Orthotopic Glioblastoma Xenografts Research Articles

EZH2 functional dichotomy in reactive oxygen species-stratified glioblastoma.

EZH2, well-known for its canonical methyltransferase activity in transcriptional repression in many cancers including glioblastoma (GBM), has an understudied non-canonical function critical for sustained tumor growth. Recent GBM consortial efforts reveal complex molecular heterogeneity for which therapeutic vulnerabilities correlated with subtype stratification remain relatively unexplored. Current enzymatic EZH2 inhibitors (EZH2inh) targeting its canonical SET domain show limited efficacy and lack durable response, suggesting that underlying differences in the non-canonical pathway may yield new knowledge. Here, we unveiled dual roles of the EZH2 CXC domain in therapeutically-distinct, reactive oxygen species (ROS)-stratified tumors. We analyzed differentially expressed genes between ROS classes by examining cis-regulatory elements as well as clustering of activities and pathways to identify EZH2 as the key mediator in ROS-stratified cohorts. Pull-down assays and CRISPR knockout of EZH2 domains were used to dissect the distinct functions of EZH2 in ROS-stratified GBM cells. The efficacy of NF-κB-inducing kinase inhibitor (NIKinh) and standard-of-care temozolomide was evaluated using orthotopic patient-derived GBM xenografts. In ROS(+) tumors, CXC-mediated co-interaction with RelB drives constitutive activation of non-canonical NF-κB2 signaling, sustaining the ROS(+) chemoresistant phenotype. In contrast, in ROS(-) subtypes, PRC2 methyltransferase activity represses canonical NF-κB. Addressing the lack of EZH2inh targeting its non-methyltransferase roles, we utilized a brain-penetrant NIKinh that disrupts EZH2-RelB binding, consequently prolonging survival in orthotopic ROS(+)-implanted mice. Our findings highlight the functional dichotomy of the EZH2 CXC domain in governing ROS-stratified therapeutic resistance, thereby advocating for the development of therapeutic approaches targeting its non-canonical activities and underscoring the significance of patient stratification methodologies.

Fiber Optic-Mediated Type I Photodynamic Therapy of Brain Glioblastoma Based on an Aggregation-Induced Emission Photosensitizer.

Glioblastoma (GBM) is one of the most lethal human malignancies. The current standard-of-care is highly invasive with strong toxic side effects, leading to poor prognosis and high mortality. As a safe and effective clinical approach, photodynamic therapy (PDT) has emerged as a suitable option for GBM. Nevertheless, its implementation is significantly impeded by the limits of light penetration depth and the firm reliance on oxygen. To overcome these challenges, herein, a promising strategy that harnesses a modified optical fiber and less oxygen-dependent Type I aggregation-induced emission (AIE) photosensitizer (PS) is developed for the first time to realize in vivo GBM treatments. The proposed AIE PS, namely TTTMN, characterized by a highly twisted molecular architecture and a bulky spacer, exhibits enhanced near-infrared emission and strong production of hydroxyl and superoxide radicals at the aggregated state, thus affording efficient fluorescence imaging-guided PDT once formulated into nanoparticles. The inhibition of orthotopic and subcutaneous GBM xenografts provides compelling evidence of the treatment efficacy of Type I PDT irradiated through a tumor-inserted optical fiber. These findings highlight the substantially improved therapeutic outcomes achieved through fiber optic-mediated Type I PDT, positioning it as a promising therapeutic modality for GBM.

EGFR and EGFRvIII coopt host defense pathways, promoting progression in glioblastoma.

Co-amplification of EGFR and EGFRvIII, a tumor-specific truncation mutant of EGFR, represent hallmark genetic lesions in glioblastoma. We used phospho-proteomics, RNA-sequencing, TCGA data and glioblastoma cell culture and mouse models to study the signal transduction mediated by EGFR and EGFRvIII. We report that EGFR and EGFRvIII stimulate the innate immune defense receptor Toll-like Receptor 2 (TLR2); and that knockout of TLR2 dramatically improved survival in orthotopic glioblastoma xenografts. EGFR and EGFRvIII activated TLR2 in a ligand-independent manner, promoting tumor growth and immune evasion. We show that EGFR and EGFRvIII cooperate to activate the Rho-associated protein kinase ROCK2, which modulated malignant progression both by activating TLR2 and WNT signaling, and through remodeling the tumor microenvironment. Together, our findings show that EGFR and EGFRvIII cooperate to drive tumor progression through ROCK2 and downstream WNT-β-catenin/TLR2 signaling pathways.

Long non-coding RNA lung cancer-associated transcript-1 promotes glioblastoma progression by enhancing Hypoxia-inducible factor 1 alpha activity.

Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia. lncRNAs induced by hypoxia in GSCs were identified by RNA-seq. Lung cancer-associated transcript-1 (LUCAT1) expression was assessed by qPCR, RNA-seq, Northern blot, single molecule FISH in GSCs, and interrogated in IvyGAP, The Cancer Genome Atlas, and CGGA databases. LUCAT1 was depleted by shRNA, CRISPR/Cas9, and CRISPR/Cas13d. RNA-seq, Western blot, immunohistochemistry, co-IP, ChIP, ChIP-seq, RNA immunoprecipitation, and proximity ligation assay were performed to investigate mechanisms of action of LUCAT1. GSC viability, limiting dilution assay, and tumorigenic potential in orthotopic GBM xenograft models were performed to assess the functional consequences of depleting LUCAT1. A new isoform of Lucat1 is induced by Hypoxia inducible factor 1 alpha (HIF1α) and Nuclear factor erythroid 2-related factor 2 (NRF2) in GSCs under hypoxia. LUCAT1 is highly expressed in hypoxic regions in GBM. Mechanistically, LUCAT1 formed a complex with HIF1α and its co-activator CBP to regulate HIF1α target gene expression and GSC adaptation to hypoxia. Depletion of LUCAT1 impaired GSC self-renewal. Silencing LUCAT1 decreased tumor growth and prolonged mouse survival in GBM xenograft models. A HIF1α-LUCAT1 axis forms a positive feedback loop to amplify HIF1α signaling in GSCs under hypoxia. LUCAT1 promotes GSC self-renewal and GBM tumor growth. LUCAT1 is a potential therapeutic target in GBM.

Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production

Glucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved single cysteine compound N-acetylcysteine (NAC) reduced GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Normal brain cells and other cancer cells showed no response to NAC. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. From analysis of the clinical proteomic tumor analysis consortium (CPTAC) database, we found that GBM cells exhibit lower expression of mitochondrial redox enzymes than four other cancers whose proteomic data are available in CPTAC. Knockdown of mitochondrial thioredoxin-2 in lung cancer cells induced NAC susceptibility, indicating the importance of mitochondrial redox enzyme expression in mitigating reductive stress. Intraperitoneal treatment of mice bearing orthotopic GBM xenografts with a two-cysteine peptide induced H2O2 in brain tumors in vivo. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

Dihydroartemisinin suppresses glioma growth by repressing ERRα-mediated mitochondrial biogenesis.

Malignant gliomas are an exceptionally lethal form of cancer with limited treatment options. Dihydroartemisinin (DHA), a sesquiterpene lactone antimalarial compound, has demonstrated therapeutic effects in various solid tumors. In our study, we aimed to investigate the mechanisms underlying the anticancer effects of DHA in gliomas. To explore the therapeutic and molecular mechanisms of DHA, we employed various assays, including cell viability, flow cytometry, mitochondrial membrane potential, glucose uptake and glioma xenograft models. Our data demonstrated that DHA significantly inhibited glioma cell proliferation in both temozolomide-resistant cells and glioma stem-like cells. We found that DHA-induced apoptosis occurred via the mitochondria-mediated pathway by initiating mitochondrial dysfunction before promoting apoptosis. Moreover, we discovered that DHA treatment substantially reduced the expression of the mitochondrial biogenesis-related gene, ERRα, in glioma cells. And the ERRα pathway is a critical target in treating glioma with DHA. Our results also demonstrated that the combination of DHA and temozolomide synergistically inhibited the proliferation of glioma cells. In vivo, DHA treatment remarkably extended survival time in mice bearing orthotopic glioblastoma xenografts. Thus, our findings suggest that DHA has a novel role in modulating cancer cell metabolism and suppressing glioma progression by activating the ERRα-regulated mitochondrial apoptosis pathway.

EXTH-67. LP-184, AN MGMT-AGNOSTIC SMALL MOLECULE, HAS POTENT SYNERGY WITH SPIRONOLACTONE TO EFFECTIVELY INHIBIT ORTHOTOPIC GBM XENOGRAFT TUMORS

Abstract Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to extend survival of patients with newly diagnosed MGMT-methylated glioblastoma (GBM). Blood-brain barrier (BBB) permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT-unmethylated GBM) are needed. Lantern Pharma is advancing LP-184, an acylfulvene prodrug activated by the enzyme PTGR1. In MGMT-expressing TMZ-resistant GBM cultures, LP-184 was found to be ~5000X more potent than TMZ with LP-184 IC50s ranging from 20-200 nM. DNA damage induced by acylfulvenes is primarily repaired via transcription-coupled nucleotide excision repair (TC-NER) mediated by ERCC complexes. ERCC3 is proteolytically degraded in response to the FDA approved BBB permeable diuretic, spironolactone (SP). We found that SP induces rapid ERCC3 degradation by up to 95% in GBM cells and validated that SP-induced ERCC3 loss led to substantial increases in LP-184 responses (DNA damage, tumor cell death) in vitro and in subcutaneous (s.c.) tumor xenografts. Co-treatment of GBM cells with LP-184 and SP resulted in a 3-6 fold decrease in LP-184 IC50s in PDX-derived (M1123 and Mayo39) and cell line (U87) cultures in vitro. Systemic SP sensitized pre-established s.c. and orthotopic (i.c.) GBM xenografts to LP-184 therapy. Pre-established s.c. U87 tumors recurred after initial regression in 5/5 animals following treatment with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals following treatment with LP-184 + SP. Pre-established M1123 i.c. xenografts treated with LP-184 + SP were ~3-fold smaller than those treated with LP-184 alone. LP-184 is a promising agent with potential clinical translation in GBM patients. Our results indicate that LP-184 is MGMT-agnostic and effective in TMZ-resistant preclinical GBM models. The first-in-human LP-184 dose escalation Phase 1A trial enrolling solid tumor patients including GBM is planned to launch Q3 2023.

EXTH-38. VELCRIN MOLECULAR GLUES INDUCE CELL DEATH IN GLIOBLASTOMAS WITH HIGH PDE3A AND SLFN12 EXPRESSION

Abstract Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase, SLFN12, and the phosphodiesterase, PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the Cancer Cell Line Encyclopedia (CCLE), including glioblastoma cell lines. We therefore aimed to characterize velcrins as novel therapeutic agents in glioblastoma. We measured PDE3A and SLFN12 expression levels in commercially available glioblastoma cells as well as glioblastoma neurospheres from the Center for Patient Derived Models at Dana Farber Cancer Institute. We identified velcrin-sensitive glioblastoma cell lines and neurospheres, and determined that velcrin response is highly correlated with PDE3A expression. We also determined that sensitive cells exhibit a reduction in nascent polypeptide formation upon velcrin treatment, which is rescued by CRISPR-induced knockout of PDE3A. To assess velcrin efficacy in vivo, we measured central nervous system and plasma levels of the velcrin, BAY 2666605, and determined that it freely crosses the blood brain barrier. We also determined that twice daily dosing of BAY 2666605 elicits full tumor regression in an orthotopic glioblastoma xenograft model, with sustained complete responses after treatment withdrawal. Lastly, we demonstrated that the velcrins BAY 2666605 and BAY 2501293 induce tumor regression in biomarker-positive subcutaneous glioblastoma patient-derived xenograft models. In summary, our data demonstrates that velcrins have anti-tumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.

Delivery versus Potency in Treating Brain Tumors: BI-907828, a MDM2-p53 Antagonist with Limited BBB Penetration but Significant In Vivo Efficacy in Glioblastoma.

MDM2-p53 inhibition may be effective in glioblastoma (GBM). This study evaluates the pharmacokinetics/pharmacodynamics of BI-907828, a potent antagonist of MDM2, in GBM, and demonstrates a translational paradigm with a focus on a unified "Delivery - Potency - Efficacy" relationship in drug development for central nervous system(CNS) tumors. BI-907828 was tested for cytotoxicity and MDM2-p53 pathway inhibition. Systemic pharmacokinetics and transport mechanisms controlling CNS distribution were evaluated in mice. BI-907828 free fractions in cell media, mouse and human specimens were measured to determine "active" unbound concentrations. Efficacy measures, including overall survival and target expression were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2-p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nmol/L. The CNS distribution of BI-907828 is limited by blood-brain barrier (BBB) efflux mediated by P-gp, resulting in a Kp,uu_brain of 0.002. Despite this seemingly "poor" BBB penetration, weekly administration of 10 mg/kg BI-907828 extended median survival of orthotopic GBM108 xenografts from 28 to 218 days (P < 0.0001). This excellent efficacy can be attributed to high potency, resulting in a limited, yet effective, exposure in the CNS. These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is limited by BBB efflux. Therefore, a comprehensive understanding of all aspects of the "Delivery - Potency - Efficacy" relationship is warranted in drug discovery and development, especially for treatment of CNS tumors.

P06.10.A CAR-ENGINEERED PRIMARY NK CELLS SHOW PROMISING EFFECTS AS TARGETED THERAPY AGAINST EGFR-POSITIVE GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GB) is the most frequent primary brain tumor and despite intensive treatment regimens including surgery, radiotherapy, and chemotherapy, it is still characterized by a dismal prognosis. Immunotherapies such as T cells or natural killer cells (NK) that carry tumor-specific chimeric antigen receptors (CAR) hold promise for the development of more effective treatment strategies. CAR-engineered derivatives of the human NK cell line NK-92 targeting EGFR or EGFRvIII showed potent antitumor activity against glioblastoma cell lines in vitro and in vivo. Nevertheless, NK-92 has some limitations compared to primary NK cells, such as the required irradiation prior to application in humans. MATERIAL AND METHODS To overcome this limitation, we evaluated the suitability of peripheral blood derived human primary NK cells (pNK) as carriers for a CAR targeting the common GB antigen epidermal growth factor receptor (EGFR) and the tumor-specific EGFR variant EGFRvIII. pNK cells from healthy donors were transduced with a lentiviral vector encoding the cetuximab-derived 225.28.z CAR (CAR-pNK) which recognizes an epitope common to both receptors. Specific tumor cell lysis by the CAR-pNK cells was then investigated in FACS-based cytotoxicity assays with GB cells expressing EGFR, EGFRvIII, or both target antigens. In addition, we tested in vivo feasibility of the CAR-pNK cells by single time intravenous injection in a subcutanous LNT-229/EGFRvIII glioblastoma xenograft model. RESULTS In comparison to non-transduced pNK cells, CAR-pNK cells displayed enhanced lysis of LNT-229 GB cells expressing EGFR and EGFRvIII. Likewise, markedly increased cytotoxicity of CAR-pNK was observed against GB cell lines such as SKMG-3 and BS153 with intrinsic EGFR gene amplification or expression of EGFRvIII, respectively. Furthermore, in a subcutaneous LNT-229/EGFRvIII glioblastoma xenograft model we observed delayed tumor growth after a single intravenous injection of CAR-pNK cells. CONCLUSION In summary, these results demonstrate promising efficacy of primary CAR-NK cells targeting EGFR and EGFRvIII both in vitro and in vivo. In ongoing work we are evaluating CAR-pNK cells in an orthotopic LNT-229 glioblastoma xenograft mouse model, which may provide the basis to translate this approach to early clinical testing.

A dual-targeting peptide for glioblastoma screened by phage display peptide library biopanning combined with affinity-adaptability analysis

The obstruction of blood–brain barrier (BBB) and the poor specific targeting are still the major obstacles and challenges of targeted nano-pharmaceutical therapy for glioblastoma (GBM) up to now. It is critical to find appropriate targeting ligands that can effectively mediate the nano-pharmaceuticals to penetrate brain capillary endothelial cells (BCECs) and then specifically bind to glioblastoma cells (GCs). Herein, a dual-targeting ligand for GBM was screened by the combination of phage display peptide library biopanning and affinity-adaptability analysis. Based on the acquisition of sub-library of peptide which exhibited the specific affinity to both BCECs and GCs, a comparison parameter of relative affinity was deliberately introduced to evaluate the relative affinity of candidate peptides to U251-MG cells and bEnd.3 cells. The optimized WTW peptide (sequenced as WTWEYTK) was provided with a high relative affinity (RU/B = 2.44), implying that its high affinity to U251-MG cells and moderate affinity to bEnd.3 cells might synergistically promote its receptor-mediated internalization and transport, the dissociation from bEnd.3, and the binding to U251-MG. The results of BBB model trials in vitro showed that the BBB penetration efficiency and GBM accumulation of WTW peptide were significantly higher than those of WSL peptide, GNH peptide, and REF peptide. Results of orthotopic GBM xenograft model assays in vivo also indicated that WTW peptide had successfully penetrated the BBB and improved accumulation in GBM. The screened WTW peptide might be the potential dual-targeting ligand to motivate the advancement of GBM targeted therapy.

Insulin feedback is a targetable resistance mechanism of PI3K inhibition in glioblastoma.

Insulin feedback is a critical mechanism responsible for the poor clinical efficacy of phosphatidylinositol 3-kinase (PI3K) inhibition in cancer, and hyperglycemia is an independent factor associated with poor prognosis in glioblastoma (GBM). We investigated combination anti-hyperglycemic therapy in a mouse model of GBM and evaluated the association of glycemic control in clinical trial data from patients with GBM. The effect of the anti-hyperglycemic regimens, metformin and the ketogenic diet, was evaluated in combination with PI3K inhibition in patient-derived GBM cells and in an orthotopic GBM mouse model. Insulin feedback and the immune microenvironment were retrospectively evaluated in blood and tumor tissue from a Phase 2 clinical trial of buparlisib in patients with recurrent GBM. We found that PI3K inhibition induces hyperglycemia and hyperinsulinemia in mice and that combining metformin with PI3K inhibition improves the treatment efficacy in an orthotopic GBM xenograft model. Through examination of clinical trial data, we found that hyperglycemia was an independent factor associated with poor progression-free survival in patients with GBM. We also found that PI3K inhibition increased insulin receptor activation and T-cell and microglia abundance in tumor tissue from these patients. Reducing insulin feedback improves the efficacy of PI3K inhibition in GBM in mice, and hyperglycemia worsens progression-free survival in patients with GBM treated with PI3K inhibition. These findings indicate that hyperglycemia is a critical resistance mechanism associated with PI3K inhibition in GBM and that anti-hyperglycemic therapy may enhance PI3K inhibitor efficacy in GBM patients.

Utilizing induced neural stem cell-based delivery of a cytokine cocktail to enhance chimeric antigen receptor-modified T-cell therapy for brain cancer.

Chimeric antigen receptor (CAR)-modified T-cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor-homing induced neural stem cells (iNSCs) are used to enhance CAR-T-cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC-secreted RANTES/IL-15 increased CAR-T-cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL-15 in combination with intravenous administration of CAR-T cells reduced established orthotopic GBM xenografts 2538-fold within the first week, followed by durable tumor remission through 60 days post-treatment. By contrast, CAR-T-cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR-T-cell therapy for brain tumors.

A Cell Type Selective YM155 Prodrug Targets Receptor-Interacting Protein Kinase 2 to Induce Brain Cancer Cell Death.

Glioblastoma (GBM) is the most prevalent and aggressive primary central nervous system (CNS) malignancy. YM155 is a highly potent broad-spectrum anti-cancer drug that was derived from a phenotypic screen for functional inhibitors of survivin expression, but for which the relevant biomolecular target remains unknown. Presumably as a result of its lack of cell-type selectivity, YM155 has suffered from tolerability issues in the clinic. Based on its structural similarity to the GBM-selective prodrug RIPGBM, here, we report the design, synthesis, and characterization of a prodrug form of YM155, termed aYM155. aYM155 displays potent cell killing activity against a broad panel of patient-derived GBM cancer stem-like cells (IC50 = 0.7-10 nM), as well as EGFR-amplified and EGFR variant III-expressing (EGFRvIII) cell lines (IC50 = 3.8-36 nM), and becomes activated in a cell-type-dependent manner. Mass spectrometry-based analysis indicates that enhanced cell-type selectivity results from relative rates of prodrug activation in transformed versus non-transformed cell types. The prodrug strategy also facilitates transport into the brain (brain-to-plasma ratio, aYM155 = 0.56; YM155 = BLQ). In addition, we determine that the survivin-suppressing and apoptosis-inducing activities of YM155 involve its interaction with receptor-interacting protein kinase 2 (RIPK2). In an orthotopic intracranial GBM xenograft model, aYM155 prodrug significantly inhibits brain tumor growth in vivo, which correlates with cell-type selective survivin-based pharmacodynamic effects.

Effective and Targeted Human Orthotopic Glioblastoma Xenograft Therapy via a Multifunctional Biomimetic Nanomedicine

Adv. Mater. 2018, 30, 1803717 DOI: 10.1002/adma.201803717 Errors were found in Figure S9 in the Supporting Information of the published article, where duplicate staining images had been included in some of the panels of the figure. The corrected figure is shown below. The following images have been replaced: Heart IV; Kidney II. The authors sincerely apologize for any inconvenience.

Oncogenic long noncoding RNA LINC02283 enhances PDGF receptor A-mediated signaling and drives glioblastoma tumorigenesis.

Long noncoding RNAs (lncRNAs) regulate the etiology of complex diseases and cancers, including glioblastoma (GBM). However, lncRNA-based therapies are limited because the mechanisms of action of many lncRNAs with their binding partners are not completely understood. We used transcriptomic and genomic data to analyze correlations between LINC02283 and PDGFRA (platelet-derived growth factor receptor A). The biological functions of the novel lncRNA were assessed in vivo using patient-derived glioma stem-like cells (GSCs), and orthotopic GBM xenografts. Immunoblotting, qRT-PCR, RNA pull down, crosslinked RNA immunoprecipitation, fluorescence in situ hybridization, and antisense oligo-mediated knockdown were performed to explore the regulation of LINC02283 on PDGFRA signaling. Expression of LINC02283 in clinical samples was assessed using pathologically diagnosed GBM patient samples. We identified a novel oncogenic lncRNA, LINC02283, that is highly expressed in the PDGFRA mutation-driven cohort of glioma patients and associated with worse prognosis. LINC02283 gene co-amplifies with the PDGFRA locus and shows high correlation with PDGFRA expression. Deprivation of LINC02283 in GSCs with PDGFRA amplification mutation, attenuated tumorigenicity and enhanced survival in orthotopic GBM xenograft models, while overexpression of LINC02283 in GSCs with wild-type PDGFRA, enhances PDGFRA signaling, and decreases survival. Further, LINC02283 interacts with PDGFRA to enhance its signaling and that of its downstream targets AKT and ERK, thus promoting oncogenesis in GBM. Our results provide strong evidence of LINC02283 as a regulator of PDGFRA oncogenic activity and GBM malignancy and support the potential of lncRNAs as possible therapeutic targets.

Mismatch between Bioluminescence Imaging (BLI) and MRI When Evaluating Glioblastoma Growth: Lessons from a Study Where BLI Suggested "Regression" while MRI Showed "Progression".

Orthotopic glioblastoma xenografts are paramount for evaluating the effect of innovative anti-cancer treatments. In longitudinal studies, tumor growth (or regression) of glioblastoma can only be monitored by noninvasive imaging. For this purpose, bioluminescence imaging (BLI) has gained popularity because of its low cost and easy access. In the context of the development of new nanomedicines for treating glioblastoma, we were using luciferase-expressing GL261 cell lines. Incidentally, using BLI in a specific GL261 glioblastoma model with cells expressing both luciferase and the green fluorescent protein (GL261-luc-GFP), we observed an apparent spontaneous regression. By contrast, the magnetic resonance imaging (MRI) analysis revealed that the tumors were actually growing over time. For other models (GL261 expressing only luciferase and U87 expressing both luciferase and GFP), data from BLI and MRI correlated well. We found that the divergence in results coming from different imaging modalities was not due to the tumor localization nor the penetration depth of light but was rather linked to the instability in luciferase expression in the viral construct used for the GL261-luc-GFP model. In conclusion, the use of multi-modality imaging prevents possible errors in tumor growth evaluation, and checking the stability of luciferase expression is mandatory when using BLI as the sole imaging modality.

The CNS-penetrating taxane drug TPI 287 potentiates antiglioma activity of the AURKA inhibitor alisertib in vivo.

Glioblastoma (GBM) has a very poor prognosis despite current treatment. We previously found cytotoxic synergy between the AURKA inhibitor alisertib and the CNS-penetrating taxane TPI 287 against GBM tumor cells in vitro. We used an orthotopic human GBM xenograft mouse model to test if TPI 287 potentiates alisertib in vivo. Western blotting, immunohistochemistry, siRNA knockdown, annexin V binding, and 3-dimensional Matrigel invasion assays were used to investigate potential mechanisms of alisertib and TPI 287 treatment interactions. Alisertib + TPI 287 combination therapy significantly prolonged animal survival compared to vehicle (p = 0.011), but only marginally compared to alisertib alone. Alisertib, TPI 287, and combined alisertib + TPI 287 reduced animal tumor volume compared to vehicle-treated controls. This was statistically significant for the combination therapy at 4 weeks (p < 0.0001). Alisertib + TPI 287 treatment decreased anti-apoptotic Bcl-2 protein levels in vivo and in vitro. Expression of the pro-apoptotic protein Bak was significantly increased by combination treatment (p < 0.0001). Pro-apoptotic Bim and Bak knockdown by siRNA decreased apoptosis by alisertib + TPI 287 in GB9, GB30, and U87 cells (p = 0.0005 to 0.0381). Although alisertib and TPI 287 significantly reduced GBM cell invasion (p < 0.0001), their combination was no more effective than TPI 287 alone. Results suggest that apoptosis is the dominant mechanism of potentiation of GBM growth inhibition by alisertib + TPI 287, in part through effects on Bcl-2 family proteins, providing a rationale for further laboratory testing of an AURKA inhibitor plus TPI 287 as a potential therapy against GBM.

Novel INHAT repressor drives glioblastoma growth by promoting ribosomal DNA transcription in glioma stem cells.

Cancer cells including cancer stem cells exhibit a higher rate of ribosome biogenesis than normal cells to support rapid cell proliferation in tumors. However, the molecular mechanisms governing the preferential ribosome biogenesis in glioma stem cells (GSCs) remain unclear. In this work, we show that the novel INHAT repressor (NIR) promotes ribosomal DNA (rDNA) transcription to support GSC proliferation and glioblastoma (GBM) growth, suggesting that NIR is a potential therapeutic target for GBM. Immunoblotting, immunohistochemical and immunofluorescent analysis were used to determine NIR expression in GSCs and human GBMs. Using shRNA-mediated knockdown, we assessed the role and functional significance of NIR in GSCs and GSC-derived orthotopic GBM xenografts. We further performed mass spectrometry analysis, chromatin immunoprecipitation, and other biochemical assays to define the molecular mechanisms by which NIR promotes GBM progression. Our results show that high expression of NIR predicts poor survival in GBM patients. NIR is enriched in the nucleoli of GSCs in human GBMs. Disrupting NIR markedly suppresses GSC proliferation and tumor growth by inhibiting rDNA transcription and pre-ribosomal RNA synthesis. In mechanistic studies, we find that NIR activates rDNA transcription to promote GSC proliferation by cooperating with Nucleolin (NCL) and Nucleophosmin 1 (NPM1), 2 important nucleolar transcription factors. Our study uncovers a critical role of NIR-mediated rDNA transcription in the malignant progression of GBM, indicating that targeting this axis may provide a novel therapeutic strategy for GBM.