bluuebunny/crossref_metadata_2025 huggingface dataset:
SCIDOT-27. TGFβ SIGNALING-INDUCED miRNA PARTICIPATES IN AUTOPHAGIC REGULATION BY TARGETING PRAS40 IN MESENCHYMAL SUBTYPE OF GLIOBLASTOMA
Authors: Xie Yingbin , Chen Luyue , Zhou Junhu , Wang Qixue , Wang Yunfei , Tan Yanli , Kang Chunsheng , Fang Chuan
Published: November 2019

Mesenchymal subtype of glioblastoma (mesGBM) is a refractory disease condition characterized by therapeutic failure and tumor recurrence, and for the past decade, no treatment option has improved the dismal prognosis of this disease. In Cancer Genome Atlas (TCGA) GBM datasets, two of the 840 transcriptomic signature genes for GBM subtyping, TGFβ1 and TGFβ receptor type II (TβRII), were exclusively upregulated in mesGBM, indicating hyperactive TGFβ signaling. In the present study, dysregulated microRNAs (miRNAs) were identified after LY2109761 (a TβRI/II inhibitor) treatment in a mesGBM-derived cell line. Importantly, several novel miRNAs that may be missing pieces of the puzzle of TGFβ signaling-mediated pathogenesis in mesGBM were identified. Novel_miR26/56/93/97/119 were validated by quantitative PCR to correlate with TGFβ signaling activity, and novel_miR56 was selected as a promising candidate for further functional verification. A proline-rich akt substrate of 40 kDa (PRAS40), which regulates autophagy initiation by targeting mTORC1, was confirmed to be a functional target of novel_miR56. Autophagy plays dichotomous roles in the determination of GBM cell fate, and in this study, TGFβ signaling-induced novel_miR56 seemed to inhibit autophagic activity in GBM and its mesenchymal subtype, resulting in the maintenance of proliferative capability. The pro-survival effect of novel_miR56 can be impeded by intentionally downregulating its expression both in vivo and in vitro. In summary, we provide novel insight into TGFβ signaling-mediated pathogenesis and targets for the development of novel therapeutic interventions for mesGBM.
SCIDOT-43. FINAL DATA ANALYSIS OF A PILOT STUDY TESTING THE EFFICACY OF USING LASER INTERSTITIAL THERMAL THERAPY (LITT) TO INDUCE TEMPORARY DISRUPTION OF THE PERITUMORAL BLOOD BRAIN BARRIER (BBB) TO IMPROVE EFFECTIVENESS OF BBB-IMPERMEANT CHEMOTHERAPY IN RECURRENT GLIOBLASTOMA
Authors: Tran David , Campian Jian , Shimony Joshua , Kim Albert , Ansstas George , Leuthardt Eric
Published: November 2019

BACKGROUND

Poor CNS drug delivery is a major limiting factor in GBM therapy. Since most recurrences occur peritumorally where infiltrating glioma cells reside, peritumoral BBB disruption may help improve drug delivery and efficacy. LITT, a minimally invasive cytoreductive treatment for brain tumors, may disrupt BBB as evidenced by post-ablation contrast enhancement.

METHODS

Patients with recurrent GBM were treated with LITT. The degree of post-LITT peritumoral BBB disruption was measured by the vascular transfer constant (Ktrans) in brain DCE-MRI. Serum levels of brain-specific enolase (BSE) were quantified as an independent measure of BBB disruption. Forty enrollments were planned with equal randomization to weekly low-dose doxorubicin, a BBB-impermeant chemotherapy, either during the BBB-permeable window (Early Dox) or after the resolution of disruption (Late Dox). The primary endpoint is 6-month PFS rate (6-PFS).

RESULTS

Peritumoral Ktrans peaked immediately post LITT, followed by a gradual decline for the next 4 weeks. Similarly, serum BSE concentrations peaked in 1–3 weeks after LITT before decreasing to baseline by 6 weeks. 41 patients were randomized to Early and Late Dox. 31 patients (14 Early and 17 Late Dox) were evaluable for the primary endpoint. MGMT promoter methylation rate was 28% and 47% in the Early and Late Dox arms, respectively. 6-PFS was 57.1% (8/14) in Early Dox compared to 35.3% (6/17) in Late Dox. PFS was 5.8 (95%CI: 4.1–6.3) months vs. 4.9 (95%CI: 2.4–6.3) months, while OS was 12.8 (95%CI: 7.8–14.5) months vs. 13.6 (95%CI: 10.2–17.1) months, for Early vs. Late Dox, respectively. The most common grade 3–4 toxicities were cytopenia observed in 6 (16%) patients.

CONCLUSIONS

LITT induces temporary peritumoral BBB disruption lasting 4–6 weeks, providing a rare opportunity to enhance local delivery of potentially efficacious but BBB-impermeant therapeutic agents. 6-PFS strongly favors dox given early during the window of BBB disruption, thus supporting the hypothesis.
RARE-46. MULTIMODAL TREATMENT OF TWO PATIENTS WITH DISSEMINATED JUVENILE PILOCYTIC ASTROCYTOMA
Authors: Gass David , Wait Scott , Jacobsen Chad
Published: November 2019

Juvenile Pilocytic Astrocytoma (JPA) is characterized by an indolent course with excellent survival if gross total resection is achieved. However, a rare subset of these low-grade neoplasms will disseminate. We present two patients with disseminated JPA and review the multimodal treatment approach required to manage this disease. CLINICAL IMPORTANCE: Disseminated JPA is a low-grade tumor with a chronic relapsing course. Radiation has previously been successful, however, the significant treatment volume needed for disease control is toxic in young children. Approaches using repeated surgical resections and multiple chemotherapy regiments to postpone time to radiation is becoming a standard approach. CLINICAL OUTCOME: Our first patient is a 7-year-old girl who had four progressions in her primary third ventricular mass in the last 2 years. Her disseminated disease has been stable throughout treatment. Initially, she underwent subtotal resection of third ventricular mass, which showed JPA, with negative BRAF v600e and negative BRAF fusion. After observation, she had her first progression and was treated with vincristine and carboplatin. Three months later she had her second progression and then treated with six cycles of selumetinib. Next, she received weekly vinblastine prior to her fourth progression. She then underwent gross total resection of her primary mass and has disease stability for five months. Our second patient is a 13-year-old male who had two progressions in last seven years. After initial subtotal resection, he had local progression and had a second resection of cerebellar mass. After four years of stability, the mass disseminated to his thoracolumbar spine. He underwent resection of spinal tumor which showed positive BRAF fusion and then treated with carboplatin and vincristine. He is showing mild response to treatment after 6 months of chemotherapy. CONCLUSION: We demonstrate that with using multimodal treatment with surgery and chemotherapy regiments, radiation for young children can be postponed.
ANGI-13. PLEXIN-B2 FACILITATES DIFFUSE GLIOMA INVASION BY REGULATING CELL ADHESION AND ACTO-MYOSIN DYNAMICS
Authors: Huang Yong , Tejero Rut , Lee Vivian , Junqueira Alves Chrystian , Foty Ramsey , Dai Guohao , Zou Hongyan , Friedel Roland
Published: November 2019

Diffuse invasion of glioblastoma (GBM) cells into brain tissue is a key factor for its high lethality. GBM cell migration is affected by functions of plexins, which are transmembrane receptors of semaphorins that regulate cell adhesion and cytoskeletal dynamics. Expression of Plexin-B2 is upregulated in GBM and correlates with malignancy. We show here that Plexin-B2 activity regulates biomechanical properties of GBM cells, promoting invasive growth. Plexin-B2 activity increased the capacity of GBM to invade as dispersed single cells by reducing the cell-cell adhesiveness between GBM cells, indicating that a major function of Plexin-B2 activity is to downregulate cell-cell adhesion systems. RNA-Seq analyses also revealed that GBM stem cells (GSC) with deletion of Plexin-B2 altered expression of genes related to cell adhesion and the matrisome, indicating compensatory mechanisms in cellular dynamics. Interestingly, in vivo intracranial transplant studies demonstrated that growth and invasion of Plexin-B2 mutant GSC was impaired, with mutant cells invading shorter distances and migrating mainly as groups of cells forming chains. Plexin-B2 mutant cells also were more likely to adhere to the vasculature, rather than to fiber tracts, suggesting altered biomechanical properties. This shift may be related to high stiffness of basal lamina of the vasculature, as Plexin-B2 KO cells have a preference for migration on stiff substrate in vitro. Intriguingly, the loss in Plexin-B2 expression also changed the distribution of the mechanosensor transction factor YAP, with high expression of Plexin-B2 correlating with increased nuclear YAP. Structure-function analyses revealed that the Ras-GAP domain as main signaling output of Plexin-B2. The Rap proteins are pleiotropic regulators of cell adhesion and actomysosin contractility. Our data also showed that overexpression of Plexin-B2 can lead to decreased levels of Rap1/Rap2. Thus, Plexin-B2 acts as a key regulator of the adhesion and contractility of GBM cells, thereby facilitating their diffuse invasion.
CBMT-06. HIGH LEVELS OF GATA-4 IN MALIGNANT MENINGIOMA LEAD TO LOWER miR-497~195 CLUSTER EXPRESSION IN TISSUE AND BLOOD
Authors: Negroni Catarina , Ercolano Emanuela , Adams Claire , Baiz Daniele , Kurian Kathreena M , Hilton David , Oliver Hanemann Clemens
Published: November 2019

Meningioma is the most common primary intracranial brain tumour, classified as benign (WHO I, 70%), atypical (WHO II, 30%) and anaplastic/malignant (WHO III, 1–3%). The 3-year recurrence rate for WHO I tumours is 40% and it is much higher in WHO II-III1. To date, meningioma classification is based only on histopathological characterization, and no circulating biomarkers have been identified to predict tumour progression2. Indeed, microRNAs are promising circulating biomarkers because they can be released from tumour cells into the blood stream via exosomes, showing potential to be used as liquid biopsies3. Previously, Cyclin D1 overexpression was suggested as a possible meningioma biomarker as it correlates with proliferative activity and tumour grade, while high levels of Cyclin E1 were shown to correlate with higher recurrence rates4,5. In this study, we showed, for the first time, that the transcription factor GATA-4 is overexpressed in malignant meningioma cells and tissues, controlling the miR-15 family (miR-15a/b-16-497-195) expression. Moreover, we proved that the miR-497~195 cluster not only regulates Cyclin D1-D2-D3 and E1 levels, but it also modulates GATA-4 expression in meningioma. Moreover, we showed that the levels of miR-497~195 cluster are significantly lower in exosomes derived from higher grade meningioma patients’ blood serum, supporting their potential as diagnostic biomarkers.
CBMT-11. HIGH MITOCHONDRIAL DNA COPY NUMBER IS ASSOCIATED WITH LONGER SURVIVAL IN YOUNG PATIENTS WITH GLIOBLASTOMA
Authors: Rousseau Audrey , Dardaud Laure-Marie , Bris Céline , Desquiret-Dumas Valérie , Boisselier Blandine , Tabouret Emeline , Mokhtari Karima , Figarella-Branger Dominique , Procaccio Vincent
Published: November 2019

Young patients (< 40 years-old) with glioblastoma (GB) may carry IDH1/2 mutations or rarely, H3F3A/HIST1H3B mutations. Some instead harbor chromosome (chr) 7 gain, chr 10 loss, and EGFR amplification (amp). IDH1/2 mutations are associated with longer survival in contrast to H3F3A/HIST1H3B mutations. Novel reliable prognostic factors are needed in GB. Mitochondria (mt) are responsible for ATP production through oxidative phosphorylation. High mtDNA copy number (CN) has been associated with variable outcomes in cancer. The mtDNA-CN was assessed by RT Q-PCR in 66 GB from patients 18 to 40 years-old. Whole mitochondrial genome sequencing was performed in 18/66 GB. 17/66 (25.8%) cases harbored IDH1-R132H mutation; 29/66 (44%) had chr 7 gain/chr 10 loss and/or EGFRamp; 4/66 (6%) harbored H3F3A/HIST1H3B mutations. The remaining cases (24.2%) had no key genetic alterations. The mtDNA/nuclearDNA ratio was subdivided into groups: Low and High according to the median (mtDNA-CN median 194.9). 33 patients displayed a low ratio (≤ median) whereas 33 displayed a high ratio (> median). The overall survival in the High vs Low group was significantly longer in the whole cohort (Log-rank test, 31.8 vs 14.2 months, p= 0.019) and the chr7+/chr10-/EGFRamp subgroup (37.8 vs 12.1 months; p= 0.0117). There was still a trend towards longer survival in the High vs Low group within the other genetic subgroups (IDH-mutant; no genetic alteration) except for the H3-mutant GB (4 patients). Analyses on 232 patients 18 to 84 years-old showed that among patients < 56 years-old, the High group (58 cases) had a longer survival compared to the Low group (59 cases) (27.3 vs 15 months; p= 0.023). High mtDNA level is linked to oxidative metabolism (which decreases tumor aggressiveness) and cell differentiation. Increasing mtDNA-CN might induce tumor cell differentiation and slow disease progression. In conclusion, mtDNA-CN may be a novel prognostic biomarker in GB.
CBMT-18. THE ROLE OF BIOMARKER CANDIDATE GELSOLIN AND ITS MICRORNAS IN GLIOBLASTOMA
Authors: Nakada Mitsutoshi , Zhang Jiakang , Furuta Taskuya , Jiapaer Shabierjiang , Tamai Sho , Sabit Hemragul , Uchida Yasuo , Ohtsuki Sumio , Terasaki Tetsuya
Published: November 2019

BACKGROUND

Among potential glioblastoma (GBM) blood biomarkers that we identified recently, we focused on gelsolin (GSN), a key regulator of actin filament disassembly. GSN was significantly lower in the blood of patients with GBM than in that of healthy controls. In this study, we analyzed the function of GSN and identified microRNAs (miRs) involved in GSN expression in GBM.

METHODS

QRT-PCR and western blot were introduced to evaluate the expression level of GSN in normal brain and GBM tissue. The localization of GSN was examined by immunohistochemistry and immunocytochemistry using human samples. The association between the expression level of GSN and progression free survival (PFS) /overall survival (OS) in GBM was assessed by Kaplan-Meier analysis. The function of GSN and its signal transduction in glioma cell lines were analyzed using small interfering RNA (siRNA) knockdown of GSN. Additionally, miRs controlling GSN expression were retrieved from databases of miRs, and miRs related to GSN expression were identified in GBM tissues.

RESULTS

The expression level of GSN was significantly lower in GBM tissues compared to normal brains. Normal astrocytes mainly expressed GSN. High expressor of GSN showed longer PFS and OS than low expressor. Proliferation and invasion in glioma cell lines were significantly promoted by siRNA for GSN accompanied with the activation of glycogen synthase kinase 3β. In GBM tissues, the expression levels of GSN and miR-654-5p, 450b-5p showed an inverse correlation. The inhibitor for miR-654-5p and miR-450b-5p accelerated GSN expression resulting reduction of proliferation.

CONCLUSION

GSN plays a role as suppressor of proliferation and invasion in GBM. miR-654-5p and miR-450b-5p which control GSN expression can be targets against GBM.
CBMT-20. SINGLE-CELL TRANSCRIPTOMICS RESOLVES CELLULAR HETEROGENEITY AND ONCOGENIC NETWORKS DURING MALIGNANT TRANSFORMATION OF PERIPHERAL NERVE SHEATH TUMORS
Authors: Man Natalie Wu Lai , Zhang Feng , Tian Weidong , Lu Richard
Published: November 2019

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann cell (SC)-derived sarcomas with strong metastatic proclivity and resistance to radiation and chemotherapy. Molecular events driving SC-to-MPNST transformation are incompletely understood. MPNST are heterogeneous tumors with a distinct microenvironment that contributes to the establishment of a tumor niche. Cancer behaviors including initiation, progression, metastasis and relapse cannot be fully defined by genetic mutations alone, but are critically dependent on intercellular communication between tumor cells and the microenvironment. However, currently, a comprehensive understanding of tumor cell lineage evolution, heterogeneity and cell-cell communication network in MPNST is lacking. A better understanding of cellular heterogeneity and tumor-initiating cells during tumorigenesis at a single-cell level will facilitate identifying molecular and cellular determinants underlying malignant transformation in MPNST. We have established a murine model of aggressive peripheral nerve malignancy with HIPPO-TAZ/YAP activation (Lats1/2-def mice), which provides a unique opportunity for dissecting mechanisms of SC malignant transformation. To identify potential PNST-driven progenitor populations, we compare single-cell transcriptome profiles of Lats1/2-def sciatic nerve tumors during pre-neoplastic and advanced stages. Our data reveal that early sciatic nerve tumors contain primarily mature SCs, whereas full-blown tumors harbor neoplastic SCs composed of different subsets: fast-proliferating, mesenchymal-like and neural crest stem cell-like cells, indicative of a developmental trajectory from differentiated SCs to tumor-like SCs with distinct molecular and transcriptional signatures. Interestingly, transformed SCs exhibit inflammation gene signature, which may suggest cell-autonomous inflammation-driven reprogramming that drives tumorigenesis. To characterize cellular heterogeneity in MPNST-like tumors, we further identify cell-cell communication networks between SC-derived tumor cells and cancer-associated stromal cells within the tumor microenvironment. In summary, our studies using single-cell transcriptomics in clinically relevant mouse models of PNST reveal tumor cellular heterogeneity and the microenvironment niche for driving SC tumorigenesis. It will provide principles for identifying potential targets or treatment strategies to cure MPNST.
CBMT-36. INITIAL EXPERIENCE WITH HYPERPOLARIZED [2-13C]PYRUVATE MR IMAGING IN PATIENTS WITH IDH-MUTANT GLIOMA
Authors: Autry Adam , Chung Brian , Chen Hsin-Yu , Gordon Jeremy , Lafontaine Marisa , Chang Susan , Villanueva-Meyer Javier , Larson Peder , Vigneron Daniel , Li Yan
Published: November 2019

INTRODUCTION

Hyperpolarized (HP) carbon-13 MR imaging allows for real-time non-invasive measurement of metabolism. Using HP [2-13C]pyruvate as a molecular probe enables the measurement of both aerobic glycolysis ([2-13C]lactate) and oxidative metabolism (such as [5-13C]glutamate and [5-13C]glutamine). This study presents the initial patient experience of acquiring HP [2-13C]pyruvate MR spectroscopic data.

METHODS

Two patients who presented with IDH-mutant gliomas (43 year-old male, grade II astrocytoma, RT/TMZ; 33 year-old female, grade III oligodendroglioma, no RT/TMZ) considered radiologically stable post resection were imaged using a 2D chemical shift imaging (CSI) sequence (15.6–18.8cm3 spatial resolution, 20s acquisition), following injection of 0.43mL/kg of 250mM HP [2-13C]pyruvate. HP [2-13C]pyruvate, [5-13C]glutamate, [5-13C]glutamine, and [2-13C]lactate resonances were quantified within a lesion voxel and contralateral normal-appearing voxel. The proton images were aligned to HP data enabled quantification of apparent diffusion coefficients (ADC), perfusion peak height (PH), and MR spectroscopy choline-to-N-acetyl-aspartate indices (CNI). ADC (nADC) and PH (nPH) values were normalized by the median value in normal-appearing white matter.

RESULTS

Proton exams revealed no Gd-enhancement lesions. For the T2-hyperintense lesion (T2L), volumeT2L=18.4/4.2cc (Patient#1/Patient#2); median ADCT2L =1.86/1.64; max CNIT2L=3.2/6.5; 90th percentile nPHT2L =1.09/2.11. HP data displayed maximum signal-to-noise ratios (SNR) for [2-13C]pyruvate, [5-13C]glutamate, [5-13C]glutamine, and [2-13C]lactate of 1360, 44, 27, and 400, respectively. The HP CSI data contained 15.9/13.7 %T2L; 15.1/4.1 %NAWM; 15.7/22.0 %GM (Patient#1/Patient#2) in selected lesion voxels, while contralateral voxels had %NAWM:%GM of Lesions demonstrated a reduced HP glutamate/pyruvate ratio [#1: 0.0089/0.0102 (lesion/normal voxel); #2: 0.0170/0.0269] and an elevated HP glutamate-to-glutamine ratio in both patients [#1: 0.81/0.66; #2: 0.84/0.66].

DISCUSSION

This first patient experience with HP [2-13C]pyruvate showed adequate SNR for quantifying HP [2-13C]pyruvate, [5-13C]glutamate, [5-13C]glutamine, and [2-13C]lactate resonances. Future studies will optimize imaging protocols and expand the patient cohort to evaluate aberrant metabolism related to IDH-mutant tumor.
CBMT-40. THE RELATIONSHIP BETWEEN GLIOMA AND THE GUT-BRAIN AXIS
Authors: Patrizz Anthony , Zorofchain Soheil , Hines Gabriella , Takayasu Takeshi , Otani Yoshihiro , Swanner Jessica , Honarpisheh Pedram , Tandon Nitin , Putluri Vasanta , Putluri Nagireddy , Priya Ganesh Bhanu , Kaur Balveen , McCullough Louise , Ballester Leomar Y , Esquenazi Yoshua
Published: November 2019

Recent studies demonstrate the potential role of the microbiome in immune-oncology, revealing specific microbial taxa can augment the effects of various therapeutic modalities against tumors. Gut dysbiosis, a disequilibrium in the host’s bacterial ecosystem, can potentially lead to overrepresentation of some bacteria and favor chronic inflammation and immunosuppression. However, the effects of microbial dysbiosis on non-gastrointestinal cancers in particular gliomas are unknown. Here, we explored the effects of glioma and Temozolomide (TMZ) on the fecal microbiome (FM) in mice (n=24) and FM and metabolome in humans (n=40). Aged C57/B6 mice were implanted with Gl261 tumor cells or vehicle and were assigned to one of the following treatment (oral) groups: vehicle, 5mg/kg TMZ or 25mg/kg TMZ beginning 14 days after surgery for 3-weeks following a 5 day on/2 day off treatment. Fecal samples were collected prior to surgery, at treatment initiation and weekly thereafter until sacrifice and sequenced for 16s RNA. Fecal samples were collected from humans with newly diagnosed glioma before resection, chemoradiation, and after chemoradiation (16s RNA, metabolomic, neurotransmitter analysis). In mice, FM beta diversity was significantly altered with glioma (p=0.003) while the alpha diversity remained unchanged. At a genus and family level analysis the relative abundance of Bacteroides (p=0.01) and Bacteroidaceae (p=0.02) was increased. Beta diversity of mice receiving 5mg/kg TMZ changed from baseline (p=0.02). Collectively, this suggests that glioma alters the FM, to what consequence remains to be explored. Alpha (Observed OTUs, p=0.029) and beta diversity (p=0.034) differences in mice correlated with survival (< 25 - >25 days). In humans, norepinephrine and 5-hydroxyindoleacetic acid were significantly lower in glioma patients at diagnosis compared to controls. Our findings demonstrate for the first time the relationship between glioma and the gut-brain axis. Understanding alterations in the FM in glioma patients may allow novel interventions and should be further investigated.
CBMT-48. TARGETING METABOLIC REPROGRAMMING WITH NANOCURCUMIN IN GLIOBLASTOMA MULTIFORME
Authors: Suhail Hamid , Ramandeep Rattan , Shailendra Giri , deCarvalho Ana , Kalkanis Steven , Ali Meser
Published: November 2019

Glioblastoma (GBM) is a highly glycolytic aggressive brain tumor characterized by increased proliferation and resistance to chemotherapy and radiotherapy. AMPK has been reported as tumor suppressor and reprograms the cellular metabolic pathways and produces a metabolic checkpoint on the cell cycle though mTORC1, p53 and other modulators involved in cell proliferation, growth, survival and autophagy. The AMPK activity is diminished in gastric, breast and ovarian tumor cells by activated PI3K-AKT pathways. Cancer cells are able to reprogram their energy metabolism to compensate their high bioenergetic demands needed for their aggressive growth and survival. Curcumin exhibits pleiotropic properties and activate MAPK and leads to suppress p53, Wnt/β-catenin, SHH and PI3K-AKT signaling pathways. Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). The absorption, biodistribution, metabolism, and elimination studies of curcumin have, unfortunately, shown only poor absorption, rapid metabolism, and elimination of curcumin as major reasons for poor bioavailability of this interesting polyphenolic compound. We have engineered a curcumin-based nanoparticle (Curc-NP) which demonstrates high water solubility. Curc-NP was effectively transported into the cells by nanoparticles through endocytosis and localized around the nuclei in the cytoplasms. In vitro studies proved that the cytotoxicity of Curc-NP is more effective against U-251 cell line in a dose-dependent manner. Systemic delivery of Curc-NP led to preferentially accumulation in an orthotopic preclinical glioma model minimizing systemic toxic effect. Multicolor microscopy images of the tumor tissue showed that Curc-NP particles were internalized inside tumor cells selectively and localized within nuclei. Curc-NP demonstrated to restore the dysregulated AMPK activity in glioma cells. Curc-NP-induced AMPK activation resulted in inhibition of oncogenic signalling pathways in glioma. Curc-NP-induced metabolic reprograming in glioma cells will be examined and the in vivo therapeutic efficacy of Curc-NP in an experimental rat model of GBM will also be evaluated.
CSIG-07. TARGETING CELL POLARITY PROTEINS FROM THE SCRIBBLE COMPLEX DISRUPTS GLIOBLASTOMA STEM CELL VIABILITY AND INVASION IN NEURAL TISSUE
Authors: Kundu Somanath , Nandhu Mohan , Longo Sharon , Longo John , Richardson Timothy , Chin Lawrence , Viapiano Mariano
Published: November 2019

Despite their molecular heterogeneity, glioblastomas (GBMs) retain a common phenotypical feature, which is their diffuse invasion through neural tissue that makes complete resection impossible. This invasive ability requires changes in cell polarity for the tumor cells to migrate following cues from their microenvironment. Cytoskeleton-associated proteins of the DLG family are part of the Scribble polarity complex that maintains stable cell-cell junctions; accordingly, these proteins are “tumor suppressors” usually downregulated in solid tumors. However, we have found that one family member, DLG5, is highly upregulated in GBM compared to other solid tumors. Results from Western blot, immunohistochemistry, and RNAseq confirmed that DLG5 is highly expressed in GBM compared to normal brain and is the predominant DLG family member in GBM stem cells (GSCs). DLG5 knockdown reduced cell viability after four days in culture, but had an immediate (24h) negative effect on cell invasion through Matrigel and brain tissue. At the molecular level DLG5 knockdown downregulated components of the Sonic-Hedgehog pathway, including PTCH1 and Gli1, as well as SHH-regulated downstream genes (CCND2, cMYC and CD44). Accordingly, DLG5-deficient GSCs showed reduced Gli1 phosphorylation in response to exogenous SHH and lacked increased invasiveness in presence of SHH compared to control GSCs. Ongoing experiments are evaluating the growth and invasion of DLG5-deficient GSCs in response to signals derived from oligodendrocyte progenitor cells, which are a major source of SHH in the postnatal brain. Taken together, our results describe for the first time the expression of DLG proteins in malignant gliomas and reveal an unexpected pro-tumoral function for the protein DLG5 that differs from its proposed tumor-suppressor role in other solid cancers. Targeting DLG5 could disrupt the ability of GSCs to respond to signals enriched in the neural microenvironment (SHH), contributing to the long-term control of residual tumor after surgical resection.
CSIG-08. EXTRACELLULAR DOMAIN MUTATIONS IN EGFR RESULT IN A HYPERSENSITIVE RECEPTOR
Authors: French Pim , de Wit Maurice , de Heer Iris , van Royen Martin , Vallentgoed Wies , Hoogstrate youri , Sillevis Smitt Peter
Published: November 2019

INTRODUCTION

The Epidermal Growth Factor Receptor (EGFR) is a driver gene that is amplified in more than half of all glioblastomas. After the initial high copy-number amplification, secondary missense mutations in the extracellular, ligand binding domain of EGFR can evolve. We here performed analysis on these mutations to better understand their function.

METHODS

Activation of EGFR-mutation constructs was measured by western blot and high-throughput quantitative image analysis using EGFR and phospho-EGFR specific antibodies.

RESULTS

Amphiregulin (AREG) is a low affinity EGFR ligand and was able to only marginally activate EGFRwt at high concentrations. In contrast however, AREG strongly activated all extracellular domain (ECD) mutation constructs (R108K, A289V and G598V). As expected, EGF-stimulation resulted in a strong activation in both EGFRwt and ECD mutation constructs. Further dose response analysis showed that in EGFRwt, all high affinity EGFR ligands (EGF, TGFa, HB-EGF and BTC) resulted in a strong activation and all low affinity ligands AREG, EREG and EPGN resulted in a markedly weaker activation. All ECD mutations however, showed strong activation towards all EGFR ligands, including the low affinity ligands (40%-80% of EGF stimulation). The constitutively active, ligand independent EGFRvIII mutation did not respond to any of the ligands and was mainly found to have an intracellular localization. Interestingly, the presence of ECD mutations and EGFRvIII are inversely correlated: in three large datasets (TCGA, Belob and Intellance 2), the majority of samples expressing EGFRvIII do not express additional ECD mutations and vice versa. ECD mutations likely affect stability of the tethered, inactive conformation thereby increasing exposure of the dimerization domain (II), resulting in increased ligand-sensitivity.

CONCLUSION

These experiments show that EGFR containing extracellular missense mutations render the receptor more sensitive to stimulation by low affinity ligands. Glioblastomas evolve to either become independent of ligand (EGFRvIII) or become ligand-hypersensitive (ECD mutations).
CSIG-12. PREX1 LINKS ABERRANT PI 3-KINASE PATHWAY SIGNALING TO MAINTENANCE OF THE NEURAL STEM CELL-LIKE PHENOTYPE OF GLIOBLASTOMA CELLS
Authors: Lorimer Ian , Lavictoire Sylvie , Jomaa Danny , Gont Alex , Jardine Karen
Published: November 2019

Virtually all primary glioblastomas have aberrant activation of the PI 3-kinase pathway through mutational inactivation of tumour suppressors such as PTEN and/or mutational activation of oncogenes. Most glioblastomas also overexpress the Rac guanine nucleotide exchange factor PREX1, which is activated by PI 3-kinase pathway signaling. To determine the role of PREX1 in mediating the effects of aberrant PI 3-kinase pathway signaling in glioblastoma, we used CRISPR/Cas9 to knockout PREX1 in patient-derived glioblastoma cells cultured under conditions that maintain their neural stem cell-like characteristics. Multiple PREX1-null glioblastoma cell clones showed reduced motility and an altered morphology, with a smaller cell body and multiple neurite-like extensions. They also had increased expression of doublecortin, a marker of committed neurogenic progenitor cells. Analysis of RNA-seq data for transcription factor usage was consistent with the acquisition of an intermediate progenitor-like phenotype in PREX1-null cells, with loss of neural stem cell-associated polycomb repressive complex 2 and DMRTA2 activity and the expression of an EOMES/TBR2 signature characteristic of intermediate progenitors. The reduced motility, altered morphology and increase in doublecortin protein levels could all be reversed by re-expression of PREX1. PREX1-null cells had a reduced ability to phosphorylate and inactivate Lgl1, which is required for the maintenance of neural stem cell-like glioblastoma cell populations in vivo. These data show that PREX1 links aberrant PI 3-kinase signaling to maintenance of the neural stem cell-like phenotype in glioblastoma cells.
CSIG-14. PROTEIN KINASE CK2 REGULATES THE EXPRESSION OF NERVE/GLIAL ANTIGEN (NG)2 IN GLIOBLASTOMA
Authors: Schmitt Beate , Sippl Christoph , Urbschat Steffi , Oertel Joachim , Laschke Matthias , Menger Michael , Ampofo Emmanuel
Published: November 2019

Nerve/glial antigen (NG)2 enhances cell proliferation, migration as well as chemoresistance and is associated with a poor clinical outcome in glioblastoma. Since regulatory mechanisms of NG2 expression are still largely unknown, we herein investigated the impact of protein kinase CK2 inhibition on NG2 expression in human glioblastoma cells. CK2 was inhibited in NG2-positive human glioblastoma cell lines (A1207, U87) and primary human glioblastoma cells by pharmacological treatment (CX-4945, TBB) or siRNA. NG2 expression was analyzed by flow cytometry, Western Blot and qRT-PCR. Cytotoxicity and viability were assessed by WST-1, LDH and BrdU assays. Scratch, sprouting and BrdU assays as well as growth curves were performed to examine cell migration and proliferation. Truncated fragments of the human NG2 promotor were generated and their transcriptional activity was assessed using reporter gene assays. The effect of CK2 inhibition on tumor growth was investigated in xenografts within the flanks of NOD/SCID mice. Finally, effects of CK2 inhibition were analyzed in primary human glioblastoma cells. We found that inhibition of CK2 significantly reduces NG2 protein levels in A1207 (19%±6.2; Mean±SD), U87 (35%±11.3) and primary human glioblastoma cells (41%) when compared to controls. Further analyses revealed that this is due to a decreased NG2 mRNA level. Of note, we identified a 200 base pair fragment, including a binding site for the CK2-dependent transcription factor SP-1. Functional assays showed no cytotoxic effects of the decreased NG2 expression after CK2 inhibition, whereas cell migration and proliferation were markedly reduced. Moreover, we found that CX-4945 treatment decreases tumor growth, which is associated with a diminished NG2 expression (56%±13.0) when compared to controls. In conclusion, we identified CK2 as a novel regulator of NG2 gene expression in human glioblastoma cells. Hence, pharmacological inhibition of CK2 may represent a novel strategy in the therapy of NG2-positive glioblastoma.
CSIG-17. SIGNALING PATHWAY-BASED CLUSTERING OF MASS CYTOMETRY DATA UNCOVERS CELLS ASSOCIATED WITH CLINICAL PROGRESSION IN GLIOBLASTOMA
Authors: Alvarado Alvaro , Alban Tyler , Nakano Ichiro , Lathia Justin , Goldman Steven , Kornblum Harley
Published: November 2019

Recent advances in single cell expression technology have allowed us to appreciate the complexity in tumoral heterogeneity of solid tumors such as glioblastoma (GBM), which remains uniformly fatal despite aggressive therapies. Currently, there is no knowledge about cell identities and functional characteristics of the different tumoral populations. To this end, we created a 28-marker mass cytometry panel composed of signaling markers, including phospho-proteins, and lineage markers that have been associated with GBM or are considered putative cancer stem cell markers. We evaluated lines established from core and edge (periphery) areas from the same patient and validated to be functionally different with RNA expression and in vivo experiments. We first clustered the cells from all samples using the signaling markers in order to establish differences based on pathway utilization, and then we evaluated the expression of all markers. We found clusters shared by cells from both regions as well as region-specific ones. In order to better characterize these, we used marker enrichment modeling (MEM) and generated machine-readable labels. These were compared to MEM labels generated after using the same signaling markers to cluster primary samples from newly diagnosed or recurrent patients. Similarly, MEM labels were obtained from patients in a clinical trial treated with capecitabine that had positive or negative responses. Clusters from edge cells were found in higher proportion in recurrent tumors and were correlated with negative responses to the chemotherapeutic agent. Initial analyses have also been done correlating immune clusters with tumor clusters in the same samples. We are currently conducting in vitro studies and analyzing additional lines from other patients in order to validate these findings. Our studies exploit single cell cytometry to underscore pathways that are preferentially use by different cell populations with the ultimate goal of establishing therapeutic efforts to extend patient survival.
CSIG-18. NERVE/GLIAL ANTIGEN (NG)2 EXPRESSION IN GLIOBLASTOMA IS REGULATED BY miR-29b-3p
Authors: Schmitt Beate , Boewe Anne , Gu Yuan , Sippl Christoph , Urbschat Steffi , Oertel Joachim , Laschke Matthias , Menger Michael , Ampofo Emmanuel
Published: November 2019

Overexpression of NG2 in human glioblastoma cells is associated with an elevated drug resistance and thereby worsens clinical outcome. However, the regulatory mechanisms of NG2 expression are largely unknown. In this study, we identified miR-29b-3p as a posttranscriptional factor of NG2 expression. The basal mRNA levels of miR-29b-3p and NG2 were detected in the NG2-positive glioblastoma cell lines A1207 and U87 by qRT-PCR. The cells were transfected with miR-29b-3p-mimic or scrambled-miR (control) and the expression of NG2 was analyzed by qRT-PCR, flow cytometry and Western blot. Reporter gene analyses of the NG2 promotor region and 3’UTR were performed to study the effect of miR-29b-3p on NG2 expression. Finally, we analyzed the mRNA levels of NG2 and miR-29b-3p in samples from glioblastoma patients. We found that the two NG2-positive glioblastoma cell lines A1207 and U87 are positive for miR-29b-3p. Transfection with miR-29b-3p-mimic reduced NG2 mRNA levels in A1207 (29%±9.9; Mean±SD) and U87 (6%±2.8), resulting in a significantly decreased NG2 protein expression in A1207 (67%±6.4) and U87 (75%±4) when compared to controls. The analysis of the 3’UTR revealed that miR-29b-3p is a posttranscriptional regulator of NG2 expression. Moreover, miR-29b-3p affects the pretranscriptional NG2 expression by diminishing SP-1-dependent NG2 promotor activity. These results were confirmed by the analysis of glioblastoma patient-derived samples, demonstrating that a high NG2 expression is associated with low levels of miR-29b-3p. In conclusion, we identified miR-29b-3p as a crucial regulator of NG2 expression in glioblastoma. Hence, targeting NG2 expression by miR-29b-3p may provide a novel therapeutic strategy to overcome drug resistance in NG2-positive glioblastoma cells.
CSIG-25. MTAP LOSS COMPROMISES DNA DAMAGE RESPONSE IN GBM CELLS
Authors: Du Changzheng , Hansen Landon , Singh Simranjit , Roso Kristen , Greer Paula , He Yiping
Published: November 2019

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic alterations in glioblastomas (GBMs), occurring in about half of all patients. Here, we demonstrated that MTAP loss compromises the proteostasis of genomic stability guardian, H2AX, via disrupting a signaling cascade of PRMT5-RNF168-SMURF2. We showed that PRMT5 sustains the expression of RNF168, an E3 ubiquitin ligase essential for cellular response to DNA damage. Suppression of PRMT5 function, as occurring in MTAP-null GBM cells, attenuates the expression of RNF168, which consequently leads to degradation of H2AX protein by a HECT-type E3 ubiquitin ligase, SMURF2. We revealed that RNF168 and SMURF2, serving as a stabilizer and destabilizer of H2AX respectively, functionally oppose each other via their dynamic interactions with H2AX. In supporting the important role of this PRMT5-RNF168-SMURF2 signaling cascade in controlling H2AX homeostasis, MTAP-null GBM cells display a compromised DNA damage response, highlighted by higher levels of DNA damage spontaneously or in response to genotoxic agents. Collectively, these results identify a novel signaling cascade that is essential to the DNA damage response, reveal the profound impact of MTAP loss on GBM cells, and suggest novel therapeutic opportunities.
CSIG-27. PRC2-INDEPENDENT FUNCTION OF EZH2/HP1BP3 COMPLEX IN GLIOMA STEM CELLS
Authors: Zhang Junxia , Yu Tianfu , Liu Ning
Published: November 2019

Glioblastoma (GBM) displays cellular and genetical heterogeneity harboring a subpopulation of glioma stem cells (GSCs). Enhancer of zeste homolog 2 (EZH2), a histone lysine methyltransferase, is the core subunit of the polycomb repressor 2 (PRC2) complex, mediates gene transcriptional repression in both normal and tumor stem cells. An oncogenic role of EZH2 as a PRC2-dependent transcriptional silencer is well established; however, non-canonical functions of EZH2 are incompletely understood. Here we found a novel oncogenic mechanism for EZH2 in a PRC2-indenpend way in GSCs. Using HPLC-MS/MS and IP assay, EZH2 bound to HP1BP3 (heterochromatin protein 1 binding protein 3), a heterochromatin-related protein, with its pre-SET domain. Overexpression of H1P3B3 enhanced the proliferation, self-renewal and temozolomide (TMZ) resistance of GBM cells. Intriguingly, H1PBP3 was up-regulated in high grade gliomas with proneural (PN) subtypes and had a high predictive value on prognosis in patients with PN gliomas. Furthermore, EZH2 and HP1BP3 co-activated the expression of WNT7B by blocking the methylation of H3K9, thereby increasing TMZ resistance and tumorigenicity of glioblastoma cells. Interestingly, inhibition of WNT7B autocrine via LGK974, a specific porcupine inhibitor, effectively reversed the TMZ resistance of both GSCs and GBM glioma cells expressing HP1BP3. Hence, targeting the PRC2-independent function of EZH2 is an effective approach to enhance the efficacy of treating GBM.
CMET-03. MANAGEMENT OF MULTIPLE BRAIN METASTASES BY STAGED SRS FOCUSING ON UTMOST RISK LESIONS
Authors: Zhen Junjie , Li Shaoqun , Lai Mingyao , wen lei , cai linbo
Published: November 2019

BACKGROUND

Patients with multiple large brain metastases (LBMs) are subject to cause intracranial hypertension, which remains a clinical difficulty so far. The present study is to investigate whether staged stereotactic radiosurgery (SRS) is a feasible solution to improve clinical symptoms and life quality in palliative management.

METHODS

Patients with brain metastases treated between 1 January 2016 and 30 March 2019 were retrospectively studied. The patients inclusion criteria included were: 1) metastatic lesions ≥ 3; 2) tumor lesions with a supratentorial invasion ≥ 3 cm or a subtentorial invasion ≥ 2 cm; 3) with neurological impairment or with a high risk for intracranial hypertension. The first stage of SRS dose regimens for utmost risk supratentorial lesions were 20-24Gy within 2-fractions (fx); whereas 24Gy/3-fx for subtentorial lesions. For the 2nd stage, the rest metastatic lesions with relatively lower risk were treated with 16-18Gy/1-fx one week after that.

RESULTS

A total of 30 patients were enrolled included in this study. The neurological symptoms were significantly relieved following 1st stage of SRS, with a median period of 3 days (2–14 days). 13 out of 30 patients were randomly selected for MR imaging two weeks after SRS. It suggested that 84.6% (11/13) of the patients were observed with a clear reduction of tumor volume. The median reduced diameter was 0.38 (0.17–0.83) cm and a median volume reduction was 3.22 (0.01–9.08) mm3. According to RANO-BM, the objective remission rate of utmost risk lesions was 100%, whereas the less critical lesions was 95.16% (59/62). One patient (3.33%) was identified with acute adverse reaction (> grade 3).

CONCLUSIONS

Staged SRS with a priority for utmost risk lesions was indicated to be an effective approach for multiple large brain metastases. Further prospective study is highly warranted.
CMET-05. EVALUATING RADIATION NECROSIS IN PATIENTS WITH INTRACRANIAL METASTASES FROM MELANOMA
Authors: Edmonds Victoria , Thomson Holly , Sharma Akanksha , Butterfield Richard , Zhang Nan , Sio Terence , Vora Sujay , Daniels Thomas , Schild Steven , Ashman Jonathan , Patel Naresh , Zimmerman Richard , Mrugala Maciej , DeLeon Thomas , Bryce Alan , Porter Alyx
Published: November 2019

BACKGROUND

Radiation is an integral component of the multidisciplinary treatment of patients with intracranial metastasis (ICM) from melanoma. The risk of radiation necrosis (RN) post-treatment can range from 5–25%. We retrospectively evaluated pre- and post-radiation clinical and radiographical characteristics in patients with ICM from melanoma to identify potential risk factors for RN.

METHODS

After IRB approval, patients with ICM from melanoma who received radiation at our institution between 2013 and 2018 were retrospectively reviewed. We recorded demographics, intracranial metastasis (burden and location), systemic therapy, resection, stereotactic radiosurgery versus whole brain radiation, and outcomes. Brain MRI was evaluated using the Response Assessment in Neuro-Oncology criteria (RANO).

RESULTS

A total of 27 patients were included in the study. RN was diagnosed in 14 patients (52%) at one month to three years following radiation. Cerebellar location (n=7) was significantly associated with RN (p=0.0058). Metastasis-associated hemorrhage was present in 7 patients, 6 of which developed RN (43% vs 8%, p=0.08). Surgical resection prior to radiation was performed in 9 patients, 7 of which developed RN (50% vs 15%, p=0.1). RN appeared to be less often diagnosed when ICM were treated with higher doses of radiation (24 Gy vs 20 Gy, p=0.07). Systemic treatment was administered prior to radiation in 25 patients and we found no relationship with systemic therapy type and RN. Presumed RN was treated in 12 of 14 patients (86%). Steroids or surgical resection were the primary treatment modalities, with laser ablation used in 1 case. Following treatment, improvement was noted in 3 patients radiographically and in 1 of these clinically.

CONCLUSIONS

This series of patients with radiotherapy-treated brain metastases from melanoma reveals cerebellar involvement, hemorrhage, and prior surgical resection as potential risk factors for radiation necrosis. Neoadjuvant systemic treatment did not appear to be a risk factor in our review.
CMET-06. AN ANALYSIS OF RESPONSE ENDPOINTS FOR BRAIN METASTASIS PATIENTS TREATED WITH STEREOTACTIC RADIOSURGERY AND PD(L)-1 INHIBITORS
Authors: Kotecha Rupesh , Miller Jacob , Kim Joseph , Juloori Aditya , Chao Samuel , Murphy Erin , Peereboom David , Mohammadi Alireza , Barnett Gene , Vogelbaum Michael , Angelov Lilyana , Suh John , Ahluwalia Manmeet
Published: November 2019

PURPOSE

Recent trials have evaluated the role of anti-PD(L)-1 inhibitors alone in patients with newly diagnosed brain metastasis, resulting in a suggested paradigm shift. In the absence of randomized comparisons, an evaluation of outcomes for similar patients treated with radiotherapy are needed to provide baseline comparative data.

METHODS

This retrospective cohort study included patients diagnosed with brain metastasis from 2010–2017 at a single tertiary care institution who received SRS and at least one anti-PD(L)-1 inhibitor. Primary endpoints included the rate of best intracranial response (either a complete [CR] or partial response [PR]), rate of intracranial benefit (percentage of patients with stable disease [SD] for at least 6 months), and overall objective response rates.

RESULTS

150 patients met the eligibility criteria for this study and the median follow-up time was 10 months (Range: 1–130 months). Only a minority of patients from this cohort would have been eligible for comparative systemic therapy alone trials: 8 (CheckMate 204, CM), 8 (ABC), and 50 patients (Yale Institutional trial, YI). Best intracranial objective response rates for these trial eligible patients were 64% (CM), 64% (ABC), and 72% (YI), respectively. The 6-month rates of intracranial benefit across patients were 75% (CM), 75% (ABC), and 93% (YI), respectively. The rates of either a CR, PR, or SD across trial eligible patients were 100% (CM), 100% (ABC), and 94% (YI), respectively. More than 50% of patients experienced out-of-field progression in each of the patient subgroups.

CONCLUSIONS

Over a seven year period, very few patients treated with SRS and anti-PD(L)-1 inhibitor therapy in a busy academic practice would have been eligible for systemic therapy alone per trial eligibility criteria. In these highly favorable subgroups, patients experience high rates of objective response, best intracranial response, and intracranial benefit after SRS and anti-PD(L)-1 therapy, representing optimal brain metastasis management.