME IN HUMAN GLIOBLASTOMA.
Authors: Bartkowiak Todd , Barone Sierra , Hayes Madeline , Roe Caroline , Sinnaeve Justine , Mistry Akshitkumar , Leelatian Nalin , Greenplate Allison , Mobley Bret , Chambless Lola , Thompson Reid , Weaver Kyle , Ihrie Rebecca , Irish Jonathan
Published: December 2020

Glioblastomas (GBM) account for up to 60% of all adult primary brain tumors. With few advances in therapeutics, median overall survival (mOS) remains at 15-months post diagnosis. Success of immunotherapy in peripheral solid tumors may offer an alternative therapeutic approach for patients with GBM tumors; however, no predictive immune features currently inform therapeutic stratification for GBM. Recently, we have identified radiographic tumor contact with the lateral ventricle (LV) as a prognostic indicator of OS, as patients with LV+ GBM survive 7 months less than patients with LV- gliomas. This disparity was independent of known prognostic factors (e.g. KPS, extent of resection). Further, we have identified a correlation between greater immune infiltration and the frequency of tumor subtypes with more favorable prognosis. We therefore hypothesized that differences in overall survival between patients with LV+ and LV- are due, in part, to a uniquely immunosuppressive microenvironment within the LV. Using 35-parameter single-cell mass cytometry (CyTOF) we profiled the immune infiltrate of human GBM tissue acquired in accordance with the Declaration of Helsinki and with the approval of the institutional review board (IRB #131870). Computational approaches (tSNE, Citrus, RAPID) correlated natural killer (NK) cell populations correlating with prognosis. NK cells made of 1–14% of the total immune infiltrate in GBM. Ninety percent of NK cells infiltrating LV- tumors were CD16+CD56dim cytotoxic NK cells (cNK). LV+ gliomas, however, were enriched in CD16+CD56bright immunoregulatory NK cells (irNK). The presence of cNK cells correlated with a 2.5-fold improvement of OS, whereas irNK cells correlated with a 2.2-fold reduction in OS. Further, 30–60% of NK cells infiltrating LV+ tumors expressed checkpoint receptors (TIGIT, TIM3, B7-H3) compared to only 10–20% in LV- tumors. These results suggest that NK cells contribute to immunosuppression in the LV and may serve as alternative targets to T cell-based therapies for GBM.
IMMU-19. TARGETING GLIOBLASTOMA IMMUNOSUPPRESSION AND TREATMENT RESISTANCE WITH IBRUTINIB IN COMBINATION WITH STEREOTACTIC RADIATION AND IMMUNE CHECKPOINT BLOCKADE
Authors: Ghose Jayeeta , Raychaudhuri Baisakhi , Liu Kevin , Jiang William , Gulati Pooja , Liu Joseph , Rajappa Prajwal , Ahluwalia Manmeet , Jacob Naduparambil , Chakravarti Arnab , Carson William , Raval Raju
Published: December 2020

BACKGROUND

Glioblastoma (GBM) is associated with systemic and intratumoral immunosuppression. Part of this immunosuppression is mediated by myeloid derived suppressor cells (MDSCs). Preclinical evidence shows that ibrutinib, a tyrosine kinase inhibitor FDA approved for use in chronic lymphocytic leukemia and known to be CNS penetrant, can decrease MDSC generation and function. Also, focal radiation therapy (RT) synergizes with anti-PD-1 therapy in mouse GBM models. Thus, we aimed to test the combination of these approaches on immune activation and survival in a preclinical immune-intact GBM mouse model.

METHODS

C57BL/6 mice intracranially implanted with the murine glioma cell line GL261-Luc2 were divided into 8 groups consisting of treatments with ibrutinib, RT (10 Gy SRS), or anti-PD-1 individually or in each combination (along with a no treatment control group). Immune cell subset changes (flow-cytometry) and animal survival (Kaplan-Meier) were assessed (n=10 mice per group).

RESULTS

Median survival of the following groups including control (28 days), ibrutinib (27 days), RT (30 days) or anti-PD-1 (32 days) showed no significant differences. However, a significant improvement in median survival was seen in mice given combinations of ibrutinib+RT (35 days), ibrutinib+anti-PD-1 (38 days), and triple therapy with ibrutinib+RT+anti-PD-1 (48 days, p < 0.05) compared to controls or single treatment groups. The reproducible survival benefit of triple combination therapy was abrogated in the setting of CD4+ and CD8+ T cell depletion. Contralateral intracranial tumor re-challenge in long-term surviving mice suggested generation of tumor-specific immune memory responses. The immune profile of the tumor microenvironment (TME) showed increased cytotoxic CD8+ T cells and decreased MDSCs and regulatory T cells in the triple combination therapy mice compared to controls.

CONCLUSION

The combination of ibrutinib, focal RT, and anti-PD-1 immune checkpoint blockade led to a significant survival benefit compared to controls in a preclinical model of GBM.