Temozolomide Resistance Research Articles

CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma.

Rationale: The large-scale genomic analysis classifies glioblastoma (GBM) into three major subtypes, including classical (CL), proneural (PN), and mesenchymal (MES) subtypes. Each of these subtypes exhibits a varying degree of sensitivity to the temozolomide (TMZ) treatment, while the prognosis corresponds to the molecular and genetic characteristics of the tumor cell type. Tumors with MES features are predominantly characterized by the NF1 deletion/alteration, leading to sustained activation of the RAS and PI3K-AKT signaling pathways in GBM and tend to acquire drug resistance, resulting in the worst prognosis compared to other subtypes (PN and CL). Here, we used the CRISPR/Cas9 library screening technique to detect TMZ-related gene targets that might play roles in acquiring drug resistance, using overexpressed KRAS-G12C mutant GBM cell lines. The study identified a key therapeutic strategy to address the chemoresistance against the MES subtype of GBM. Methods: The CRISPR-Cas9 library screening was used to discover genes associated with TMZ resistance in the U87-KRAS (U87-MG which is overexpressed KRAS-G12C mutant) cells. The patient-derived GBM primary cell line TBD0220 was used for experimental validations in vivo and in vitro. Chromatin isolation by RNA purification (ChIRP) and chromatin immunoprecipitation (ChIP) assays were used to elucidate the silencing mechanism of tumor suppressor genes in the MES-GBM subtype. The small-molecule inhibitor EPIC-0412 was obtained through high-throughput screening. Transmission electron microscopy (TEM) was used to characterize the exosomes (Exos) secreted by GBM cells after TMZ treatment. Blood-derived Exos-based targeted delivery of siRNA, TMZ, and EPIC-0412 was optimized to tailor personalized therapy in vivo. Results: Using the genome-wide CRISPR-Cas9 library screening, we found that the ERBIN gene could be epigenetically regulated in the U87-KRAS cells. ERBIN overexpression inhibited the RAS signaling and downstream proliferation and invasion effects of GBM tumor cells. EPIC-0412 treatment inhibited tumor proliferation and EMT progression by upregulating the ERBIN expression both in vitro and in vivo. Genome-wide CRISPR-Cas9 screening also identified RASGRP1(Ras guanine nucleotide-releasing protein 1) and VPS28(Vacuolar protein sorting-associated protein 28) genes as synthetically lethal in response to TMZ treatment in the U87-KRAS cells. We found that RASGRP1 activated the RAS-mediated DDR pathway by promoting the RAS-GTP transformation. VPS28 promoted the Exos secretion and decreased intracellular TMZ concentration in GBM cells. The targeted Exos delivery system encapsulating drugs and siRNAs together showed a powerful therapeutic effect against GBM in vivo. Conclusions: We demonstrate a new mechanism by which ERBIN is epigenetically silenced by the RAS signaling in the MES subtype of GBM. Restoration of the ERBIN expression with EPIC-0412 significantly inhibits the RAS signaling downstream. RASGRP1 and VPS28 genes are associated with the promotion of TMZ resistance through RAS-GDP to RAS-GTP transformation and TMZ efflux, as well. A quadruple combination therapy based on a targeted Exos delivery system demonstrated significantly reduced tumor burden in vivo. Therefore, our study provides new insights and therapeutic approaches for regulating tumor progression and TMZ resistance in the MES-GBM subtype.

The Inflammatory Characteristics of Symptomatic Glioma Associated With Poor Prognosis and Chemoresistance via Tumor Necrosis Factor Signaling Pathway.

Among gliomas, the most common primary malignant brain tumor, incidental gliomas account for 2.5%-5% of cases. The controversy over whether to pursue immediate treatment or adopt a wait-and-see approach remains, and more molecular and immunological evidence is needed for definitive treatment decisions. Total RNA sequencing (RNA-seq) data and single cell RNA sequencing (scRNA-seq) data were retrospectively analyzed to compare the molecular and immunological tumor microenvironment differences between incidental glioma and symptomatic glioma samples. These were classified using symptom data from The Cancer Genome Atlas (TCGA) and public dataset. RNA-seq analysis of the GBMLGG dataset identified 343 genes upregulated in symptomatic glioma and 118 in incidental glioma, with 104 common genes upregulated in symptomatic glioma across both the TCGA and Chinese Glioma Genome Atlas (CGGA) datasets. Enrichment analysis revealed that these 104 genes in symptomatic glioma were significantly associated with immunological pathways. scRNA-seq analysis of glioma revealed 11 cell types, including T cells, myeloid cells, and oligodendrocytes, with the tumor necrosis factor (TNF) signaling pathway strongly influencing other cell types, particularly myeloid cells. Enrichment and survival analyses showed that TNF signaling is associated with temozolomide resistance and poorer prognosis in glioma patients. The findings suggest that symptomatic glioma enhances inflammatory responses linked to poor prognosis and chemoresistance. This supports the hypothesis that immediate treatment of incidental glioma may improve patient outcomes over a wait-and-see approach.

Revisiting Temozolomide's role in solid tumors: Old is gold?

Temozolomide is an imidazotetrazine with a long history in oncology especially for the high grade malignant glioma and metastatic melanoma. However, last year's new indications for its use are added. Its optimum pharmacodynamic profile, its ability to penetrate the blood-brain barrier, the existence of methylation of MGMT in solid tumors which enhances its efficacy, the identification of new agents that can overcome temozolomide's resistance, the promising role of temozolomide in turning immune cold tumors to hot ones, are leading to expand its use in other solid tumors, giving oncologists an additional tool for the treatment of advanced and aggressive neoplasms.

RNASE4 promotes malignant progression and chemoresistance in hypoxic glioblastoma via activation of AXL/AKT and NF-κB/cIAPs signaling pathways.

Glioblastoma (GBM) is the most malignant brain tumor frequently characterized by a hypoxic microenvironment. In this investigation, we unveiled unprecedented role of Ribonuclease 4 (RNASE4) in GBM pathogenesis through integrative methodologies. Leveraging The Cancer Genome Atlas (TCGA) dataset and clinical specimens from normal brain tissues, low- and high-grade gliomas, alongside rigorous in vitro and in vivo functional analyses, we identified a consistent upregulation of RNASE4 correlating with advanced GBM pathological stages and poor clinical survival outcomes. Functional assays corroborated the pivotal influences of RNASE4 on key tumorigenic processes such as cell proliferation, migration, invasion, stemness properties and temozolomide (TMZ) resistance. Further, Gene Set Enrichment Analysis (GSEA) illuminated the involvement of RNASE4 in modulating epithelial-mesenchymal transition (EMT) via activation of AXL, AKT and NF-κB signaling pathways. Furthermore, recombinant human RNASE4 (hRNASE4)-mediated NF-κB activation through IκBα phosphorylation and degradation could result in the upregulation of inhibitors of apoptosis proteins (IAPs), such as cIAP1, cIAP2, and SURVIVIN. Notably, treating RNASE4-induced TMZ-resistant cells with the SURVIVIN inhibitor YM-155 significantly restored cellular sensitivity to TMZ therapy. Herein, this study positions RNASE4 as a potent prognostic biomarker and therapeutic target, offering new insights into molecular pathogenesis of GBM and new avenues for future therapeutic interventions.

A multi-institutional phase I study of acetazolamide with temozolomide in adults with newly diagnosed MGMT-methylated malignant glioma.

A significant unmet need exists for the treatment of glioblastoma, IDH-wildtype (GBM). Preclinical work shows that acetazolamide sensitizes GBM to temozolomide (TMZ) by overcoming TMZ resistance due to BCL-3-dependent upregulation of carbonic anhydrase. Acetazolamide is Food and Drug Administration-approved for the treatment of altitude sickness. Drug repurposing enables the application of drugs to diseases beyond initial indications. This multi-institutional, open-label, phase I trial examined a combination of acetazolamide and TMZ in patients with MGMT promoter-methylated high-grade glioma. A total of 24 patients (GBM, IDH-wildtype = 22; Grade 4 astrocytoma, IDH-mutant = 1; Grade 3 astrocytoma, IDH-mutant = 1) were accrued over 17 months. All patients received oral acetazolamide (250mg BID for 7 days increased to 500mg BID for Days 8-21 of each 28-day cycle) during the adjuvant phase of TMZ for up to 6 cycles. No patient had a dose-limiting toxicity. Adverse events were consistent with known sequelae of acetazolamide and TMZ. In the 23 WHO Grade 4 patients, the median overall survival (OS) was 30.1 months and the median progression-free survival was 16.0 months. The 2-year OS was 60.9%. In total 37% of the study population had high BCL-3 staining and trended toward shorter OS (17.2 months vs N.R., P = .06). The addition of acetazolamide is safe and tolerable in GBM patients receiving standard TMZ. Survival results compare favorably to historical data from randomized trials in patients with MGMT promoter-methylated GBM and support examination of acetazolamide in a randomized trial. BCL-3 expression is a potential biomarker for prognosis in GBM or for patients more likely to benefit from TMZ.

A chloromethyl-triazole fluorescent chemosensor for O6-methylguanine DNA methyltransferase.

Fluorescent chemosensors offer a direct means of measuring enzyme activity for cancer diagnosis, predicting drug resistance, and aiding in the discovery of new anticancer drugs. O6-methylguanine DNA methyltransferase (MGMT) is a predictor of resistance towards anticancer alkylating agents such as temozolomide. Using the fluorescent molecular rotor, 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), we synthesized, and evaluated a MGMT fluorescent chemosensor derived from a chloromethyl-triazole covalent inhibitor, AA-CW236, a non-pseudosubstrate of MGMT. Our fluorescence probe covalently labelled the MGMT active site C145, producing a 18-fold increase in fluorescence. Compared to previous fluorescent probes derived from a substrate-based inhibitor, our probe had improved binding and reaction rate. Overall, our chloromethyl triazole-based fluorescence MGMT probe is a promising tool for measuring MGMT activity to predict temozolomide resistance.

Diterpenoid honatisine overcomes temozolomide resistance in glioblastoma by inducing mitonuclear protein imbalance through disruption of TFAM-mediated mtDNA transcription

BackgroundGlioblastoma (GBM) represents as the most formidable intracranial malignancy. The systematic exploration of natural compounds for their potential applications in GBM therapy has emerged as a pivotal and fruitful avenue of research. PurposeIn the present study, a panel of 96 diterpenoids was systematically evaluated as a repository of potential antitumour agents. The primary objective was to discern their potency in overcoming resistance to temozolomide (TMZ). Through an extensive screening process, honatisine, a heptacyclic diterpenoid alkaloid, emerged as the most robust candidate. Notably, honatisine exhibited remarkable efficacy in patient-derived primary and recurrent GBM strains. Subsequently, we subjected this compound to comprehensive scrutiny, encompassing GBM cultured spheres, GBM organoids (GBOs), TMZ-resistant GBM cell lines, and orthotopic xenograft mouse models of GBM cells. ResultsOur investigative efforts delved into the mechanistic underpinnings of honatisine's impact. It was discerned that honatisine prompted mitonuclear protein imbalance and elicited the mitochondrial unfolded protein response (UPRmt). This effect was mediated through the selective depletion of mitochondrial DNA (mtDNA)-encoded subunits, with a particular emphasis on the diminution of mitochondrial transcription factor A (TFAM). The ultimate outcome was the instigation of deleterious mitochondrial dysfunction, culminating in apoptosis. Molecular docking and surface plasmon resonance (SPR) experiments validated honatisine's binding affinity to TFAM within its HMG-box B domain. This binding may promote phosphorylation of TFAM and obstruct the interaction of TFAM bound to heavy strand promoter 1 (HSP1), thereby enhancing Lon-mediated TFAM degradation. Finally, in vivo experiments confirmed honatisine's antiglioma properties. Our comprehensive toxicological assessments underscored its mild toxicity profile, emphasizing the necessity for a thorough evaluation of honatisine as a novel antiglioma agent. ConclusionIn summary, our data provide new insights into the therapeutic mechanisms underlying honatisine's selective inducetion of apoptosis and its ability to overcome chemotherapy resistance in GBM. These actions are mediated through the disruption of mitochondrial proteostasis and function, achieved by the inhibition of TFAM-mediated mtDNA transcription. This study highlights honatisine's potential as a promising agent for glioblastoma therapy, underscoring the need for further exploration and investigation.

Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

Gene signatures associated with prognosis and chemotherapy resistance in glioblastoma treated with temozolomide.

Background: Glioblastoma (GBM) prognosis remains extremely poor despite standard treatment that includes temozolomide (TMZ) chemotherapy. To discover new GBM drug targets and biomarkers, genes signatures associated with survival and TMZ resistance in GBM patients treated with TMZ were identified. Methods: GBM cases in The Cancer Genome Atlas who received TMZ (n = 221) were stratified into subgroups that differed by median overall survival (mOS) using network-based stratification to cluster patients whose somatic mutations affected genes in similar modules of a gene interaction network. Gene signatures formed from differentially mutated genes in the subgroup with the longest mOS were used to confirm their association with survival and TMZ resistance in independent datasets. Somatic mutations in these genes also were assessed for an association with OS in an independent group of 37 GBM cases. Results: Among the four subgroups identified, subgroup four (n = 71 subjects) exhibited the longest mOS at 18.3months (95% confidence interval: 16.2, 34.1; p = 0.0324). Subsets of the 86 genes that were differentially mutated in this subgroup formed 20-gene and 8-gene signatures that predicted OS in two independent datasets (Spearman's rho of 0.64 and 0.58 between actual and predicted OS; p < 0.001). Patients with mutations in five of the 86 genes had longer OS in a small, independent sample of 37 GBM cases, but this association did not reach statistical significance (p = 0.07). Thirty-one of the 86 genes formed signatures that distinguished TMZ-resistant GBM samples from controls in three independent datasets (area under the curve ≥ 0.75). The prognostic and TMZ-resistance signatures had eight genes in common (ANG, BACH1, CDKN2C, HMGA1, IFI16, PADI4, SDF4, and TP53INP1). The latter three genes have not been associated with GBM previously. Conclusion: PADI4, SDF4, and TP53INP1 are novel therapy and biomarker candidates for GBM. Further investigation of their oncologic functions may provide new insight into GBM treatment resistance mechanisms.

Neovascularization directed by CAVIN1/CCBE1/VEGFC confers TMZ-resistance in glioblastoma

Acquisition of resistance to temozolomide (TMZ) poses a significant challenge in glioblastoma (GBM) therapy. Neovascularization, a pivotal process in tumorigenesis and development, remains poorly understood in its contribution to chemoresistance in GBMs. This study unveils aberrant vascular networks within TMZ-resistant (TMZ-R) GBM tissues and identifies the extracellular matrix (ECM) protein CCBE1 as a potential mediator. Through in vivo and in vitro experiments involving gain and loss of function assessments, we demonstrate that high expression of CCBE1 promotes hyper-angiogenesis and orchestrates partial endothelial-to-mesenchymal transition (EndMT) in human microvascular endothelial cells (HCMEC/d3) within GBM. This is likely driven by VEGFC/Rho signaling. Intriguingly, CCBE1 overexpression substantially fails to promote tumor growth, but endows resistance to GBM cells in a vascular endothelial cell-dependent manner. Mechanically, the constitutive phosphorylation of SP1 at Ser101 drives the upregulation of CCBE1 transcription in TMZ resistant tumors, and the excretion of CCBE1 depends on caveolae associated protein 1 (CAVIN1) binding and assembling. Tumor cells derived CCBE1 promotes VEGFC maturation, activates VEGFR2/VEGFR3/Rho signaling in vascular endothelial cells, and ultimately results in hyper-angiogenesis in TMZ-R tumors. Collectively, the current study uncovers the cellular and molecular basis of abnormal angiogenesis in a chemo resistant microenvironment, implying that curbing CCBE1 is key to reversing TMZ resistance.

Carmofur prevents cell cycle progression by reducing E2F8 transcription in temozolomide-resistant glioblastoma cells

Sphingolipid metabolism is dysregulated in many cancers, allowing cells to evade apoptosis through increased sphingosine-1-phosphate (S1P) and decreased ceramides. Ceramidases hydrolyze ceramides to sphingosine, which is phosphorylated by sphingosine kinases to generate S1P. The S1P allows cells to evade apoptosis by shifting the equilibrium away from ceramides, which favor cell death. One tumor type that exhibits a shift in the sphingolipid balance towards S1P is glioblastoma (GBM), a highly aggressive brain tumor. GBMs almost always recur despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Understanding sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target dysregulation of sphingolipid metabolism in GBM. Carmofur, a derivative of 5-fluorouracil, inhibits acid ceramidase (ASAH1), a key enzyme in the production of S1P, and is in use outside the USA to treat colorectal cancer. We find that the mRNA for ASAH1, but not other ceramidases, is elevated in recurrent GBM. When TMZ-resistant GBM cells were treated with carmofur, decreased cell growth and increased apoptosis were observed along with cell cycle perturbations. RNA-sequencing identified decreases in cell cycle control pathways that were specific to TMZ-resistant cells. Furthermore, the transcription factor and G1 to S phase regulator, E2F8, was upregulated in TMZ-resistant versus parental GBM cells and inhibited by carmofur treatment in TMZ-resistant GBM cells, specifically. These data suggest a possible role for E2F8 as a mediator of carmofur effects in the context of TMZ resistance. These data suggest the potential utility of normalizing the sphingolipid balance in the context of recurrent GBM.

Identification of potential glioma drug resistance target proteins based on ultra-performance liquid chromatography-mass spectrometry differential proteomics.

In this study, to screen for candidate markers of temozolomide (TMZ) resistance in glioblastoma, we artificially established TMZ drug-resistant glioblastoma (GBM) cell lines, U251-TMZ and U87-TMZ. In the U251-TMZ and U87-TMZ cell lines, we screened and analyzed differentially expressed proteins using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) differential proteomics. Compared with the U251 and U87 control cell lines, 95 differential proteins were screened in the U251-TMZ and U87-TMZ cell lines, of which 28 proteins were upregulated and 67 proteins were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the co-upregulated proteins showed that most of the differentially expressed proteins were located in the cytoplasm and were significantly upregulated in the biological processes related to vesicular transport in the intimal system and inflammatory response mediated by myeloid leukocytes. Seven candidates were identified as potential GBM markers of TMZ resistance. Combined with existing research findings, our study supports that UAP1L1 and BCKDK are promising potential markers of TMZ resistance in GBM. This is important for further understanding the molecular mechanisms that drive the development and enhancement of TMZ resistance.

Unveiling the Influence of Tumor Microenvironment and Spatial Heterogeneity on Temozolomide Resistance in Glioblastoma Using an Advanced Human In Vitro Model of the Blood‐Brain Barrier and Glioblastoma (Small 52/2023)

Unveiling the Influence of Tumor Microenvironment and Spatial Heterogeneity on Temozolomide Resistance in Glioblastoma Using an Advanced Human In Vitro Model of the Blood‐Brain Barrier and Glioblastoma (Small 52/2023)

Interaction of SENP6 with PINK1 Promotes Temozolomide Resistance in Neuroglioma Cells via Inducing the Mitophagy

Interaction of SENP6 with PINK1 Promotes Temozolomide Resistance in Neuroglioma Cells via Inducing the Mitophagy

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.

RPL22L1, a novel candidate oncogene promotes temozolomide resistance by activating STAT3 in glioblastoma

Aggressiveness and drug resistance are major challenges in the clinical treatment of glioblastoma (GBM). Our previously research reported a novel candidate oncogene ribosomal protein L22 like 1 (RPL22L1). The aim of this study was to elucidate the potential role and mechanism of RPL22L1 in progression and temozolomide (TMZ) resistance of GBM. Online database, tissue microarrays and clinical tissue specimens were used to evaluate the expression and clinical implication of RPL22L1 in GBM. We performed cell function assays, orthotopic and subcutaneous xenograft tumor models to evaluate the effects and molecular mechanisms of RPL22L1 on GBM. RPL22L1 expression was significantly upregulated in GBM and associated with poorer prognosis. RPL22L1 overexpression enhanced GBM cell proliferation, migration, invasion, TMZ resistance and tumorigenicity, which could be reduced by RPL22L1 knockdown. Further, we found RPL22L1 promoted mesenchymal phenotype of GBM and the impact of these effects was closely related to EGFR/STAT3 pathway. Importantly, we observed that STAT3 specific inhibitor (Stattic) significantly inhibited the malignant functions of RPL22L1, especially on TMZ resistance. RPL22L1 overexpressed increased combination drug sensitive of Stattic and TMZ both in vitro and in vivo. Moreover, Stattic effectively restored the sensitive of RPL22L1 induced TMZ resistance in vitro and in vivo. Our study identified a novel candidate oncogene RPL22L1 which promoted the GBM malignancy through STAT3 pathway. And we highlighted that Stattic combined with TMZ therapy might be an effective treatment strategy in RPL22L1 high-expressed GBM patients.

Overcoming chemotherapy resistance in low-grade gliomas: A computational approach.

Low-grade gliomas are primary brain tumors that arise from glial cells and are usually treated with temozolomide (TMZ) as a chemotherapeutic option. They are often incurable, but patients have a prolonged survival. One of the shortcomings of the treatment is that patients eventually develop drug resistance. Recent findings show that persisters, cells that enter a dormancy state to resist treatment, play an important role in the development of resistance to TMZ. In this study we constructed a mathematical model of low-grade glioma response to TMZ incorporating a persister population. The model was able to describe the volumetric longitudinal dynamics, observed in routine FLAIR 3D sequences, of low-grade glioma patients acquiring TMZ resistance. We used the model to explore different TMZ administration protocols, first on virtual clones of real patients and afterwards on virtual patients preserving the relationships between parameters of real patients. In silico clinical trials showed that resistance development was deferred by protocols in which individual doses are administered after rest periods, rather than the 28-days cycle standard protocol. This led to median survival gains in virtual patients of more than 15 months when using resting periods between two and three weeks and agreed with recent experimental observations in animal models. Additionally, we tested adaptive variations of these new protocols, what showed a potential reduction in toxicity, but no survival gain. Our computational results highlight the need of further clinical trials that could obtain better results from treatment with TMZ in low grade gliomas.

EXTH-12. CYTOCHROME C OXIDASE SUBUNIT 4 ISOFORM 1 MEDIATES ONCOLYTIC HERPES SIMPLEX VIRUS-1 EFFICACY IN HIGH-GRADE GLIOMA

Abstract RATIONALE Outcomes for patients with high-grade glioma (HGG) are dismal despite standard of care surgical resection, radiation, and temozolomide (TMZ) chemotherapy. RESULTS from our Phase I trial of pediatric patients with recurrent HGG suggest oncolytic herpes simplex virus-1 (oHSV) is safe with promising efficacy. However, optimization of oHSV therapy is needed to achieve more durable responses. Based on our prior work with matched pair HGG cell lines and patient-derived xenografts (PDXs) with and without acquired TMZ resistance, oHSV efficacy is enhanced in TMZ-resistant cells with no difference in oHSV entry receptor (CD111) expression. Microarray transcriptomic data of four PDXs with and without TMZ resistance revealed downregulation of interferon (IFN) responses. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1) is expressed in recurrent HGG samples and cell lines. While COX4I1 positively regulates influenza virus replication, no study has investigated the role of COX4I1 in mediating oncolytic virotherapy efficacy. HYPOTHESIS We hypothesized that COX4I1 expression mediates HGG sensitivity to oHSV through suppression of IFN signaling. METHODS Using previously published models, oHSV cytotoxicity was assessed in vitro using Alamar Blue and CellTiter-Glo assays. oHSV infectivity and CD111 protein expression was assessed by flow cytometry. COX4I1 protein expression was measured by immunoblot. mRNA expression of IFN-stimulated genes was assessed by qPCR. RESULTS COX4I1 overexpression increased oHSV cytotoxicity and infectivity compared to empty vector controls. Short hairpin RNA knockdown of COX4I1 decreased oHSV cytotoxicity and infectivity in TMZ-resistant cells compared to pLKO controls. IFN-stimulated genes IFNB1, TMEM173, DDX58, IFIH1, and OAS1 were all significantly decreased in COX4I1 overexpressing cells and TMZ-resistant cells compared to parental lines. CONCLUSIONS Overexpression of COX4I1 sensitizes HGG cells to oHSV, and COX4I1 knockdown de-sensitizes TMZ-resistant HGG cells to oHSV, suggesting a novel role of COX4I1 in mediating HGG sensitivity to oHSV.

DDDR-14. DEVELOPMENT OF A NOVEL CYP17A1-TARGETED INHIBITOR FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a highly aggressive and invasive brain tumor, resistant to current standard treatments, including temozolomide (TMZ) and radiotherapy. Despite these interventions, GBM's intrinsic drug resistance often renders it incurable, thus highlighting the dire need for novel therapeutic strategies. In this study, we used ultra-high pressure liquid chromatography-mass spectrometry to examine the levels of neurosteroids in tumor specimens from primary and recurrent GBM patients. This led to the discovery of differential expression patterns, most notably in androgen, including dehydroepiandrosterone and testosterone. Our results indicated androgen over-production weakens the GBM's sensitivity to TMZ by inducing post-translational modification of Sp1 protein, which aids in DNA repair. Interestingly, Sp1 activity is further enhanced by histone deacetylase (HDAC) 6, which in turn diminishes the DNA damage caused by TMZ. In addition, proteomic analysis revealed an overexpression of cytochrome (CYP) 17A1, particularly in recurrent GBM, which led us to hypothesize that a drug inhibiting both CYP17A1 and HDAC6 could potentially serve as a viable treatment for TMZ-resistant GBM. To validate this, the drug was tested in vitro on U87MG-R cells and patient-derived Pt#3 TMZ-resistant cells, demonstrating the therapeutic efficacy based on cell viability assays. In vivo testing in the TMZ-resistant GBM orthotopic mouse model also showed promising anti-tumor activity, with treated mice demonstrating increased survival times compared to controls. In conclusion, our findings suggest that this drug targeting both CYP17A1 and HDAC6 presents a potential breakthrough in GBM-targeted therapy, addressing the pervasive issue of TMZ resistance.

EXTH-83. BIRC3 AS A PREDICTOR OF OUTCOME AND A NEW THERAPEUTIC TARGET IN TMZ RESISTANT GLIOBLASTOMA TUMORS

Abstract Glioblastoma (GBM) is an incurable malignant brain tumor, with a highly unfavorable prognosis. Apart from MGMT promoter methylation and TMZ response, molecular biomarkers associated with therapeutic response or survival prognosis are still lacking in GBM. Previously, using NAD(P)H-fluorescence lifetime imaging, a new drug screening precision medicine ex-vivo approach, we perturbed patients-derived vital GBM tumor explants (GB-EXPs) by TMZ treatment and obtained a stratification in TMZ responder (Res) and non-responder (Non-Resp) tumors, identifying several genes differentially expressed between the two groups. To validate the approach we enlarged our glioblastoma case study. Transcriptome analysis was performed to characterize TMZ Res and Non-Resp cases. In-silico and functional cellular investigations were carried on to explore the biomarker's efficiency. The majority of the genes found dysregulated in the previous study, showed the same directional regulation of expression and we confirmed, in TMZ-resistant samples the statistically significant upregulation of BIRC3, a strong inhibitor of apoptosis. The TCGA dataset provided an opportunity to fully assess the BIRC3 mRNA expression complementarity with the MGMT status as a predictor of outcome. BIRC3 was validated as a prognostic factor of survival also in large French and Italian glioblastoma cohorts. Most importantly, AZD5582 drug, which is a specific antagonist of BIRC3, combined with TMZ treatment, is able to reverse the TMZ resistance, in glioblastoma cell lines, by restoring apoptosis, and in GB-EXPs that were resistant to TMZ treatment. BIRC3 represents a potential biomarker of prognosis and a predictor of TMZ treatment response in GBM and complements MGMT status as an outcome predictor. The level of BIRC3 expression could stratify patients who will benefit from AZD5582 or its combination with TMZ. Our study provides a potentially new treatment approach and valuable leads to determine whether functional precision medicine combined with BIRC3 expression assessment become a standard tool in glioblastoma clinical oncology.