bluuebunny/crossref_metadata_2025 huggingface dataset:
DDIS-27. TARGETING ATP DOCKING AND P35/P25 BINDING OF CDK5 IN GLIOMA STEM CELLS USING SYNTHETIC INHIBITORS
Authors: Mukherjee Subhas , Schiltz Gary , Clutter Matt , Mishra Rama , Olson Cheryl , Dunder Bilge , Sharma Nitya , Markwell Steven , Brat Daniel
Published: November 2019

The survival advantage of glioma stem cells (GSCs) represents a critical mechanism for growth, therapy resistance and recurrence in glioblastoma. So far, targeting GSCs has not been highly specific, since these cells co-opt the normal developmental signaling pathways. We have demonstrated that the activated CDK5-CREB1 signaling axis regulates GSC self-renewal and also promotes radiation-resistance. Thus targeting CDK5 signaling is highly rational, yet there are challenges. Most of the available CDK5 inhibitors also target other CDKs non-specifically. In collaboration with The Center for Molecular Evolution and Drug Discovery, we are developing novel CDK5 inhibitors that are highly potent and specific. METHODOLOGY: The CKD5-p25 crystal structure (pdb code 1UNL) was used to conduct a virtual high throughput screen (vHTS). A library of 10 million commercially available compounds which had been filtered to ensure they possessed good drug-like properties was screened against the crystal structure. The top 33 compounds based on their predicted target binding, synthetic feasibility and availability were tested in an in vitro kinase assay to measure CDK5 inhibition. RESULTS: Of the 33 potential hit, 11 compounds showed a CDK5 inhibition of < 50 µM. These 11 hits represent 4 distinct chemical scaffolds. Two of them have IC50 < 1 µM, with one compound having an IC50 < 0.4 µM. The vHTS and subsequent in vitro testing have therefore confirmed the identification of several new series of potent CDK5 hit compounds. We are now characterizing the kinase selectivity of our different hit series and evaluating their activity in cell-based assays. This will help focus efforts on the most promising 1–2 scaffolds for further medicinal chemistry optimization to improve the compounds’ potency, selectivity and brain penetration. Ultimately, our optimized compounds will be tested in GBM models to demonstrate their effectiveness in inhibiting CDK5 as a new approach for treating GBM.
DDIS-32. MEK INHIBITORS INDUCES NEURONAL DIFFERENTIATION IN EGFR AMPLIFIED GLIOMA STEM LIKE CELLS
Authors: Balasubramaniyan Veerakumar , Park Soon , Piao Yuji , Martínez Emmanuel , Dong Jianwen , Mittal Sandeep , Khan Sabbir , Zhang Ze-yan , Sulman Erik , DeGroot John
Published: November 2019

The median survival for patients with glioblastoma (GBM) is 12–15 months highlighting the need for better therapeutic strategies for this deadly disease. Genomic and epigenomic sequencing analysis at the single cell level have identified multiple genomic aberrations as potential targets for therapeutic intervention in GBM. EGFR and PDGFR amplification are evident in nearly 40% and 12% of human GBM, respectively. Although the first and second-generation EGFR tyrosine kinase small molecule inhibitors failed to show long term therapeutic benefit in GBM patients, multiple factors such as incorrect patient selection, acquired resistance, and drug-target heterogeneity may all lead to clinical failure of targeted therapies. Although the multilevel genomic characterization of gliomas are increasing, the clinical translation of these findings is beginning to unravel. In this study, we attempted to correlate the genomic variations using an unbiased high throughput drug screen using primary glioma stem-like cell (GSCs) as our model system. An unbiased high-throughput screen utilizing our GSC models identified that glioblastoma cells harboring focal EGFR amplification are sensitive to mitogen-activated protein kinase (MEK) inhibitors. MEK inhibition induced apoptosis in EGFR amplified cells at low concentration. RNA sequence analysis of cells treated with MEK inhibitors revealed upregulation of genes related to neuronal differentiation and down regulation of MEK target genes in MEK sensitive glioma stem cells. Additionally, RNA sequencing of GSCs with acquired MEK inhibitor resistance demonstrated an upregulation of oncogenic transcription factor ETS Variant Gene 1 (ETV1) as a mediator of resistance. Overall our data suggest that the MEK inhibition in combination with ETV inhibitors could be a potential therapeutic target for a subset of GBM patients.
DDIS-33. SELECTIVE AND COMPLETE INHIBITION OF mTORC1 BY BI-STERIC mTOR INHIBITORS DRIVES THERAPEUTIC RESPONSE IN GLIOBLASTOMA
Authors: Wen Fan Qi , Lorenzana Edward , Luo Xujun , Lea Robin , Aksoy Ozlem , An Zhenyi , Ozawa Tomoko , Wassarman Douglas , Shokat Kevan , Aggen James , Smith Jaqueline A M , Singh Mallika , Butowski Nicholas , Raleigh David , Weiss William
Published: November 2019

Activation of phosphatidylinositol 3-kinase (PI3K)/PTEN pathway and oncogenic signaling via the mechanistic target of rapamycin (mTOR) occur in a majority of high-grade glial brain tumors. Allosteric mTOR inhibitors, such as rapamycin and other rapalogs, incompletely block mTORC1 by reducing phosphorylation of some substrates, including S6K1, but not 4EBP1. In contrast, ATP-competitive inhibitors, such as sapanisertib, fully inhibit mTORC1. However these inhibitors are also active against mTORC2 and lipid kinases, likely enhancing toxicity. A new class of selective mTORC1 inhibitor that interacts with both the ATP- and FKBP12/FRB-binding sites has been developed, which we term ‘bi-steric’. The prototype bi-steric inhibitor, RapaLink-1, blocks phosphorylation of many mTORC1 substrates, including 4EBP1. Importantly, RapaLink-1 showed improved efficacy in glioblastoma models in vivo as compared to rapamycin or sapanisertib (Fan et al., Cancer Cell 2017). Revolution Medicines has developed novel next-generation bi-steric mTORC1-selective inhibitors that exhibit potent and selective (>10-fold) inhibition of mTORC1 over mTORC2 in vitro in cell line models. Two of these compounds, RM-001 and RM-006, showed sustained blockade of mTORC1 signaling, including dephosphorylation of 4EBP1, following weekly ip dosing in an orthotopic U87MG-Luc model of glioblastoma. Repeated weekly administration of these agents resulted in significantly greater anti-tumor efficacy, as assessed via tumor burden (bioluminescence imaging) and overall survival in comparison to daily sapanisertib and the rapalog everolimus, and weekly dosing of the first generation bi-steric RapaLink-1, all at maximally tolerated doses. In summary, our data demonstrate that bi-steric mTORC1 selective inhibitors elict marked anti-tumor efficacy at doses that are well tolerated in a preclinical model of glioblastoma. Our study emphasizes the importance of mTOR as a central target in glioblastoma, and showcases the therapeutic potential of novel and selective clinical bi-steric mTORC1 inhibitors under development as investigational new drugs.
DRES-02. PARYLATION OF MGMT BY PARP IS REQUIRED FOR MGMT MEDIATED TEMOZOLOMIDE-INDUCED O6-METHYLGUANINE REPAIR: A NOVEL MECHANISM OF MGMT ACTION
Authors: Wu Shaofang , Gao Feng , Koul Dimpy , Yung Alfred
Published: November 2019

Temozolomide (TMZ) is the standard chemotherapy for malignant gliomas (MG), and resistance to this drug is mediated by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Epigenetic silencing of the MGMT gene by promoter methylation in about 40% patients is associated with loss of MGMT expression that compromises DNA repair leading to favorable response to TMZ therapy. Understanding the mechanism of MGMT mediated repair and modulating MGMT activity will enhance TMZ activity in MGMT unmethylated MG. Here we report a novel mode of regulation of MGMT protein activity by Poly-ADP-ribose polymerase (PARP). We found that PAPR physically interacts with MGMT and PARylates MGMT in response to TMZ treatment. We further showed PARylation of MGMT by PAPR is required for MGMT binding to DNA and to remove O6-methylguanine adducts in damaged DNA induced by TMZ. All 4 PARP inhibitors (trapping and non-trapping) tested (Talazoparib, Pamiparib, Veliparib, Olaparib) can inhibit PARP-MGMT binding, PARylation of MGMT, binding to DNA and subsequent removal of O6 - lesions in damaged DNA. We showed combination of PARP inhibitor with TMZ potentiated TMZ cytotoxicity in both MGMT methylated and unmethylated Glioma stem cell lines, but more profoundly in unmethylated group in vitro and in vivo. PARP inhibition acted as a double-edged sword in MGMT unmethylated MG: blocking BER/SSBR pathway to repair TMZ induced N7-MetG and O3-MetA, and more importantly, suppressing PARP-mediated PARylation of MGMT and thus silencing MGMT activity to repair O6-MetG, resulting in augmented cytotoxicity. This is the first study to show that PARylation of MGMT by PARP is required for repairing TMZ-induced O6-methylguanine adducts, and inhibition of MGMT PARylation abolishes MGMT function and renders sensitization to TMZ treatment. This finding provides a rationale for combining TMZ/CCNU and PARP inhibitors in MGMT unmethylated MG patients to enhance the benefit of adjuvant chemotherapy.
DRES-04. LONG NON-CODING RNAS IN GLIOBLASTOMA TUMOR RECURRENCE AND THERAPY RESISTANCE
Authors: Stackhouse Christian , Yancey Gillespie G , Willey Christopher
Published: November 2019

Recurrence of therapy resistant Glioblastoma multiforme (GBM) is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We are modeling acquired radiation resistance using 8 radiation-sensitive GBM patient-derived xenolines (PDX) made resistant by six irradiation series (6x2Gy each) in vivo. In 4 resistance-induced PDX, MGMT (O6-methylguanine–DNA methyltransferase) protein expression increased over original isogenic PDX. Paradoxically, temozolomide screening of orthotopic radiation-resistant PDX with increased MGMT expression revealed increased, not decreased chemo-sensitivity. This suggests an unanticipated mechanism associating acquired radiation resistance and alkylating chemotherapy sensitivity. RNA-seq data of long non-coding RNAs (lncRNAs) has revealed associations with patient overall survival and age at diagnosis. Out of 24,076 lncRNAs, 5 are significantly differentially expressed with regard to patient overall survival and age at diagnosis. The functions of lnc-ZNF117-1, lnc-DCUN1D4-1, and LINC01397 are unknown, but they are enriched in brain over other tissues. Tissue specific expression and function are unknown for lnc-TBL1XR1-5, but it is highly expressed in HepG2 (hepatocellular carcinoma) as well as in non-functioning pituitary adenomas (NFPAs). The lnc-CDH17-1 transcript is also highly expressed in NFPAs. All 5 of these lncRNAs have complex secondary and tertiary structures, but their physiologic or pathologic functions are unknown. LncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. Deep sequencing of total RNA isolated from intracranial radiation–sensitive/–resistant PDX to relate transcriptional programs and therapy resistance is underway. These data will be paired with kinomic profiling of matched tumors to elucidate basal signaling modality changes between radiation–sensitive/–resistant tumors. Analysis of differentially expressed lncRNA will be used to predict lncRNA structure/function, elucidating druggable mechanisms mediated by lncRNAs.
DRES-10. TEMOZOLOMIDE-ASSOCIATED HYPERMUTATION DETECTED WITH A GENE PANEL SIGNATURE IMPROVES IMMUNE RESPONSE IN GLIOBLASTOMA
Authors: Daniel Paul , Meehan Brian , Sabri Siham , Sarkaria Jann , Rak Janusz , Abdulkarim Bassam
Published: November 2019

Glioblastoma (GBM) is the most common and deadly type of malignant brain cancer in adults. While current standard of care which combines resection, radiation therapy (RT) and Temozolomide (TMZ) effectively eliminates primary disease, recurrence is inevitable, occurs rapidly following treatment and is ultimately lethal due to limited therapeutic opportunities of recurrent GBM. Hypermutation has been reported to occur in a subset of both low and high-grade gliomas and emerges after exposure to TMZ. Mutational inactivation and loss of mismatch repair (MMR) gene expression lead to the accumulation of single nucleotide polymorphisms throughout the genome. To date, the gain of hypermutation and subsequent therapeutic responses are still largely unknown. We hypothesized that hypermutant (HM) and non-hypermutant (NH) tumors represent two recurrent GBM subtypes, which has distinct therapeutic vulnerabilities. In addition, given the lack of concordance between microsatellite instability (MSI) and occurrence of hypermutation in GBM, we sought to derive a limited gene panel which can be used as surrogate biomarker for hypermutation following TMZ to replace whole exome sequencing (WES). Using public datasets, we demonstrated that recurrent GBM can be clustered into two subtypes: HM and NH. We used matched primary and recurrent GBM datasets to derive a gene panel signature, which is uniquely mutated at recurrence in HM GBM and confirmed the specificity of this panel in an independent dataset. Furthermore, we utilized patient derived xenograft (PDX) models to generate pre-clinical models and demonstrated that HM recurrent GBM are more immune responsive while NH recurrent GBM maintained sensitivity to a range of alternate chemotherapies such as cisplatin and RT. Finally, we demonstrated that this signature is represented in exosomes and can be enriched by use of tumor specific antibody capture methods to improve the sensitivity of hypermutation detection in liquid biopsy.
EPID-13. IDENTIFICATION OF PROGNOSTIC MARKERS IN A COHORT OF CONSECUTIVE NON-SELECTED GLIOBLASTOMA PATIENTS RECEIVING STANDARD THERAPY
Authors: Regner Michaelsen Signe , Urup Thomas , Grunnet Kirsten , Jarle Christensen Ib , Lassen Ulrik , Skovgaard Poulsen Hans
Published: November 2019

Glioblastoma is treated by standard with maximal surgical resection followed by radiation therapy with concomitant and adjuvant temozolomide. Still, median overall survival (OS) for glioblastoma is around 15 months, although the prognosis varies greatly. To improve the treatment, stratification of glioblastoma patients according to favorable and poor prognostic groups is needed. The aim of this study was to develop a prognostic model for glioblastoma patients treated with standard therapy outside clinical trials. The study included 680 consecutive, non-selected glioblastoma patients with WHO performance status (PS) 0–2 administered postoperative with standard first-line therapy between years 2005–2016 at the University Hospital of Copenhagen, Denmark. At disease recurrence, patients were mostly treated by reoperation and/or bevacizumab combination therapy. Patients were retrospectively evaluated by reviewing of medical records, histopathological descriptions and imaging analyses. The cohort had a median progression-free survival (PFS) of 7.5 months, median OS of 15.7 months and median OS from recurrence of 7.4 months. Comparative analyses of survival endpoints dependent on year of diagnosis did not reveal any statistically significant improvement from 2005 and forth. Factors significantly associated with worse prognosis in univariate and multivariate analyses were for OS, low extent of primary surgery, increased age, multifocal disease, increased PS and O6-methylguanine DNA methyltransferase (MGMT) tumor methylation. For PFS, multifocal disease, increased PS, and MGMT tumor methylation were significantly associated with worse prognosis. Gender, tumor location, tumor size, comorbidity score and site of tumor relapse were not significantly associated with any survival endpoints. Based on study results, a predictive model for survival will be generated able to calculate survival probability for the individual glioblastoma patient. Such a model could prove useful for stratification in clinical scientific protocols and for making glioblastoma treatment more individualized. Updated results will be presented.
EPID-26. SEX-SPECIFIC GLIOMA MOLECULAR SIGNATURES
Authors: Claus Elizabeth , Cannataro Vincent , Gaffney Stephen , Townsend Jeffrey
Published: November 2019

Many genetic and environmental factors have been explored in epidemiological studies of glioma risk but the magnitude of effect of these factors on gliomagenesis is uncertain. A number of constitutional genes associated with glioma risk, many noted to be related to telomere function, have been identified. High-dose ionizing radiation has consistently been associated with an increased glioma risk while a history of allergies and atopy is inversely associated with risk. Results have been inconsistent for most other factors studied but whether this lack of evidence is due to study limitations or a true lack of risk factors is unclear. Using 901 glioma exomes from The Cancer Genome Atlas (TCGA) we estimate the intensity of positive selection on gene variants, and order them by their cancer effect size, quantifying the contribution of gene variants to the somatic evolution of glioma. Examination of subsets of tumor exomes from males and females showed similar patterns in genes mutated but some differences in variant profile for a given gene including PIK3CA suggesting a possible differential response to treatment. Furthermore, these cancer effect sizes enable quantification of the degree to which COSMIC-defined processes associated with genetic pathways and environmental environmental factors within the somatic evolution of tumors contribute mutations that are highly selected, driving gliomagenesis. The results confirm the major roles of previously reported mutations in IDH1, TP53, EGFR, and PIK3CA, but also suggest a large cancer effect for low-prevalence genes including BRAF. Age-related mutagenesis was the primary molecular signature identified for all glioma while homologous recombination disfunction was an important signature for males but not females. Although additional signatures remain to be identified and less common exposures difficult to access, these data suggest a profile of glioma mutations that are caused primarily from endogenous rather than exogenous factors.
EPID-29. THE INFLUENCE OF SOCIOECONOMIC FACTORS AND CANCER CARE RECEIVED AT DIFFERENT FACILITIES ON OUTCOME IN PATIENTS WITH GLIOBLASTOMA
Authors: Li Terry , Patel Palak , Chou Janice , Patel Amie , Crispo Sylvia , Utate Minerva , Kurz Sylvia
Published: November 2019

BACKGROUND

Younger age and higher performance status are strong positive predictors of longer survival in patients with high-grade gliomas. In many cancers, disparities in race, gender, socioeconomic background, and immigration status were shown to influence outcome. Only few studies have investigated the impact of healthcare disparities in patients with glioblastoma, with conflicting results. We hypothesized that patient-specific and healthcare system-related factors influence the utilization of healthcare and impact outcome in glioblastoma patients.

METHODS

This is a retrospective observational study in glioblastoma patients receiving care at a NCI-designated Comprehensive Cancer Center (CCC) or a City Hospital (CH). Patient information including demographics, insurance status, molecular characteristics, treatment details, and survival was retrieved from the medical record.

RESULTS

59 patients were included (CCC n=47, PCH n=12). There were 22 females and 37 males. Median age was 63.0 years at CCC, 54.0 years at CH (p = 0.023). The two groups significantly differed by race (p=0.004), insurance status (p< 0.001), median income by zip code (p=0.015), extent of resection (p=0.001), receiving radiotherapy (p=0.014), and receiving chemotherapy (p< 0.001). Median overall survival (mOS) was longer at CCC (17.9 months) versus at CH (4.4 months) (p=0.021). Univariate Cox regression analyses found significantly better survival for insured vs. uninsured patients (HR 0.035, 95% CI 0.002–0.611, p=0.022) and for married/domestic partnership (M/DP) vs. non-M/DP patients (HR 0.352, 95% CI 0.141–0.879, p=0.025). Care facility, race, income, and treatment received did not emerge as significant predictors. Multivariate analysis controlling for facility and age at diagnosis showed improved survival for M/DP versus non-M/DP patients (HR 0.267, 95% CI 0.103–0.691, p=0.006). CONCLUSION(S): Insurance status and marital status emerged as significant contributors to survival. They may serve as surrogate markers for socioeconomic class and social support. Differences in Cancer Care received at different facilities may influence outcome as well.
EPID-33. GENDER-SPECIFIC PROBABILISTIC ATLASES OF GLIOBLASTOMA REVEAL IMPACT OF TUMOR LOCATION ON PROGRESSION FREE SURVIVAL
Authors: Beig Niha , Ismail Marwa , Saeed Bamashmos Anas , Statsevych Volodymyr , Hill Virginia , Madabhushi Anant , Ahluwalia Manmeet , Tiwari Pallavi
Published: November 2019

BACKGROUND

Recent epidemiological studies suggest that gender differences in Glioblastoma (GBM) influence the prognostic outcome of patients, and thus should be considered for patient management. In this feasibility study, we developed population atlases of pre-treatment MRI lesions to evaluate whether progression free survival (PFS) in gender-specific GBM patients have a spatial proclivity to hemispheric or lobe-specific locations.

METHODS

203 pre-operative MRI sequences (1.5T/3T T1w, T2w, FLAIR scans, multi-center) of GBM (125 males [m] & 78 females [f]) from TCIA (n=130), Ivy-GAP (n=32) and participating institution (n=41), along with PFS (median cut-off =189 days [m], 78 days [f]) were analysed. Frequency atlases (improved PFS [i-PFS] & worse PFS [ω-PFS]) of tumor occurrence in T2/FLAIR hyperintense regions were developed for each gender, by averaging voxel intensities across all patients. To compute significant differences (p-value< 0.05), voxel-based analysis of differential involvement (ADIFFI) based on two-tailed Fisher’s exact test was performed on (a) m: i-PFS (n= 61) vs m: ω-PFS (n= 62), and (b) f: i-PFS (n= 39) vs f: ω-PFS (n= 39) atlases. Prominent clusters were identified and mapped to LONI Probabilistic Brain Atlas (LPBA40) parcellations to provide anatomic localization for each gender’s PFS.

RESULTS

ADIFFI analysis revealed that m: i-PFS tumors were more often localized to the inferior frontal gyrus of left hemisphere with a frequency of 20% whereas m: ω-PFS were more often localized to the right insular cortex (p< 0.005). f: i-PFS tumors were more often localized to the right hemisphere in the parietal and temporal lobe, while f: ω-PFS had 15.7% occurrence in the superior and middle frontal gyri of the right hemisphere (p< 0.03).

CONCLUSION

Our analysis suggests spatial proclivity of gender-specific PFS to hemispheric and lobe-specific locations in the brain. Gender-specific prognostic models and nomograms may be more accurate than unisex models and nomograms for GBM patients.
EXTH-06. DOWN-REGULATION OF PD-L1 VIA FKBP5 LOWERED BY A CYCLOOXYGENASE-2 INHIBITOR IN GSCs AND GBM CELLS MAY BE ATTRIBUTABLE TO ENHANCE ANTITUMOR EFFECTS OF IMMUNOTHERAPY
Authors: Yamaguchi Izumi , Nakajima Kohei , Shono Kenji , Mizobuchi Yoshifumi , Fujihara Toshitaka , Kitazato Keiko , Takagi Yasushi
Published: November 2019

BACKGROUND

Antitumor therapies targeting programmed cell death-1 (PD-1)/its ligand-1 (PD-L1) are influential at present stage. However, in glioblastoma (GBM), the expression of PD-L1 is variable and the role of anti-PD-1 antibody therapy is still unclear. The high expression of PD-L1 affects cell proliferation and invasion in GBM cells. As COX-2 modulates PD-L1 expression in cancer cells, we tested our hypothesis that a COX-2 inhibitor, celecoxib may play a role on anti-PD-1 antibody treatment via down-regulation of PD-L1.

METHODS

Six weeks old male C57BL/6 mice subjected to intracranial injection of mice glioma stem cells (GSCs) were randomly divided into four treatment groups; vehicle control (VC), celecoxib, anti PD-1 antibody or the combination of celecoxib and anti-PD-1 antibody groups and examined antitumor effects. To verify the mechanisms underlying antitumor effects, mice GSCs and human GBM cells were used.

RESULTS

Compared to each single treatment in the glioma model, the combination therapy of anti PD-1 antibody and celecoxib significantly decreased the tumor volume and improved the survival period. Importantly, the high expression of PD-L1 in the glioma model, mice GSCs and human GBM cells was decreased by celecoxib. Interestingly, the reduction of PD-L1 was associated with post-transcriptional regulation of co-chaperone FK506-binding protein 5 (FKBP5) by celecoxib. The combination therapy of anti PD-1 antibody with celecoxib could be a promising therapeutic strategy targeting PD-L1 in GSCs and GBM.

CONCLUSIONS

Down-regulation of PD-L1 via FKBP5 by celecoxib may play a role on the antitumor effects under the overwhelmed expression of PD-L1.
EXTH-10. THE ACTIVATION AND SENSITIZATION OF GLIOBLASTOMA CELLS VIA COLD ATMOSPHERIC PLASMA TREATMENT
Authors: Sherman Jonathan , Yan Dayun , Gjika Eda , Keidar Michael
Published: November 2019

BACKGROUND

Treatment of glioblastoma multiforme (GBM) continues to remain a challenge using conventional treatment. Through an in vitro study we assessed the efficacy of our novel cold atmospheric plasma technology (CAP) to sensitize GBM cells to temozolomide (TMZ).

METHODS

The CAP jet is formed through the discharge (Pk-Pk: 5.8 kV) between a ring grounded cathode and a central anode and with He flow through a glass tube. The discharge process is driven by an AC high voltage (3.16 kV) with a frequency of 12.5 kHz. Human glioblastoma (U87MG) cells were cultured in DMEM supplemented by 1% (v/v) penicillin and streptomycin solution and 10% (v/v) FBS. CAP was delivered to U87 cells in a 96-well plate for 1 min in combination with 10 and 15 μM H2O2. The cell viability was measured by using the MTT assay. We then tested TMZ concentrations of 10 and 50 uM. Cell viability was monitored with the Cell Titer Glo 2.0. luminescent assay. All experiments were performed in triplicate and were independently repeated at least 3 times.

RESULTS

We identified an activation state of U87MG cells after the plasma treatment. This activation state resulted in GBM cells sensitized to reactive species identified by decreased cell viability after treatment with H2O2 as compared to the H2O2 treatment alone (p< 0.005). In addition, the plasma-activated cells were sensitized to TMZ. Cells treated with CAP in combination with TMZ displayed decreased cell viability at TMZ concentrations of (10 uM) (p< 0.05) and (50 uM) (p< 0.005) as compared to TMZ alone.

CONCLUSIONS

This study demonstrates the activation phenomenon on GBM cells via direct CAP treatment. Due to this activation, the GBM cells were sensitized to both H2O2 and TMZ identified via decreased cell viability. Future work looks to assess this effect of cell activation/sensitization with chemotherapy plus radiation treatment.
EXTH-27. ACTIVATING THE IMMUNITY WITHIN THE TUMOR USING VIROIMMUNOTHERAPY: DELTA-24-RGD ONCOLYTIC ADENOVIRUS ARMED WITH THE IMMUNOPOSITIVE REGULATOR GITRL
Authors: Rivera-Molina Yisel , Fueyo Juan , Jiang Hong , Nguyen Teresa , Ho Shin Dong , Youssef Gilbert , Fan Xuejun , Gumin Joy , Alonso Marta M , Phadnis Sheetal , Lang Frederick , Gomez-Manzano Candelaria
Published: November 2019

Based on promising results of recent clinical trials using oncolytic viruses, virotherapy is evolving as an alternative to treat patients with malignant glioma. Our group developed the oncolytic adenovirus Delta-24-RGD (DNX-2401) that is being tested, alone or in combination with anti-PD1, in clinical trials for recurrent glioblastoma (NCT00805376; NCT01956734; NCT02798406). The results suggest that, besides the expected oncolytic effect, the injection of the pathogen initiated, in a subset of patients, an anti-tumoral immunity that led to 20% of long-term survivors (3.5–5 years). To further enhance this effect, we have armed Delta-24-RGD to express the co-stimulatory ligand GITRL, and generated Delta-24-GREAT. The intracranial injection of Delta-24-GREAT prolonged the survival of GL261 glioma-bearing immunocompetent mice when compared to Delta-24-RGD treatment (P=0.002, log-rank test). Delta-24-GREAT treatment resulted in enhanced frequency of tumor-infiltrating lymphocytes: T lymphocytes (CD45+/CD3+) and cytotoxic T lymphocytes (CD45+CD3+CD8+). Functional studies performed by culturing splenocytes from Delta-24-GREAT-treated mice with glioma cells and analyzing secretion of Th1 cytokines, such as IL2 and IFN-γ, showed that lymphocytes recognized not only viral antigens but also tumoral antigens, suggesting the triggering of anti-tumoral immunity. Of interest, Delta-24-GREAT treatment resulted in an antigen-restricted anti-tumor memory effect and in the generation of central immune memory. Thus, rechallenging the survivor mice from the first experiment with a second implantation of glioma cells did not lead to tumor growth, and we detected an increased frequency of central memory CD8+ T cells (CD45+CD62L+). However, survivor mice developed lethal tumors when implanted intracranially with B16/F10 melanoma cells, strongly indicating that the developed immune response was specific for GL261 glioma antigens. This is a novel approach using an oncolytic adenovirus expressing GITRL to target cancer and to stimulate the immunity within the tumor. Our data strongly indicate that this type of strategy may be further developed to treat patients with malignant glioblastoma.
EXTH-46. ALISERTIB POTENTIATES THE EFFECT OF CABOZANTINIB AND VANDETANIB AGAINST GLIOBLASTOMA CELLS
Authors: Williams Brian , Zumbar Cory , Mifsud Caroline , Smith Kendall , Sak Muge , Lehman Norman
Published: November 2019

Glioblastoma is the most common primary malignant brain tumor in adults and has a poor prognosis with current standard of care. Aurora-A (AURKA) is a serine-threonine kinase critical for mitotic progression through its role in centrosome maturation and mitotic spindle assembly. Vascular endothelial growth factor (VEGF) is a regulator of tumor angiogenesis and vasculogenesis. Both AURKA and VEGF are commonly over-expressed in glioblastoma. The AURKA inhibitor alisertib and inhibitors of VEGF have been shown to inhibit glioblastoma cell proliferation in vitro and in vivo, and both have been used in clinical trials for glioblastoma. Here we tested the ability of alisertib to potentiate the effects of the VEGF receptor (VEGFR) inhibitors cabozantinib and vandetanib using colony formation assays in U1242 and U87 glioblastoma cell lines. Chou-Talalay and Bliss independence models demonstrated synergistic growth inhibition in some cases. Annexin V binding assays were performed to assess the extent that apoptosis could account for the inhibitory effects in U87 cells, and a concentration of alisertib that did not cause apoptosis as a single agent was found to increase the induction of apoptosis caused by cabozantinib. These results support the rationale for further in vitro and in vivo studies of the combined use of alisertib and VEGFR inhibitors, including possible future clinical trials.
EXTH-54. MULTIVALENT TARGETED PROTEINS FOR GLIOBLASTOMA TREATMENT
Authors: Sharma Puja , Herpai Denise , Rossmeisl John , Tatter Stephen , Debinski Waldemar
Published: November 2019

A heterogeneous and infiltrating tumor like glioblastoma (GBM) cannot be effectively treated with a single-targeted therapy. Hence, targeting several GBM-associated factors that are responsible for the disease progression and/or recurrence at once is a way to address the issue of tumor heterogeneity. Building on a Phase I clinical trial of a cytotoxic cocktail targeting IL-13-RA2 and EphA2 receptors showing dramatic anti-tumor responses in dogs with spontaneous gliomas, we have developed multivalent single pharmaceutical agents that target four specific to GBM receptors: IL-13RA2, EphA2, EphA3, and EphB2. The combined expression of these receptors spans almost 100% of the tumor microenvironment of pathobiological importance, such as tumor initiating cells, infiltrating cells, neo-vasculature, and GBM-infiltrating cells of monocytic origin like macrophages. Variants of this multivalent protein termed QUAD 2.0 and 3.0 contain an IgG1 scaffold, ephrinA5, which is a ligand for the EphA2, EphA3, and EphB2 receptors, and IL-13.E13K, a mutated version of interleukin 13 (IL-13) binding preferentially to IL-13RA2. In QUAD 3.0, a cysteine was added to the C-terminal end of QUAD 2.0 to allow effective conjugation to cytotoxic agents/labels. All QUAD variants bound effectively to the four receptors using ELISA with KD values of 100–200 pM for EphA2, EphA3, and IL-13RA2, and 700 pM-4 nM for EphB2, and downregulated the EphA2 and EphA3 receptors in GBM cells. QUAD 2.0 and 3.0 were conjugated to a modified form of Pseudomonas Exotoxin A and a modified doxorubicin. The conjugates were highly cytotoxic to established and primary GBM cells with IC50s < 1.0 nM. Intracranial injections (1.3 mM, 5µL) of the conjugates were well tolerated in mice. Thus, targeting multiple receptors with a single multivalent protein is possible. One additional advantage in using these targeted cytotoxins is that they produce immunogenic cell death and can be combined with other approaches activating the immune system.
EXTH-58. INHIBITION OF DNA TOPOISOMERASE 1 AND POLY(ADP-RIBOSE) POLYMERASE SYNERGISTICALLY INDUCES CELL DEATH IN GLIOBLASTOMA WITH PTEN LOSS
Authors: Butler Madison , Su Yu-Ting , Hwang Lee , Marzi Laetitia , Gilbert Mark , Pommier Yves , Wu Jing
Published: November 2019

BACKGROUND

Glioblastoma is known for its aggressive behavior and resistance to most available treatments. Topoisomerase 1 (Top1) functions by relaxing DNA supercoiling to facilitate replication. The novel Top1 inhibitor, LMP400, traps the enzyme and prevents its normal function, particularly in highly proliferative cells, resulting in DNA damage. Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair responses triggered by Top1 inhibition. Phosphatase and tensin homolog (PTEN) loss, a frequent occurrence in glioblastoma, promotes DNA damage repair deficiency. We hypothesize that PTEN loss presents a vulnerability to combined induction of DNA damage and inhibition of repair mechanisms.

METHODS

Human glioblastoma cells were treated with LMP400 and/or Olaparib, a PARP inhibitor. Effects on cell proliferation and cell death were determined using a Beckman Coulter cell viability analyzer and colony formation assays. Synergism was calculated using COMPUSYN software. Cell cycle analysis was performed by FACS with EdU/DAPI staining, and Western blotting of lysate from drug-treated cells was performed to explore mechanisms of cytotoxicity.

RESULTS

LMP400 inhibited cell proliferation with an EC50 of 8.0 nM in U251 cells, and combination of LMP400 and Olaparib had a synergistic cytotoxic effect. Combination treatment enhanced activation of ATM-Chk2 and ATR-Chk1 pathways involved in DNA damage response signaling and increased S phase arrest. Increased protein expression of gamma-H2AX, cleaved caspase 3, and cleaved PARP was observed in combination-treated cells compared to single agents. Cell viability assays using isogenic glioblastoma cell lines with and without PTEN indicate that PTEN loss increases sensitivity to combination treatment.

CONCLUSION

Our results suggest that LMP400 and Olaparib combined treatment synergistically induces glioblastoma cell death, partially through inducing DNA damage, cell cycle arrest, and apoptosis. Mechanistic studies to investigate this vulnerability in glioblastoma with PTEN loss are ongoing. These results may lead to an effective personalized therapy for a molecularly defined subset of glioblastoma patients.
EXTH-61. CELECOXIB IMPROVES OUTCOME OF PATIENTS TREATED WITH TUMOR TREATING FIELDS
Authors: Wong Eric , Xu Oliver , Riley Mercedes , Swanson Kenneth
Published: November 2019

Tumor Treating Fields (TTFields) are known to cause mitotic catastrophe resulting in aberrant mitotic exit. However, there is accumulating evidence that the clinical effectiveness of TTFields requires immune competence. Exposure of cells to TTFields induced signs of cellular stress including cytoskeletal changes, surface expression of calreticulin and secretion of HMGB1 suggesting that TTFields may promote anti-tumor immunity that leads to subsequent tumor regression. Many tumor-protective mechanisms require immunosuppressive cell types within the tumor microenvironment whose development and suppressive functions are supported by prostaglandin E2 (PGE2). The rate limiting enzyme for PGE2 production is Cyclooxygenases-2 (COX-2) whose upregulation has been correlated with poor survival in glioma patients. Therefore, we reasoned that reduction of PGE2 production within the tumor microenvironment might improve response to TTFields therapy. Previous data from our group indicated that dexamethasone use interfered with TTFields-efficacy. When our population having low dexamethasone usage (n=42) was dichotomized according to celecoxib use, those without celecoxib (n = 14) had a median overall survival (OS) of 8.1 months while those receiving celecoxib had a median OS of 12.9 months (chi-square 5.42, p=0.002, HR = 2.1, 95% CI = 1.19–5.59). Patients not receiving celecoxib were treated with TTFields for a median of 3.8 months while those receiving celecoxib were treated for a median of 8.2 months (Chi-square = 6.75, p = 0.01, HR = 2.3, CI = 1.33–6.47). These data suggest that the addition of celecoxib improved the efficacy of TTFields against glioblastoma in this cohort. However, celecoxib did not sensitize TTFields-induced killing of cells in vitro suggesting an indirect mechanism of action. Together with our in vitro data, the results support an immune mechanism of action underlying TTFields’ anti-tumor efficacy. These findings concerning the effects of celecoxib on patient outcomes need to be confirmed in a prospective clinical trial.
EXTH-64. EVALUATION THE COMBINATION EFFECT OF RADIATION AND FOCUSED ULTRASOUND IN TREATMENT OF BRAIN TUMOR MODEL ANIMALS
Authors: Huang Chiungyin , Wei Kuochen
Published: November 2019

The standard treatment of malignant brain tumor is surgical resection followed by adjuvant chemo/radiotherapy. However, in spite of these aggressive treatments, tumor may still recur and resulting in poor survival. In solid tumors, the hypoxia condition presented in most of tumor area may contribute to treatment failure in both adjuvant therapies. It had been proven that systemic delivered oxygen may not sufficient for ionizing radiation to produce cytotoxic effect. A recent study reported using focused ultrasound combined with oxygen-contained microbubbles prior to radiation significantly increased the oxygen content in breast cancers and statistically improved animal survival. Therefore, providing sufficient oxygen in tumor area may be beneficial for radiation therapy. To study the potential role of such strategy for brain tumor treatment, we used focused ultrasound to treat brain tumor bearing animals before radiation therapy. As compared with non-ultrasound treatment groups, the tumor growth rate was significantly inhibited, and the animal survival is prolonged. Furthermore, we found focused ultrasound combined with non-oxygen-containing microbubbles also represented similar results, demonstrate the focused ultrasound induced cerebral vascular permeability increment may also contribute to the radiation therapy effect for brain tumor treatment.
EXTH-65. PLASMA AND CEREBROSPINAL FLUID PHARMACOKINETICS COMPARISON OF BRAF AND MEK INHIBITORS FOLLOWING SINGLE AND EXTENDED ADMINISTRATION IN A PRE-CLINICAL NON-HUMAN PRIMATE MODEL
Authors: Lester McCully Cynthia , Gross Andrea , Cruz Garcia Rafael , Shankarappa Priya , Odabas Arman , Rodgers Louis , Peer Cody , Figg William , Widemann Brigitte , Warren Katherine
Published: November 2019

BACKGROUND

Aberrations in the MAPK signaling pathway are present in adult and pediatric CNS tumors. The BRAF and MEK inhibitors Dabrafenib, Vemurafenib, Selumetinib and Trametinib, alone or in combination, have demonstrated clinical efficacy. However, minimal CSF penetration has been demonstrated in pre-clinical models with single dose administration. CSF penetration of these agents was compared following single and extended oral administration in a nonhuman CSF Lateral Reservoir model to determine if CSF penetration increases at steady state.

METHODS

Agents were administered orally, to each subject (n=9), in single or multiple doses, as serial single agent studies, with washout periods between studies. The following dosages (Human equivalent dose HED) were utilized in the studies: Single Dosage-Selumetinib 2.5 mg/kg (50.0 mg/m2), Dabrafenib 8.0 mg/kg (160 mg/m2), Vemurafenib 26 mg/kg (520 mg/m2), Trametinib 0.06 and 0.12 mg/kg (1.2 and 2.4 mg/m2). Extended dosages-Selumetinib 2.5 mg/kg BID x 3 days (50.0 mg/m2), Dabrafenib 4.0 mg/kg BID x 2 days (160 mg/m2/day), Vemurafenib 26 mg/kg BID x 3 days (520 mg/m2), Trametinib 0.05 mg/kg x 10 days BID (1.0mg/m2). Paired serial plasma and CSF samples were collected. Agents were quantified by uHPLC-MS/MS. Pharmacokinetic parameters were calculated via noncompartmental methods. Plasma exposure reported as Area Under the Curve (AUC) and CSF penetration as ratio of CSF:plasma AUC. Statistical significance determined by t-test.

RESULTS

Single administration AUCinf/D (hr*ng/ml/mg) vs. extended administration AUCtau/D (hr*ng/ml/mg) for each agent: Vemurafenib-Plasma 30.9–351.4 vs.29.2–379.7, CSF-non-quantifiable; Dabrafenib-Plasma 29.3–102.6 vs.3.4–58.2, CSF-0.12–0.54 vs 0.04–0.19, % CSF:Plasma-0.3–0.84 vs.0.32–1.3; Trametinib-Plasma AUC indeterminable, CSF-non-quantifiable; Selumetinib-Plasma 69.8–133.0 vs.26.5–81.2; CSF-AUC indeterminable vs. 0.37–0.86, % CSF:Plasma-Indeterminable vs. 0.78–1.4. T-test: A downward trend in Dabrafenib and Selumetinib plasma AUC and a marked upward trend in Selumetinib CSF AUC.

CONCLUSIONS

With the exception of Selumetinib, the CSF exposure of BRAF and MEK inhibitors studied did not markedly increase following extended dosing.
EXTH-67. IDENTIFICATION AND DEVELOPMENT OF NEURAL ECM-BINDING LAMPREY ANTIBODIES (VARIABLE LYMPHOCYTE RECEPTORS) THAT TARGET GLIOBLASTOMA
Authors: Umlauf Benjamin , Clark Paul , Lajoie Jason , Georgieva Julia , Bremner Samantha , Herrin Brantley , Shusta Eric , Kuo John
Published: November 2019

INTRODUCTION

The median survival of glioblastoma (GBM) patients remains less than two years even with state-of-the-art treatment. Current targeted GBM therapies demonstrate initial therapeutic benefit; however, patients relapse due to therapeutic selection of treatment resistant GBM cellular populations. Therefore, we propose targeting pathologic disruption of the blood brain barrier (BBB) via exposure of neural ECM, rather than disease markers, to overcome therapy-resistant GBM.

METHODS

We identify Variable Lymphocyte Receptors (VLRs, the antigen recognition system used by lamprey) that demonstrate high specificity for neural ECM. Candidate VLRs underwent further refinement using in vitro binding assays and ex vivo tissue staining. Utilizing pathologic disruption of BBB as an approach for targeting GBM was confirmed in vivo using intracranial murine glioblastoma models.

RESULTS

The lead neural ECM-binding VLR candidate, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to other tissues. P1C10 demonstrates nanomolar affinity for in vitro derived neural ECM, and preferentially accumulates at intracranial GL261 and U87 lesions in murine GBM models. Finally, administration of P1C10-targeted doxorubicin-loaded liposomes significantly extends the survival of mice bearing intracranial U87 GBM.

CONCLUSIONS

We identified VLRs that bind neural ECM, and demonstrate their utility for delivering compounds and nanoparticles to sites of GBM induced blood brain barrier disruption. This novel strategy allows for targeting therapeutics via the underlying physiology of GBM rather than relying on cellular disease markers that are often lost in patients that relapse after targeting therapies.
GENE-14. BMP4 INDUCES TO ASYMMETRIC CELL DIVISION IN HUMAN GLIOMA STEM-LIKE CELLS
Authors: Koguch Motofumi , Izumi Hideki , Nakahara Yukiko , Ito Hiroshi , Wakamiya Tomihiro , Yoshioka Fumitaka , Ogata Atsushi , Inoue Inoue Kohei , Masuoka Jun , Abe Tatsuya
Published: November 2019

Glioblastoma (GBM) is one of the human malignant tumors with a high recurrence rate and the poorest prognosis. Therefore, a new treatment strategy is required. It has also come to be thought that cancer is a heterogeneous, which composes of cancer stem cells (CSCs), which have self-renewal ability, multipotency and treatment resistance. Although various strategies targeting glioma stem-like cells (GSCs) have been studied, a median survival remains to be less than 2 years. In this study, we focused on strategies targeting GSCs through induction of differentiation using BMP4. We examined the expression of CD133, a cancer stem cell marker, under BMP4-treatment in GSCs using flowcytometry analysis, western blotting and qPCR. We also examined immunofluorescent staining of GSCs to study cell division. The treatment of BMP4 caused downregulation of CD133 expression in GSCs. In addition, BMP4-treatment induced to asymmetric cell division in GSCs. Tumor sphere assay showed that BMP4 suppresses self-renewal ability. These findings may provide a new perspective how BMP4 reduces the tumorigenicity of GSCs.
GENE-16. NEXT GENERATION SEQUENCING IDENTIFIES IDH-1, ATRX, TP53 MUTATION AND MYCN AMPLIFICATION IN GLIOBLASTOMA WITH EXTENSIVE SUBARACHNOID SPREAD: A CASE REPORT
Authors: Sang Lee Kyu , Choe Gheeyoung
Published: November 2019

Glioblastoma showing primarily extensive subarachnoid spreading has much worse prognosis than intracortical glioblastoma. Nevertheless, their genetic pathogenesis is still unknown due to its low incidence. Present report is a rare case of glioblastoma with primitive neuroectodermal tumor (PNET)-like feature, showing extensive subarachnoid spread, as well as the distinguishing result of its molecular analysis using next generation sequencing (NGS). A 37-year-old male presented with a new onset, severe headache. Magnetic resonance imaging test revealed a diffuse infiltrative mass in the right frontal lobe. The tumor was also spread broadly in subarachnoid space. The patient underwent craniotomy and tumorectomy. Microscopically, glioblastoma showed PNET-like feature. Tumor cells had high nuclear/cytoplasmic ratios, oval, slightly elongated or angulated hyperchromatic nuclei, markedly increased mitotic activity, and obvious apoptotic bodies. Immunohistochemical test showed partly positivity of GFAP, CD56 and synaptophysin. For comprehensive genomic profiling, we assessed NGS based on the SNUBH Brain v1.0 (Macrogen, Seoul, South Korea) panel. This panel is designed to include all genes known to be somatically altered in human brain tumor. IDH-1 (p.Arg132His) mutation and two frame shift genetic alteration including ATRX (p.Tyr2208fs) and TP53 (p.Pro301fs) were found. Additionally, MYCN (2p24.3) amplification was identified. Estimated MYCN copy number was 18. Despite the post-operative therapy including temozolomide and radiotherapy, the tumor aggressively progressed and the patient expired nine months after the first symptom onset. Although there have been several previous reports of MYCN amplification in glioblastoma with PNET-like feature, this is the first case report which finding in glioblastoma, IDH-mutant type. IDH-1 mutation and younger age are usually associated with a better prognosis in glioblastoma patients. However, our case may suggest that when MYCN amplification coexists in IDH-mutant type, the tumor behavior could be contrarily aggressive.