Patient-derived Glioblastoma Multiforme Cells Research Articles

IMMU-29. B7-H3-TARGETED CAR-T AND CAR-NK CELLS AGAINST HUMAN GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM) is the most lethal primary brain tumor, necessitating novel therapies. Chimeric antigen receptor (CAR) T-cell therapy has recently shown efficacy against GBM. However, this approach is costly and time-consuming due to its personalized nature. Alternatively, CAR-transduced natural killer (NK) cells offer a potential “off-the-shelf” immunotherapy option, as they do not cause graft-versus-host disease. This study aimed to assess the anti-GBM efficacy of CAR T and NK cells targeting B7-H3, a protein highly expressed in GBM. To achieve this, CAR T cells targeting B7-H3 were developed using known anti-B7-H3 scFv sequences. Additionally, cord blood-derived NK cells were transduced with the B7-H3 CAR. Both types of CAR-modified cells were tested for their anti-GBM activity in vitro. Moreover, their anti-tumor effects were evaluated in an in vivo xenograft model using patient-derived GBM cells. The results demonstrated that both B7-H3 CAR T cells and CAR NK cells exhibited significant cytotoxicity against patient-derived GBM cells in vitro. Furthermore, intracranial injection of these CAR-modified cells into the xenograft model significantly reduced tumor growth. In conclusion, B7-H3 targeted CAR T cells and cord blood-derived CAR NK cells both show potential in eliminating GBM cells, suggesting promising avenues for future GBM treatment strategies.

DDDR-21. MPT0G521 AS A DUAL INHIBITOR OF LSD1 AND HDAC: A POTENTIAL THERAPEUTIC AGENT AGAINST PARENTAL AND TEMOZOLOMIDE-RESISTANT GLIOBLASTOMA MULTIFORME

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is an aggressive brain tumor with limited treatment options and poor prognosis. Resistance to temozolomide (TMZ), the standard chemotherapy for GBM, further complicates treatment. This study investigates the effects of MPT0G521, a dual inhibitor of LSD1 and HDAC, on both parental and TMZ-resistant GBM cells to determine its potential as a therapeutic agent. MATERIALS AND METHODS TMZ and MPT0G521 were introduced in our study. Human GBM cell lines A172 and patient-derived primary GBM cells Pt#3, along with their TMZ-resistant counterparts, were cultured under specified conditions. Assays conducted include cell proliferation (MTT), colony formation, cell cycle analysis via flow cytometry, and western blot analysis for protein expression. Statistical significance was evaluated using ANOVA. RESULTS MPT0G521 exhibited significant anti-proliferative activity and toxicity against both parental and TMZ-resistant GBM cells in a dose- and time-dependent manner. Treatment with MPT0G521 led to reduced cell viability and colony formation. Cell cycle analysis revealed G2M phase arrest and increased subG1 phase, indicating cell cycle disruption and apoptosis. Western blot analysis showed upregulation of histone H3 methylation and acetylation, confirming the dual inhibitory action of MPT0G521 on LSD1 and HDAC. Additionally, MPT0G521 modulated markers of cell cycle and apoptosis, including increased levels of cleaved caspase-3 and PARP. CONCLUSION MPT0G521 demonstrates potential as an effective therapeutic agent against GBM by inhibiting cell proliferation, inducing cell cycle arrest, and promoting apoptosis in both parental and TMZ-resistant cells. These findings support further investigation of MPT0G521 as a promising treatment for GBM, particularly in cases resistant to standard chemotherapy.

CCRG-02. MICRORNA TARGETING LOGIC IN DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMA

Abstract microRNA (miR)-124 is one of the main drivers of mitotic exit during neurogenesis, which is recapitulated in miR-9/9* and -124 (miR-9/9*-124)-mediated direct neuronal reprogramming. In the reprogramming of various cell types, miR-9/9*-124 induce non-neuronal cell fate erasure before activating the neuronal program. Regardless of the different starting transcriptome landscapes, a core gene regulatory network (GRN) is always targeted by miR-9/9*-124, with cell cycle progression as the most significant pathway. We reason that miR-9/9*-124 can induce cell fate erasure in cancerous cells by repressing similar GRNs, using glioblastoma multiforme (GBM) as a paradigm. Mechanistic insights into GBM fate erasure by miR-9/9*-124 while inducing the postmitotic state would facilitate the understanding of GRNs that govern the tumorigenic identity of GBMs and potentially offer novel therapeutic implications. Indeed, miR-9/9*-124 readily induces mitotic exit in patient-derived GBM cell lines, represses GBM cell fates, and promotes neuronal identity over time as assessed by RNA-seq. Mice receiving GBM intracerebral xenografts with miR-9/9*-124 survived significantly longer than those receiving non-specific control miRNA. Global profiling of miRNA targeting activity by AGO2-eCLIP reveals that miR-124 binds to transcripts enriched in G1/S transition and mitogen response. Conversely, miR-506—a miR-124 homolog—despite of sharing identical seed sequences, has significantly less targeting activity and fails to stop cell cycle progression. We are currently dissecting how the difference in the miRNA 3’ sequence renders contrasting biological outcomes. Preliminary data suggests that the length of miRNA alone alters targeting specificity to cell cycle transcripts. However, different permutations of miRNA 3’ sequence, though sufficient to induce mitotic exit, are not all competent to achieve neuronal reprogramming, indicating that mitotic exit and neurogenic activities of miR-124 could be decoupled. We hope that understanding the grammar logic of miRNA targeting would provide deeper insights into how miRNAs can be leveraged to manipulate GBM identity.

Reprogramming Glioblastoma Cells into Non-Cancerous Neuronal Cells as a Novel Anti-Cancer Strategy.

Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell's fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

MODL-38. DEFINING THE ROLE OF DAAM2 R414W MUTATION IN GLIOMAGENESIS AND GBM GROWTH

Abstract Each year in the United States, more than 13,000 people are diagnosed with glioblastoma multiforme (GBM), and more than 10,000 are estimated to succumb to the disease, making GBM the most common and lethal primary adult brain tumor. Despite advancing therapies, GBM patient survival has not improved over the decades, so it is critical to better understand glioma etiology. The majority of GBM cases arise from a sporadic accumulation of somatic mutations, but approximately 5-10% of gliomas are classified as “familial”, occurring clustered with hereditary cancer predispositions. These familial gliomas are caused by germline mutations inherited from parent to offspring, and only a few of these mutations have been directly linked to the development of GBM. We recently discovered five germline mutations in Daam2 (Dishevelled associated activator of morphogenesis 2) from GBM patients with a history of familial glioma. To examine the role of these mutations in GBM, we overexpressed these mutations in patient-derived GBM cells, which were then orthotopically xenografted into immunodeficient mice. We identified that one of these mutations, Daam2-R414W, promotes tumor cell proliferation both in vitro and in vivo. To further understand the role of the Daam2-R414W mutation in GBM, we generated a knock-in immunocompetent mouse model that harbors the Daam2-R414W mutation. In these mutant mice, we found increased proliferation of glial progenitor cells in the subventricular zone compared to Daam2 wildtype mice, suggesting the potential of the Daam2-R414W mutation to influence glial progenitor expansion to stimulate glioma initiation. Future experiments aim to answer whether this mutation promotes GBM growth in an immunocompetent model and to decipher the mechanism relating the Daam2-R414W mutation to GBM pathology. These studies will shed light on the repercussions of a human developmental gene variant and its potential to disrupt normal cellular machinery to promote malignancy and GBM development.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.

A Novel Patient-Personalized Nanovector Based on Homotypic Recognition and Magnetic Hyperthermia for an Efficient Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the deadliest brain tumor, characterized by an extreme genotypic and phenotypic variability, besides a high infiltrative nature in healthy tissues. Apart from very invasive surgical procedures, to date, there are no effective treatments, and life expectancy is very limited. In this work, an innovative therapeutic approach based on lipid-based magnetic nanovectors is proposed, owning a dual therapeutic function: chemotherapy, thanks to an antineoplastic drug (regorafenib) loaded in the core, and localized magnetic hyperthermia, thanks to the presence of iron oxide nanoparticles, remotely activated by an alternating magnetic field. The drug is selected based on ad hoc patient-specific screenings; moreover, the nanovector is decorated with cell membranes derived from patients' cells, aiming at increasing homotypic and personalized targeting. It is demonstrated that this functionalization not only enhances the selectivity of the nanovectors toward patient-derived GBM cells, but also their blood-brain barrier in vitro crossing ability. The localized magnetic hyperthermia induces both thermal and oxidative intracellular stress that lead to lysosomal membrane permeabilization and to the release of proteolytic enzymes into the cytosol. Collected results show that hyperthermia and chemotherapy work in synergy to reduce GBM cell invasion properties, to induce intracellular damage and, eventually, to prompt cellular death.

The addition of arginine deiminase potentiates Mithramycin A-induced cell death in patient-derived glioblastoma cells via ATF4 and cytochrome C

BackgroundArginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from Streptococcus pyogenes (SpyADI) has proven activity against GBM in preclinical studies. The good safety profile of SpyADI renders this agent an ideal combination partner for cytostatic therapy.MethodsIn this study, we combined the antineoplastic antibiotic Mithramycin A (MitA) with SpyADI to boost single-agent activity and analyzed underlying response mechanisms in-depth.ResultsMitA monotherapy induced a time- and dose-dependent cytotoxicity in eight patient-derived GBM cell lines and had a radiosensitizing effect in all but one cell line. Combination treatment boosted the effects of the monotherapy in 2D- and 3D models. The simultaneous approach was superior to the sequential application and significantly impaired colony formation after repetitive treatment. MitA monotherapy significantly inhibited GBM invasiveness. However, this effect was not enhanced in the combination. Functional analysis identified SpyADI-triggered senescence induction accompanied by increased mitochondrial membrane polarization upon mono- and combination therapy. In HROG63, induction of lysosomes was seen after both monotherapies, indicative of autophagy. These cells seemed swollen and had a more pronounced cortically formed cytoskeleton. Also, cytochrome C and endoplasmatic reticulum-stress-associated proteins ATF4 and Calnexin were enhanced in the combination, contributing to apoptosis. Notably, no significant increases in glioma-stemness marker were seen.ConclusionsTherapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials.Graphical

Identification of Dysregulated microRNAs in Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor with a poor median survival of less than 15 months. However, clinical strategies and effective therapies are limited. Here, we found that the second-generation small molecule multi-CDK inhibitor AT7519 is a potential drug for GBM treatment according to high-throughput screening via the Approved Drug Library and Clinical Compound Library (2718 compounds). We found that AT7519 significantly inhibited the cell viability and proliferation of U87MG, U251, and patient-derived primary GBM cells in a dose-dependent manner. Furthermore, AT7519 also inhibited the phosphorylation of CDK1/2 and arrested the cell cycle at the G1-S and G2-M phases. More importantly, AT7519 induced intrinsic apoptosis and pyroptosis via caspase-3-mediated cleavage of gasdermin E (GSDME). In the glioblastoma intracranial and subcutaneous xenograft assays, tumor volume was significantly reduced after treatment with AT7519. In summary, AT7519 induces cell death through multiple pathways and inhibits glioblastoma growth, indicating that AT7519 is a potential chemical available for GBM treatment.

Inhibition of MZF1/c-MYC Axis by Cantharidin Impairs Cell Proliferation in Glioblastoma

Myeloid zinc finger 1 (MZF1), also known as zinc finger protein 42, is a zinc finger transcription factor, belonging to the Krüppel-like family that has been implicated in several types of malignancies, including glioblastoma multiforme (GBM). MZF1 is reportedly an oncogenic gene that promotes tumor progression. Moreover, higher expression of MZF1 has been associated with a worse overall survival rate among patients with GBM. Thus, MZF1 may be a promising target for therapeutic interventions. Cantharidin (CTD) has been traditionally used in Chinese medicine to induce apoptosis and inhibit cancer cell proliferation; however, the mechanism by which CTD inhibits cell proliferation remains unclear. In this study, we found that the expression of MZF1 was higher in GBM tissues than in adjacent normal tissues and low-grade gliomas. Additionally, the patient-derived GBM cells and GBM cell lines presented higher levels of MZF1 than normal human astrocytes. We demonstrated that CTD had greater anti-proliferative effects on GBM than a derivative of CTD, norcantharidin (NCTD). MZF1 expression was strongly suppressed by CTD treatment. Furthermore, MZF1 enhanced the proliferation of GBM cells and upregulated the expression of c-MYC, whereas these effects were reversed by CTD treatment. The results of our study suggest that CTD may be a promising therapeutic agent for patients with GBM and suggest a promising direction for further investigation.

MicroRNA and mRNA Expression Changes in Glioblastoma Cells Cultivated under Conditions of Neurosphere Formation.

Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. The study of the pathogenesis of GBM, as well as the development of targeted oncolytic drugs, require the use of actual cell models, in particular, the use of 3D cultures or neurospheres (NS). During the formation of NS, the adaptive molecular landscape of the transcriptome, which includes various regulatory RNAs, changes. The aim of this study was to reveal changes in the expression of microRNAs (miRNAs) and their target mRNAs in GBM cells under conditions of NS formation. Neurospheres were obtained from both immortalized U87 MG and patient-derived BR3 GBM cell cultures. Next generation sequencing analysis of small and long RNAs of adherent and NS cultures of GBM cells was carried out. It was found that the formation of NS proceeds with an increase in the level of seven and a decrease in the level of 11 miRNAs common to U87 MG and BR3, as well as an increase in the level of 38 and a decrease in the level of 12 mRNA/lncRNA. Upregulation of miRNAs hsa-miR: -139-5p; -148a-3p; -192-5p; -218-5p; -34a-5p; and -381-3p are accompanied by decreased levels of their target mRNAs: RTN4, FLNA, SH3BP4, DNPEP, ETS2, MICALL1, and GREM1. Downregulation of hsa-miR: -130b-5p, -25-5p, -335-3p and -339-5p occurs with increased levels of mRNA-targets BDKRB2, SPRY4, ERRFI1 and TGM2. The involvement of SPRY4, ERRFI1, and MICALL1 mRNAs in the regulation of EGFR/FGFR signaling highlights the role of hsa-miR: -130b-5p, -25-5p, -335-3p, and -34a-5p not only in the formation of NS, but also in the regulation of malignant growth and invasion of GBM. Our data provide the basis for the development of new approaches to the diagnosis and treatment of GBM.

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and β-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.

Genomic and Transcriptomic Differences at the Pyruvate Kinase M (PKM) Locus in Patient-Derived Glioblastoma Multiforme (GBM) Cell Lines

Glioblastoma multiforme (GBM), a particularly devastating primary CNS tumor, alters the expression and activity of multiple metabolic genes to enhance its ability to grow and divide, buffer against oxidative stress, and resist apoptosis. The shift from oxidative metabolism in the mitochondria to aerobic glycolysis is known as the Warburg effect, a phenomenon mediated by gene expression changes like the isoform switch at the pyruvate kinase muscle-type (PKM) locus from PKM1 to PKM2. Novel pharmaceuticals targeting this tumor-specific isoform have entered clinical trials, but the effect of genetic or transcriptomic background on the effectiveness of these drugs is unknown. The purpose of this work is to characterize the PKM locus at the level of DNA and mRNA.Our lab has previously collected and curated patient-derived GBM cell lines and characterized them with high-throughput transcriptome sequencing (RNA-seq). Herein, RNA-seq reads were mapped to the genome. Limiting analysis to the PKM locus, variant calling including single nucleotide polymorphism (SNP) calling is performed, and absolute expression and relative isoform abundance is measured and compared across lines.In this analysis of 16 GBM cell lines, multiple SNPs are identified that are predicted to be deleterious to the function of the protein. Additionally, concordant with previous findings in astrocytomas, the PKM2 isoform is the dominant pyruvate kinase transcript in all cell lines profiled. One cell line, in particular, had substantially higher PKM expression compared to the other cell lines.Previous studies have queried the mutational burden of PKM in other cancer types, but no mutational analysis has been performed on GBM. This work suggests genetic variation at this important locus. Targeting tumor-specific metabolism can increase the effectiveness of current standard-of-care therapies while leaving normal tissue intact. This characterization of the PKM locus at the level of DNA variants and gene expression will inform planned future work using a novel pharmaceutical targeting PKM2.K.J. Lee: None. C. Willey: None.

Oxyphyllanene B overcomes temozolomide resistance in glioblastoma: Structure–activity relationship and mitochondria-associated ER membrane dysfunction

The identification of novel therapeutic candidates from natural products for the development of chemoresistant glioblastoma multiforme (GBM) treatment has been a highly significant and effective strategy. Sesquiterpenes are a class of naturally occurring 15-carbon isoprenoid compounds, and several types of sesquiterpenes have the ability to induce growth inhibition and apoptosis in a variety of cancer cell lines. In the present study, 56 sesquiterpenes of five types, namely, eudesmane-type (I) (1-24), eremophilane-type (II) (25-32), cadinane-type (III) (33-41), guaiane-type (IV) (42-49), and oplopanone-type (V) (50-56), were screened for their antiglioma activity, structure-activity relationship analysis (SAR), and underlying mechanism based on patient-derived recurrent GBM strains, patient-derived GBM cell sphere, GBM organoid (GBO) models, and temozolomide (TMZ)-resistant GBM cell lines. We found that compound 12 (oxyphyllanene B, OLB) showed the most potent antiglioma activity, and we confirmed that OLB could induce apoptosis in a time- and dose-dependent manner in TMZ-resistant GBM cells and GBOs. SAR announced that the presence of an α, β-unsaturated carbonyl moiety was likely to enhance cytotoxic activities. Mechanistic studies demonstrated that OLB induced abnormal changes in ER and mitochondria-associated membrane (MAM) networks, which triggered ER stress, mitochondrial dysfunction, and apoptosis. Furthermore, our findings suggested that OLB-triggered PACS2 activation might form a committed step to disrupt ER-mitochondria communication and showed for the first time that the expression levels of PACS2 might positively correlate with the progression and chemotherapy resistance of glioma. Our results indicated that OLB might be a promising candidate for treating TMZ-resistant GBM cells by activating PACS2, which triggered a crucial event to promote the disruption of ER-mitochondria communication and overcome chemotherapy resistance of GBM.

CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression.

Simple SummaryGlioblastoma multiforme (GBM) is the most common and lethal primary malignant cancer of the central nervous system with a median patient survival of ~15 months. It has been reported that particularly nerve/glial antigen (NG)2-positive GBM is associated with an aggressive clinical phenotype and poor prognosis. Based on our latest findings, that protein kinase CK2 is a crucial regulator of NG2 expression in pericytes, we investigated the effect of CK2 inhibition by CX-4945 as well as CK2 KO on NG2 expression in human GBM cells. We found that CK2 inhibition suppresses proliferation and migration of different NG2-positive GBM cells. In silico analyses revealed a positive correlation between the mRNA expression of the two proteins. Moreover, we verified the decreased expression of NG2 in patient-derived GBM cells after CX-4945 treatment. These novel insights into the molecular signaling of NG2-positive GBM demonstrate that CX-4945 may represent a promising drug for future GBM therapy.Nerve/glial antigen (NG)2 expression crucially determines the aggressiveness of glioblastoma multiforme (GBM). Recent evidence suggests that protein kinase CK2 regulates NG2 expression. Therefore, we investigated in the present study whether CK2 inhibition suppresses proliferation and migration of NG2-positive GBM cells. For this purpose, CK2 activity was suppressed in the NG2-positive cell lines A1207 and U87 by the pharmacological inhibitor CX-4945 and CRISPR/Cas9-mediated knockout of CK2α. As shown by quantitative real-time PCR, luciferase-reporter assays, flow cytometry and western blot, this significantly reduced NG2 gene and protein expression when compared to vehicle-treated and wild type controls. In addition, CK2 inhibition markedly reduced NG2-dependent A1207 and U87 cell proliferation and migration. The Cancer Genome Atlas (TCGA)-based data further revealed not only a high expression of both NG2 and CK2 in GBM but also a positive correlation between the mRNA expression of the two proteins. Finally, we verified a decreased NG2 expression after CX-4945 treatment in patient-derived GBM cells. These findings indicate that the inhibition of CK2 represents a promising approach to suppress the aggressive molecular signature of NG2-positive GBM cells. Therefore, CX-4945 may be a suitable drug for the future treatment of NG2-positive GBM.

Rapid 3D Bioprinting of Glioblastoma Model Mimicking Native Biophysical Heterogeneity.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor characterized by high cellular and molecular heterogeneity, hypervascularization, and innate drug resistance. Cellular components and extracellular matrix (ECM) are the two primary sources of heterogeneity in GBM. Here, biomimetic tri-regional GBM models with tumor regions, acellular ECM regions, and an endothelial region with regional stiffnesses patterned corresponding to the GBM stroma, pathological or normal brain parenchyma, and brain capillaries, are developed. Patient-derived GBM cells, human endothelial cells, and hyaluronic acid derivatives are used to generate a species-matched and biochemically relevant microenvironment. This in vitro study demonstrates that biophysical cues are involved in various tumor cell behaviors and angiogenic potentials and promote different molecular subtypes of GBM. The stiff models are enriched in the mesenchymal subtype, exhibit diffuse invasion of tumor cells, and induce protruding angiogenesis and higher drug resistance to temozolomide. Meanwhile, the soft models demonstrate enrichment in the classical subtype and support expansive cell growth. The three-dimensional bioprinting technology utilized in this study enables rapid, flexible, and reproducible patient-specific GBM modeling with biophysical heterogeneity that can be employed by future studies as a tunable system to interrogate GBM disease mechanisms and screen drug compounds.

Targeting glioblastoma using a novel peptide specific to a deglycosylated isoform of brevican.

Glioblastoma multiforme (GBM) is the most common and deadliest form of brain tumor and remains amongst the most difficult cancers to treat. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein, is upregulated in high-grade glioma cells, including GBM. A Bcan isoform lacking most glycosylation, dg-Bcan, is found only in GBM tissues. Here, dg-Bcan is explored as a molecular target for GBM. In this study, we screened a d-peptide library to identify a small 8-amino acid dg-Bcan-Targeting Peptide (BTP) candidate, called BTP-7 that binds dg-Bcan with high affinity and specificity. BTP-7 is preferentially internalized by dg-Bcan-expressing patient-derived GBM cells. To demonstrate GBM targeting, we radiolabeled BTP-7 with 18F, a radioisotope of fluorine, and found increased radiotracer accumulation in intracranial GBM established in mice using positron emission tomography (PET) imaging. dg-Bcan is an attractive molecular target for GBM, and BTP-7 represents a promising lead candidate for further development into novel imaging agents and targeted therapeutics.

Ablation of neuropilin-1 improves the therapeutic response in conventional drug-resistant glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly proliferative and locally invasive cancer with poor prognosis and a high recurrence rate. Although anti-VEGF (vascular endothelial growth factor) therapy offers short-term benefit to GBM patients, this approach fails as the tumor develops into a more invasive and drug-resistant phenotype and ultimately recurs. Recently, both glioma stemlike cells (GSCs) and brain tumor-initiating cells (BTICs) have been implicated in GBM recurrence and its resistance to therapy. We observed that patient-derived GBM cells expressing shRNAs of VEGF or neuropilin-1 (NRP-1) attenuate cancer stem cell markers, inhibit the tumor-initiating cell's neurosphere-forming capacity, and migration. Furthermore, both VEGF and NRP-1 knockdown inhibit the growth of patient-derived GBM xenografts in both zebrafish and mouse models. Interestingly, NRP-1-depleted patient-derived GBM xenografts substantially prolonged survival in mice compared to that of VEGF depletion. Our results also demonstrate that NRP-1 ablation of patient-derived GBM cells improves the sensitivity of TMZ and enhances the overall survival of the respective tumor-bearing mice. This improved outcome may provide insight into the inhibition of GBM progression and effective treatment strategies by targeting NRP-1 in addition to chemotherapy and radiotherapy.