Therapeutic Target In Glioblastoma Research Articles

Lactate dehydrogenase A regulates tumor-macrophage symbiosis to promote glioblastoma progression

Abundant macrophage infiltration and altered tumor metabolism are two key hallmarks of glioblastoma. By screening a cluster of metabolic small-molecule compounds, we show that inhibiting glioblastoma cell glycolysis impairs macrophage migration and lactate dehydrogenase inhibitor stiripentol emerges as the top hit. Combined profiling and functional studies demonstrate that lactate dehydrogenase A (LDHA)-directed extracellular signal-regulated kinase (ERK) pathway activates yes-associated protein 1 (YAP1)/ signal transducer and activator of transcription 3 (STAT3) transcriptional co-activators in glioblastoma cells to upregulate C-C motif chemokine ligand 2 (CCL2) and CCL7, which recruit macrophages into the tumor microenvironment. Reciprocally, infiltrating macrophages produce LDHA-containing extracellular vesicles to promote glioblastoma cell glycolysis, proliferation, and survival. Genetic and pharmacological inhibition of LDHA-mediated tumor-macrophage symbiosis markedly suppresses tumor progression and macrophage infiltration in glioblastoma mouse models. Analysis of tumor and plasma samples of glioblastoma patients confirms that LDHA and its downstream signals are potential biomarkers correlating positively with macrophage density. Thus, LDHA-mediated tumor-macrophage symbiosis provides therapeutic targets for glioblastoma.

Exosome-transmitted podoplanin promotes tumor-associated macrophage-mediated immune tolerance in glioblastoma.

Glioblastoma is the most frequent and aggressive primary brain tumor, characterized by rapid disease course and poor treatment responsiveness. The abundance of immunosuppressive macrophages in glioblastoma challenges the efficacy of novel immunotherapy. Bulk RNA-seq and single-cell RNA-seq of glioma patients from public databases were comprehensively analyzed to illustrate macrophage infiltration patterns and molecular characteristics of podoplanin (PDPN). Multiplexed fluorescence immunohistochemistry staining of PDPN, GFAP, CD68, and CD163 were performed in glioma tissue microarray. The impact of PDPN on macrophage immunosuppressive polarization was investigated using a co-culture system. Bone marrow-derived macrophages (BMDMs) and OT-II T cells isolated from BALB/c and OT-II mice respectively were co-cultured to determine T-cell adherence. Pathway alterations were probed through RNA sequencing and western blot analyses. Our findings demonstrated that PDPN is notably correlated with the expression of CD68 and CD163 in glioma tissues. Additionally, macrophages phagocytosing PDPN-containing EVs (EVsPDPN ) from GBM cells presented increased CD163 expression and augmented secretion of immunoregulatorycytokine (IL-6, IL-10, TNF-α, and TGF-β1). PDPN within EVs was also associated with enhanced phagocytic activity and reduced MHC II expression in macrophages, compromising CD4+ T-cell activation. This investigation underscores that EVsPDPN derived from glioblastoma cells contributes to M2 macrophage-mediated immunosuppression and is a potential prognostic marker and therapeutic target in glioblastoma.

NCDN is a Potential Biomarker and Therapeutic Target for Glioblastoma.

Background: Glioblastoma (GBM) is a type of central nervous system malignancy. In our study, we determined the effect of NCDN in GBM patients through The Cancer Genome Atlas (TCGA) data analysis, and studied the effects of NCDN on GBM cell function to estimate its potential as a therapeutic target. Methods: Gene expression profiles of glioblastoma cohort were acquired from TCGA database and analyzed to look for central genes that may serve as GBM therapeutic targets. Then the cell function of NCDN in glioblastoma cell was explored through in vitro cell experiments. Results: Through gene ontology (GO) analysis, weighted gene co-expression network analysis (WGCNA), and survival analysis, we identified three key genes (NCDN, PAK1 and SPRYD3) associated with poor prognosis in glioblastoma. In vitro experiments showed impaired cell migration, apoptosis, and cell cycle arrest in NCDN knockdown cells. Conclusion: NCDN affects the progress and prognosis of glioblastoma by promoting cell migration and inhibiting apoptosis.

Integrated Gene Expression Data-Driven Identification of Molecular Signatures, Prognostic Biomarkers, and Drug Targets for Glioblastoma.

Glioblastoma (GBM) is a highly prevalent and deadly brain tumor with high mortality rates, especially among adults. Despite extensive research, the underlying mechanisms driving its progression remain poorly understood. Computational analysis offers a powerful approach to explore potential prognostic biomarkers, drug targets, and therapeutic agents for GBM. In this study, we utilized three gene expression datasets from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) associated with GBM progression. Our goal was to uncover key molecular players implicated in GBM pathogenesis and potential avenues for targeted therapy. Analysis of the gene expression datasets revealed a total of 78 common DEGs that are potentially involved in GBM progression. Through further investigation, we identified nine hub DEGs that are highly interconnected in protein-protein interaction (PPI) networks, indicating their central role in GBM biology. Gene Ontology (GO) and pathway enrichment analyses provided insights into the biological processes and immunological pathways influenced by these DEGs. Among the nine identified DEGs, survival analysis demonstrated that increased expression of GMFG correlated with decreased patient survival rates in GBM, suggesting its potential as a prognostic biomarker and preventive target for GBM. Furthermore, molecular docking and ADMET analysis identified two compounds from the NIH clinical collection that showed promising interactions with the GMFG protein. Besides, a 100 nanosecond molecular dynamics (MD) simulation evaluated the conformational changes and the binding strength. Our study highlights the potential of GMFG as both a prognostic biomarker and a therapeutic target for GBM. The identification of GMFG and its associated pathways provides valuable insights into the molecular mechanisms driving GBM progression. Moreover, the identification of candidate compounds with potential interactions with GMFG offers exciting possibilities for targeted therapy development. However, further laboratory experiments are required to validate the role of GMFG in GBM pathogenesis and to assess the efficacy of potential therapeutic agents targeting this molecule.

Systematic analysis based on the cuproptosis-related genes identifies ferredoxin 1 as an immune regulator and therapeutic target for glioblastoma

Glioblastoma multiforme (GBM) is recognized as the prevailing malignant and aggressive primary brain tumor, characterized by an exceedingly unfavorable prognosis. Cuproptosis, a recently identified form of programmed cell death, exhibits a strong association with cancer progression, therapeutic response, and prognostic outcomes. However, the specific impact of cuproptosis on GBM remains uncertain. To address this knowledge gap, we obtained transcriptional and clinical data pertaining to GBM tissues and their corresponding normal samples from various datasets, including TCGA, CGGA, GEO, and GTEx. R software was utilized for the analysis of various statistical techniques, including survival analysis, cluster analysis, Cox regression, Lasso regression, gene enrichment analysis, drug sensitivity analysis, and immune microenvironment analysis. Multiple assays were conducted to investigate the expression of genes related to cuproptosis and their impact on the proliferation, invasion, and migration of glioblastoma multiforme (GBM) cells. The datasets were obtained and prognostic risk score models were constructed and validated using differentially expressed genes (DEGs) associated with cuproptosis. To enhance the practicality of these models, a nomogram was developed.Patients with glioblastoma multiforme (GBM) who were classified as high risk exhibited a more unfavorable prognosis and shorter overall survival compared to those in the low risk group. Additionally, we specifically chose FDX1 from the differentially expressed genes (DEGs) within the high risk group to assess its expression, prognostic value, biological functionality, drug responsiveness, and immune cell infiltration. The findings demonstrated that FDX1 was significantly upregulated and associated with a poorer prognosis in GBM. Furthermore, its elevated expression appeared to be linked to various metabolic processes and the susceptibility to chemotherapy drugs. Moreover, FDX1 was found to be involved in immune cell infiltration and exhibited correlations with multiple immunosuppressive genes, including TGFBR1 and PDCD1LG2. The aforementioned studies offer substantial assistance in informing the chemotherapy and immunotherapy approaches for GBM. In summary, these findings contribute to a deeper comprehension of cuproptosis and offer novel perspectives on the involvement of cuproptosis-related genes in GBM, thereby presenting a promising therapeutic strategy for GBM patients.

Zotiraciclib (TG02) for newly diagnosed glioblastoma in the elderly or for recurrent glioblastoma: The EORTC 1608 STEAM trial

BackgroundZotiraciclib (TG02) is an oral multi-cyclin dependent kinase (CDK) inhibitor thought to inhibit tumor growth via CDK-9-dependent depletion of survival proteins such as c-MYC and MCL-1 which are frequently overexpressed in glioblastoma. MethodsEORTC 1608 (NCT03224104) (STEAM) had a three parallel group (A,B,C) phase Ib, open-label, non-randomized, multicenter design in IDH wild-type newly diagnosed glioblastoma or anaplastic astrocytoma. Groups A and B explored the maximum tolerated dose (MTD) of TG02 in elderly patients, in combination with hypofractionated radiotherapy alone (group A) or temozolomide alone (group B), according to O6-methylguanine DNA methyltransferase promoter methylation status determined centrally. Group C explored single agent activity of TG02 at first relapse after temozolomide chemoradiotherapy with a primary endpoint of progression-free survival at 6 months (PFS-6). Tumor expression of CDK-9, c-MYC and MCL-1 was determined by immunohistochemistry. ResultsThe MTD was 150 mg twice weekly in combination with radiotherapy alone (group A) or temozolomide alone (group B). Two dose-limiting toxicities were observed at 150 mg: one in group A (grade 3 seizure), one in group B (multiple grade 1 events). Main toxicities included neutropenia, gastrointestinal disorders and hepatotoxicity. PFS-6 in group C was 6.7%. CDK-9, c-MYC and MCL-1 were confirmed to be expressed and their expression was moderately cross-correlated. High protein levels of MCL-1 were associated with inferior survival. ConclusionsTG02 exhibits overlapping toxicity with alkylating agents and low single agent clinical activity in recurrent glioblastoma. The role of CDK-9 and its down-stream effectors as prognostic factors and therapeutic targets in glioblastoma warrants further study.

Flavonoids Regulate Redox-Responsive Transcription Factors in Glioblastoma and Microglia.

The tumor microenvironment (TME) has emerged as a valuable therapeutic target in glioblastoma (GBM), as it promotes tumorigenesis via an increased production of reactive oxygen species (ROS). Immune cells such as microglia accumulate near the tumor and its hypoxic core, fostering tumor proliferation and angiogenesis. In this study, we explored the therapeutic potential of natural polyphenols with antioxidant and anti-inflammatory properties. Notably, flavonoids, including fisetin and quercetin, can protect non-cancerous cells while eliminating transformed cells (2D cultures and 3D tumoroids). We tested the hypothesis that fisetin and quercetin are modulators of redox-responsive transcription factors, for which subcellular location plays a critical role. To investigate the sites of interaction between natural compounds and stress-responsive transcription factors, we combined molecular docking with experimental methods employing proximity ligation assays. Our findings reveal that fisetin decreased cytosolic acetylated high mobility group box 1 (acHMGB1) and increased transcription factor EB (TFEB) abundance in microglia but not in GBM. Moreover, our results suggest that the most powerful modulator of the Nrf2-KEAP1 complex is fisetin. This finding is in line with molecular modeling and calculated binding properties between fisetin and Nrf2-KEAP1, which indicated more sites of interactions and stronger binding affinities than quercetin.

CREB-induced LINC00473 promotes chemoresistance to TMZ in glioblastoma by regulating O6-methylguanine-DNA-methyltransferase expression via CEBPα binding

Temozolomide (TMZ) offers substantial therapeutic benefits for glioblastoma (GB), yet its efficacy is hindered the development of chemoresistance. The role of long non-coding RNAs (lncRNAs) in tumorigenesis and chemoresistance has garnered great attention in studies on TMZ resistance. This study aimed to reveal the role of LINC00473 in TMZ chemoresistance and the underlying mechanism in GB. The expression of LINC00473 in TMZ-resistant and TMZ-sensitive GB cells was investigated using qPCR analysis. The role of LINC00473 in regulating TMZ resistance in GB cells was analyzed using the CCK-8 assay, colony formation assay, and flow cytometry. The next steps included assessing if LINC00473 is regulated by CREB and whether LINC00473 promotes chemoresistance through MGMT regulation via CEBPα. Further, chemoresistance delivery between cells via exosomal LINC00473 was validated in vitro and in vivo. Results showed that LINC00473 levels were elevated in TMZ-resistant cells upon CREB activation, and the lncRNA promoted the chemoresistance of GB cells through the upregulation of MGMT expression. Mechanistically, LINC00473 regulated the MGMT expression by binding to CEBPα. The highly-expressed LINC00473 packaged in exosomes transferred chemoresistance to the adjacent TMZ-sensitive GB cells. In conclusion, a novel CREB/LINC00473/CEBPα/MGMT pathway was revealed in the GB TMZ-resistance formation. In addition, an exosome-based mechanism of chemoresistance transmission was revealed, suggesting that LINC00473 could be used as a novel therapeutic target for GB.

Schnurri-3 drives tumor growth and invasion in cancer cells expressing interleukin-13 receptor alpha 2

Interleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/β-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.

MODL-02. DEVELOPMENT OF RODENT GLIOBLASTOMA MODELS OF MGMT-MEDIATED TEMOZOLOMIDE RESISTANCE

Abstract O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine DNA damage lesions generated by alkylators such as temozolomide (TMZ), the standard chemotherapeutic for glioblastoma (GBM) treatment. High MGMT expression leads to TMZ resistance and is correlated with lower survival, making it an important prognostic biomarker and potential therapeutic target. Xenograft models with human GBM cell lines require implantation into immunodeficient animals, precluding the study of MGMT-dependent alkylator sensitivity in the setting of an intact tumor immune microenvironment. In this study, we developed rat and mouse glioma models of MGMT overexpression and characterized response to alkylator therapy. RG2 rat glioma and SB28 mouse glioma cells, which have low endogenous MGMT levels, were transduced with cDNA expressing FLAG-tagged MGMT to generate an isogenic pair. RG2 MGMT+ and SB28 MGMT+ cells showed robust expression of MGMT, which was effectively abrogated by MGMT inhibitors. In vitro cell viability assays showed that MGMT overexpression resulted in increased resistance to TMZ compared to parental cells, and this resistance was reversed when cells were concurrently treated with the MGMT inhibitor lomeguatrib. To model MGMT overexpression in vivo, luciferase-expressing RG2 MGMT+ cells were implanted into the brains of syngeneic Fischer 344 rats. Rats were treated with TMZ at 50mg/kg daily x 5 days via intraperitoneal injection. Tumor growth was measured by bioluminescence imaging and rats were monitored for survival and toxicity. RG2 MGMT+ tumors were highly resistant to TMZ, as treatment did not improve survival or delay tumor growth. Overall, our newly developed isogenic rat and mouse glioma models recapitulate MGMT-dependent TMZ sensitivity and provide useful preclinical tools for studying MGMT as a biomarker and potential therapeutic target in GBM. In addition, these syngeneic models facilitate studies on the immune modulation of chemotherapeutic agents in the context of MGMT expression and allows for testing of chemoimmunotherapy combinations.

DDDR-08. DISCOVERY OF ER STRESS-INDUCING COMPOUNDS FOR GBM: A PROMISING APPROACH TOWARDS THERAPEUTIC DEVELOPMENT

Abstract The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses encountered by tumor cells, such as hypoxia, acidosis, reactive oxygen species and even radiation and chemotherapy. In response to increased unfolded or mis-folded proteins in the endoplasmic reticulum (ER), the UPR initiates specific cell signaling pathways aiming to alleviate ER stress and restore ER proteostasis which include increasing the expression of chaperones to aid in protein folding, enhancing protein degradation and reducing global protein synthesis. However, if homeostasis cannot be restored, the UPR switches from survival to cell death. The UPR is a relatively novel therapeutic target for glioblastoma (GBM), and specifically GBM stem-like cells (GSCs) which maintain tumor heterogeneity, drive GBM tumor growth and therapy resistance. Here we tested a series of curcumin derivatives against three GSC lines and identified a trimethoxy bis-chalcone which promotes robust GSC death with an average IC50 of just below 300 nM, a 100-fold increase in activity compared to curcumin. Furthermore, this bis-chalcone was non-toxic to normal human mesenchymal stem cells. Western blot analysis indicated that the bis-chalcone induced robust activation of UPR pro-death protein C/EBP Homologous Protein (CHOP) while simultaneously decreasing the expression of pro-survival chaperone GRP78/Bip. Lastly our bis-chalcone induced caspase 7 expression and PARP cleavage indicating apoptosis. Most ER stress inducing agents promote both the survival, upregulation of Grp78, and cell death arms of the UPR. Here we identified a compound that only promotes UPR cell death signaling and is highly toxic to radiation and temozolomide resistant GSCs while demonstrating no toxicity to non-tumor cells. This is a promising lead compound for the development of UPR targeted GSC/GBM therapeutics.

CTNI-34. ZOTIRACICLIB (TG02) FOR NEWLY DIAGNOSED GLIOBLASTOMA IN THE ELDERLY OR FOR RECURRENT GLIOBLASTOMA: FINAL RESULTS OF THE EORTC 1608 STEAM TRIAL

Abstract BACKGROUND Zotiraciclib (TG02) is an oral multi-cyclin dependent kinase (CDK) inhibitor that is supposed to suppress tumor growth via CDK-9-dependent depletion of survival proteins such as c-MYC and MCL-1 which are frequently overexpressed in glioblastoma. METHODS EORTC 1608 (NCT03224104) (STEAM) had a three parallel group (A,B,C) phase Ib, open-label, non-randomized, multicenter design. Groups A and B explored the maximum tolerated dose (MTD) of TG02 in elderly patients with IDH1R132H-non-mutant newly diagnosed glioblastoma or anaplastic astrocytoma, in combination with hypofractionated radiotherapy alone (group A) or temozolomide alone (group B), based on O6-methylguanine DNA methyltransferase promoter methylation status determined centrally. Group C explored single agent activity of TG02 in IDH1R132H-non-mutant glioblastoma or anaplastic astrocytoma at first relapse after temozolomide chemoradiotherapy with a primary endpoint of progression-free survival at 6 months. Secondary objectives included efficacy, quality of life, and safety. Expression of CDK-9, c-MYC and MCL-1 was determined by immunohistochemistry. RESULTS The MTD was 150 mg twice weekly in combination with radiotherapy alone (group A) or temozolomide alone (group B) in elderly patients. Two dose-limiting toxicities were observed at 150 mg, one in group A (grade 3 seizure) and one in group B (multiple grade 1 events). Main toxicities included neutropenia, gastrointestinal disorders, and hepatotoxicity. Progression-free survival at 6 months in group C was 6.7%. CDK9 and its indirect targets, c-MYC and MCL-1, were confirmed to be expressed and their expression was correlated, supporting a regulatory role for CDK-9. The protein levels of CDK-9 and MCL-1 correlated with overall survival. CONCLUSION TG02 exhibits overlapping toxicity with alkylating agents and low clinical activity as a single agent. Larger randomized trials are required to explore activity in combination with RT or TMZ. The role of CDK-9 and its down-stream effectors as prognostic factors and therapeutic targets in glioblastoma warrants further study.

Oncogenic Activities of Tribbles1 (TRIB1) Pseudokinase Overexpressed in GBM are Mediated by Protein-Protein Interactions

Glioblastoma (GBM) is the most aggressive form of glioma with a low 5-year survival rate. The current treatments are inadequate and crippled by therapy resistance. Therefore, there is an unmet need to identify druggable therapeutic targets in GBM. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold to initiate Ubiquitin Proteasome System-mediated degradation of its substrates. We and others have found that TRIB1 activates the canonical MAPK and Akt signaling cascades. Previous reports also suggest that TRIB1 contributes to chemotherapy resistance in various cancers. Therefore, we evaluated oncogenic roles of TRIB1 in GBM cells and its contribution to therapy resistance. Patient-centered reverse translational approach was utilized to identify novel therapeutic targets. To this end, TRIB1 was identified by statistical association (Cox regression analysis) of the patient-derived gene expression profiling data publicly available from TCGA GBM cohort. TRIB1 was functionally validated in vitro by generating stable overexpression cell lines (patient-derived) by antibiotic selection. Conditional knockdown of TRIB1 was achieved by doxycycline induction. Protein-protein interactions were evaluated by co-immunoprecipitation. Protein levels were detected by western blotting. Changes in tumor volume and overall survival (OS) were calculated. The mRNA profiling of TCGA GBM cohort revealed that increased TRIB1 gene expression was associated with worse OS of GBM patients [HR = 1.3 (1.0-1.5); P = 0.019]. The same analyses in our institutional cohort revealed a similar association. Mice bearing TRIB1 transgene overexpressing tumors had the increased tumor volume and shorter OS compared to empty vector control at the end of experiment. Overexpression of TRIB1 increased the phosphorylation/activation of ERK and Akt in patient-derived primary cell lines. Akt but not ERK activation was decreased after TRIB1 knockdown. TRIB1 bound directly to ERK and Akt in these cells. TRIB1 also formed a complex with p53, COP1 and HDAC1 in patient-derived primary cell lines. This protein-protein interaction was independent of TP53 mutation status. Our data suggest that TRIB1 overexpressed in GBM executes various oncogenic functions through interaction with different proteins. Activating ERK signaling, can induce cell proliferation. Similarly, by activating Akt it can cause prosurvival effects. Finally, by associating with HDAC1 and COP1, TRIB1 can modulate p53 function. All these protein-protein interactions ultimately contribute to chemoradiotherapy resistance in GBM cells. We are currently developing small molecule inhibitors targeting the above-mentioned interactions of TRIB1 to overcome therapeutic resistance.

MicroRNA-92b targets tumor suppressor gene FBXW7 in glioblastoma.

Glioblastoma (GBM) is a highly aggressive and lethal primary brain tumor. Despite limited treatment options, the overall survival of GBM patients has shown minimal improvement over the past two decades. Factors such as delayed cancer diagnosis, tumor heterogeneity, cancer stem cell survival, infiltrative nature of GBM cells, metabolic reprogramming, and development of therapy resistance contribute to treatment failure. To address these challenges, multitargeted therapies are urgently needed for improved GBM treatment outcomes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Dysregulated miRNAs have been identified in GBM, playing roles in tumor initiation, progression, and maintenance. Among these miRNAs, miR-92b (miRNA-92b-3p) has been found to be overexpressed in various cancers, including GBM. However, the specific target genes of miR-92b and its therapeutic potential in GBM remain poorly explored. Samples encompassed T98G, U87, and A172 human GBM cell lines, GBM tumors from Puerto Rican patients, and murine tumors. In-situ hybridization (ISH) assessed miR-92b expression in patient tumors. Transient and stable transfections modified miR-92b levels in GBM cell lines. Real-time PCR gauged gene expressions. Caspase 3 and Trypan Blue assays evaluated apoptosis and viability. Bioinformatics tools (TargetScanHuman 8.0, miRDB, Diana tools, miRWalk) predicted targets. Luciferase assays and Western Blots validated miRNA-target interactions. A subcutaneous GBM Xenograft mouse model received intraperitoneal NC-OMIs or miR92b-OMIs encapsulated in liposomes, three-times per week for two weeks. Analysis utilized GraphPad Prism 8; statistical significance was assessed using 2-tailed, unpaired Student's t-test and two-way ANOVA as required. This study investigated the expression of miR-92b in GBM tumors compared to normal brain tissue samples, revealing a significant upregulation. Inhibition of miR-92b using oligonucleotide microRNA inhibitors (OMIs) suppressed GBM cell growth, migration, and induced apoptosis, while ectopic expression of miR-92b yielded opposite effects. Systemic administration of liposomal-miR92b-OMIs in GBM xenograft mice resulted in reductions in tumor volume and weight. Subsequent experiments identified F-Box and WD Repeat Domain Containing 7 (FBXW7) as a direct target gene of miR-92b in GBM cells. FBXW7 acts as a tumor suppressor gene in various cancer types, and analysis of patient data demonstrated that GBM patients with higher FBXW7 mRNA levels had significantly better overall survival compared to those with lower levels. Taken together, our findings suggest that the dysregulated expression of miR-92b in GBM contributes to tumor progression by targeting FBXW7. These results highlight the potential of miR-92b as a therapeutic target for GBM. Further exploration and development of miR-92b-targeted therapies may offer a novel approach to improve treatment outcomes in GBM patients.

P17.12.B FEASIBILITY OF PROLYL OLIGOPEPTIDASE (PREP) AS TUMOR BIOMARKER IN GLIOBLASTOMA PATIENTS: A PILOT STUDY

Abstract BACKGROUND Prolyl oligopeptidase (PREP) is a serine protease that hydrolyzes biologically active oligopeptides on their carboxyl side. PREP has been connected with multiple physiological and pathological conditions. Moreover, increased PREP activities have been found in tumors, although its involvement in the modulation of cancer mechanism is still unclear. Thus, the identification of PREP and clarification of its clinical significance are important for improving the diagnosis and treatment of brain tumors. The purpose of this study was to analyze the expression of PREP and its diagnostic and prognostic value in patients with Glioblastoma (GBM). Materials and METHODS In this study, GBM patients and controls were enrolled and clinical data and PREP expression, from serum and biopsies, were collected. The relationships between PREP, Protein phosphatase 2A (PP2A), STAT3 and Transforming growth factor-β (TGF- β) were also analyzed from clinical data. RESULTS Our study found that PREP was significantly upregulated in the serum samples and in biopsy from GBM patients compared with controls. This upregulation was negatively correlated with survival, but significantly correlated with age and Mindbomb Homolog-1 Index (MIB1). Also, PREP expression was inversely correlated with tumor suppressor PP2A in biopsy from GBM patients, while PREP protein levels resulted to be positively correlated with STAT3 and TGF-β protein expression. CONCLUSION s: The detection of PREP in serum and biopsies have the potential to serve as diagnostic and prognostic biomarker in GBM patient. The findings may provide diagnostic and therapeutic target for GBM.

DYRK1A-mediated PLK2 phosphorylation regulates the proliferation and invasion of glioblastoma cells.

Polo-like kinases (PLKs) are a family of serine-threonine kinases that exert regulatory effects on diverse cellular processes. Dysregulation of PLKs has been implicated in multiple cancers, including glioblastoma (GBM). Notably, PLK2 expression in GBM tumor tissue is lower than that in normal brains. Notably, high PLK2 expression is significantly correlated with poor prognosis. Thus, it can be inferred that PLK2 expression alone may not be sufficient for accurate prognosis evaluation, and there are unknown mechanisms underlying PLK2 regulation. In the present study, it was demonstrated that dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) interacts with and phosphorylates PLK2 at Ser358. DYRK1A-mediated phosphorylation of PLK2 increases its protein stability. Moreover, PLK2 kinase activity was markedly induced by DYRK1A, which was exemplified by the upregulation of alpha-synuclein S129 phosphorylation. Furthermore, it was found that phosphorylation of PLK2 by DYRK1A contributes to the proliferation, migration and invasion of GBM cells. DYRK1A further enhances the inhibition of the malignancy of GBM cells already induced by PLK2. The findings of the present study indicate that PLK2 may play a crucial role in GBM pathogenesis partially in a DYRK1A-dependent manner, suggesting that PLK2 Ser358 may serve as a therapeutic target for GBM.

Identification and validation of SOCS1/2/3/4 as potential prognostic biomarkers and correlate with immune infiltration in glioblastoma.

Suppressor of cytokine signalling (SOCS) 1/2/3/4 are involved in the occurrence and progression of multiple malignancies; however, their prognostic and developmental value in patients with glioblastoma (GBM) remains unclear. The present study used TCGA, ONCOMINE, SangerBox3.0, UALCAN, TIMER2.0, GENEMANIA, TISDB, The Human Protein Atlas (HPA) and other databases to analyse the expression profile, clinical value and prognosis of SOCS1/2/3/4 in GBM, and to explore the potential development mechanism of action of SOCS1/2/3/4 in GBM. The majority of analyses showed that SOCS1/2/3/4 transcription and translation levels in GBM tissues were significantly higher than those in normal tissues. qRT-PCR, western blotting (WB) and immunohistochemical staining were used to verify that SOCS3 was expressed at higher mRNA and protein levels in GBM than in normal tissues or cells. High SOCS1/2/3/4 mRNA expression was associated with poor prognosis in patients with GBM, especially SOCS3. SOCS1/2/3/4 were highly contraindicated, which had few mutations, and were not associated with clinical prognosis. Furthermore, SOCS1/2/3/4 were associated with the infiltration of specific immune cell types. In addition, SOCS3 may affect the prognosis of patients with GBM through JAK/STAT signalling pathway. Analysis of the GBM-specific protein interaction (PPI) network showed that SOCS1/2/3/4 were involved in multiple potential carcinogenic mechanisms of GBM. In addition, colony formation, Transwell, wound healing and western blotting assays revealed that inhibition of SOCS3 decreased the proliferation, migration and invasion of GBM cells. In conclusion, the present study elucidated the expression profile and prognostic value of SOCS1/2/3/4 in GBM, which may provide potential prognostic biomarkers and therapeutic targets for GBM, especially SOCS3.

Chi3l1 Is a Modulator of Glioma Stem Cell States and a Therapeutic Target in Glioblastoma.

Chi3l1 is a modulator of glioma stem cell states that can be targeted to promote differentiation and suppress growth of glioblastoma.

The Membrane Protein Sortilin Is a Potential Biomarker and Target for Glioblastoma.

Glioblastoma (GBM) is a devastating brain cancer with no effective treatment, and there is an urgent need for developing innovative biomarkers as well as therapeutic targets for better management of the disease. The membrane protein sortilin has recently been shown to participate in tumor cell invasiveness in several cancers, but its involvement and clinical relevance in GBM is unclear. In the present study, we explored the expression of sortilin and its potential as a clinical biomarker and therapeutic target for GBM. Sortilin expression was investigated by immunohistochemistry and digital quantification in a series of 71 clinical cases of invasive GBM vs. 20 non-invasive gliomas. Sortilin was overexpressed in GBM and, importantly, higher expression levels were associated with worse patient survival, pointing to sortilin tissue expression as a potential prognostic biomarker for GBM. Sortilin was also detectable in the plasma of GBM patients by enzyme-linked immunosorbent assay (ELISA), but no differences were observed between sortilin levels in the blood of GBM vs. glioma patients. In vitro, sortilin was detected in 11 brain-cancer-patient-derived cell lines at the anticipated molecular weight of 100 kDa. Interestingly, targeting sortilin with the orally bioavailable small molecule inhibitor AF38469 resulted in decreased GBM invasiveness, but cancer cell proliferation was not affected, showing that sortilin is targetable in GBM. Together, these data suggest the clinical relevance for sortilin in GBM and support further investigation of GBM as a clinical biomarker and therapeutic target.

Histone acetylation gene-based biomarkers as novel markers of the immune microenvironment in glioblastoma.

Glioblastoma (GBM) is a primary malignant tumour with high intracranial morbidity, high malignancy and poor prognosis. Abnormal changes in histone acetylation are closely related to the occurrence and development of cancer. However, there is still a lack of systematic research on histone acetylation in GBM. Whole-transcriptome sequencing data and clinical data of GBM patients were obtained through the TCGA database. Single-cell RNA-sequencing (scRNA-seq) data from GBM patients were obtained from GSE146711 in the Gene Expression Omnibus database. Cell descending fractionation was first performed for scRNA-seq on GBM. The CellChat and PROGENy scores explore the impact of the histone acetylation pathway in GBM on intercellular chat and tumour pathways. The AddModuleScore function evaluates the enrichment score of histone acetylation in cells and divides them into high-histone acetylation and low-histone acetylation groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the differential genes between different histone acetylation states, and the biological processes and pathways that may be affected by histone acetylation were evaluated. Based on this, a prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) analysis, and survival analysis was performed to evaluate its prognostic performance. Finally, we also analysed the main effects of the constructed histone acetylation-related model on GBM immune infiltration by multiple methods, and analysed the main mutation data of its different subgroups. GBM samples mainly include seven large cell populations: oligodendrocyte precursor cells (OPCs), myeloid, neoplastic, oligodendrocytes, astrocytes, vascular and neurons. Cellchat and ProgenY scores revealed that in GBM tumours, histone acetylation interacts closely with multiple immune cells and tumour pathways. GO and KEGG analyses revealed the main impact proteins and pathway correlates of histone acetylation. Five histone acetylation genes were screened using LASSO analysis and a prognostic model was constructed. The results revealed that prognostic models were significant in the prognostic stratification of patients in both the training and validation groups of GBM patients. Immune infiltration analysis revealed that the mechanism of histone acetylation in GBM may be related to the immune infiltration of multiple effector immune cells. Our histone acetylation-based biomarkers are closely associated with immune microenvironmental infiltration and functional mutations in multiple tumour pathways in GBM. This suggests that histone acetylation may reveal microscopic alterations in the tumour microenvironment, and may provide potential evidence and a research basis for the development of novel therapeutic targets for GBM. On this basis, a novel perspective on the spatial biology and immunological understanding of GBM is provided.