Glioblastoma Stem-like Cells Research Articles

The Antitumor Natural Compound Falcarindiol Disrupts Neural Stem Cell Homeostasis by Suppressing Notch Pathway.

Neural stem cells (NSCs) are undifferentiated, multi-potent cells that can give rise to functional neurons and glial cells. The disruption in NSC homeostasis and/or the impaired neurogenesis lead to diverse neurological diseases, including depression, dementia, and neurodegenerative disorders. Falcarindiol (FAD) is a polyacetylene found in many plants, and FAD shows the cytotoxicity against breast cancers and colon cancers. However, there is no research on the consequence of FAD treatment in normal stem cells. Here, we suggest that FAD has anticancer roles against glioblastoma cells by inducing the differentiation of glioblastoma stem-like cells, as well as activating apoptosis pathway in glioblastoma cells. On the other hand, we also show that FAD has detrimental effects by disrupting the maintenance of normal NSCs and altering the balance between self-renewal and differentiation of NSCs.

Kr\xfcppel-like factor 9 and histone deacetylase inhibitors synergistically induce cell death in glioblastoma stem-like cells

BackgroundThe dismal prognosis of patients with glioblastoma (GBM) is attributed to a rare subset of cancer stem cells that display characteristics of tumor initiation, growth, and resistance to aggressive treatment involving chemotherapy and concomitant radiation. Recent research on the substantial role of epigenetic mechanisms in the pathogenesis of cancers has prompted the investigation of the enzymatic modifications of histone proteins for therapeutic drug targeting. In this work, we have examined the function of Krüppel-like factor 9 (KLF9), a transcription factor, in chemotherapy sensitization to histone deacetylase inhibitors (HDAC inhibitors).MethodsSince GBM neurosphere cultures from patient-derived gliomas are enriched for GBM stem-like cells (GSCs) and form highly invasive and proliferative xenografts that recapitulate the features demonstrated in human patients diagnosed with GBM, we established inducible KLF9 expression systems in these GBM neurosphere cells and investigated cell death in the presence of epigenetic modulators such as histone deacetylase (HDAC) inhibitors.ResultsWe demonstrated that KLF9 expression combined with HDAC inhibitor panobinostat (LBH589) dramatically induced glioma stem cell death via both apoptosis and necroptosis in a synergistic manner. The combination of KLF9 expression and LBH589 treatment affected cell cycle by substantially decreasing the percentage of cells at S-phase. This phenomenon is further corroborated by the upregulation of cell cycle inhibitors p21 and p27. Further, we determined that KLF9 and LBH589 regulated the expression of pro- and anti- apoptotic proteins, suggesting a mechanism that involves the caspase-dependent apoptotic pathway. In addition, we demonstrated that apoptosis and necrosis inhibitors conferred minimal protective effects against cell death, while inhibitors of the necroptosis pathway significantly blocked cell death.ConclusionsOur findings suggest a detailed understanding of how KLF9 expression in cancer cells with epigenetic modulators like HDAC inhibitors may promote synergistic cell death through a mechanism involving both apoptosis and necroptosis that will benefit novel combinatory antitumor strategies to treat malignant brain tumors.

R-spodin2 enhances canonical Wnt signaling to maintain the stemness of glioblastoma cells

BackgroundAs newly identified Wnt enhancer, R-spondin gene family members have been linked to various cancers; however, their role in isocitrate dehydrogenase-wildtype subtype of human glioblastoma (GBM) cells remains unknown.MethodsHuman U87 and U251 cell lines were used to perform the experiments. GBM stem-like cells were enriched in stem cell growth media and induced to differentiate using retinoid acid or growth factor deprivation. Wnthigh and Wntlow subpopulations were isolated and evaluated by MTS, sphere formation, transwell migration and xenograft formation assays.ResultsR-spondin 2 but not R-spondin 3 potentiates Wnt/β-catenin signaling in GBM cell lines. While R-spondin 2 does not affect cell growth, it induces the expression of pluripotent stem cell markers in combination with Wnt3A. GBM stem-like cells are endowed with intrinsic high activity of β-catenin signaling, which can be further intensified by R-spondin 2. In addition, R-spondin2 promotes stem cell self-renewal and suppresses retinoid acid- or growth factor deprivation-induced differentiation, indicating R-spondin 2 maintains stem cell traits in GBM. On the other hand, we identify subpopulations of GBM cells that show distinctive responsiveness to Wnt/β-catenin signaling. Interestingly, Wnthigh and Wntlow cells display distinctive biologic properties. Moreover, Wnthigh cell-inoculated xenografts exhibit enhanced tumorigenicity and increased expression levels of R-spondin 2 compared to Wntlow cell-inoculated xenografts.ConclusionOur study reveals a novel regulatory mechanisms underlying the over-activation of β-catenin-mediated signaling in the pathogenesis of GBM.

Proscillaridin A exerts anti-tumor effects through GSK3\u03b2 activation and alteration of microtubule dynamics in glioblastoma

Glioblastoma (GBM) is characterized by highly aggressive growth and invasive behavior. Due to the highly lethal nature of GBM, new therapies are urgently needed and repositioning of existing drugs is a promising approach. We have previously shown the activity of Proscillaridin A (ProA), a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase (NKA) pump, against proliferation and migration of GBM cell lines. ProA inhibited tumor growth in vivo and increased mice survival after orthotopic grafting of GBM cells. This study aims to decipher the mechanism of action of ProA in GBM tumor and stem-like cells. ProA displayed cytotoxic activity on tumor and stem-like cells grown in 2D and 3D culture, but not on healthy cells as astrocytes or oligodendrocytes. Even at sub-cytotoxic concentration, ProA impaired cell migration and disturbed EB1 accumulation at microtubule (MT) plus-ends and MT dynamics instability. ProA activates GSK3β downstream of NKA inhibition, leading to EB1 phosphorylation on S155 and T166, EB1 comet length shortening and MT dynamics alteration, and finally inhibition of cell migration and cytotoxicity. Similar results were observed with digoxin. Therefore, we disclosed here a novel pathway by which ProA and digoxin modulate MT-governed functions in GBM tumor and stem-like cells. Altogether, our results support ProA and digoxin as potent candidates for drug repositioning in GBM.

RND1 regulates migration of human glioblastoma stem-like cells according to their anatomical localization and defines a prognostic signature in glioblastoma.

Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ–). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, RND1 in PVZ GSCs compared to CT GSCs. Overexpression of RND1, dramatically inhibited PVZ GSC migration and conversely, downregulation of RND1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of RND1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of RND1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of RND1, we identified an RND1low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the RND1low signature as key targets genes to impede tumor relapse after treatment.

FK506 Attenuates the MRP1-Mediated Chemoresistant Phenotype in Glioblastoma Stem-Like Cells.

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

MRI tracking of polyethylene glycol-coated superparamagnetic iron oxide-labelled placenta-derived mesenchymal stem cells toward glioblastoma stem-like cells in a mouse model

Mesenchymal stem cells (MSCs) that display homing and infiltration properties towards tumor cells are a promising cellular targeting vector for brain tumor therapy but are limited to local-regional delivery in current preclinical models. Here, we investigated whether placenta-derived MSCs (P-MSCs) are a superior cellular vector for systemic targeting of glioblastoma stem-like cells (GSCs), with an imaging modality to real-time monitor the trafficking P-MSCs to glioblastoma sites. Results demonstrated that P-MSCs had greater migratory activity towards GSCs and across blood–brain barrier compared with bone marrow-derived MSCs, and this activity was enhanced by hypoxia precondition. Chemokine ligand 5 was identified as a chemoattractant responsible for the glioblastoma tropism of P-MSCs. Polyethylene glycol-coated superparamagnetic iron oxide (PEG–SPIO) was synthesized for cellular labelling and imaging P-MSCs, displaying high cellular uptake and no cytotoxic effect on P-MSCs cell proliferation or stemness property. The homing effects of intravenously administered PEG–SPIO-labelled P-MSCs towards intracerebral GSCs were able to be detected in mice models through T2-weighted magnetic resonance imaging (MRI). This study suggests the possibility of innovative systemic P-MSC-based cell therapy for aggressive GSCs, developing a state-of-the-art theranostic technique for real-time tracking of therapeutic P-MSCs tumor infiltration through cellular MRI.

Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells.

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with "replication factories" and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro These data identify RS as a cancer stem cell-specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060-71. ©2018 AACR.

Combined HDAC and Bromodomain Protein Inhibition Reprograms Tumor Cell Metabolism and Elicits Synthetic Lethality in Glioblastoma.

Purpose: Glioblastoma remains a challenge in oncology, in part due to tumor heterogeneity.Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary model systems.Results: Based on a transcriptome and subsequent gene set enrichment analysis (GSEA), we show by using clinically validated compounds that the combination of histone deacetylase (HDAC) inhibition and bromodomain protein (BRD) inhibition results in pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like glioblastoma cells. Transcriptome-based GSEA analysis suggests that metabolic reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an integrated stress response, originating from the endoplasmic reticulum. This results in an increase in proapoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of antiapoptotic Mcl-1 protein. Pharmacologic, utilizing the FDA-approved drug sorafenib, and genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor growth, and that triple therapy involving the clinically validated compounds panobinostat, OTX015, and sorafenib further enhances these effects, culminating in a significant regression of tumors in vivoConclusions: Overall, these results warrant clinical testing of this novel, efficacious combination therapy. Clin Cancer Res; 24(16); 3941-54. ©2018 AACR.

FGFR1/FOXM1 pathway: a key regulator of glioblastoma stem cells radioresistance and a prognosis biomarker

Glioblastoma are known to be aggressive and therapy-resistant tumors, due to the presence of glioblastoma stem cells inside this heterogeneous tumor. We investigate here the involvement of FGFR1 in glioblastoma stem-like cells (GSLC) radioresistance mechanisms. We first demonstrated that the survival after irradiation was significantly diminished in FGFR1-silenced (FGFR1-) GSLC compared to control GSLC. The transcriptome analysis of GSLCs FGFR1(-) showed that FOX family members are differentially regulated by FGFR1 inhibition, particularly with an upregulation of FOXN3 and a downregulation of FOXM1. GSLC survival after irradiation was significantly increased after FOXN3 silencing and decreased after FOXM1 inhibition, showing opposite effects of FGFR1/FOX family members on cell response to ionizing radiation. Silencing FGFR1 or FOXM1 downregulated genes involved in mesenchymal transition such as GLI2, TWIST1, and ZEB1 in glioblastoma stem-like cells. It also dramatically reduced GSLC migration. Databases analysis confirmed that the combined expression of FGFR1/FOXM1/MELK/GLI2/ZEB1/TWIST1 is significantly associated with patients overall survival after chemo-radiotherapy treatment. All these results, associated with our previous conduced ones with differentiated cells, clearly established that FGFR1-FOXM1 dependent glioblastoma stem-like cells radioresistance pathway is a central actor of GBM treatment resistance and a key target to inhibit in the aim to increase the sensitivity of GBM to the radiotherapy.

Inhibition of autophagy increases susceptibility of glioblastoma stem cells to temozolomide by igniting ferroptosis

The role of autophagy in cancer onset and progression appears still controversial. On one hand, autophagy allows cancer cell to survive in unfavorable environmental conditions, on the other hand, once internal energy resources are exhausted, it leads to cell death. In addition, autophagy interpheres with cell cycle progression, de facto exerting a cytostatic activity. Hence, it represents an important target for anticancer therapy. For example, temozolomide (TMZ), of use for glioblastoma (GBM) treatment, appears as capable of inducing autophagy partially inhibiting cancer cell proliferation. However, GBM, a very aggressive brain tumor with poor prognosis even after surgery and radio-chemotherapy, invariably recurs and leads to patient death. Since cancer stem cells have been hypothesized to play a role in refractory/relapsing cancers, in the present work we investigated if autophagy could represent a constitutive cytoprotection mechanism for glioblastoma stem-like cells (GSCs) and if the modulation of autophagic process could affect GBM growth and survival. Thus, in the present study we first evaluated the relevance of autophagy in GBM tumor specimens, then its occurrence in GSCs and, finally, if modulation of autophagy could influence GSC response to TMZ. Our results suggested that, in vitro, the impairing autophagic process with quinacrine, a compound able to cross the blood-brain barrier, increased GSC susceptibility to TMZ. Death of GSCs was apparently due to the iron dependent form of programmed cell death characterized by the accumulation of lipid peroxides called ferroptosis. These results underscore the relevance of the modulation of autophagy in the GSC survival and death and suggest that triggering of ferroptosis in GSCs could represent a novel and important target for the management of glioblastoma.

The XPO1 Inhibitor Selinexor Inhibits Translation and Enhances the Radiosensitivity of Glioblastoma Cells Grown In Vitro and In Vivo.

Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSC) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of clinically relevant XPO1 inhibitor selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. On the basis of γH2AX foci and neutral comet analyses, selinexor inhibited the repair of radiation-induced DNA double-strand breaks in GSCs, suggesting that the selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to selinexor and radiation. Treatment of mice bearing orthotopic xenografts with selinexor decreased tumor translational efficiency as determined from polysome profiles. Although selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves the global inhibition of gene translation. Mol Cancer Ther; 17(8); 1717-26. ©2018 AACR.

Restriction of Replication of Oncolytic Herpes Simplex Virus with a Deletion of γ34.5 in Glioblastoma Stem-Like Cells.

Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the γ34.5 genes responsible for neurovirulence. However, loss of γ34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to replication and cell killing by oHSV with deletion of the γ34.5 genes (γ34.5- oHSV), while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV-1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after γ34.5- oHSV infection of GSCs but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2α and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of γ34.5- oHSV. The difference in degrees of permissivity between GSCs and ScGCs to γ34.5- oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer-selective replication and oncolytic activity. γ34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are nonpermissive for γ34.5- HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of γ34.5- HSV. We have identified a cell type-specific innate response to HSV-1 that limits oncolytic activity in glioblastoma.

Combinatorial Effects of VEGFR Kinase Inhibitor Axitinib and Oncolytic Virotherapy in Mouse and Human Glioblastoma Stem-Like Cell Models.

Purpose: Glioblastoma (GBM), a fatal brain cancer, contains a subpopulation of GBM stem-like cells (GSCs) that contribute to resistance to current therapy. Angiogenesis also plays a key role in GBM progression. Therefore, we developed a strategy to target the complex GBM microenvironment, including GSCs and tumor vasculature.Experimental Design: We evaluated the cytotoxic effects of VEFGR tyrosine kinase inhibitor (TKI) axitinib in vitro and then tested antitumor efficacy of axitinib in combination with oncolytic herpes simplex virus (oHSV) expressing antiangiogenic cytokine murine IL12 (G47Δ-mIL12) in two orthotopic GSC-derived GBM models: patient-derived recurrent MGG123 GSCs, forming vascular xenografts in immunodeficient mice; and mouse 005 GSCs, forming syngeneic tumors in immunocompetent mice.Results: GSCs form endothelial-like tubes and were sensitive to axitinib. G47Δ-mIL12 significantly improved survival, as did axitinib, while dual combinations further extended survival significantly compared with single therapies alone in both models. In MGG123 tumors, axitinib was effective only at high doses (50 mg/kg), alone and in combination with G47Δ-mIL12, and this was associated with greatly decreased vascularity, increased macrophage infiltration, extensive tumor necrosis, and PDGFR/ERK pathway inhibition. In the mouse 005 model, antiglioma activity, after single and combination therapy, was only observed in immunocompetent mice and not the T-cell-deficient athymic mice. Interestingly, immune checkpoint inhibition did not improve efficacy.Conclusions: Systemic TKI (axitinib) beneficially combines with G47Δ-mIL12 to enhance antitumor efficacy in both immunodeficient and immunocompetent orthotopic GBM models. Our results support further investigation of TKIs in combination with oHSV for GBM treatment. Clin Cancer Res; 24(14); 3409-22. ©2018 AACR.

BMP9 counteracts the tumorigenic and pro-angiogenic potential of glioblastoma.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor, with a strong ability to disseminate and invade the surrounding parenchyma. In addition, a subpopulation of GBM stem cells has been reported to possess the ability to transdifferentiate into tumor-derived endothelial cells (TDECs), supporting the resistance to anti-angiogenic treatments of newly formed blood vessels. Bone Morphogenetic Protein 9 (BMP9) is critically involved in the processes of cancer cell differentiation, invasion and metastasis, representing a potential tool in order to impair the intrinsic GBM aggressiveness. Here we demonstrate that BMP9 is able to trigger the activation of SMADs in patient-derived GBM cells, and to strongly inhibit proliferation and invasion by reducing the activation of PI3K/AKT/MAPK and RhoA/Cofilin pathways, respectively. Intriguingly, BMP9 treatment is sufficient to induce a strong differentiation of GBM stem-like cells and to significantly counteract the already reported process of GBM cell transdifferentiation into TDECs not only in in vitro mimicked TDEC models, but also in vivo in orthotopic xenografts in mice. Additionally, we describe a strong BMP9-mediated inhibition of the whole angiogenic process engaged during GBM tumor formation. Based on these results, we believe that BMP9, by acting at multiple levels against GBM cell aggressiveness, can be considered a promising candidate, to be further developed, for the future therapeutic management of GBM.

Abstract 4171: Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb

Abstract Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness and migration. Using a GSC orthotopic brain tumor model, we tested the hypothesis that location within the brain microenvironment influences the radiosensitivity of GBM cells. At 35 days after implantation of CD133+ NSC11 GSCs into the right striatum of nude mice, tumor cells are detectable throughout the striatum, the corpus callosum and the olfactory bulb. To evaluate radioresponse of tumor cells in each brain region, mice were injected with the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase and allows for the identification of proliferating cells. Specifically, NSC11 brain tumor xenografts received a single dose of 10Gy, which results in an approximate 13-day survival advantage, followed by 3 daily intraperitoneal (IP) injections of CldU initiated every 4 days following irradiation. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the corpus callosum, striatum, and olfactory bulb. When CldU injections were initiated 4 days post-10Gy, 4% of tumor cells in the corpus callosum, 0.75% in the striatum, and 35% in the olfactory bulb were CldU positive. These values can be compared to 55%, 53%, and 56% CldU positive tumor cells in the corpus callosum, striatum, and olfactory bulb, respectively, of the sham irradiated tumors. These data indicate that by 4 days after irradiation significantly more tumor cells in the olfactory bulb were proliferating than in the corpus callosum or striatum. CldU injections initiated 12 days post-10Gy resulted in 7% of tumor cells in the corpus callosum, 4% in the striatum, and 60% in the olfactory bulb being CldU positive. Finally, when CldU delivery was begun 20 days post-10Gy approximately 17% of tumor cells in the corpus callosum, 15% in the striatum, and 85% in the olfactory bulb were CldU positive. These data suggest that after irradiation, GBM tumor cells located in the olfactory bulb recover and begin to proliferate sooner than those in the striatum or corpus callosum. As a measure of radiation-induced DNA double strand breaks, γH2AX foci were quantified in the tumor cells located in the corpus callosum, striatum, and olfactory bulb on day 35 post-implantation of NSC11 cells. At 0.5h after irradiation, no difference was detected in the mean number of γH2AX foci per tumor cell in the corpus callosum, striatum, or olfactory bulb; no consistent differences were detected out to 6h post-irradiation. While the mechanism remains to be defined, analysis of tumor cell proliferation indicates a preferential radioresistance of GBM cells located in the olfactory bulb. Citation Format: Cindy R. Timme, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4171.

Abstract 4444: The XPO1 inhibitor Selinexor attenuates global translation and enhances the radiosensitivity of glioblastoma cells grown in vitro and in vivo

Abstract Radiation therapy remains a primary treatment modality for glioblastoma (GBM). To identify novel targets for GBM radiosensitization, the radiation-induced translatome was defined for a series of glioblastoma stem-like cells (GSCs). Analysis of the radiation-induced translatome of GSCs identified an interacting network in which Exportin 1 (XPO1) serves as a major hub protein. XPO1 transports a variety of proteins and RNAs from the nucleus to the cytoplasm via the nuclear pore complex and is dysregulated in multiple types of cancers, including GBM. To determine whether XPO1 provides a target for GBM radiosensitization, we defined the effects of the clinically relevant XPO1-inhibitor Selinexor (KPT-330) on the radiosensitivity of GSC lines. Using clonogenic survival analysis, Selinexor was shown to enhance the in vitro radiosensitivity of GSCs but not normal fibroblast cell lines. Because XPO1 mediates the nuclear export of ribosomal subunits, we tested the effect of Selinexor on gene translation using polysomes profiles to calculate translational efficiency and O-propargyl-puromycin (OPP) incorporation to measure de novo protein synthesis. Selinexor significantly reduced translational efficiency in GSCs beginning at 1-6h after treatment, which was accompanied by a decrease in protein synthesis. Moreover, Selinexor exposure increased in 18S and 5S rRNA accumulation in the nucleus with corresponding decreases in the cytoplasm suggesting that the inhibition of rRNA export plays a role in the Selinexor-mediated decrease in protein synthesis. In contrast to the tumor cells, Selinexor had no effect on protein synthesis in normal fibroblast cell lines and astrocytes. To determine the effect of XPO1 inhibition on GBM cells in vivo, mice bearing orthotopic xenografts initiated from a GSC line (NSC11) were treated with Selinexor (20 mg/kg) and tumors were collected over 48 hours. Polysome profiles generated from these orthotopic xenografts indicated that Selinexor treatment resulted in a decrease in translational efficiency with the greatest effect at 24 hours after treatment. These results suggest that consistent with in vitro data, Selinexor inhibits global translation in GBM cells grown under orthotopic, in vivo conditions. To determine the effect of XPO1 inhibition on radioresponse in vivo, mice bearing orthotopic xenografts were treated with Selinexor in combination with fractionated radiation. Whereas Selinexor alone had no effect on survival, the XPO1 inhibitor enhanced radiation-induced prolongation of animal survival with an apparent dose enhancement factor of 2. These results suggest that Selinexor delivered in combination with radiotherapy may improve the effectiveness of GBM treatment. Citation Format: Amy Wahba, John W. O'Neill, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. The XPO1 inhibitor Selinexor attenuates global translation and enhances the radiosensitivity of glioblastoma cells grown in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4444.

Abstract 413: Emerging principles in synthetic lethality in glioblastoma

Abstract Synthetic lethality occurs when mutations in two otherwise nonessential genes are combined to cause lethality. Because cancer is a disease of genetic alteration, synthetic lethality has been heralded as a method to identity candidate therapeutic targets, e.g., where a target gene could be "synthetic lethal" with a cancer driver mutation. To define synthetic lethal relationships in glioblastoma (GBM), we have performed multiple focused-set and genome-wide CRISPR-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs) and nontransformed human neural progenitor cells. Because GSCs isolates likely represent a sub-clone of the original tumor and we can determine GSCs' genetic and epigenetic makeup, it is possible to address the concept of synthetic lethality for GBM. To this end, we recently performed comprehensive CRISPR-Cas9 retests of all scoring GBM lethal genes (>900) from screens in three patient isolates with different and overlapping genetic drivers. We then performed secondary retests of high-priority gene targets in 13 GSC harboring various alterations commonly found in GBMs, e.g., EGFRamp, NF1mut, PIK3CAmut, PTENloss/mut, TP53mut, etc. The results were surprising, first in what we did not find. We failed to find synthetic lethal targets for TP53loss/mut, RB1mut, or TERT expression, suggesting that synthetic lethal relationships for these alterations may not exist for GBM. Second, NF1mut interactors defined a broader class of synthetic lethal targets with general overactivity of the RTK/Ras pathway, which can arise from various activating lesions. Third, candidate synthetic lethal relationships can be observed, but, so far, only with EGFRamp, MYC/MYCNamp, and PTEN/PIK3CA alterations. Thus, in general our results suggest that the majority of synthetic lethal relationships in GBM arise from oncogenic activation of the RTK/Ras and PI-3 kinase pathway or amplification of MYC/MYCN. (Synthetic lethal targets will be revealed and discussed at the meeting.) Citation Format: Pia Hoellerbauer, Sonali Arora, Megan Kufeld, Lucas Carter, Emily J. Girard, Heather Feldman, Philip Corrin, James M. Olson, Patrick J. Paddison. Emerging principles in synthetic lethality in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 413.

Abstract 1906: PFKFB4, much more than just a glycolytic gene

Abstract Glioblastoma is one of the most aggressive primary brain tumors in adults, with a dismal median overall survival of only 14 months after diagnosis. Glioblastoma stem-like cells (GSCs) are particularly resistant to current therapies, capable of self-renewal and tumour initiation and are hence thought to be major contributors to patient relapse. A kinome and phosphatome wide screen revealed glycolysis gene 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) as an important candidate gene for GSC survival. Notably, silencing PFKFB4 in an orthotopic xenograft mouse model of glioblastoma completely rid the mice of the tumor. Here we show a brand new function of PFKFB4, independent of its role in glycolysis - namely in the regulation of HIF1α, which is upregulated in GSCs. Gene expression profiling revealed a downregulation in HIF1α target genes in PFKFB4-silenced GSCs, and HIF1α protein levels are also dramatically reduced upon silencing. Mass spectrometric analysis of immunoprecipitated PFKFB4 protein revealed a novel interaction partner, F-box only protein 28 (FBXO28), an E3 ubiqutin ligase. Co-immunoprecipation assays show that FBXO28 forms an SCF multi-protein ubiquitin ligase complex with CUL1 and SKP1. Ubiquitylation studies of HIF1α show that PFKFB4 stabilizes this crucial protein in GSCs by preventing its targeting by FBXO28 for proteasomal degradation. These new findings, coupled with its cancer specific expression in a variety of tumor entities, makes PFKFB4 a compelling target. Citation Format: Emma L. Phillips, Frederic Bethke, Jörg Balss, Stefan Pusch, Stefan Christen, Martina Schnölzer, Antje Habel, David Capper, Andreas Von Deimling, Sarah-Maria Fendt, Peter Lichter, Violaine Goidts. PFKFB4, much more than just a glycolytic gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1906.

Abstract LB-328: Influence of radiation on the evolution of orthotopic xenografts initiated from glioblastoma stem-like cells

Abstract Glioblastoma (GBM) is the most common and malignant primary adult brain cancer with a median survival of 15 months despite treatment with surgical resection followed by chemo-radiotherapy. By examining changes that occur following irradiation of brain tumor xenografts initiated from glioma stem-like cells (GSCs), this study seeks to better understand the impact of radiotherapy on GBM evolution. Therefore, we intracranially implanted CD133+ NSC11 cells, a well-documented GSC line, into nude mice. After 21 days, bioluminescence imaging was performed to confirm the presence of tumor prior to randomization into control and radiation therapy groups (3x5Gy). Following treatment, brain samples were collected at various time points out to morbidity to investigate changes in tumor morphology and histology. Further, tumors from morbid mice (control and 3x5Gy) were collected for viral integration site analysis (VISA). Initial survival analysis demonstrated a significant survival advantage for mice undergoing radiation therapy (+34.2 days) compared to controls. On gross examination of morphology at morbidity, brains bearing irradiated tumors contained tumor tissue that was softer, more adherent to the skull, and more likely to efface olfactory bulb(s) than control tumor brains. H&E staining demonstrated that irradiated tumors were more dense/compact and did not grow as infiltrative as tumors from control mice. These histological changes were followed up with VISA to determine whether certain clones were responsible for this difference. VISA revealed that irradiated tumors were heterogeneously enriched for clones with different integration sites when compared to control tumors and in vitro tumor lines. Heat map analysis of the number of clones present in each sample demonstrated that control tumors harbor fewer clones than in vitro lines and that radiated tumors harbor the fewest clones of all. Initial data also revealed that various doses of radiation (3x5Gy or 10x5Gy) can provide very similar selection pressures to limit the number of clones. Our results demonstrate that radiation, a treatment component for almost all glioblastoma patients, can have wide-ranging effects on the evolution of this dynamic tumor. In particular, the pressures imposed by radiation treatment seem to lead to the selection of a reduced number of clones. This selection may have future implications for tumor evolution and the treatment of recurrent GBM. Citation Format: Joseph H. McAbee, Barbara H. Rath, Kevin A. Camphausen, Philip J. Tofilon. Influence of radiation on the evolution of orthotopic xenografts initiated from glioblastoma stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-328.