Glioblastoma Cell Death Research Articles

DNAR-07. ATR INHIBITORS IN COMBINATION WITH RADIATION AND TEMOZOLOMIDE INCREASE CELL DEATH OF PATIENT-DERIVED GLIOBLASTOMA CELLS

Abstract Glioblastoma is treated with surgery to remove as much as the tumour as possible then radiation therapy (RT) and temozolomide (TMZ) to cause extensive DNA damage in any remaining tumour cells, triggering cell death by apoptosis. However, glioblastoma cells respond by upregulating the DNA damage response (DDR) triggering DNA repair that contributes to treatment resistance. Progression then generally occurs rapidly leaving patients with a poor prognosis and survival. We previously showed that gartisertib, an inhibitor of ATM- and Rad3-Related protein (ATR), a serine/threonine kinase that triggers DNA repair, sensitises glioblastoma cells to TMZ and RT. We extend this investigation to brain-penetrant ATR inhibitors elimusertib (BAY1895344) and ceralasertib (AZD6738). Patient-derived glioblastoma cell lines were treated with a dose response of ATRi (elimusertib and ceralasertib) combined with a clinically relevant dose of TMZ (35uM) and/or RT (2Gy). A human astrocyte cell line was also treated with a dose response of single agent elimusertib and ceralasertib. Cells were incubated for 7 days and assessed for cell viability through an MTT assay or cell-cell Incucyte analysis. Both elimusertib and ceralasertib as single agents reduced glioblastoma cell growth, with elimusertib being more potent compared to ceralasertib. Elimusertib had greater potency in causing cell death of the glioblastoma cell lines than a human astrocyte cell line. However, there was less difference between glioblastoma and astrocyte cell death when treated with ceralasetib. When combined with RT and/or TMZ, elimusertib once again was more potent in reducing glioblastoma cell growth compared to ceralasertib. We have expanded our in vitro data showing that various ATR inhibitors (elimusertib, ceralasertib and previously gartisertib) are able to reduce the growth of glioblastoma cells in combination with RT and TMZ treatment. This provides further support for initiating a clinical trial of ATR inhibition in combination with standard treatment for patients with glioblastoma.

Cell death in glioblastoma and the central nervous system.

Glioblastoma is the commonest and deadliest primary brain tumor. Glioblastoma is characterized by significant intra- and inter-tumoral heterogeneity, resistance to treatment and dismal prognoses despite decades of research in understanding its biological underpinnings. Encompassed within this heterogeneity and therapy resistance are severely dysregulated programmed cell death pathways. Glioblastomas recapitulate many neurodevelopmental and neural injury responses; in addition, glioblastoma cells are composed of multiple different transformed versions of CNS cell types. To obtain a greater understanding of the features underlying cell death regulation in glioblastoma, it is important to understand the control of cell death within the healthy CNS during homeostatic and neurodegenerative conditions. Herein, we review apoptotic control within neural stem cells, astrocytes, oligodendrocytes and neurons and compare them to glioblastoma apoptotic control. Specific focus is paid to the Inhibitor of Apoptosis proteins, which play key roles in neuroinflammation, CNS cell survival and gliomagenesis. This review will help in understanding glioblastoma as a transformed version of a heterogeneous organ composed of multiple varied cell types performing different functions and possessing different means of apoptotic control. Further, this review will help in developing more glioblastoma-specific treatment approaches and will better inform treatments looking at more direct brain delivery of therapeutic agents.

THE EPIGENETIC REGULATORY PROTEIN CBX2 AS A NOVEL THERAPEUTIC TARGET FOR GLIOBLASTOMA

Abstract AIMS Glioblastoma (GBM) is the deadliest and most common form of brain tumour. The average length of survival for GBM patients remains low, in part, due to a lack of targeted therapeutics. Identification and validation of novel therapeutic targets is therefore crucial. Chomobox 2 (CBX2; a component of polycomb repressive complex 1 (PRC1)), represses gene expression and has a pro-oncogenic role in several aggressive cancer subtypes. CBX2 expression is elevated in GBM, however, very little is known about its role in this deadly disease. We aim to assess the phenotypic and gene expression effects of preventing CBX2-chromatin interaction in patient-derived GBM cell models, and to determine the functional mechanisms by which CBX2 promotes GBM progression; thereby determining its potential as a novel therapeutic target. METHOD Crucial to impactful target validation is the use of translationally relevant models, this study utilises a 3D spheroid model, using patient-derived cells, which more closely recapitulates the in vivo tumour microenvironment. GBM cell monolayers and spheroids were treated with SW2_152F, a selective CBX2 inhibitor. The phenotypic effects of CBX2 inhibition were assessed by cell count, CellTiter-Glo viability assays, fluorescence microscopy, and flow cytometry. Genes and biological pathways differentially regulated following CBX2 inhibition were assessed by RNA-sequencing and Gene Set Enrichment Analysis (GSEA). RESULTS Inhibition of CBX2 induced cell death and reduced cell viability in both 2D and 3D GBM cultures. RNA-seq and GSEA of SW2_152F-treated cells identified dysregulation of gene signatures involved in pro-oncogenic MYC and E2F signalling pathways, and dysregulation of genes associated with G2/M cell cycle progression. CONCLUSION Preventing CBX2-chromatin interaction induces GBM cell death. Further research will identify CBX2- chromatin binding sites to characterise the activity and direct regulatory role of CBX2 in GBM. Furthermore, we will assess the phenotypic and gene expression effects of CBX2 inhibition and depletion on ex vivo maintained GBM tissue biopsies.

Dual genes manipulation enhanced chemotherapy potentiates antitumor immunity based on extracellular vesicle system for glioblastoma treatment

Glioblastoma is one of the most malignant tumors in the central nervous system, which the conventional therapeutic modalities fail to cure the disease but improve the treatment outcome slightly. In the past decades, immune checkpoint blockade (ICB) and cytokine-regulated immunotherapy have ushered in a new era for glioblastoma treatment. However, the immunosuppressive tumor microenvironment hinders and opposite the therapeutic outcomes. Therefore, the increased importance and necessary for better modality in urgent. Moreover, many novel methods and strategies are searching for more effectively therapeutic outcomes. In this study, the glioblastoma cells derived extracellular vesicles (EVs) were adopted as a delivery platform for the chemotherapy agent (doxorubicin, Dox), gene editing platform of CD47 downregulation and IL-9 overexpression (EVs@Dox/sgCD47/IL-9), due to its excellent blood brain barrier (BBB) crossing rate and specifically targeting capability. The as-prepared EVs@Dox/sgCD47/IL-9 was capable to deliver the Dox and gene editing platform into glioblastoma tissue and was assimilated by cancer cells efficiently, where the Dox induce the immunogenic cell death (ICD) of glioblastoma cells, the CRISPR-Cas9 platform down-regulated the expression of CD47 and the over-expression system up-regulated the expression of IL-9, both enhanced the immunity of biological system. Collectively, EVs@Dox/sgCD47/IL-9 demonstrated remarkable anti-tumor effects and reshaped the immunosuppressive tumor microenvironment that polarized the tumor-associated macrophages and activated CD8+ T cells with good biosafety. Thus, it establishes a strong foundation for the development of more effective strategies against glioblastoma.

P15-27 Does activation of N9 microglia by thimerosal increase GL261 glioblastoma cell death?

P15-27 Does activation of N9 microglia by thimerosal increase GL261 glioblastoma cell death?

Combinatorial targeting of glutamine metabolism and lysosomal-based lipid metabolism effectively suppresses glioblastoma

Antipsychotic drugs have been shown to have antitumor effects but have had limited potency in the clinic. Here, we unveil that pimozide inhibits lysosome hydrolytic function to suppress fatty acid and cholesterol release in glioblastoma (GBM), the most lethal brain tumor. Unexpectedly, GBM develops resistance to pimozide by boosting glutamine consumption and lipogenesis. These elevations are driven by SREBP-1, which we find upregulates the expression of ASCT2, a key glutamine transporter. Glutamine, in turn, intensifies SREBP-1 activation through the release of ammonia, creating a feedforward loop that amplifies both glutamine metabolism and lipid synthesis, leading to drug resistance. Disrupting this loop via pharmacological targeting of ASCT2 or glutaminase, in combination with pimozide, induces remarkable mitochondrial damage and oxidative stress, leading to GBM cell death invitro and invivo. Our findings underscore the promising therapeutic potential of effectively targeting GBM by combining glutamine metabolism inhibition with lysosome suppression.

Melanocortin Receptor Agonist Bremelanotide Induces Cell Death and Growth Inhibition in Glioblastoma Cells via Suppression of Survivin Expression.

Glioblastoma is the most aggressive form of brain tumor and has a dismal prognosis; therefore, novel therapeutic approaches based on the mechanisms underlying its aggressive nature are urgently required. A growing body of evidence suggests that neurotransmitters play a key role in modulating the biology of glioblastoma; however, the role of melanocortins remains unclear. The effects of bremelanotide, a melanocortin receptor agonist, alone or in combination with chemotherapeutic agents, on survivin expression and cell viability were investigated in human glioblastoma cell lines. Bremelanotide reduced survivin expression and induced cell death in glioblastoma cells at concentrations that were not toxic to normal human cells, and both of these effects were canceled in the presence of an antagonist of melanocortin receptors 3 and 4. Bremelanotide-induced cell death was prevented by the forced over-expression of survivin in glioblastoma cells, suggesting that bremelanotide induces glioblastoma cell death by inhibiting the expression of survivin. Bremelanotide also promoted cell death induced by chemotherapeutic agents, such as temozolomide and osimertinib. The present results identified melanocortin receptors 3 and 4 as novel and viable therapeutic targets for glioblastoma. Activation of these receptors by bremelanotide may inhibit the expression of survivin, thereby sensitizing glioblastoma cells to cell death.

5-Aminolevulinic acid-mediated photodynamic therapy in combination with kinase inhibitor lapatinib enhances glioblastoma cell death

5-Aminolevulinic acid (ALA) is an intraoperative imaging agent approved for protoporphyrin IX (PpIX) fluorescence-guided resection of glioblastoma (GBM). It is currently under clinical evaluation for photodynamic therapy (PDT) after the completion of GBM surgery. We previously showed that lapatinib, a clinical kinase inhibitor of epidermal growth factor receptor 1 & 2 (EGFR and HER2), enhanced PpIX fluorescence in a panel of GBM cell lines by blocking ABCG2 (ATP-binding cassette super-family G member 2)-mediated PpIX efflux, which suggests its potential for improving ALA for GBM surgery and PDT. Here we show that lapatinib enhanced PDT-induced cytotoxicity by promoting GBM cell death with the induction of apoptosis followed by necrosis. While the induction of tumor cell apoptosis was massive and rapid in the H4 cell line with no detectable Bcl-2 and a low level of Bcl-xL, it was delayed and much less in extent in A172, U-87 and U-118 cell lines with higher levels of pro-survival Bcl-2 family proteins. Lapatinib treatment alone neither reduced GBM cell viability nor had any significant effect on EGFR downstream signaling. Its enhancement of ALA–PDT was largely due to the increase of intracellular PpIX particularly in the mitochondria, resulting in the activation of mitochondria-mediated apoptosis in H4 cells. Our present study demonstrates that lapatinib inhibits ABCG2-mediated PpIX efflux and sensitizes GBM cells to ALA–PDT by inducing tumor cell death.

The Fluorinated NAD Precursors Enhance FK866 Cytotoxicity by Activating SARM1 in Glioblastoma Cells.

Glioblastoma, a formidable brain tumor characterized by dysregulated NAD metabolism, poses a significant therapeutic challenge. The NAMPT inhibitor FK866, which induces NAD depletion, has shown promise in controlling tumor proliferation and modifying the tumor microenvironment. However, the clinical efficacy of FK866 as a single drug therapy for glioma is limited. In this study, we aim to disrupt NAD metabolism using fluorinated NAD precursors and explore their synergistic effect with FK866 in inducing cytotoxicity in glioblastoma cells. The synthesized analogue of nicotinamide riboside (NR), ara-F nicotinamide riboside (F-NR), inhibits nicotinamide ribose kinase (NRK) activity in vitro, reduces cellular NAD levels, and enhances FK866's cytotoxicity in U251 glioblastoma cells, indicating a collaborative impact on cell death. Metabolic analyses reveal that F-NR undergoes conversion to fluorinated nicotinamide mononucleotide (F-NMN) and other metabolites, highlighting the intact NAD metabolic pathway in glioma cells. The activation of SARM1 by F-NMN, a potent NAD-consuming enzyme, is supported by the synergistic effect of CZ-48, a cell-permeable SARM1 activator. Temporal analysis underscores the sequential nature of events, establishing NAD depletion as a precursor to ATP depletion and eventual massive cell death. This study not only elucidates the molecular intricacies of glioblastoma cell death but also proposes a promising strategy to enhance FK866 efficacy through fluorinated NAD precursors, offering potential avenues for innovative therapeutic interventions in the challenging landscape of glioblastoma treatment.

The Proteasome Inhibitor Marizomib Evokes Endoplasmic Reticulum Stress and Promotes Apoptosis in Human Glioblastoma Cells.

Proteasomes play an important role in the physiology of cancer cells, and inhibition of their activity may be used as a promising therapeutic strategy against glioblastoma (GBM). Although certain proteasome inhibitors (PIs) have been approved for the treatment of other malignancies, they have limited effectiveness against GBM due to low brain bioavailability. Marizomib (MZB) is an irreversible, second-generation proteasome inhibitor, which unlike other PIs can penetrate through the blood-brain barrier, making it a promising therapeutic tool in brain malignancies. The antitumor activity of MZB was investigated in LN229 and U118 cells. The MTT test and the ATP-based assay were performed to evaluate cytotoxicity. Flow cytometry analysis was used to determine the apoptotic death of GBM cells. Luminescent assays were used to assess levels of reactive oxygen species (ROS) and the activity of caspase 3/7. RT-qPCR and Western blot analyses were used to determine gene and protein expressions. Marizomib decreased the viability and caused apoptotic death of GBM cells. The proapoptotic effect was accompanied by activation of caspase 3 and overexpression of cl-PARP, Noxa, Cyt C, and DR5. Moreover, treatment with MZB triggered endoplasmic reticulum (ER) stress, as shown by increased expressions of GRP78, IRE1α, p-EIF2α, p-SAPK/JNK, CHOP, ATF6α, and ATF4. On the contrary, overproduction of ROS or increased expressions of ERO1α, LC3 II, Beclin 1, and ATG5 were not detected, suggesting that neither oxidative stress nor autophagy were involved in the process of MZB-induced cell death. Thus, marizomib represents a potentially promising compound for facilitating further progress in brain cancer therapy.

Astrocytes and the tumor microenvironment inflammatory state dictate the killing of glioblastoma cells by Smac mimetic compounds

Smac mimetic compounds (SMCs) are small molecule drugs that sensitize cancer cells to TNF-α-induced cell death and have multiple immunostimulatory effects through alterations in NF-κB signaling. The combination of SMCs with immunotherapies has been reported to result in durable cures of up to 40% in syngeneic, orthotopic murine glioblastoma (GBM) models. Herein, we find that SMC resistance is not due to a cell-intrinsic mechanism of resistance. We thus evaluated the contribution of GBM and brain stromal components to identify parameters leading to SMC efficacy and resistance. The common physiological features of GBM tumors, such as hypoxia, hyaluronic acid, and glucose deprivation were found not to play a significant role in SMC efficacy. SMCs induced the death of microglia and macrophages, which are the major immune infiltrates in the tumor microenvironment. This death of microglia and macrophages then enhances the ability of SMCs to induce GBM cell death. Conversely, astrocytes promoted GBM cell growth and abrogated the ability of SMCs to induce death of GBM cells. The astrocyte-mediated resistance can be overcome in the presence of exogenous TNF-α. Overall, our results highlight that SMCs can induce death of microglia and macrophages, which then provides a source of death ligands for GBM cells, and that the targeting of astrocytes is a potential mechanism for overcoming SMC resistance for the treatment of GBM.

Folic-Acid-Conjugated Poly (Lactic-Co-Glycolic Acid) Nanoparticles Loaded with Gallic Acid Induce Glioblastoma Cell Death by Reactive-Oxygen-Species-Induced Stress.

Glioblastoma (GBM) conventional treatment is not curative, and it is associated with severe toxicity. Thus, natural compounds with anti-cancer properties and lower systemic toxicity, such as gallic acid (GA), have been explored as alternatives. However, GA's therapeutic effects are limited due to its rapid metabolism, low bioavailability, and low permeability across the blood-brain barrier (BBB). This work aimed to develop poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) modified with folic acid (FA), as its receptor is overexpressed in BBB and GBM cells, for GA delivery to enhance its therapeutic efficacy. The preparation of NPs was optimized by a central composite design (CCD). The obtained NPs showed physicochemical features suitable for drug internalization in BBB and tumor cells (sizes below 200 nm, monodispersity, and negative surface charge) and the ability to maintain a slow and sustained release for 40 days. In vitro studies using a human GBM cell line (U215) revealed the NPs' ability to accumulate in the target cells, further promoting GA antiproliferative activity by inducing the production of intracellular reactive oxygen species (ROS). Furthermore, GA encapsulation in the developed nanosystems conferred higher protection to healthy cells.

Investigating the Effects of Dorema hyrcanum Root Extracts on Selective Induction of Programmed Cell Death in Glioblastoma, Ovarian Cancer and Breast Cancer Cell Lines.

Despite remarkable advances, cancer has remained the second cause of death, which shows that more potent novel compounds should be found. Ethnobotanical compounds have a long history of treating diseases, and several approved chemotherapeutic compounds were isolated from plants. The research aimed to evaluate the cytotoxic effects of Dorema hyrcanum root extract on ovarian, breast, and glioblastoma cells while examining its selectivity towards normal cells. Additionally, the study is directed to investigate cell death mechanisms, delineate modes of cell death, and explore intracellular ROS production. Cytotoxic effects of alcoholic, dichloromethane, and petroleum ether fractions of Dorema hyrcanum were investigated on cancer and normal cells by using MTT assay, and the concentration around IC50 values was used for flow cytometric assessment of apoptosis, evaluation of the expression of selected genes via RT-qPCR and production of ROS. Methanolic extract exhibited the highest cytotoxicity, impacting A2780CP and MDA-MB-231. All fractions showed comparable effects on U251 cells. Notably, extracts displayed higher IC50 values in normal HDF cells, indicating cancer cell specificity. Flow cytometry revealed induction of apoptosis and non-apoptotic death in all three cancer cell lines. QPCR results showed upregulation of related genes, with RIP3K prominently increased in U251 glioblastoma. The DCFH-DA assay demonstrated ROS induction by the PE fraction exclusively in A2780CP cells after 30 minutes and up to 24 hours. Dorema hyrcanum root extracts exhibited potent anti-tumor effects against all studied cell lines. The methanolic extract demonstrated the highest cytotoxicity, particularly against A2780CP and MDA-MB-231 cells. Importantly, all fractions displayed selectivity for cancer cells over normal HDF cells. Unique modes of action were observed, with the petroleum ether fraction inducing significant non-apoptotic cell death. These findings suggest promising therapeutic potential for Dorema hyrcanum in cancer treatment with subject to further mechanistic studies.

LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Nitric oxide-dependent cell death in glioblastoma and squamous cell carcinoma via prodeath mitochondrial clustering

Besides the fission–fusion dynamics, the cellular distribution of mitochondria has recently emerged as a critical biological parameter in regulating mitochondrial function and cell survival. We previously found that mitochondrial clustering on the nuclear periphery, or monopolar perinuclear mitochondrial clustering (MPMC), accompanies the anticancer activity of air plasma-activated medium (APAM) against glioblastoma and human squamous cell carcinoma, which is closely associated with oxidant-dependent tubulin remodeling and mitochondrial fragmentation. Accordingly, this study investigated the regulatory roles of nitric oxide (NO) in the anticancer activity of APAM. Time-lapse analysis revealed a time-dependent increase in NO accompanied by MPMC. In contrast, APAM caused minimal increases in MPMC and NO levels in nontransformed cells. NO, hydroxyl radicals, and lipid peroxide levels increased near the damaged nuclear periphery, possibly within mitochondria. NO scavenging prevented tubulin remodeling, MPMC, perinuclear oxidant production, nuclear damage, and cell death. Conversely, synthetic NO donors augmented all the prodeath events and acted synergistically with APAM. Salinomycin, an emerging drug against multidrug-resistant cancers, had similar NO-dependent effects. These results suggest that APAM and salinomycin induce NO-dependent cell death, where MPMC and oxidative mitochondria play critical roles. Our findings encourage further investigations on MPMC as a potential target for NO-driven anticancer agents against drug-resistant cancers.

In situ administration of STING-activating hyaluronic acid conjugate primes anti-glioblastoma immune response

Glioblastoma (GBM) is an aggressive brain tumor, with a highly immunosuppressive tumor immune microenvironment (TIME). In this work, we investigated the use of the STimulator of INterferon Genes (STING) pathway as an effective means to remodel the GBM TIME through the recruitment of both innate and adaptive immune cell populations. Using hyaluronic acid (HA), we developed a novel polymer-drug conjugate of a non-nucleotide STING agonist (MSA2), called HA-MSA2 for the in situ treatment of GBM. In JAWSII cells, HA-MSA2 exerted a greater increase of STING signaling and upregulation of STING-related downstream cyto-/chemokines in immune cells than the free drug. HA-MSA2 also elicited cancer cell-intrinsic immunostimulatory gene expression and promoted immunogenic cell death of GBM cells. In the SB28 GBM model, local delivery of HA-MSA2 induced a delay in tumor growth and a significant extension of survival. The analysis of the TIME showed a profound shift in the GBM immune landscape after HA-MSA2 treatment, with higher infiltration by innate and adaptive immune cells including dendritic, natural killer (NK) and CD8 T cell populations. The therapeutic potential of this novel polymer conjugate warrants further investigation, particularly with other chemo-immunotherapeutics or cancer vaccines as a promising combinatorial therapeutic approach.

Abstract LB021: BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation

Abstract The excessive expression of antiapoptotic members in the B-cell leukemia/lymphoma-2 (BCL-2) family proteins emerges as a noteworthy oncogenic target in cancer cells. Employing a multistep virtual screening and filtering approach that integrates structure and ligand-based drug design, we aimed to identify novel and potent BCL-2 inhibitors from a sizable small molecule database, comprising over 210,000 compounds. Subsequently, seven selected molecules underwent rigorous biological activity tests in human cancer cell lines and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays. Four of these compounds exhibited notable inhibitory effects on cancer cell proliferation and led to an increase in apoptotic cell fractions. Among the identified compounds, BAU-243 displayed a substantial affinity for Bcl-2, demonstrating efficacy in reducing glioblastoma (GBM) cell proliferation, including cancer-initiating cells. Notably, in contrast to the selective Bcl-2 inhibitor ABT-199, BAU-243 induced autophagic cell death in GBM cells. In vivo investigations using orthotopic brain tumor models revealed a significant reduction in tumor growth with BAU-243 compared to both the vehicle group and animals treated with ABT-199. Computational modeling simulations, in concordance with in vitro findings, proposed that BAU-243 activates autophagic cell death by disrupting the Beclin 1:Bcl-2 complex. This positions BAU-243 as a promising small molecule for the treatment of GBM. Citation Format: Seyma Calis, Berna Dogan, Asuman Celebi, Ozlem Yapicier, Turker Kilic, Eda Tahir Turanli, Timucin Avsar, Serdar Durdagi. BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB021.

New Salicylanilide Derivatives and Their Peptide Conjugates as Anticancer Compounds: Synthesis, Characterization, and In Vitro Effect on Glioblastoma.

Pharmacologically active salicylanilides (2-hydroxy-N-phenylbenzamides) have been a promising area of interest in medicinal chemistry-related research for quite some time. This group of compounds has shown a wide spectrum of biological activities, including but not limited to anticancer effects. In this study, substituted salicylanilides were chosen to evaluate the in vitro activity on U87 human glioblastoma (GBM) cells. The parent salicylanilide, salicylanilide 5-chloropyrazinoates, a 4-aminosalicylic acid derivative, and the new salicylanilide 4-formylbenzoates were chemically and in vitro characterized. To enhance the internalization of the compounds, they were conjugated to delivery peptides with the formation of oxime bonds. Oligotuftsins ([TKPKG]n, n = 1-4), the ligands of neuropilin receptors, were used as GBM-targeting carrier peptides. The in vitro cellular uptake, intracellular localization, and penetration ability on tissue-mimicking models of the fluorescent peptide derivatives were determined. The compounds and their peptide conjugates significantly decreased the viability of U87 glioma cells. Salicylanilide compound-induced GBM cell death was associated with activation of autophagy, as characterized by immunodetection of autophagy-related processing of light chain 3 protein.

492 Novel BH3 Mimetic Bcl-2 Inhibitor Promotes Autophagic Cell Death and Reduces in Vivo Glioblastoma Tumor Growth

INTRODUCTION: Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma, the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. METHODS: In silico studies resulted in 500,000 molecules being screened with computer-aided drug design methods and the molecule with the highest binding affinity was selected and named BAU-243. Cell viability, IC50 value, tumor formation potential, tumor spheroid formation potential, tumor invasion studies were investigated following BAU-243 application. The molecular changes caused by Bcl-2 inhibition were demonstrated by quantitative PCR, western blot and immunofluorescence methods. In in vivo studies, glioma tumors were transplanted intracranially into Nod Scid Gamma(NSG) mice using the mouse xenograft model. RESULTS: Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall glioblastoma cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. CONCLUSIONS: Results indicated that BAU-243 activates autophagic cell death by disrupting Beclin 1:Bcl-2 complex, and may serve as a potential small molecule for the treatment of glioblastoma.

DNA damage-regulated autophagy modulator 1 prevents glioblastoma cells proliferation by regulating lysosomal function and autophagic flux stability

Glioblastoma (GBM) is the most aggressive and life-threatening brain tumor, characterized by its highly malignant and recurrent nature. DNA damage-regulated autophagy modulator 1 (DRAM-1) is a p53 target gene encoding a lysosomal protein that induces macro-autophagy and damage-induced programmed cell death in tumor growth. However, the precise mechanisms underlying how DRAM-1 affects tumor cell proliferation through regulation of lysosomal function and autophagic flux stability remain incompletely understood. We found that DRAM-1 expressions were evidently down-regulated in high-grade glioma and recurrent GBM tissues. The upregulation of DRAM-1 could increase mortality of primary cultured GBM cells. TEM analysis revealed an augmented accumulation of aberrant lysosomes in DRAM-1-overexpressing GBM cells. The assay for lysosomal pH and stability also demonstrated decreasing lysosomal membrane permeabilization (LMP) and impaired lysosomal acidity. Further research revealed the detrimental impact of lysosomal dysfunction, which impaired the autophagic flux stability and ultimately led to GBM cell death. Moreover, downregulation of mTOR phosphorylation was observed in GBM cells following upregulation of DRAM-1. In vivo and in vitro experiments additionally illustrated that the mTOR inhibitor rapamycin increased GBM cell mortality and exhibited an enhanced antitumor effect.