Glioblastoma Multiforme Cell Proliferation Research Articles

The Power of Synergism: A Novel Drug Combination for Improved Therapy of Glioblastoma

AbstractOne of the biggest obstacles in treating glioblastoma multiforme (GBM) is the plasticity and adaptability of GBM cells, which renders monotherapy ineffective. Novel drug combinations that target multiple pathways are required to overcome this challenge. In the current study, the PI3K/AKT/mTOR pathway is identified as a crucial component in GBM cell survival using the KEGG database and literature search. Hence, it is hypothesized that a potential synergistic therapy can be achieved by targeting different stages of this pathway by combining disulfiram (DSF) and 7‐ethyl‐10‐hydroxycamptothecin (SN‐38). The efficacy of this drug combination is evaluated using 2D and 3D in vitro assays, which exhibited a significant synergism with 5 × 104 times lower IC50 than that of temozolomide (TMZ), the gold‐standard drug. The combination treatment increases intracellular ROS (reactive oxygen species) production, and ROS inhibition by using N‐Acetyl Cysteine (NAC) reduced the cytotoxicity substantially, indicating that ROS played a crucial role in the synergistic cytotoxicity. The combination treatment inhibited GBM cell proliferation, migration, and stem cell marker expression. Mechanistically, the DSF+SN‐38 combination is found to increase p53 expression, inhibit PI3K signaling, and activate caspase 9. Altogether, this study demonstrates that the DSF+SN‐38 combination can present a promising therapeutic strategy for treating GBM.

Exploring the Therapeutic Effects of Radix Astragalus on Glioblastoma Multiforme Cell Culture

Objective: Glioblastomas, recognized for their aggressive and invasive nature, present a daunting challenge for effective treatment. At present, available therapies are not entirely efficacious in combating this primary brain tumor. In light of this, the present study aims to explore novel therapeutic strategies by investigating the antitumorigenic potential of Radix astragalus against Glioblastoma Multiforme (GBM) cell-lines in vitro. Methods: To accomplish this, the study employed a comprehensive research methodology that involved evaluating GBM cell viability and proliferation through MTT and LDH assays. Additionally, the study analyzed the oxidative burden of the GBM cell-line by measuring total antioxidant capacity (TAC), total oxidant status (TOS) and malondialdehyde (MDA) values, in order to further investigate the effects of Radix astragalus extracts. These advanced techniques allowed us to gain an in-depth understanding of the intricate molecular interactions between GBM cells and the extracts, paving the way for the development of novel therapies that can effectively combat this highly malignant disease. Results: The findings of this study demonstrate the remarkable antitumorigenic activity of Radix astragalus, as it significantly reduced cell viability in the GBM cell-line. Moreover, the extract inhibited the oxidative stress of GBM cells, which is a crucial factor that contributes to the development and progression of GBMs. Furthermore, the antioxidant assays revealed increased antioxidant activity in Radix astragalus-treated GBM cells compared to normal GBM cells. These findings offer a promising solution for treating GBMs, which are notoriously challenging to treat. Conclusion: The remarkable antitumorigenic activity of Radix astragalus identified in this study presents a novel avenue for cancer treatment research, with potential applications in the development of efficacious treatments against malignant brain tumors. Further exploration of the potential of Radix astragalus is warranted to pave the way for innovative therapies that can effectively combat GBMs.

Autophagy-related CMTM6 promotes glioblastoma progression by activating Wnt/β-catenin pathway and acts as an onco-immunological biomarker.

Glioblastoma multiforme (GBM) is identified as one of the most prevalent and malignant brain tumors, characterized by poor treatment outcomes and a limited prognosis. CMTM6, a membrane protein, has been found to upregulate the expression of programmed cell death 1 ligand 1 protein (PD-L1) and acts as an immune checkpoint inhibitor by inhibiting the programmed death 1 protein/PD-L1 signaling pathway. Recent research has demonstrated a high expression of CMTM6 in GBM, suggesting its potential role in influencing the pathogenesis and progression of GBM, as well as its association with immune cell infiltration in the tumor microenvironment. However, the underlying mechanism of CMTM6 in GBM requires further investigation. Data from cancer patients in The Cancer Genome Atlas, Gene Expression Omnibus and Chinese Glioma Genome Atlas cohorts were consolidated for the current study. Through multi-omics analysis, the study systematically examined the expression profile of CMTM6, epigenetic modifications, prognostic significance, biological functions, potential mechanisms of action and alterations in the immune microenvironment. Additionally, the study investigated CMTM6 expression in GBM cell lines and normal cells using reverse transcription PCR and western blot analysis. The impact of CMTM6 on GBM cell proliferation, migration and invasion was evaluated using a combination of cell counting kit-8 assay, clone formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, wound healing assay and Transwell assay. In order to explore the mechanism of CMTM6, the Wnt/β-catenin signaling pathway and autophagy-related genes were further verified through western blot analysis. CMTM6 is highly expressed in multiple tumors, particularly GBM. CMTM6 has been shown to be a valuable diagnostic and prognostic biomarker by various bioinformatics approaches. Additionally, CMTM6 plays a pivotal role in the pathogenesis of cancer, specifically GBM, by modulating various biological processes such as DNA methyltransferase expression, RNA modification, copy number variation, genomic heterogeneity, tumor stemness and DNA methylation. The findings of the experiment indicate a significant correlation between elevated CMTM6 expression and the proliferation, invasion, migration and autophagy of GBM cells, with potential key roles mediated through the Wnt/β-catenin signaling pathway. Furthermore, CMTM6 is implicated in modulating tumor immune cell infiltration and is closely linked to the expression of various immune checkpoint inhibitors and immune modulators, particularly within the context of GBM. High levels of CMTM6 expression also enhance the responsiveness of GBM patients to radiotherapy and chemotherapy, thereby offering valuable insights for guiding treatment strategies for GBM. Autophagy-related CMTM6 is highly expressed in various types of cancer, especially GBM, and it can regulate GBM progression through the Wnt/β-catenin signaling pathway and is capable of being used as an underlying target for the diagnosis, treatment selection and prognosis of patients with GBM.

PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-β and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta(PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. Thefunctional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-b and suppressing Wnt/b-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

Rhein inhibits cell proliferation of glioblastoma multiforme cells by regulating the tgfß and apoptotic signaling pathways

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a plant metabolite found in rhubarbs. It inhibits cell proliferation and stimulates apoptosis in in vivo and in vitro. However, research into the molecular mechanisms of action is insufficient for recommending it as a therapeutic agent. Therefore, this study aims to investigate the antiproliferative, apoptotic, and antimetastatic effects of rhein by targeting the TGF-β signaling pathway, and apoptotic pathway in glioblastoma cells (U87 GBM). In this study, the XTT assay was utilized to determine cell viability, the colony formation assay to measure cell survival and proliferation, RT-qPCR for the analysis of gene expressions, and ELISA for the detection of proteins. U87 GBM cells were treated with varying concentrations of rhein (5-100 µM) in a time-dependent manner (24, 48 h), after which the percentage of cell viability was calculated. The colony formation assay was performed by treating cells with the IC50 dose of rhein. According to the XTT assay, the IC50 dose of rhein was determined as 10 µM at 24 h. The ability to form colonies was significantly decreased in the cells of the treatment group. According to the gene expression analysis, rhein increased the mRNA levels of CASP3, -8, -9, BAX, and TGF-β1 genes, while a notable decrease was observed in the BCL-2, SMAD2, SMAD3, and TIMP1 genes. In conclusion, it was determined that rhein induces apoptosis via the non-canonical TGF-β pathway.

FOXP3 promote the progression of glioblastoma via inhibiting ferroptosis mediated by linc00857/miR-1290/GPX4 axis

The oncogenic properties of members belonging to the forkhead box (FOX) family have been extensively documented in different types of cancers. In this study, our objective was to investigate the impact of FOXP3 on glioblastoma multiforme (GBM) cells. By conducting a screen using a small hairpin RNA (shRNA) library, we discovered a significant association between FOXP3 and ferroptosis in GBM cells. Furthermore, we observed elevated levels of FOXP3 in both GBM tissues and cell lines, which correlated with a poorer prognosis. FOXP3 was found to promote the proliferation of GBM cells by inhibiting cell ferroptosis in vitro and in vivo. Mechanistically, FOXP3 not only directly upregulated the transcription of GPX4, but also attenuated the degradation of GPX4 mRNA through the linc00857/miR-1290 axis, thereby suppressing ferroptosis and promoting proliferation. Additionally, the FOXP3 inhibitor epirubicin exhibited the ability to impede proliferation and induce ferroptosis in GBM cells both in vitro and in vivo. In summary, our study provided evidences that FOXP3 facilitates the progression of glioblastoma by inhibiting ferroptosis via the linc00857/miR-1290/GPX4 axis, highlighting FOXP3 as a potential therapeutic target for GBM.

Nuclear autoantigenic sperm protein facilitates glioblastoma progression and radioresistance by regulating the ANXA2/STAT3 axis.

Although radiotherapy is a core treatment modality for various human cancers, including glioblastoma multiforme (GBM), its clinical effects are often limited by radioresistance. The specific molecular mechanisms underlying radioresistance are largely unknown, and the reduction of radioresistance is an unresolved challenge in GBM research. We analyzed and verified the expression of nuclear autoantigenic sperm protein (NASP) in gliomas and its relationship with patient prognosis. We also explored the function of NASP in GBM cell lines. We performed further mechanistic experiments to investigate the mechanisms by which NASP facilitates GBM progression and radioresistance. An intracranial mouse model was used to verify the effectiveness of combination therapy. NASP was highly expressed in gliomas, and its expression was negatively correlated with the prognosis of glioma. Functionally, NASP facilitated GBM cell proliferation, migration, invasion, and radioresistance. Mechanistically, NASP interacted directly with annexin A2 (ANXA2) and promoted its nuclear localization, which may have been mediated by phospho-annexin A2 (Tyr23). The NASP/ANXA2 axis was involved in DNA damage repair after radiotherapy, which explains the radioresistance of GBM cells that highly express NASP. NASP overexpression significantly activated the signal transducer and activator of transcription 3 (STAT3) signaling pathway. The combination of WP1066 (a STAT3 pathway inhibitor) and radiotherapy significantly inhibited GBM growth invitro and invivo. Our findings indicate that NASP may serve as a potential biomarker of GBM radioresistance and has important implications for improving clinical radiotherapy.

Lycorine hydrochloride interferes with energy metabolism to inhibit chemoresistant glioblastoma multiforme cell growth through suppressing PDK3.

Glioblastoma multiforme (GBM) is the highest grade of glioma. Tumours, including GBM, possess reprogrammed metabolism, such as altered aerobic glycolysis and aberrant energy production. Lycorine hydrochloride (LH) was extracted from the bulb of Lycoris radiata. The previous study indicated that LH exerts antiviral, anti-inflammatory and antitumour effects. However, the effect of LH on GBM and the underlying molecular mechanism remain unclear. Our study revealed that LH restrained chemoresistant GBM cells growth by inhibiting PDK3 expression in vitro and in vivo. Functionally, LH inhibited the proliferation and invasive capacity of chemoresistant GBM cells in dose-dependent manner. Metabolomics and cellular energy analyses showed that LH decreased extracellular acidification rates while increased oxidative respiration and ROS levels. Mechanistically, LH inhibits the growth of GBM chemoresistant cells by regulating the expression of apoptosis-related proteins, while overexpression of of PDK3 can reverse the antitumor effect of LH. In conclusion, our study revealed that LH could reprogramme cell energy metabolism, including aerobic glycolysis suppression and oxidative phosphorylation hyperactivation by inhibiting PDK3. PDK3 may be a candidate therapeutic target for chemoresistant GBM treatment with LH.

PVT1 promotes proliferation and macrophage immunosuppressive polarization through STAT1 and CX3CL1 regulation in glioblastoma multiforme.

This study aimed to investigate the role of plasmacytoma variant translocation 1 (PVT1), a long non-coding RNA, in glioblastoma multiforme (GBM) and its impact on the tumor microenvironment (TME). We assessed aberrant PVT1 expression in glioma tissues and its impact on GBM cell growth invitro and invivo. Additionally, we investigated PVT1's role in influencing glioma-associated macrophages. To understand PVT1's role in cell growth and the immunosuppressive TME, we performed a series of comprehensive experiments. PVT1 was overexpressed in GBM due to copy number amplification, correlating with poor prognosis. Elevated PVT1 promoted GBM cell proliferation, while its downregulation inhibited growth invitro and invivo. PVT1 inhibited type I interferon-stimulated genes (ISGs), with STAT1 as the central hub. PVT1 correlated with macrophage enrichment and regulated CX3CL1 expression, promoting recruitment and M2 phenotype polarization of macrophages. PVT1 localized to the cell nucleus and bound to DHX9, enriching at the promoter regions of STAT1 and CX3CL1, modulating ISGs and CX3CL1 expression. PVT1 plays a significant role in GBM, correlating with poor prognosis, promoting cell growth, and shaping an immunosuppressive TME via STAT1 and CX3CL1 regulation. Targeting PVT1 may hold therapeutic promise for GBM patients.

Ultra High‐Plex Spatial Proteogenomic Investigation of Giant Cell Glioblastoma Multiforme Immune Infiltrates Reveals Distinct Protein and RNA Expression Profiles

AbstractBackgroundThe advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin‐fixed paraffin‐embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post‐translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”.MethodHere we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high‐plex digital quantitation of proteins (147‐plex) and RNA (whole transcriptome,>18,000‐plex) from a single FFPE sample. We demonstrated high concordance, R>0.85, and minor change in sensitivity (<11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies.ResultsWe observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in‐depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post‐translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods.ConclusionsWithin our dataset, we observed >2‐fold higher protein expression levels of phospho‐GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho‐Thr231 Tau was >2‐fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology.

JAK3 Inhibition Regulates Stemness and Thereby Controls Glioblastoma Pathogenesis.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, effective treatment options for which still remain elusive. The current treatment procedure of maximal resection followed by chemotherapy has proved to be grossly insufficient to prevent disease progression and death. Despite best efforts, the maximum survival post-diagnosis is a mere 1.5 years. Therefore, there is a huge unmet clinical need to find effective therapeutic procedures to prevent the pathogenesis and relapse of GBM. Small-molecule inhibitors of signaling pathways are an attractive option to prevent various types of tumors. However, no effective small-molecule inhibitors have been successful against GBM in clinical trials. Various signaling pathways are altered and an array of signaling molecules, transcription factors (TFs), and epigenetic modifying factors have been implicated in the pathogenesis of GBM. JAK-STAT pathway alteration is an important contributor to GBM pathogenesis and relapse. Many small-molecule inhibitors of JAKs, or STAT TFs, especially JAK2 and STAT3, have been assessed for their anti-tumor activity in GBM. However, no definitive success so far has been achieved. Herein, by using two small-molecule inhibitors of JAK3, we show that they are quite effective in inhibiting GBM cell proliferation and neurosphere formation, downregulating their stemness character, and inducing differentiation into neuronal origin cells. The effect of a single treatment with the drugs, both in a serum-containing differentiation medium and in a proliferation medium containing EGF and FGF, was really strong in limiting GBM cell growth, suggesting a potential therapeutic application for these JAK inhibitors in GBM therapy.

Silencing GMPPB Inhibits the Proliferation and Invasion of GBM via Hippo/MMP3 Pathways.

Glioblastoma multiforme (GBM) is a highly aggressive malignancy and represents the most common brain tumor in adults. To better understand its biology for new and effective therapies, we examined the role of GDP-mannose pyrophosphorylase B (GMPPB), a key unit of the GDP-mannose pyrophosphorylase (GDP-MP) that catalyzes the formation of GDP-mannose. Impaired GMPPB function will reduce the amount of GDP-mannose available for O-mannosylation. Abnormal O-mannosylation of alpha dystroglycan (α-DG) has been reported to be involved in cancer metastasis and arenavirus entry. Here, we found that GMPPB is highly expressed in a panel of GBM cell lines and clinical samples and that expression of GMPPB is positively correlated with the WHO grade of gliomas. Additionally, expression of GMPPB was negatively correlated with the prognosis of GBM patients. We demonstrate that silencing GMPPB inhibits the proliferation, migration, and invasion of GBM cells both in vitro and in vivo and that overexpression of GMPPB exhibits the opposite effects. Consequently, targeting GMPPB in GBM cells results in impaired GBM tumor growth and invasion. Finally, we identify that the Hippo/MMP3 axis is essential for GMPPB-promoted GBM aggressiveness. These findings indicate that GMPPB represents a potential novel target for GBM treatment.

Glioblastoma cellular MAP4K1 facilitates tumor growth and disrupts T effector cell infiltration.

MAP4K1 has been identified as a cancer immunotherapy target. Whether and how cancer cell-intrinsic MAP4K1 contributes to glioblastoma multiforme (GBM) progression remains unclear. We found that MAP4K1 was highly expressed in the glioma cells of human GBM specimens. High levels of MAP4K1 mRNA were prevalent in IDH-WT and 1p/19q non-codeletion gliomas and correlated with poor prognosis of patients. MAP4K1 silencing inhibited GBM cell proliferation and glioma growth. Transcriptome analysis of GBM cells and patient samples showed that MAP4K1 modulated cytokine‒cytokine receptor interactions and chemokine signaling pathway, including IL-18R and IL-6R Importantly, MAP4K1 loss down-regulated membrane-bound IL-18R/IL-6R by inhibiting the PI3K-AKT pathway, whereas MAP4K1 restoration rescued this phenotype and therefore GBM cell proliferation. MAP4K1 deficiency abolished GBM cell pro-proliferation responses to IL-18, suggesting an oncogenic role of MAP4K1 via the intrinsic IL-18/IL-18R pathway. In addition, GBM cell-derived MAP4K1 impaired T-cell migration and reduced CD8+ T-cell infiltration in mouse glioma models. Together, our findings provide novel insight into the pathological significance of GBM cell-intrinsic MAP4K1 in driving tumor growth and immune evasion by remodeling cytokine-chemokine networks.

BCL2L13 promotes mitophagy through DNM1L-mediated mitochondrial fission in glioblastoma

There is an urgent need for novel diagnostic and therapeutic strategies for patients with Glioblastoma multiforme (GBM). Previous studies have shown that BCL2 like 13 (BCL2L13) is a member of the BCL2 family regulating cell growth and apoptosis in different types of tumors. However, the clinical significance, biological role, and potential mechanism in GBM remain unexplored. In this study, we showed that BCL2L13 expression is significantly upregulated in GBM cell lines and clinical GBM tissue samples. Mechanistically, BCL2L13 targeted DNM1L at the Ser616 site, leading to mitochondrial fission and high mitophagy flux. Functionally, these alterations significantly promoted the proliferation and invasion of GBM cells both in vitro and in vivo. Overall, our findings demonstrated that BCL2L13 plays a significant role in promoting mitophagy via DNM1L-mediated mitochondrial fission in GBM. Therefore, the regulation and biological function of BCL2L13 render it a candidate molecular target for treating GBM.

NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

Glioblastoma multiforme (GBM) is the most frequent and lethal cancer derived from the central nervous system, of which the mesenchymal (MES) subtype seriously influences the survival and prognosis of patients. NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) serves an important role in the carcinogenesis and progression of various types of cancer; however, the specific mechanism underlying the regulatory effects of NQO1 on GBM is unclear. Thus, the present study aimed to explore the role and mechanism of NQO1 in GBM progression. The results of bioinformatics analysis and immunohistochemistry showed that high expression of NQO1 was significantly related to the MES phenotype of GBM and shorter survival. In addition, MTT, colony formation, immunofluorescence and western blot analyses, and lung metastasis model experiments suggested that silencing NQO1 inhibited the proliferation and metastasis of GBM cells invitro and invivo. Furthermore, western blotting showed that the activity of the PI3K/Akt/mTOR signaling pathway was revealed to be inhibited by downregulation of NQO1 expression, whereas it was enhanced by overexpression of NQO1. Notably, co‑immunoprecipitation and ubiquitination experiments suggested that Snail was considered an important downstream target of NQO1 in GBM cells. Snail knockdown could eliminate the promoting effect of ectopic NQO1 on the proliferation and invasion of GBM cells, and reduce its effects on the activity of PI3K/Akt/mTOR signaling pathway. These results indicated that NQO1 could promote GBM aggressiveness by activating the PI3K/Akt/mTOR signaling pathway in a Snail‑dependent manner, and NQO1 and its relevant pathways may be considered novel targets for GBM therapy.

TMEM2 induces epithelial-mesenchymal transition and promotes resistance to temozolomide in GBM cells

Glioblastoma multiforme (GBM) is the most common intracranial malignant tumor and is notorious for its poor prognosis. An important element in the short overall survival of GBM patients is the lack of understanding the pathogenesis and progression of tumor and deficiency biomarkers that can be used for early diagnosis and therapeutic sensitivity monitoring. Studies have shown that transmembrane protein 2 (TMEM2) is participated in tumorigenesis of various human tumors, including rectal and breast cancers. Although Qiuyi Jiang et al. have reported that TMEM2 combined with IDH1/2 and 1p19q can predict the survival time of glioma patients based on bioinformatics, its expression and biological role of glioma remain unclear. In our study, we investigated the effect of TMEM2 expression level on glioma malignancy in public datasets and an independent internal dataset. We revealed TEMM2 expression was higher in GBM tissues than in non-tumor brain tissues (NBT). Moreover, the increase in TMEM2 expression level was closely related to tumor malignancy. The survival analysis showed that TMEM2 high expression reduces survival time in all glioma patients, including GBM and LGG patients. Subsequent experiments demonstrated that knockdown TMEM2 inhibited proliferation of GBM cells. In addition, we analyzed TMEM2 mRNA levels in different GBM subtypes, and demonstrated that TMEM2 expression was upregulated in mesenchymal subtype. Meanwhile, bioinformatics analysis and transwell assay indicated that knockdown TMEM2 suppressed epithelial-mesenchymal transition (EMT) in GBM. Importantly, Kaplan–Meier analysis demonstrated that TMEM2 high expression reduced the treatment response to TMZ in GBM patients. Knockdown of TMEM2 alone did not reduce apoptosis GBM cells, but significant apoptotic cells were observed in the group treated with additional TMZ. These studies may contribute to improving the accuracy of early diagnosis and evaluating the effectiveness of TMZ treatment in GBM patients.

Flubendazole exhibits anti-glioblastoma effect by inhibiting STAT3 and promoting cell cycle arrest

Glioblastoma multiforme (GBM) belongs to most aggressive and invasive primary brain tumor in adults whose prognosis and survival remains poor. Potential new treatment modalities include targeting the cytoskeleton. In our study, we demonstrated that repurposed drug flubendazole (FLU) significantly inhibits proliferation and survival of GBM cells. FLU exerted its effect by affecting microtubule structure and our results also suggest that FLU influences tubulins expression to a certain degree. Moreover, FLU effects decreased activation of STAT3 and also partially inhibited its expression, leading to upregulation of p53 signaling pathway and subsequent cell cycle arrest at G2/M phase as well as caspase-dependent cell death in GBM cells. These results suggest FLU as a promising agent to be used in GBM treatment and prompting further testing of its effects on GBM.

Abstract 3100: The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM)

Abstract Glioblastoma multiforme (GBM) is an aggressive stage IV brain cancer that is difficult to detect, typically inoperable, and associated with a poor prognosis. Challenges in targeting GBM arise from the involvement of various pro-survival pathways, being comprised of four different cellular states, and displaying heterogeneity between each tumor. Marketed FDA-approved drugs for GBM have demonstrated limited efficacy, toxicity issues, resistance, and poor BBB penetrance. Further probing into pathway breakdowns led to the discovery of the DYRKs (dual-specificity tyrosine-regulated kinase), CLKs (CDC-like kinases), PDGFRA/B (platelet-derived growth factor receptor), and PI3Kα (phosphoinositide 3-kinase) involvement and overexpression. The upregulation of these kinases are found to be prominently involved in GBM cell proliferation, survival, replication, aggression, metastasis, and resistance. Herein, we believe that the development of pleiotropic competitive inhibitors for the treatment of GBM could be a promising therapeutic option based on literature precedence and current data from research. DYR726, a water-soluble, pleiotropic inhibitor [IC50 PDGFRA/B/PI3Kα/CLk2/3DYRK1a/2/3 ( 53.0/48.0/6.2/15.13/137/4/38.6/11.2 nM)] has demonstrated in vitro cellular efficacy across various GBM cell lines. When treated in non-cancerous cell lines, DYR726 displayed an optimal therapeutic window, which is not observed in any glioma drugs currently in clinical trials. At 1µM concentrations, complete dissociation of the formation of neural stem cells and neurosphere growth in glioma cells are observed. Reduction in metastasis and proliferation are found when treated with DYR726 over three weeks. In vitro efficacy against current approved FDA treatment options and clinical candidates has been extensively tested across numerous GBM cell lines. When benched against one of the best PI3K kinase inhibitors in the clinic, Buparlisib, DYR726 was comparable, indicating a disruption in the RTK signaling survival pathway that is commonly associated with GBM. DYR726 performed favorably against Avapritinib (clinical trials for gliomas are beginning at Stanford) and when treated against FDA-approved combination treatment Dabrafenib+Trametinib (for 2% of glioma patients), it performed identically, suggesting that DYR726 can inhibit the cells at the same rate as the new FDA drug. Within patient-derived cell lines, it behaves identically to Buparlisib, and DYR726 has been shown to outperform AstraZenecas’ Osimertinib (which was significantly less potent) and Tafinlars’ Dabrafenib (displayed no effect). Based on the significance of efficacy, this series is on the CRUK Glasgow Cancer Center grant renewal as a potential Phase 0 within the next five years. Citation Format: Alessandra Fistrovich, Vasudah Tandon, Carly Cabel, Laura Basantes, Curtis Thorne, William Montfort, Sourav Banerjee, Christopher Hulme. The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3100.

Abstract 4310: Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles

Abstract The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin-fixed paraffin-embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post-translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”. Here we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (147-plex) and RNA (whole transcriptome, > 18,000-plex) from a single FFPE sample. We demonstrated high concordance, R > 0.85, and minor change in sensitivity (<11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies. We observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post-translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Within our dataset, we observed >2-fold higher protein expression levels of phospho-GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho-Thr231 Tau was >2-fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology. Citation Format: Shilah Bonnett, Alyssa Rosenbloom, Giang Ong, Mark Conner, Daniel Newhouse, Felicia New, Hiromi Sato, Chi Pha, Saskia Ilcisin, John Lyssand, Gary Geiss, Joseph M. Beechem. Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4310.

MAPK4 predicts poor prognosis and facilitates the proliferation and migration of glioma through the AKT/mTOR pathway.

Mitogen-activated protein kinase 4 (MAPK4) is an atypical member of the mitogen-activated protein kinase (MAPK) family. We report here that MAPK4 is overexpressed in glioma. The clinical significance, biological roles and underlying molecular mechanisms through which MAPK4 acts in glioma remain unclear. Analysis of MAPK4 expression and associated survival in glioma patients was performed based on data obtained from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases and confirmed in human glioma tissue by immunohistochemistry. MAPK4 function and pathway enrichment were analyzed through Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO). The viability and migration ability of MAPK4-silenced glioblastoma multiforme (GBM) cells were evaluated using CCK8 and transwell assays, respectively, and cell cycle and apoptosis analyses were performed using flow cytometry. Immunoblotting was used to analyze the protein level in MAPK4 knockdown glioma cells. We also analyzed the correlation of MAPK4 expression with immune infiltration and immune checkpoints in glioma. MAPK4 was overexpressed in IDH wild-type (wt) and 1p/19q non-codeletion gliomas. MAPK4 expression predicted poor prognosis of glioma patients. MAPK4 was significantly related to functional states, including stemness, metastasis, cell cycle, differentiation and proliferation, in glioma at single-cell resolution. MAPK4 silencing inhibited proliferation and migration and induced G1 cell cycle arrest in glioma cells via the AKT/mTOR pathway. In vivo, MAPK4 knockdown markedly suppressed the growth of primary glioma. In addition, MAPK4 expression correlated negatively with the infiltration of plasmacytoid DC cells, CD8+ T cells and T helper cells. Moreover, MAPK4 expression correlated positively with expression of the main immunoinhibitor checkpoint molecules and some chemokines in glioma. MAPK4 functions as a prognostic indicator in glioma and promotes the proliferation and migration of GBM cells through the AKT/mTOR pathway. MAPK4 may participate in immune infiltration and the expression of immune checkpoints in the glioma microenvironment.