Glioblastoma Multiforme Patients Research Articles

Identification of candidate biomarkers for GBM based on WGCNA

Glioblastoma multiforme (GBM), the most aggressive form of primary brain tumor, poses a considerable challenge in neuro-oncology. Despite advancements in therapeutic approaches, the prognosis for GBM patients remains bleak, primarily attributed to its inherent resistance to conventional treatments and a high recurrence rate. The primary goal of this study was to acquire molecular insights into GBM by constructing a gene co-expression network, aiming to identify and predict key genes and signaling pathways associated with this challenging condition. To investigate differentially expressed genes between various grades of Glioblastoma (GBM), we employed Weighted Gene Co-expression Network Analysis (WGCNA) methodology. Through this approach, we were able to identify modules with specific expression patterns in GBM. Next, genes from these modules were performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using ClusterProfiler package. Our findings revealed a negative correlation between biological processes associated with neuronal development and functioning and GBM. Conversely, the processes related to the cell cycle, glomerular development, and ECM-receptor interaction exhibited a positive correlation with GBM. Subsequently, hub genes, including SYP, TYROBP, and ANXA5, were identified. This study offers a comprehensive overview of the existing research landscape on GBM, underscoring the challenges encountered by clinicians and researchers in devising effective therapeutic strategies.

Temozolomide Resistance Mechanisms: Unveiling the role of Translesion DNA Polymerase Kappa in Glioblastoma Spheroids in vitro.

Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Polκ) has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Polκ in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Polκ knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 µM); (ii) demonstrates significant cytotoxicity (25-200 μM); (iii) exerts antiproliferative effects (≤ 25 μM) and promotes cell cycle arrest (G2/M phase) in Polκ KO spheroids when compared to WTE counterparts. Furthermore, Polκ KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Polκ KO than in WTE spheroids. Collectively, the results suggest that TLS Polκ plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Polκ emerges as a potential therapeutic target for GBM patients.

Upregulation of HAS2 promotes glioma cell proliferation and chemoresistance via c-myc

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant human brain tumor. Although comprehensive therapies, including chemotherapy and radiotherapy following surgery, have shown promise in prolonging survival, the prognosis for GBM patients remains poor, with an overall survival rate of only 14.6 months. Chemoresistance is a major obstacle to successful treatment and contributes to relapse and poor survival rates in glioma patients. Therefore, there is an urgent need for novel strategies to overcome chemoresistance and improve treatment outcomes for human glioma patients. Recent studies have shown that the tumor microenvironment plays a key role in chemoresistance. Our study demonstrates that upregulation of HAS2 and subsequent hyaluronan secretion promotes glioma cell proliferation, invasion, and chemoresistance in vitro and in vivo through the c-myc pathway. Targeting HAS2 sensitizes glioma cells to chemotherapeutic agents. Additionally, we found that hypoxia-inducible factor HIF1α regulates HAS2 expression. Together, our findings provide insights into the dysregulation of HAS2 and its role in chemoresistance and suggest potential therapeutic strategies for GBM.

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.

STAT1 mediated downregulation of the tumor suppressor gene PDCD4, is driven by the atypical cadherin FAT1, in glioblastoma

STAT1 (Signal Transducer and Activator of Transcription 1), belongs to the STAT protein family, essential for cytokine signaling. It has been reported to have either context dependent oncogenic or tumor suppressor roles in different tumors. Earlier, we demonstrated that Glioblastoma multiforme (GBMs) overexpressing FAT1, an atypical cadherin, had poorer outcomes. Overexpressed FAT1 promotes pro-tumorigenic inflammation, migration/invasion by downregulating tumor suppressor gene, PDCD4. Here, we demonstrate that STAT1 is a novel mediator downstream to FAT1, in downregulating PDCD4 in GBMs. In-silico analysis of GBM databases as well as q-PCR analysis in resected GBM tumors showed positive correlation between STAT1 and FAT1 mRNA levels. Kaplan-Meier analysis showed poorer survival of GBM patients having high FAT1 and STAT1 expression. SiRNA-mediated knockdown of FAT1 decreased STAT1 and increased PDCD4 expression in glioblastoma cells (LN229 and U87MG). Knockdown of STAT1 alone resulted in increased PDCD4 expression. In silico analysis of the PDCD4 promoter revealed four putative STAT1 binding sites (Site1-Site4). ChIP assay confirmed the binding of STAT1 to site1. ChIP-PCR revealed decrease in the binding of STAT1 on the PDCD4 promoter after FAT1 knockdown. Site directed mutagenesis of Site1 resulted in increased PDCD4 luciferase activity, substantiating STAT1 mediated PDCD4 inhibition. EMSA confirmed STAT1 binding to the Site 1 sequence. STAT1 knockdown led to decreased expression of pro-inflammatory cytokines and EMT markers, and reduced migration/invasion of GBM cells. This study therefore identifies STAT1 as a novel downstream mediator of FAT1, promoting pro-tumorigenic activity in GBM, by suppressing PDCD4 expression.

Temozolomide-associated blepharoconjunctivitis: a case report

BackgroundTemozolomide (TMZ) is an effective oral alkylating agent used in treating glioblastoma multiforme (GBM) and high-grade gliomas. It works by introducing methyl groups into DNA, inhibiting cell division. A case of blepharoconjunctivitis linked to the administration of TMZ is detailed in this report.Case presentationWe present a case of a 58-year-old African-American man diagnosed with GBM. Following adjuvant TMZ treatment, he developed blepharoconjunctivitis, characterized by eyelid and conjunctival inflammation. Symptoms included eyelid swelling, crusting, and conjunctival discharge, which were promptly resolved with topical steroid cream and eye drops.ConclusionsReports specifically linking TMZ to blepharoconjunctivitis are limited. The exact mechanism remains unclear but may involve inflammation extending from blepharitis to the conjunctiva. Healthcare providers must recognize and manage ophthalmic complications promptly. This case report highlights blepharoconjunctivitis associated with TMZ use in a GBM patient. While TMZ is an effective treatment, ophthalmic side effects can occur.

Diagnostic and Prognostic Value of Circulating DNA Fragments in Glioblastoma Multiforme Patients.

Novel blood-circulating molecules, as potential biomarkers for glioblastoma multiforme (GBM) diagnosis and monitoring, are attracting particular attention due to limitations of imaging modalities and invasive tissue biopsy procedures. This study aims to assess the diagnostic and prognostic values of circulating cell-free DNA (cfDNA) in relation to inflammatory status in GBM patients and to determine the concentration and average size of DNA fragments typical of tumour-derived DNA fractions. Preoperative plasma samples from 40 patients (GBM 65.0 ± 11.3 years) and 40 healthy controls (HC 70.4 ± 5.4 years) were compared. The cfDNA concentrations and lengths were measured using the electrophoresis platform, and inflammatory indices (NLR, PLR, LMR, and SII) were calculated from complete blood cell analysis. More fragmented cfDNA and 4-fold higher 50-700 bp cfDNA concentrations were detected in GBM patients than in healthy controls. The average cfDNA size in the GBM group was significantly longer (median 336 bp) than in the HC group (median 271 bp). Optimal threshold values were 1265 pg/μL for 50-700 bp cfDNA (AUC = 0.857) and 290 bp for average cfDNA size (AUC = 0.814). A Kaplan-Meier survival curves analysis also demonstrated a higher mortality risk in the GBM group with a cut-off >303 bp cfDNA. This study is the first to have revealed glioblastoma association with high levels of cfDNA > 1000 pg/μL of 50-700 bp in length, which can be aggravated by immunoinflammatory reactivity.

Identification of Novel Molecular and Clinical Biomarkers of Survival in Glioblastoma Multiforme Patients: A Study Based on The Cancer Genome Atlas Data.

Glioblastoma multiforme (GBM) is the most prevalent type of brain tumour; although advancements in treatment have been made, the median survival time for GBM patients has persisted at 15 months. This study is aimed at investigating the genetic alterations and clinical features of GBM patients to find predictors of survival. GBM patients' methylation and gene expression data along with clinical information from TCGA were retrieved. The most overrepresented pathways were identified independently for each omics dataset. From the genes found in at least 30% of these pathways, one gene that was identified in both sets was further examined using the Kaplan-Meier method for survival analysis. Additionally, three groups of patients who started radio and chemotherapy at different times were identified, and the influence of these variations in treatment modality on patient survival was evaluated. Four pathways that seemed to negatively impact survival and two with the opposite effect were identified. The methylation status of PRKCB was highlighted as a potential novel biomarker for patient survival. The study also found that treatment with chemotherapy prior to radiotherapy can have a significant impact on patient survival, which could lead to improvements in clinical management and therapeutic approaches for GBM patients.

Relevance of Thymic Stromal Lymphopoietin on the Pathogenesis of Glioblastoma: Role of the Neutrophil

Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.Graphical Protumoral activity of TSLP. Neutrophils (derived from GBM patients) and GBM cells (under EGF stimulus) not only produce TSLP but also express its receptor. TSLP induces PDL1 expression and decreases apoptosis on both GBM cells and neutrophils. TSLP also increases proliferation and satellite development on GBM cells, whereas favors more neutrophil infiltration by increasing IL8 production.

100 The Impact of Race and Social Determinants of Health on Clinical Outcome of Glioblastoma Multiforme Patients Over a Decade.

OBJECTIVES/GOALS: While the evolving treatment paradigm for Glioblastoma (GBM) leverages different modalities to improve outcomes, treatment access might be limited by cost and disparities. This study explores the influence of race and social determinants of health (SDoH) on healthcare access and outcomes of GBM patients in a large metropolitan area over a decade. METHODS/STUDY POPULATION: Our institution’s tumor registry (2009-2019) was queried to identify our GBM cohort. Data were supplemented by electronic health records to include demographics, outcome, NCI Comorbidity Index, and the Agency for Healthcare Research and Quality (AHRQ) socioeconomic status (SES) index. RESULTS/ANTICIPATED RESULTS: Of the 559 GBM records, 361 unique patients met the inclusion criteria, and 43% were Non-White. Non-White patients predominantly comprised the lowest AHRQ SES index quartile and had longer hospital stays (LOS; p<0.001). White patients accounted for 61% of privately insured patients (p<0.001). Private insurance (p= 0.02) and age < 65 years (p= 0.039) were associated with a higher rate of home discharge. Patients diagnosed with GBM in the emergency department were more likely to be discharged to acute rehab than home (p<0.001). At 2 years, privately insured patients had longer OS (HR= 1.46; p= 0.04). DISCUSSION/SIGNIFICANCE: In contrast to previous studies, the study demonstrates that GBM affected a higher proportion of Non-White patients. Our data show that SDoH influences multiple outcomes in GBM patients. Efforts to identify and correct these barriers are needed to improve the care of all GBM patients.

Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Predominance of MGMT promoter methylation among Pakistani glioblastoma patients.

Glioblastoma multiforme (GBM), the most prevalent subgroup of neuroepithelial tumors, is characterized by dismal overall survival (OS). Several studies have linked O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation to OS in GBM patients. However, MGMT methylation frequencies vary geographically and across ethnicities, with limited data for South Asian populations, including Pakistan. This study aimed to analyze MGMT promoter methylation in Pakistani GBM patients. Consecutive primary GBM patients diagnosed ≥ 18 years-of-age, with no prior chemotherapy or radiotherapy history, were retrospectively selected. DNA was isolated from formalin-fixed-paraffin-embedded tissues. MGMT promoter methylation was analyzed using methylation-specific PCR. Clinical, pathological, and treatment data were assessed using Fisher's exact/Chi-squared tests. OS was calculated using Kaplan-Meier analysis in SPSS 27.0.1. The study included 48 GBM patients, comprising 38 (79.2%) males and 10 (20.8%) females. The median diagnosis age was 49.5 years (range 18-70). MGMT methylation was observed in 87.5% (42/48) of all cases. Patients with MGMT methylation undergoing radiotherapy or radiotherapy plus chemotherapy exhibited significantly improved median OS of 7.2 months (95% CI, 3.7-10.7; P < 0.001) and 16.9 months (95% CI, 15.9-17.9; P < 0.001), respectively, compared to those undergoing surgical resection only (OS: 2.2 months, 95% CI, 0.8-3.6). This is the first comprehensive study highlighting a predominance of MGMT methylation in Pakistani GBM patients. Furthermore, our findings underscore the association of MGMT methylation with improved OS across diverse treatment modalities. Larger studies are imperative to validate our findings for better management of Pakistani GBM patients.

Abstract 5585: Genetic variants of IDH and EGFR as diagnostic and prognostic biomarkers in glioblastoma multiforme: A case-control study in the Jordanian Arab population

Abstract Background: Glioblastoma multiforme (GBM) represents a highly aggressive primary brain tumor with poor prognosis. Mutation in the Isocitrate dehydrogenase (IDH1) gene and epidermal growth factor (EGFR) amplification are key diagnostic markers for GBM. However, the prognostic role of genetic variants of these genes remains unclear. Therefore, we aimed to investigate the association between specific single nucleotide polymorphisms (SNPs) for IDH and EGFR genes, and their impact on the risk and prognosis of GBM patients within the Jordanian Arab population. Methods: We carried out a case-control study involving 63 GBM patients and 226 healthy controls at King Abdullah University Hospital in Jordan. Genomic DNA was extracted from formalin-fixed and paraffin-embedded tissue for GBM patients and blood samples for controls. IDH1 (rs121913500C&amp;gt;T), IDH2 (rs11540478G&amp;gt;A), EGFR (rs231677G&amp;gt;A), and EGFR (rs1468727C&amp;gt;T) SNPs were genotyped and analyzed using the Sequenom iPLEX assay sequencing technique. Overall survival (OS) was analyzed between SNPs using the univariable Cox proportional hazard model. The Cancer Genome Atlas (TCGA-GBM) cohort of 585 patients were analyzed for IDH1, IDH2, and EGFR alterations with OS, disease-free survival (DFS), and progression-free survival (PFS). Single-cell RNA analysis was performed on the following scRNA datasets from the Gene Expression Omnibus (GEO): GSE163108 (n=25,013 cells), and GSE102130 (n=3,321 cells) . Results: Our study revealed a significant difference in genotype frequency between GBM cases and controls for IDH1 rs121913500C&amp;gt;T, and EGFR rs1468727C&amp;gt;T SNPs. Univariable Cox model showed that the G/G genotype of rs231677G&amp;gt;A (EGFR) SNP was associated with better OS in the codominant model (HR: 0.24, 95% CI: 0.08-0.73, p-value=0.012), and rs11540478G&amp;gt;A (IDH2) SNP (HR: 0.02, 95% CI: 0.0-0.29, p-value=0.005) showing significantly better prognosis compared to the A/G genotype. In the TCGA validation cohort, the altered group of IDH1 were significantly associated with better OS (HR: 0.35, 95% CI: 0.24-0.50, p-value&amp;lt;0.001), DFS (HR: 0.34, 95% CI: 0.23-0.50, p-value&amp;lt;0.001) , and PFS (HR: 0.43, 95% CI: 0.30-0.62, p-value&amp;lt;0.001). The scRNA analysis showed predominant expression of IDH1, IDH2, and EGFR by malignant cells, oligodendrocyte precursor cells, and macrophages. Conclusion: Our investigation into the genetic landscape of GBM within the Jordanian population suggest that specific genetic variants, particularly in IDH1 genes and EGFR, may serve as diagnostic and prognostic biomarkers for GBM. Additionally, IDH1 alterations were associated with better prognostic outcomes. Single-cell RNA analysis further illuminates the predominant expression of IDH1, IDH2, and EGFR by malignant cells which can be used as potential avenues for targeted therapies based on genetic profiles. Citation Format: Ayah N. Al-Bzour, Sohaib M. Al-Khatib, Mohammad N. Almajali, Tariq A. Jarrad. Genetic variants of IDH and EGFR as diagnostic and prognostic biomarkers in glioblastoma multiforme: A case-control study in the Jordanian Arab population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5585.

Abstract 6355: Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC)

Abstract Background: Glioblastoma Multiforme (GBM), primarily arising in the brain's frontal and temporal lobes, is a devastating malignancy originating from astrocytes. Despite treatment advancements, the prognosis remains bleak, with a median survival of 15-18 months and a 10% five-year survival rate.Our study explored a novel targeted approach to combat this challenge. uPARAP is an endocytic receptor expressed at low levels on selected mesenchymal cell types but highly overexpressed on the majority of Soft-Tissue- and Bone Sarcomas. It internalizes fragments of collagen and transfers them to the lysosome for degradation. Methods &amp; Results: First, we analyzed formalin-fixed paraffin-embedded (FFPE) tumors from GBM patients through immunohistochemistry (IHC) staining. Interestingly, a majority of the samples showed strong but varying uPARAP expression. In parallel, we detected and quantified uPARAP receptor expression in GBM cell lines by flow cytometry, underscoring its potential as a therapeutic target.Next, we generated an antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), a potent antimitotic agent. Confocal microscopy showed rapid internalization of the ADC in several GBM cell lines in vitro. Cellular cytotoxicity was demonstrated in Incucyte and Alamar Blue in vitro assays. Finally, anti-tumor activity of the uPARAP-ADC was studied in a GBM patient-derived xenograft (PDX) model in vivo, where it resulted in significant tumor regression with a tumor growth inhibition (TGI) of 92 and 97% at 3 and 6 mg/kg, respectively. Conclusions: Our findings show that uPARAP is overexpressed on GBM tumors and that a uPARAP-targeted ADC has anti-tumor activity in a PDX model of GBM. This research provides initial evidence that uPARAP-ADCs could become a novel treatment option for this highly aggressive disease. Citation Format: Ida Gregersen, Cecillie R. Løkke, Jette B. Lange, Pernille Barkholt, Christophé Côme, Tina Broberg, Marianne M. Petersen, Carmel M. Lynch, Dominik Mumberg, Niels Behrendt, Lars H. Engelholm. Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6355.

Abstract 4465: Galectin-1 regulates CD147 and downstream pathways critical for glioma progression

Abstract Galectin-1 (Gal-1) is an important protein for glioma progression and glioma stem cell maintenance. Gal-1 may act through CD147, a potential Gal-1 receptor. CD147, also known as Basigin or extracellular matrix metalloproteinase inducer (EMMPRIN), is a transmembrane glycoprotein highly expressed in various cancer cells. An analysis utilizing the Gliovis database and encompassing 667 Glioblastoma Multiforme (GBM) patients segregated based on median CD147 expression levels delineated cohorts with above-median (n=339) and below-median (n=328) CD147 expression. Kaplan-Meier survival curves unveiled a significant association between heightened CD147 expression and reduced overall survival (OS) in this patient population. Experimental approaches employing immunoblotting and immunohistochemistry techniques on shGal-1 treated cell lysates and mouse brain samples exhibited decreased CD147 expression compared to respective controls. Additionally, immunocytochemical studies corroborated an augmented colocalization of Gal-1 and CD147, with a subsequent reduction in this association following shGal-1 treatment. Next, we used AC-73, a selective CD147 inhibitor, to determine the levels of CD147 and Gal-1. Treatment with 20μM AC-73 and siRNA for CD147 showed reduced CD147 levels in GSC33 and GSC20 cells while maintaining unaltered Gal-1 expression. Inhibiting CD147 did not affect Gal-1 levels, further suggesting Gal-1 acts upstream. CD147 activates matrix metalloproteinase-9 (MMP9), and CD147 knockdown reduced MMP9 levels, elucidating a Gal-1-CD147-MMP9 pathway. Overall, Gal-1 appears to act upstream of CD147 to regulate MMP9, integrins, and other factors important for glioma progression. Further elucidation of these pathways will provide insights into glioma biology and potential therapeutic targets. Citation Format: Maheedhara R. Guda, Andrew J. Tsung, Swapna Asuthkar, Kiran Velpula. Galectin-1 regulates CD147 and downstream pathways critical for glioma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4465.

Abstract 2632: A new paradigm for brain tumors treatment: Guadecitabine as a fundamental player to improve tumor immune response

Abstract Background:Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor refractory to standard treatments and to immunotherapy with immune-checkpoint inhibitors (ICI) (PMID: 35626018). Interestingly, melanoma brain metastases (MM-BM), sharing the same niche as GBM, often respond to current ICI immunotherapies (PMID: 36521332). The role of epigenetic modifications in regulating GBM cellular proliferation, invasion, and prognosis, as well as their impact on the cross-talk between GBM and immune cells in the tumor milieu, has become evident (PMID: 34361090). Consequently, manipulating the epigenome has emerged as a therapeutic opportunity in GBM. Methods:Microarray transcriptional and methylation profiles, followed by gene set enrichment and IPA analyses, were conducted to study the differences in constitutive expression profiles between GBM and MM-BM cells compared to extracranial MM cells. Additionally, the modulatory effects of the DNA hypomethylating agent (DHA) guadecitabine on different tumor cells were investigated. The prognostic relevance of DHA-modulated genes was assessed through Cox analysis in a TCGA GBM patients' cohort. Results:The most notable differences between constitutive GBM and MM-BM cells lie in the enrichment of biological processes associated with tumor growth, invasion, and extravasation, as well as the inhibition of MHC class II antigen processing/presentation in GBM cells. Guadecitabine treatment appeared to shrink these differences, shifting the phenotype of GBM cells towards the more immunogenic MM-BM profile. This was achieved through promoter hypomethylation and subsequent up-regulation of genes mainly associated with the activation, proliferation, and migration of T and B cells, along with MHC class II antigen processing/presentation. Among DHA-modulated genes in GBM, 7.6% exhibited significant prognostic relevance. Moreover, a substantial set of immune-related upstream regulators (URs) were commonly modulated by DHA in GBM, MM-BM, and/or MM cells: DHA-activated URs enriched biological processes mainly involved in the regulation of cytokines and chemokines production, inflammatory response, and Type I/II/III IFN-mediated signaling. DHA-inhibited URs were associated with metabolic and proliferative pathways. Conclusions:Epigenetic remodeling of GBM by DHA represents a promising strategy to enhance the efficacy of cancer immunotherapy, supporting the rationale for developing new combinatorial approaches for the treatment of brain malignancies. Citation Format: Maria Fortunata Lofiego, Francesca Piazzini, Francesca Pia Caruso, Francesco Marzani, Laura Solmonese, Emma Bello, Fabrizio Celesti, Maria Claudia Costa, Teresa Maria Noviello, Roberta Mortarini, Andrea Anichini, Michele Ceccarelli, Sandra Coral, Anna Maria Di Giacomo, Michele Maio, Alessia Covre. A new paradigm for brain tumors treatment: Guadecitabine as a fundamental player to improve tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2632.

Abstract 5594: Exploring genetic determinants: A comprehensive analysis of SERPINB family variants and prognosis in Jordanian glioblastoma multiforme patients

Abstract Background: Glioblastoma multiforme (GBM) is a major concern with high fatality rate. In Jordan, the incidence of GBM has notably increased, emphasizing the urgency for population-specific research. Serpins are serine proteinase inhibitors, with several Serpins being overexpressed in cancer cells however the exact mechanism by which they affect GBM progression remains unclear. Thus, we aim to analyze the single-nucleotide polymorphism (SNP) of SERPINB11 and its association with GBM survival. Methods: A cohort of 63 GBM patients recruited from King Abdullah University Hospital (KAUH) in Jordan, underwent genomic DNA extraction, polymorphism analysis, and overall survival (OS) assessments. The Serpin B family were validated using The Cancer Genome Atlas (TCGA-GBM) cohort of 160 patients. We constructed a risk-score using the principal component analysis for the following Serpin genes: Serpinb3, Serpinb5, Serpinb6, Serpinb11, and Serpinb12, and patients were grouped into high- vs. low-risk based on median cutoff. Univariable Cox models were used to study the survival outcomes, differential expression analysis between the high- and low-risk groups was carried out to identify the differentially expressed genes (DEGs), gene ontology (GO and tumor microenvironment analyses were carried out. Results: In our primary cohort, we identified a significant association between rs4940595 (SERPINB11) SNP and survival, with the G/T- genotype showing worse prognosis compared to the G/G-T/T genotype in the over dominant model (HR: 2.75, 95% CI: 1.29-5.88, p-value=0.009). In the TCGA validation-cohort, alterations in the SERPINB3gene showed significantly worse OS (Median: 9.53 vs. 14.3, p-value=0.044) compared to the no alteration group. Univariable Cox showed worse PFS outcomes with higher SERPINB5 (HR: 1.67, 95% CI: 1.15-2.43, p-value=0.007) and SERPINB6 expression (HR: 1.44, 95% CI: 1.06-1.96, p-value=0.021). A Serpin B 5-gene risk score was constructed revealing significant association with IDH mutation status and a trend towards worse PFS in the high-risk group. Upregulated DEGs showed GO enrichment in cytokine regulation and production, positive regulation of leukocyte activation and MAPK cascade. While the downregulated DEGs were enriched in forebrain development, and negative regulation of neuron differentiation. The high-risk group showed a significantly higher infiltration of M2 macrophages and activated mast cells. Conclusion: Our findings showed a significant role of the Serpin B family with GBM survival in the Jordanian population. Molecular analyses showed potential mechanisms underlying these associations. Our exploration of SERPINB family and associated SNPs provides valuable insights into the molecular landscape of GBM, paving the way for potential targeted interventions and personalized treatment strategies. Citation Format: Sohaib Al-Khatib, Mohammad Nitham Almajali, Ayah Al-Bzour, Joud Al-Ramadneh, Laila Sa'd, Noor Othman. Exploring genetic determinants: A comprehensive analysis of SERPINB family variants and prognosis in Jordanian glioblastoma multiforme patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5594.

Current approaches in glioblastoma multiforme immunotherapy.

Glioblastoma multiform (GBM) is the most prevalent CNS (central nervous system) tumor in adults, with an average survival length shorter than 2years and rare metastasis to organs other than CNS. Despite extensive attempts at surgical resecting, the inherently permeable nature of this disease has rendered relapse nearly unavoidable. Thus, immunotherapy is a feasible alternative, as stimulated immune cells can enter into the remote and inaccessible tumor cells. Immunotherapy has revolutionized patient upshots in various malignancies and might introduce different effective ways for GBM patients. Currently, researchers are exploring various immunotherapeutic strategies in patients with GBM to target both the innate and acquired immune responses. These approaches include reprogrammed tumor-associated macrophages, the use of specific antibodies to inhibit tumor progression and metastasis, modifying tumor-associated macrophages with antibodies, vaccines that utilize tumor-specific dendritic cells to activate anti-tumor T cells, immune checkpoint inhibitors, and enhanced T cells that function against tumor cells. Despite these findings, there is still room for improving the response faults of the many currently tested immunotherapies. This study aims to review the currently used immunotherapy approaches with their molecular mechanisms and clinical application in GBM.

Hsp70 and Calcitonin Receptor Protein in Extracellular Vesicles from Glioblastoma Multiforme: Biomarkers with Putative Roles in Carcinogenesis and Potential for Differentiating Tumor Types.

Glioblastoma multiforme (GBM) is a malignancy of bad prognosis, and advances in early detection and treatment are needed. GBM is heterogenous, with varieties differing in malignancy within a tumor of a patient and between patients. Means are needed to distinguish these GMB forms, so that specific strategies can be deployed for patient management. We study the participation of the chaperone system (CS) in carcinogenesis. The CS is dynamic, with its members moving around the body in extracellular vesicles (EVs) and interacting with components of other physiological systems in health and disease, including GBM. Here, we describe the finding of high amounts of Hsp70 (HSPA1A) and the calcitonin receptor protein (CTR) in EVs in patients with GBM. We present a standardized protocol for collecting, purifying, and characterizing EVs carrying Hsp70 and CTR in plasma-derived EVs from patients with GBM. EVs from GBM patients were obtained just before tumor ablative surgery (T0) and 7 days afterwards (T1); Hsp70 was highly elevated at T0 and less so at T1, and CTR was greatly increased at T0 and reduced to below normal values at T1. Our results encourage further research to assess Hsp70 and CTR as biomarkers for differentiating tumor forms and to determine their roles in GBM carcinogenesis.

Involvement of microglia-expressed MS4A6A in the onset of glioblastoma.

Glioblastoma multiforme (GBM) represents the deadliest form of brain tumour, characterized by its low survival rate and grim prognosis. Cytokines released from glioma-associated microglia/macrophages are involved in establishing the tumour microenvironment, thereby crucially promoting GBM progression. MS4A6A polymorphism was confirmed to be associated with neurodegenerative and polymorphism disease pathobiology, but whether it participates in the regulation of GBM and the underlying mechanisms is still not elucidated. Here, we found that MS4A6A was significantly upregulated in GBM patient samples. The results from the single-cell RNA-sequencing (scRNA-seq) database and immunostaining demonstrated the specific expression of MS4A6A in microglial cells. In vitro, microglial overexpression of MS4A6A stimulated the proliferation and migration of glioblastoma cells. Moreover, high MS4A6A mRNA expression was related to poor prognosis in GBM patients. Our study highlights the potential of MS4A6A as a promising biomarker for GBM, which may provide novel strategies for its prevention, diagnosis and treatment.