Glioma Stem-like Cells Research Articles

Xihuang Pill-destabilized CD133/EGFR/Akt/mTOR cascade reduces stemness enrichment of glioblastoma via the down-regulation of SOX2

BackgroundOur previous study found that XHP could induce GBM cells to undergo apoptosis. A lot of evidence suggests that glioma stem-like cells (GSCs) are key factors that contribute to disease progression and poor prognosis of glioblastoma multiforme (GBM). Traditional Chinese medicine has been applied in clinical practice as a complementary and alternative therapy for glioma. PurposeTo evaluate the effect and the potential molecular mechanism of Xihuang pill (XHP) on GSCs. MethodsUPLC-QTOF-MS analysis was used for constituent analysis of XHP. Using network pharmacology and bioinformatics methods, a molecular network targeting GSCs by the active ingredients in XHP was constructed. Cell viability, self-renewal ability, apoptosis, and GSC markers were detected by CCK-8 assay, tumor sphere formation assay and flow cytometry, respectively. The interrelationship between GSC markers (CD133 and SOX2) and key proteins of the EGFR/Akt/mTOR signaling pathway was evaluated using GEPIA and verified by western blot. A GBM cell line stably overexpressing Akt was constructed using lentivirus to evaluate the role of Akt signaling in the regulation of glioma stemness. The effect of XHP on glioma growth was analyzed by a subcutaneously transplanted glioma cell model in nude mice, hematoxylin-eosin staining was used to examine pathological changes, TUNEL staining was used to detect apoptosis in tumor tissues, and the expression of GSC markers in tumor tissues was identified by western blot and immunofluorescence. ResultsBioinformatics analysis showed that 55 matched targets were related to XHP targets and glioma stem cell targets. In addition to causing apoptosis, XHP could diminish the number of GBM 3D spheroids, the proportion of CD133-positive cells and the expression level of GSC markers (CD133 and SOX2) in vitro. Furthermore, XHP could attenuate the expression of CD133, EGFR, p-Akt, p-mTOR and SOX2 in GBM spheres. Overexpression of Akt significantly increased the expression level of SOX2, which was prohibited in the presence of XHP. XHP reduced GSC markers including CD133 and SOX2, and impeded the development of glioma growth in xenograft mouse models in vivo. ConclusionWe demonstrate for the first time that XHP down-regulates stemness, restrains self-renewal and induces apoptosis in GSCs and impedes glioma growth by down-regulating SOX2 through destabilizing the CD133/EGFR/Akt/mTOR cascade.

Pharmacogenomic profiling reveals molecular features of chemotherapy resistance in IDH wild-type primary glioblastoma

BackgroundAlthough temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application.MethodsWe stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n = 29) and sensitive (n = 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy.ResultsIn vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P = 1.12e−4 for progression-free survival (PFS) and 3.63e−4 for overall survival (OS)). Moreover, we found that elevated gene expression of EGR4, PAPPA, LRRC3, and ANXA3 was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration in PTEN, EGFR, and CDKN2A/B was more frequent in TMZ-sensitive samples (Fisher’s exact P = 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P = 4.58e−4 for PFS and 3.66e−4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage (http://www.wang-lab-hkust.com:3838/TMZEP).ConclusionsWe identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.

Feedforward loop between IMP1 and YAP/TAZ promotes tumorigenesis and malignant progression in glioblastoma.

YAP/TAZ have been identified as master regulators in malignant phenotypes of glioblastoma (GBM); however, YAP/TAZ transcriptional disruptor in GBM treatment remains ineffective. Whether post-transcriptional dysregulation of YAP/TAZ improves GBM outcome is currently unknown. Here, we report that insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IGF2BP1 or IMP1) is upregulated in mesenchymal GBM compared with proneural GBM and correlates with worse patient outcome. Overexpression of IMP1 in proneural glioma stem-like cells (GSCs) promotes while IMP1 knockdown in mesenchymal GSCs attenuates tumorigenesis and mesenchymal signatures. IMP1 binds to and stabilizes m6A-YAP mRNA, leading to activation of YAP/TAZ signaling, depending on its m6A recognition and binding domain. On the other hand, TAZ functions as enhancer for IMP1 expression. Collectively, our data reveal a feedforward loop between IMP1 and YAP/TAZ maintaining GBM/GSC tumorigenesis and malignant progression and a promising molecular target in GBM.

Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production

Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. Here, we investigated the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in 24(S),25-epoxycholesterol (EPC). EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. Critical for its application in the brain, we report an MM0299 derivative that is orally bioavailable, brain-penetrant, and induces the production of EPC in orthotopic GBM tumors but not normal mouse brain. These studies have implications for the development of an LSS inhibitor to treat GBM or other neurologic indications.

Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma.

Epidermal growth factor receptor (EGFR) amplification is found in nearly 40%-50% of glioblastoma cases. Several EGFR inhibitors have been tested in glioblastoma but have failed to demonstrate long-term therapeutic benefit, presumably because of acquired resistance. Targeting EGFR downstream signaling with mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitors would be a more effective approach to glioblastoma treatment. We tested the therapeutic potential of MEK1/2 inhibitors in glioblastoma using 3D cultures of glioma stem-like cells (GSCs) and mouse models of glioblastoma. Several MEK inhibitors were screened in an unbiased high-throughput platform using GSCs. Cell death was evaluated using flow cytometry and Western blotting (WB) analysis. RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and WB analysis were used to identify and validate neuronal differentiation. Unbiased screening of multiple MEK inhibitors in GSCs showed antiproliferative and apoptotic cell death in sensitive cell lines. An RNA-seq analysis of cells treated with trametinib, a potent MEK inhibitor, revealed upregulation of neurogenesis and neuronal differentiation genes, such as achaete-scute homolog 1 (ASCL1), delta-like 3 (DLL3), and neurogenic differentiation 4 (NeuroD4). We validated the neuronal differentiation phenotypes in vitro and in vivo using selected differentiation markers (β-III-tubulin, ASCL1, DLL3, and NeuroD4). Oral treatment with trametinib in an orthotopic GSC xenograft model significantly improved animal survival, with 25%-30% of mice being long-term survivors. Our findings demonstrated that MEK1/2 inhibition promotes neuronal differentiation in glioblastoma, a potential additional mechanism of action of MEK1/2 inhibitors. Thus, MEK inhibitors could be efficacious in glioblastoma patients with activated EGFR/MAPK signaling.

CHRDL1 Regulates Stemness in Glioma Stem-like Cells

Glioblastoma (GBM) still presents as one of the most aggressive tumours in the brain, which despite enormous research efforts, remains incurable today. As many theories evolve around the persistent recurrence of this malignancy, the assumption of a small population of cells with a stem-like phenotype remains a key driver of its infiltrative nature. In this article, we research Chordin-like 1 (CHRDL1), a secreted protein, as a potential key regulator of the glioma stem-like cell (GSC) phenotype. It has been shown that CHRDL1 antagonizes the function of bone morphogenic protein 4 (BMP4), which induces GSC differentiation and, hence, reduces tumorigenicity. We, therefore, employed two previously described GSCs spheroid cultures and depleted them of CHRDL1 using the stable transduction of a CHRDL1-targeting shRNA. We show with in vitro cell-based assays (MTT, limiting dilution, and sphere formation assays), Western blots, irradiation procedures, and quantitative real-time PCR that the depletion of the secreted BMP4 antagonist CHRDL1 prominently decreases functional and molecular stemness traits resulting in enhanced radiation sensitivity. As a result, we postulate CHRDL1 as an enforcer of stemness in GSCs and find additional evidence that high CHRDL1 expression might also serve as a marker protein to determine BMP4 susceptibility.

GAS5 attenuates the malignant progression of glioma stem-like cells by promoting E-cadherin.

It has been widely reported that glioma stem-like cells (GSCs) serve a crucial role in the malignant progression of glioma. In particular, recent studies have reported that long non-coding RNAs (lncRNAs) are closely associated with glioma development. However, the underlying molecular regulatory mechanistic role of GSCs remains poorly understood. The present study established two highly malignant glioma stem-like cell lines from clinical surgical specimens. In these, it was found that the lncRNA growth arrest-specific 5 (GAS5) expression was downregulated in GSCs and high-grade glioma tissues, compared with normal human astrocyte cells (NHAs) and normal brain tissues, respectively, which also showed a positive correlation with patient survival. Functional assays revealed that knocking down GAS5 expression promoted the proliferation, invasion, migration, stemness, and tumorigenicity of GSGs, while suppressing their apoptosis. Mechanistically, GAS5 directly sponged miR-23a, which in turn functioned as an oncogene by inhibiting E-cadherin, through the assays of reverse transcription-quantitative PCR (RT-qPCR) and luciferase reports. In addition, rescue experiments demonstrated that GAS5 could promote the expression and function of E-cadherin in a miR-23a-dependent manner. Collectively, these data suggest that GAS5 functions as a suppressor in GSCs by targeting the miR-23a/E-cadherin axis, which may be a promising therapeutic target against glioma.

DNAR-05. A ROLE FOR TAZ/YAP IN DNA DAMAGE REPAIR IN GLIOBLASTOMA

Abstract Previous studies have shown that glioma stem-like cells (GSCs) evade conventional cytotoxic therapies and repopulate as recurrent tumors. A fuller understanding of the molecular mechanisms underlying the biology and therapy resistance of GSCs is required. Our group has shown that TAZ, a transcriptional co-factor is highly expressed in about 70% of glioblastomas (GBMs). TAZ and its paralog YAP are oncogenic drivers of brain tumor progression. GSCs overexpressing TAZ undergo a proneural (PN) to mesenchymal (MS) subtype transition, which is accompanied by aggressive phenotypes such as increased grade and treatment resistance in GSC xenografts. Although the oncogenic functions of TAZ/YAP are well established, the exact molecular mechanisms underlying TAZ/YAP mediated cell fate transition and therapy resistance in GBM are not fully understood. Here, we present novel functional interaction between TAZ/YAP and DNA-dependent protein kinase (DNA-PK) in response to exposure to ionizing radiation in GSCs. Mass-spectrometric analyses of TAZ-binding proteins showed that DNA-PK complex composed of the catalytic subunit (DNA-PKcs) and a Ku heterodimer of Ku70 and K80 interacted with TAZ. Immunoprecipitation of DNA-PK showed interaction of both YAP as well. Interaction of YAP/TAZ with DNA-PK complex and TEAD was enhanced by ionizing radiation. DNA-PKcs phosphorylated both YAP and TAZ, and phosphorylation level of these proteins were increased after irradiation, consistent with an increased interaction of these proteins. Intriguingly, siRNA knockdown of YAP and TAZ resulted in inactivation of DNA-PKcs, although protein stability of DNA-PKcs was not affected. Pharmacological inhibition of TAZ/YAPTEAD resulted in increased DNA damage and suppressed cell proliferation, and this effect was synergized by irradiation. We conclude that TAZ and YAP are integral components of DNA damage response and that pharmacological targeting of TAZ/YAP/TEAD pathway can overcome radiation resistance in GSCs.

CNSC-12. NEURONAL DIFFERENTIATION DRIVES THE ANTI-TUMOR EFFICACY OF MEK INHIBITION IN GLIOBLASTOMA

Abstract Multiple genomic aberrations and distinct molecular subtypes of glioblastoma (GBM) have been identified through multi-omics analyses including single-cell genomic and epigenomic analysis. Epidermal Growth Factor Receptor (EGFR) amplification is found in nearly 50% of GBM tumors. Several EGFR inhibitors have been tested in GBM but failed to demonstrate long-term therapeutic benefit, presumably due to target heterogeneity, escape signal pathways, and acquired resistance. Therefore, we investigated downstream signaling, mitogen-activated protein kinase (MEK1/2) as a potential therapeutic target for GBM. We utilized an unbiased high-throughput screening with a panel of glioma stem-like cells (GSCs) and identified that GBM cells harboring EGFR amplification showed anti-proliferative and apoptotic cell death (sensitive) to multiple MEK inhibitors. RNA-seq analysis of cells pre- and post-treatment with trametinib, a potent MEK inhibitor, revealed an upregulation of neurogenesis and neuronal differentiation genes such as β-III tubulin, ASCL1, DLL1, DLL3, NeuroD4, PAX6 and NCAM1. In addition, downregulation of the several MEK targets including erythroblast transformation specific (ETS) family genes, particularly ETV5 was observed. We demonstrated that MEK inhibition increased neuronal differentiation demonstrated by immunofluorescence, western blot, and RT-qPCR experiments using selected differentiation markers (β-III tubulin, ASCL1, DLL3, and NeuroD4). In addition, ETV-5 knockout in GSCs by CRISPR/Cas-9 promoted the expression of neuronal differentiation genes. Oral treatment of trametinib in an orthotopic GSC xenograft model significantly improved animal survival, in which 25-30% of mice are long-term survivors. This was accompanied by decreased MEK/ERK signal and cell proliferation in tumor tissues. Thus, we demonstrated here that MEK1/2 inhibition by trametinib induces neuronal differentiation mediated through downregulation of ETV-5 in GBM, a potential novel mechanism of action of MEK1/2 signal inhibition.

TMET-30. ENHANCED RADIOSENSITIZATION OF GLIOMAS BY METABOLIC TARGETING OF SALVAGE PATHWAY OF NAD SYNTHESIS THROUGH NAMPT INHIBITION

Abstract BACKGROUND Pathway-specific targeted agents have failed to improve outcome in patients with high grade gliomas. Tumor recurrence is driven by glioma heterogeneity and emergence of therapy-resistant subpopulations. We have previously reported that metabolic targeting of NAMPT, the rate limiting enzyme in the NAD salvage synthesis pathway, circumvents glioma heterogeneity and induces cytotoxicity by mitochondrial membrane depolarization and generation of reactive oxygen species. Based on these data, we examined the potential for NAMPT inhibition to synergize with ionizing radiation to enhance glioma cytotoxicity in vitro and in vivo. METHODS Using a panel of genetically heterogeneous patient-derived glioma stem-like cells (GSC), we examined the effects of NAD targeting using KPT9274, a potent NAMPT inhibitor, in combination with ionizing radiation on cell proliferation and clonogenicity using Cell Titre Glo assay, colony formation assays and secondary sphere formation. Changes in DNA damage-related proteins were visualized by immunofluorescence assay and confocal microscopy. Effects on gene expression and protein levels were assessed using western blot, reverse phase protein array (RPPA) and RNA Seq. RESULTS NAMPT inhibition reduced cell proliferation rate, self-renewal and clonogenic capacity in vitro. Combining KPT-9274 with ionizing radiation caused synergistic inhibition of secondary sphere and colony formation of GSC. RPPA and RNASeq results showed downregulation of several DNA repair proteins which was further confirmed using immunofluorescence staining indicating a crucial role for these proteins in glioma resistance to therapies. Results of ongoing in vivo studies to assess the PK/PD, drug toxicity and survival benefits of this combination therapy in orthotopic GSC intracranial glioma mouse model will be reported. CONCLUSIONS Our results suggest that metabolic targeting through NAMPT inhibition sensitizes glioma cells to radiotherapy. Ongoing studies delineating the mechanistic basis of these effects and their impact in vivo as a potential rationale for clinical assessment will be reported.

TMIC-12. COMPLEMENT SIGNALING IN HYPOXIC NICHES OF THE GLIOMA MICROENVIRONMENT

Abstract Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.

PATH-09. SUBTYPE-ASSOCIATED RNA SPLICING CONTROLLED BY GLIOMA DRIVER MUTATIONS REGULATES GLIOMAGENESIS

Abstract Dysregulated RNA alternative splicing (AS) is a hallmark of cancer and has been studied in cancers including glioma. However, whether the dysregulated AS and the regulating RNA-binding proteins (RBPs) drive gliomagenesis and the role of glioma driver mutations in shaping the RBP/AS landscape have not been studied. Here we describe two AS-defined glioma subtypes that correlate with IDH1 mutation, tumor grade, and patient prognosis. Oncogenic AS isoforms are exclusively associated with AS subtype 2, IDH1 wild-type gliomas. Modulation of representative AS isoforms or subtype-associated RBPs regulate glioma stem-like cell (GSC) tumorgenicity. With a human induced pluripotent stem cell-derived glioma model system, we demonstrate that specific glioma driver mutations are involved in shaping the subtype-associated AS. Lastly, we target a sub2-associated RBP with anti-sense oligonucleotides to inhibit GSC tumorigenicity. Our data establish relationships between glioma driver mutations and AS programs and identify dysregulated RBP/AS network as a therapeutic vulnerability.

EXTH-67. PRECLINICAL ASSESSMENT OF THE EFFICACY OF MPT0B640, AN ORALLY BIOAVAILABLE SMALL MOLECULE HSP90 INHIBITOR, AGAINST GLIOMAS

Abstract BACKGROUND Heat-shock protein 90 (HSP90), an evolutionary conserved protein, is a key member of the epichaperome complex involved in maintenance of protein homeostasis during non-stress and stress conditions. It is overexpressed in gliomas and supports tumor cell survival and resistance. Hsp90 inhibition can impact several pathways essential for glioma cell survival and overcome tumor heterogeneity. Here, we examined the activity and toxicity profile of MPT0B640, an orally bioavailable Hsp90 inhibitor, against gliomas and its ability to cross the blood-brain barrier (BBB). METHODS Using genetically heterogeneous glioma stem-like cell (GSC) lines, we determined the effects of MPT0B640 on cell proliferation (Cell Titre Glo assay), apoptosis induction and cell cycle changes (flowcytometry; AnnexinV/PI staining), and alterations in protein expressions (western blot and reverse-phase protein array, RPPA assays). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed in non-tumor bearing mice to assess BBB permeability and antitumor effects of MPT0B640. RESULTS MPT0B640 inhibited proliferation of both adherent (LN229, A172, U251-HF) and patient-derived GSCs (GSC262, GSC811, GSC11, GSC23, GSC20 and GSC272) in a dose- and time-dependent fashion with an IC50 range of 50-1000 nM. MPT0B640-treated cells underwent apoptosis and/or cell cycle arrest with downregulation of pro-survival signal kinases, phospho-Akt, p-ERK1/2 and p-S6. PK/PD studies showed increased HSP70 expression (a marker of Hsp90 inhibition) and detectable levels of drug in brain specimens from non-tumor bearing mice after a single dose of MPT0B640, indicating BBB permeability and target inhibition of the agent. Results of ongoing animal studies assessing drug toxicity and animal survival with MPT0B640 will be presented. CONCLUSIONS MPT0B640 is a potent HSP90 inhibitor that has potent antitumor activity against genetically heterogeneous gliomas in vitro, effectively crosses the intact BBB and demonstrates target inhibition warranting further investigation for treatment of gliomas.

Targeting Nuclear Pore Complex to Radiosensitize Glioblastoma Stem Cells

<h3>Purpose/Objective(s)</h3> Glioblastoma is the most prevalent and lethal primary intrinsic tumor in the central nervous system. Glioblastoma stem cells (GSCs) reprogram the transcriptional and metabolic machinery to mediate therapeutic resistance to radiation. Understanding the regulatory mechanism of GSCs may inform novel approaches to improve the clinical outcome for glioblastoma patients. Nuclear pore complex (NPC) is an evolutionarily conserved structure that maintains the traffic between the nucleus and cytoplasm. In current study, we aim to interrogate the aberrant regulation of NPC in GSCs and develop novel targeted approach to sensitize GSC to radiation. <h3>Materials/Methods</h3> Patient-derived GSCs were validated using functional assays. Transcriptional analysis of GSCs and matched differentiated glioblastoma cells (DGCs) were performed using RNA sequencing to identify aberrant regulatory pathways upregulated in GSCs. The clinical impact of target genes on patient survival was assessed using the glioblastoma dataset from The Cancer Genome Atlas (TCGA). Quantitative RT-PCR and Western blotting were performed to assess the relative expression between GSCs and DGCs. Radiation sensitivity was measured using Cs-137 irradiator. Validation of the stem-like function of target genes was performed using RNA interference followed by <i>in vitro</i> neurosphere formation assay and <i>in vivo</i> tumorigenesis with xenograft mouse model. <h3>Results</h3> 27 of the total 31 NPC genes were found to be upregulated in the primary glioblastoma patient specimens compared to non-tumor brain tissue in the TCGA dataset. Among them, a systematic approach combining transcriptional profiling and clinical validation linked nucleoporin 98 (NUP98) to a nexus between GSC phenotype and radiation resistance. GSCs preferentially express NUP98, which is essential for GSC maintenance and tumorigenesis <i>in vitro</i> and <i>in vivo</i>. RNA sequencing demonstrated that NUP98 regulates the expression of key DNA repair genes, including BRCA1, BRCA2, and RAD51. NUP98 forms a complex with the transcription factor P65, and directly stimulates the active transcription of genes involved in homologous recombination. Attenuation of NUP98 or P65 expression leads to the accumulation of unrepaired intrinsic DNA damage and sensitizes GSC to the treatment of ionizing radiation. Clinically, overexpression of NUP98 informs poor clinical outcome among patients with glioblastoma. <h3>Conclusion</h3> Combining transcriptional discovery and clinical validation, we discovered that NUP98, an essential NPC component, maintains GSC proliferation and tumorigenesis through modulating the transcriptional control of homologous recombination pathway. Our results demonstrated a novel therapeutic approach of sensitizing GSC to radiation through targeting the nexus between aberrant transcriptional regulation and therapeutic resistance in stem-like glioma cells.

Enhanced Radiation-Sparing Effects of Ultra-High Dose Rate Proton Radiation (FLASH-RT) in a Human Induced Pluripotent Stem Cell-Derived Cerebral Organoid Model

<h3>Purpose/Objective(s)</h3> Superior normal tissue sparing with similar antitumoral effectiveness has been observed in preclinical animal models following delivery of radiation at ultra-high dose rates (FLASH-RT), relative to conventional dose rates (CONV-RT). Clinical application of FLASH-RT could reduce devastating radiation-related side effects for patients with aggressive brain tumors such as glioblastoma; however, the FLASH effect must be established in representative models of human systems before its implementation in the clinic. Our objective is to measure relevant response outcomes to proton FLASH-RT using advanced cerebral organoid (CO) models of normal and diseased human brain tissue, correspondingly generated with human induced pluripotent stem cells and patient-derived glioma stem-like cells (GSCs). <h3>Materials/Methods</h3> Mature COs were irradiated to 9 Gy using conventional (CONV, 0.2 Gy/s) and FLASH (100 Gy/s) dose rates using a proton beam and prepared for histological analysis at 30 days post-irradiation. Spatial distribution of hypoxia was visualized in COs by incubating for 2 hours with pimonidazole prior to preparation for histologic analysis. Image analysis was performed with QuPath. Lactate dehydrogenase (LDH) release, a marker of cell death, was sequentially quantified. COs implanted with GSCs expressing a luciferase reporter were co-cultured for 14 days and irradiated as above. Tumor cell proliferation was tracked via luciferase expression. <h3>Results</h3> At 2- and 4-days post-irradiation, there was an increase in LDH release for CONV versus FLASH-radiated COs (p<0.01, p<0.01 respectively) which normalized after 9 days. At 30 days, when specific cell populations in FLASH-radiated COs were histologically compared to CONV-radiated COs, there were higher numbers of proliferating neural progenitor cells (SOX2+/Ki67+, p<0.01), mature neurons (NeuN+, p=0.03), and deep cortical surface layer neurons (SATB2+, p=0.01). The spatial distribution of neural progenitor cells and mature neurons overlapped with hypoxic regions of the CO (pimonidazole+). A trend towards lower expression of activated microglia (IBA1+/CD68+) in FLASH-irradiated COs compared to CONV-radiated COs was noted (p=0.12). A 48.6% versus 51.2% reduction in tumor proliferation in FLASH versus CONV-irradiated GSC-laden-COs was observed at 4d post-irradiation (p=0.98). <h3>Conclusion</h3> We report preliminary findings of normal tissue toxicity reduction and iso-effective tumor response in a human-derived brain model following proton FLASH-RT. Potential protective effects following FLASH-RT to be further validated include preservation of specific cell populations within hypoxic niches including progenitor and differentiated neurons with a corresponding reduction in chronic neuroinflammation.

Inhibitory Effects of Acetyloctadecadienediynoic Acid Isolated from Leaves of Dendropanax morbiferus on Viability and Self-Renewal Activity of Glioma Stem-Like Cells.

Glioblastoma (GBM) is one of the most dangerous brain tumors in humans. The median survival of patients with GBM is <18 months. Glioma stem-like cells (GSCs), a small subpopulation of cells with stem cell-like characteristics found within GBM, are regarded as the main cause of GBM malignancy. Therefore, targeting GSCs presents an important therapeutic strategy for reducing the aggressiveness of tumors. In this study, we examined effects of (9Z,16S)-16-O-acetyl-9,17-octadecadiene-12,14-diynoic acid (AODA), a diacetylenic carboxylic acid isolated from leaves of Dendropanax morbiferus, on viability and self-renewal activity of GSCs. AODA substantially decreased GSC growth, causing apoptotic cell death as assessed by Annexin V/PI staining and morphological alterations by optical diffraction tomography. Interestingly, treatment with AODA suppressed ''stem-like features'' in vitro by limiting dilution assays and real-time polymerase chain reaction analysis. In addition, Western blotting revealed that AODA treatment decreased expression levels of phosphorylated AKT and phosphorylated ERK in GSC11 cells. Taken together, our results indicate that AODA could be considered a new therapeutic candidate to target GSCs.

Folate enzyme MTHFD2 links one-carbon metabolism to unfolded protein response in glioblastoma

The mitochondrial folate enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) has shown oncogenic roles in various cancers and may have non-metabolic functions. This study investigated the role of MTHFD2 in glioblastoma pathogenesis. We find that MTHFD2 expression is enriched in gliomas by analysing public databases and clinical specimens. RNA interference (RNAi) and inhibitor of MTHFD2 hamper the proliferation of glioblastoma and induce apoptosis in cell lines, glioma stem-like cells (GSCs) and patient-derived xenografts (PDX). Metabolomic analyses show that MTHFD2 depletion suppresses the central carbon metabolic pathways, including glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle. GSEA reveals a novel non-metabolic function of MTHFD2 in association with the unfolded protein response (UPR). MTHFD2 depletion activates the PERK/eIF2α axis which contributes to translation inhibition and apoptosis; these effects are attenuated by a PERK inhibitor. Mechanistically, MTHFD2 may be linked to UPR via the post-transcriptionally regulation of chaperone protein GRP78. In conclusion, MTHFD2 could be a promising therapeutic target for glioblastoma. Besides its canonical role, MTHFD2 may contribute to glioblastoma pathogenesis via UPR, highlighting a newly identified functional link between one-carbon metabolism and cell stress response.

P12.04.A Exosomes from glioma associated sphere forming cells induce a transition of invasive phenotype via transfer of EMP2 and CA9

Abstract Background Glioblastoma multiforme (GBM) mostly occurs local recurrence at normal parenchyme adjacent tumor despite of conventional treatment. Glioma stem like cells (GSC) forming intratumoral heterogeneity within the GBM acquired the microenvironmental adaptation by inter-exosomal contents exchange between heterogenic cells. In addition, GSC has an invasive potential as like human GBM. Therefore, we investigate whether exosomal proteins of GSC affect the normal tissue invasion in GBM. Material and Methods Exosomes were isolated by Size-Exclusion method from conditioned media and validated by Electron microscope and Immunoblot assay. Exosomal proteomics were examined with Liquid Chromatography-Mass Spectrometry (LC/MS). To produce the fluorescent exosome, bi-cistron vectors were cloned with shRNA and CD63-GFP. To identify the effect of tranfected exosome, the isolated exosomes were treated to recipient cells and examined the invasion by 3D invasion assay and mouse intracranial model. Results Firstly, we dichotomized two groups following tumor invasion at matrigel assay and GSC derived orthotopic mouse model. CSC2 and X01 GSCs revealed highly invasive phenotype whereas 83NS and 528NS GSCs did not. Exosome was isolated in each group and identified by CD63 expression or electron microscopy. In proteomics analysis, hypoxia, extracellular matrix organization, GTPase cycle related proteins were enriched in highly invasive cell’s exosome. Among them, we focused the carbonic anhydrase IX (CA9) and the epithelial membrane protein 2 (EMP2) on its permissive role to glioblastoma invasion respectively. CA9 and EMP2 mRNA and protein levels were verified in GSCs and their exosomes and the high expression levels were detected in CSC2 and X01 compared to the low one in 83NS and 528NS GSCs. To evaluate the effects of CA9 and EMP2 on exosome mediated invasion potential, viral bi-cistron vectors was composed with the target gene knockdown and the CD63 fluorescence was used to detect intracellular exosome transfer. Interestingly, the decreased expression of phosphorylated FAK, a key invasive marker, was observed after Lentiviral mediated CA9- and EMP2-knockdown in highly invasive CSC2. To identify whether CA9 and EMP2 proteins are the intracellular effector protein responsible for exosome mediated glioma invasion, the donor exosomes (Exo-CSC2-sh-CA9 and Exo-CSC2-EMP2, after Lentiviral transfection to CSC2s) were isolated and treated to the non invasive 528NS cells as recipient cells. In 3D invasion assay, Exo-CSC2-shCA9 or Exo-CSC2-shEMP2 mediated tumor invasion was significantly decreased at 528NS GSCs compared to Exo-CSC2-shEV. These features were found at mouse intracranial model as well. Conclusion Together with these, we conclude that exosome derived from GSCs induces a transition of invasive phenotype via transfer of EMP2 and CA9 proteins.

Glioblastoma CD105+ cells define a SOX2− cancer stem cell-like subpopulation in the pre-invasive niche

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. Glioma stem like cells (GSC) represent the highest cellular hierarchy in GBM and have a determining role in tumor growth, recurrence and patient prognosis. However, a better definition of GSC subpopulations, especially at the surgical resection margin, is warranted for improved oncological treatment options. The present study interrogated cells expressing CD105 (CD105+) specifically within the tumor front and the pre-invasive niche as a potential GSC subpopulation. GBM primary cell lines were generated from patients (n = 18) and CD105+ cells were isolated and assessed for stem-like characteristics. In vitro, CD105+ cells proliferated and enriched in serum-containing medium but not in serum-free conditions. CD105+ cells were characterized by Nestin+, Vimentin+ and SOX2−, clearly distinguishing them from SOX2+ GCS. GBM CD105+ cells differentiated into osteocytes and adipocytes but not chondrocytes. Exome sequencing revealed that GBM CD105+ cells matched 83% of somatic mutations in the Cancer cell line encyclopedia, indicating a malignant phenotype and in vivo xenotransplantation assays verified their tumorigenic potential. Cytokine assays showed that immunosuppressive and protumorigenic cytokines such as IL6, IL8, CCL2, CXCL-1 were produced by CD105+ cells. Finally, screening for 88 clinical drugs revealed that GBM CD105+ cells are resistant to most chemotherapeutics except Doxorubicin, Idarubicin, Fludarabine and ABT-751. Our study provides a rationale for targeting tumoral CD105+ cells in order to reshape the tumor microenvironment and block GBM progression.

Exosomal miR-155-5p derived from glioma stem-like cells promotes mesenchymal transition via targeting ACOT12

Tumor-associated exosomes play essential roles in intercellular communication and the foundation of cancer microenvironment in glioma. Many mRNAs, microRNAs (miRNAs) and proteins contained in tumor-associated exosomes can be transferred to recipient cells and contribute to the progression of tumor. Nevertheless, the cellular communication between malignant cells with different heterogeneities or characteristics and resultant tumor progression are still unclear in glioma. Here, we show that exosomes released from glioma stem-like cells (GSCs) contain a significant increasing level of miR-155-5p and could be horizontally transferred to surrounding glioma cells. High expression of miR-155-5p in plasma exosomes from patients was associated with glioma diagnosis and grading. Mechanically, we found that miR-155-5p markedly reduced the expression of acetyl-CoA thioesterase 12 (ACOT12), which played as a tumor suppressor in glioma. Furthermore, mesenchymal transition was significantly promoted in glioma cells treated with GSCs-derived exosomes. In conclusion, GSCs-derived exosomal miR-155-5p play a critical role in glioma progression and facilitating tumor aggressive growth by targeting ACOT12 and promoting mesenchymal transition. Exosomal miR-155-5p is also a potential predictive biomarker for glioma, which may provoke the development of novel diagnostic and therapeutic strategies against glioma.