Virtual Screening-Guided Discovery of Small-Molecule CHI3L1 Inhibitors with Functional Activity in Glioblastoma Spheroids.

Chitinase-3-like protein 1 (CHI3L1), a glycoprotein implicated in inflammation, fibrosis, and cancer, has emerged as a potential therapeutic target for glioblastoma (GBM). CHI3L1 contributes to tumor progression and immune evasion by promoting STAT3 signaling and mesenchymal transition. To identify small molecule CHI3L1 inhibitors, a structure-based 3D pharmacophore model was developed and applied to virtually screen over 4.4 million compounds from the Enamine collection. Following multi-tiered filtering, 35 candidates were selected for experimental evaluation. Binding validation via microscale thermophoresis (MST) confirmed dose-dependent CHI3L1 interactions for two compounds, 8 and 39, with dissociation constants (Kd) of 6.8 μM and 22 μM, respectively. These affinities were further supported by surface plasmon resonance (SPR), which yielded Kd values of 5.69 μM for compound 8 and 17.09 μM for compound 39. In 3D GBM spheroid models, compound 8 significantly reduced spheroid viability and attenuated phospho-STAT3 levels, consistent with CHI3L1 pathway disruption. These findings identify two promising scaffolds and support the utility of pharmacophore-guided virtual screening for discovering functionally active ligands targeting CHI3L1 in GBM.

CHI3L1-targeted small molecules as glioblastoma therapies: Virtual screening-based discovery, biophysical validation, pharmacokinetic profiling, and evaluation in glioblastoma spheroids.

Glioblastoma (GBM) remains the most aggressive primary brain malignancy with a 10% three- year survival rate. Chitinase-3-like protein 1 (CHI3L1) has emerged as a critical factor in the progression of GBM progression, invasion, and treatment resistance. However, small molecule inhibitors targeting CHI3L1 are largely unexplored. Microscale thermophoresis (MST) investigation of the direct binding potential of reported CHI3L1 modulators ( K284, G721-0282, CHI3L1-IN-1 ) revealed modest to undetectable direct CHI3L1 binding affinity. Herein, pharmacophore-based virtual screening of in-house library resulted in the discovery of G28 as the most potent small molecule CHI3L1 binder reported to date. The CHI3L1 binding affinity of G28 was validated using MST and surface plasmon resonance (SPR). To evaluate the GBM-modulatory potential of G28 , we conducted comprehensive pharmacokinetic and 3D spheroid studies alongside established CHI3L1 modulators. G28 demonstrated outstanding bioavailability and low toxicity, addressing key limitations faced by previous CHI3L1-targeted strategies. Notably, in 3D GBM spheroid models, G28 significantly outperformed reported CHI3L1 small molecule modulators, showing the most pronounced dose-dependent reductions in spheroid weight, migration, and viability. These findings position G28 as the most promising CHI3L1-targeted small molecule to date and a compelling candidate for GBM therapeutic development.

Lead optimization of a CHI3L1 inhibitor for Glioblastoma: Enhanced target engagement, pharmacokinetics, and efficacy in 3D spheroid models.

CHI3L1 predicted in malignant entities is associated with glioblastoma immune microenvironment

Great interest persists in useful therapeutic targets in glioblastoma (GBM). Deregulation of microRNAs (miRNAs) expression has been associated with cancer formation through alterations in gene targets. In this study, we reported the role of miR-101 in human glioblastoma stem cells (GSCs) and the potential mechanisms. Quantitative real-time PCR showed that miR-101 expression was decreased in GSCs. Overexpression of miR-101 reduced the proliferation, migration, invasion, and promoted apoptosis of GSCs. One direct target of miR-101, the transcription factor Kruppel-like factor 6 (KLF6), was identified using the Dual-Luciferase Reporter Assay System, which mediated the tumor suppressor activity of miR-101. This process was coincided with the reduced expression of Chitinase-3-like protein 1 (CHI3L1) whose promoter could be bound with and be promoted by KLF6 demonstrated by luciferase assays and chromatin immunoprecipitation assays. The downregulation of CHI3L1 led to the inactivation of MEK1/2 and PI3K signal pathways. Furthermore, nude mice carrying the tumors of overexpressed miR-101 combined with knockdown of KLF6 produced the smallest tumors and showed the highest survival rate. Our findings provided a comprehensive analysis of miR-101 and further defining it as a potential therapeutic candidate for GBM.

The neurofibromin-1 tumor suppressor gene (NF1) is altered in approximately 20% of sporadic glioblastoma (GBM) cases. NF1 deficient GBM frequently shows a mesenchymal gene expression signature, suggesting a relationship between NF1 status and the tumor microenvironment. To identify changes in the production of secreted cytokines/chemokines in NF1 deficient glioma, we applied cytokine arrays to conditioned media from a panel of three GBM cell lines after siRNA-mediated NF1 knockdown. We identified increased secretion of platelet-derived growth factor AA (PDGF-AA), chitinase-3-like protein 1 (CHI3L1), interleukin-8 (IL-8), and endoglin (ENG) in different subsets of these cell lines. Secretion was associated with induction of the corresponding messenger RNA, suggesting a mechanism involving transcriptional upregulation. By contrast, in non-transformed immortalized normal human astrocytes, PDGF-AA secretion was increased upon NF1 knockdown, while secreted CHI3L1, ENG, and IL-8 were reduced or unchanged. Analysis of The Cancer Genome Atlas confirmed a relationship between glioma NF1 status and ENG and CHI3L1 in tumor samples. Overall, this study identifies candidate changes in secreted proteins from NF1 deficient glioma cells that could influence the tumor microenvironment, and suggests a direct link between NF1 loss and increased tumor cell production of CHI3L1 and endoglin, two factors implicated in mesenchymal identity in glioblastoma.

Current targeted therapies for glioblastoma multiforme (GBM) fail to significantly improve clinical outcome. Identifying new molecular targets driving GBM tumorigenesis is imperative. Our previous study demonstrated that STK17A, a serine-threonine kinase in the death-associated protein family, is a bona fide p53 target gene. In silico analyses indicated that STK17A is highly overexpressed in GBM patients in a tumor grade-dependent manner. Furthermore, high STK17A expression correlated with poor clinical outcome and decreased survival of patients from multiple datasets. This correlation was independent of age, tumor subtype and known biomarkers such as EGFR, NF1, and IDH, suggesting STK17A may contribute to GBM development and progression. In vitro experiments confirmed increased mRNA and protein expression of STK17A in GBM cells compared to immortalized normal human astrocytes and other cancer types. ShRNA mediated STK17A knockdown in GBM cells decreased cell survival and sensitized cells to genotoxic stress. In addition, STK17A knockdown led to reduced tumor cell proliferation and clonogenicity, suggesting a tumor-promoting role for STK17A in GBM. Interestingly, STK17A depletion resulted in a cell morphological change from a spindle-like phenotype to a phenotype with a flattened, enlarged and more rounded shape that was associated with induction of actin stress fibers. This cytoskeleton remodeling was associated with impaired cell migration and invasion. In contrast STK17A overexpressing cells displayed a pronounced needle-like elongated phenotype. In addition genome-wide expression analysis of STK17A knockdown GBM cells revealed regulation of genes involved in glycolysis including PKM2, PGAM1 and HK2, suggesting that STK17A may also promote tumor growth and survival through regulating metabolism. Small molecule inhibitors that block the kinase activity of STK17A decreased cell survival of GBM cells cultured under both serum and serum-free conditions. Further investigation is required to understand the precise role of STK17A in GBM. Citation Format: Pingping Mao, Mary P. Jardine, Gilbert J. Rahme, Eric C. Yang, Janice Tam, Anita Kodali, Bijesh Biswal, Camilo E. Fadul, Arti B. Gaur, Mark A. Israel, Alexandre Pletnev, Michael Spinella. STK17A is a potential therapeutic target in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2014-4605

Epigenetically controlled processes play an important role in development but also in the formation and progression of many common diseases. Molecular signatures are increasingly incorporated into cancer classifications and DNA methylation-based analyses of brain tumors including glioma is currently the gold standard. Glioblastoma (GBM), CNS WHO grade 4, is characterized by a very diffuse infiltrative growth into the adjacent brain parenchyma. Due to these highly invasive but also genetically heterogeneous and plastic cellular phenotypes, standard treatment, including maximum resection, radio- and chemotherapy, has limited efficacy. It can be assumed that intertumoral or molecular heterogeneity of gliomas at the transcriptome level is caused by altered epigenetic changes within the individual tumor cells. Considering this, we hypothesize that tumor cell invasion being one of the major hallmarks of highly malignant gliomas can be associated with altered DNA methylation and therefore transcript expression. To analyze this, we isolated fractions of glioma cells with increased invasive properties and performed Illumina® array-based methylation profiling as well as RNA sequencing analysis to identify methylation-driven, invasion-promoting genes. Among other, we focused on Chitinase-3-like protein 1 (CHI3L1) as a candidate gene with significantly decreased promoter methylation in invasive cell fractions and overall strong transcript expression in high-grade gliomas. Differential expression and methylation correlation of TCGA data combined with our own dataset revealed that CHI3L1 expression is dependent on the methylation status of at least three CpG sites within the gene promoter region. We validated these findings using several in vitro experimental settings and propose an upstream regulatory mechanism in which single CpG DNA demethylation within the CHI3L1 promoter facilitates transcription factor binding resulting in gene expression.

STAT3 tyrosine705 phosphorylation (p-STAT3, Tyr705), a molecular hub for several signal transduction pathways of glioma, plays a central role in glioblastoma (GBM) carcinogenesis and progression. However, it is still controversial whether p-STAT3 expression is associated with the clinical outcome of patients with glioblastoma. Such evidence would contribute to further illustrate whether STAT3 inhibition is suitable for clinical treatment. Here, we examined the expression of p-STAT3 in the tumor tissues from 90 patients with newly diagnosed supratentorial GBM via immunohistochemical technique and evaluated the influences of its expression on progression-free survival (PFS) and overall survival (OS) using the Kaplan-Meier curve and COX proportional hazards regression model. Immunohistochemical assay indicated increased expression of p-STAT3 in GBM tissue compared to adjacent normal brain tissue without p-STAT3 expression. There were no observed associations between p-STAT3 expression and patients' gender (P = 0.660), age (P = 0.867) or preoperative Karnofsky Performance Status (KPS) (P = 0.121). Univariate survival analysis revealed significant correlations of high p-STAT3 expression with shorter PFS (P = 0.012) and OS (P = 0.009). Multivariate survival analysis confirmed high p-STAT3 expression as a significant prognostic indicator for shorter PFS (HR 2.158, P = 0.019) and OS (HR 2.120, P = 0.031), independent of age, KPS and chemoradiotherapy. In summary, the high percentage of p-STAT3 positive tumor cells is a significant independent prognostic indicator for poor clinical outcome in patients with GBM, in addition to advanced age, poor performance status and nonstandard chemoradiotherapy, suggesting that STAT3 might be as a promising therapeutic target in GBM.

Purpose: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor, with a median survival of less than two years despite maximal therapy. Human ether a gò-gò-related-1 gene (hERG) encodes a voltage-dependent K+ channel found overexpressed in GBM cell lines, and linked to aberrant proliferation in other cancers. We analyzed hERG expression in a glioblastoma stem-like cell (GSC)-derived tumor model and a clinically annotated human GBM tissue microarray (TMA) to determine correlation with patient survival. Additionally, since all FDA-approved drugs undergo cardiotoxicity profiling that includes hERG inhibition, GBM patient survival was also analyzed based on whether a patient received hERG inhibitory drugs and their hERG expression levels. Methods: GSC sphere cultures were isolated from patient GBMs by marker-neutral culture in stem cell media, and validated with progenitor markers, multipotent differentiation, and orthotopic xenograft formation by implanting 2x105 GSCs into right striata of NOD-SCID mice. Tumor xenografts (10-14 weeks) were verified by MRI and animals were sacrificed when moribund. Histology and immunohistochemistry were performed on paraffin-embedded sections for hERG expression and tumor proliferation. Sphere-forming assays with hERG blockers (E-4031, phenytoin) were performed using two low hERG-expressing and two high hERG-expressing GSC lines to test for drug sensitivity. A GBM TMA of 115 patients linked to clinical data was used to correlate hERG expression with patient survival. Clinical data was analyzed to determine if patient survival was affected by incidental administration of drugs with hERG inhibition activity, and the correlative effect of patient GBM hERG expression levels. Results: hERG expression was upregulated in GBM xenografts with a higher Ki-67 proliferative index. High hERG-expressing GSC lines showed a 50% reduction in sphere formation when treated with known hERG inhibitors at their known hERG channel inactivation IC50 concentrations (E-4031 7.7nM, phenytoin 100uM). GBM TMA analysis showed significantly worse survival for GBM patients with high hERG expression versus low hERG expression (p= 0.0168). In addition, patients who incidentally received at least one hERG blocker drug had significantly better survival compared to patients who did not receive hERG blockers (p=0.0004). Subgroup analysis showed that GBM patients with high hERG expression who also received a hERG blocker had significantly improved survival (p=0.0495);however, there was no significant difference in survival for low hERG-expressing GBM patients who received hERG blocker drugs (p=0.5482). Conclusions: In this study, we showed that GBM xenografts with higher hERG expression had higher proliferation rates, and treatment with two known hERG inhibitors (phenytoin, E-4031) at IC50 concentrations for inactivating hERG channel activity also inhibited sphere formation in high hERG expressing GSC lines. Furthermore, TMA analyses showed that patients with high hERG-expressing GBMs who received hERG inhibitory drugs had significantly better survival. This data suggest that already FDA-approved drugs with hERG inhibition activity should be tested in patients with high hERG-expressing GBMs. Citation Format: Kelli B. Pointer, Fang Liu, David K. Jones, Paul A. Clark, Gail A. Robertson, John S. Kuo. Human ether-a-go-go-related-1 gene (hERG) K+ channel as a prognostic marker and therapeutic target for glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B15.

Glioblastoma (GBM) is the most aggressive and lethal form of primary brain tumor, often characterized by resistance to conventional therapies and a poor clinical prognosis. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells; however, TRAIL resistance remains a significant obstacle in GBM treatment. SETD5, a histone methyl transferase with emerging roles in chromatin remodeling and gene regulation, has been implicated in neurodevelopmental disorders and cancer, but remains poorly characterized in glioma biology. This study investigates the role of SETD5 in modulating TRAIL resistance and explores its potential as a therapeutic target in GBM. Publicly available datasets (TCGA, GTEx, GEPIA2, UALCAN, Gliovis, and GSCA) were used to assess SETD5 expression, mutation, and promoter methylation in GBM. Associations with clinical parameters, molecular subtypes, immune infiltration, and functional states were analyzed using tools like cBioPortal, CancerSEA, MEXPRESS, and STRING. siRNA-mediated knockdown of SETD5 was performed in U87 and LN229 GBM cell lines, followed by TRAIL treatment. Western blotting assessed changes in apoptotic and survival pathways, while wound healing assays evaluated cell migration. Expression of deubiquitinases (USP5, USP8, and USP10) was also measured. SETD5 was significantly upregulated in GBM compared to normal brain tissues across multiple databases and was especially elevated in the Mesenchymal and Classical subtypes. CNV analyses revealed alterations in SETD5 that correlated with immunosuppressive cell infiltration. SETD5 expression was positively correlated with survival pathways and negatively correlated with cytotoxic immune infiltration. TRAIL treatment induced SETD5 expression in a dose-dependent manner. Knockdown of SETD5 enhanced TRAIL-mediated apoptosis, reduced expression of survival markers (p-AKT, P-ERK, NF-κB), and increased pro-apoptotic proteins (Cytochrome c, SMAC). SETD5 depletion also downregulated USP5, USP8, and USP10, suggesting its role in stabilizing oncogenic proteins via deubiquitination. SETD5 plays a critical role in promoting TRAIL resistance and GBM survival by regulating apoptotic pathways, immune evasion, and deubiquitinase expression. Moreover knockdown of SETD along with TRAIL treatment leads to down regulation NFκB which a major promoter for cell survival. Its inhibition sensitizes glioma cells to TRAIL-induced apoptosis, identifying SETD5 as a potential therapeutic target. Targeting SETD5 could represent a novel strategy to overcome TRAIL resistance and enhance the efficacy of GBM therapies. Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-17871-9.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

BackgroundImmunotherapy, especially checkpoint inhibitors targeting PD-1 or PD-L1, has revolutionized cancer therapy. However, PD-1/PD-L1 inhibitors have not been investigated thoroughly in glioblastoma (GBM). Studies have shown that polymerase 1 and transcript release factor (PTRF/Cavin-1) has an immune-suppressive function in GBM. Thus, the relationship between PTRF and PD-L1 and their role in immune suppression requires further investigation in GBM.MethodsWe used public databases and bioinformatics analysis to investigate the relationship between PTRF and PD-L1. We next confirmed the predicted relationship between PTRF and PD-L1 in primary GBM cell lines by using different experimental approaches. RIP-Seq, RIP, ChIP, and qRT-PCR were conducted to explore the molecular mechanism of PTRF in immunosuppression.ResultsWe found that PTRF stabilizes lncRNA NEAT1 to induce NF-κB and PD-L1 and promotes immune evasion in GBM. PTRF was found to correlate with immunosuppression in the public GBM databases. PTRF increased the level of PD-L1 in primary cell lines from GBM patients. We carried out RIP-Seq of GBM cells and found that PTRF interacts with lncRNA NEAT1 and stabilizes its mRNA. PTRF also promoted the activity of NF-κB by suppressing UBXN1 expression via NEAT1 and enhanced the transcription of PD-L1 through NF-κB activation. Finally, PTRF promoted immune evasion in GBM cells by regulating PD-1 binding and PD-L1 mediated T cell cytotoxicity.ConclusionsIn summary, our study identified the PTRF-NEAT1-PD-L1 axis as a novel immune therapeutic target in GBM.

New targeted therapies are urgently needed in glioblastoma (GBM). IL13Rα2 receptor is considered a highly specific receptor for GBM cells and has been proposed for therapeutic purposes. Here, we aimed to identify the protein interaction network of IL13Rα2 and its mediators in GBM searching for novel associated therapeutic targets. We investigated the IL13Rα2 protein interaction network in U251 GBM cells using immunoprecipitation followed by mass spectrometry. Different cell lines were used for validation. Two glioblastoma cell lines were used for testing proliferation, adhesion, migration and invasion. Claramine, a PTP1B inhibitor, was tested to study inhibition of GBM cells growth in cultured cells and in animal experiments. In vivo experiments with NSG mice were performed after treatment with PTP1B inhibitors. Using a proteomic approach, we identified the association of the PTP1B phosphatase with IL13Rα2 in U251 GBM cells. Although GBM cell lines were positive for PTP1B, the highest PTP1B expression was found in GBM clinical samples. Moreover, “in silico” dataset analysis showed PTP1B association with poor outcome in GBM patients. PTP1B silencing inhibited the IL-13-promoted activation of Src (Tyr419), AKT and ERK1/2 in all the cell lines tested causing a significant effect on IL-13-mediated migration and invasion, and proliferation at a lower extent, of GBM cells. Therefore, PTP1B-silencing reverted most of the pro-metastatic capacities of the IL-13/ IL13Rα2 signaling axis. Further immunoprecipitation of PTP1B showed its association with a large number of NF-κB and NOTCH-related proteins in GBM cells. Both pathways are highly relevant for cancer proliferation and invasion. Claramine, a selective inhibitor of PTP1B, caused similar results to PTP1B silencing, inhibiting the dephosphorylation of Src Tyr527, migration and invasion in GBM. Moreover, treatment with Claramine caused a complete regression of GBM xenografts in NSG mice and total inhibition of liver metastatic growth in nude mice inoculated with colorectal cancer cells. In conclusion, our results provide new insights into the mechanisms underlying IL13Rα2-driven GBM aggressiveness after identifying PTP1B as a critical mediator of IL-13/IL13Rα2 signaling. A PTP1B inhibitor such as Claramine showed a great value as a therapeutic candidate in GBM. PTP1B inhibitors might be highly effective to prevent GBM progression and invasion. Citation Format: Ruben Bartolomé, Marta Jaén, Miranda Burdiel, Irina V. Balyasnikova, Ignacio Casal. Protein-tyrosine phosphatase 1B (PTP1B) as a therapeutic target in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 253.