A multivariate retrospective analysis of high-grade gliomas: Survival and prognostic factors.

OBJECTIVES: High-grade gliomas (HGGs) are highly malignant, aggressive, and have a high incidence and mortality rate. The aim of this study was to investigate survival outcomes and prognostic factors in patients with HGGs. METHODS: In this retrospective study, a total of 159 patients with histologically confirmed HGGs were included. The recruitment period was from January 2011 to December 2019. We evaluated patient demographic data, tumor characteristics, treatment methods, immunocytochemistry results, overall survival (OS) time, and progression-free survival (PFS) time using Kaplan-<>Meier survival analysis with log-rank testing. Additionally, we employed Cox regression analysis to identify independent factors associated with survival outcomes. RESULTS: Kaplan-Meier survival analysis revealed that the 1-, 2-, and 5-years OS rates were 81.8%, 50.3%, and 12.6%, respectively. Similarly, the 1-, 2-, and 5-years PFS rates were 50.9%, 22.4%, and 3.1%, respectively. The median OS duration was 35.0 months. The univariate analysis indicated that postoperative pathological classification, grade, and age were significantly associated with patient outcomes (p < 0.01). Among the patients, 147 received concurrent chemoradiotherapy, while 12 did not. The immunohistochemical markers of ki-67, MGMT, IDH1R132H, and p53 demonstrated statistically significant differences in their prognostic impact (p = 0.001, p = 0.020, p = 0.003, and p = 0.021, respectively). In conclusion, we found that grades, age, pathological classification, ki-67, MGMT, and IDH1R132H expression were statistically significantly associated with PFS (p < 0.01, p = 0.004, p = 0.003, p = 0.001, p = 0.036, and p = 0.028). Additionally, immunohistochemical expressions of TRIB3 and AURKA were significantly higher in patients with shorter survival (p = 0.015 and p = 0.023). CONCLUSIONS: Tumor grade and the use of concurrent chemoradiotherapy after surgery were independent prognostic factors that significantly influenced patient survival. Additionally, tumor grade and MGMT expression were found to be independent factors affecting progression-free survival (PFS). Notably, the expression of TRIB3 and AURKA was higher in patients with poor survival outcomes.

Stanniocalcin-1 promotes temozolomide resistance of glioblastoma through regulation of MGMT.

Temozolomide (TMZ) resistance is a major challenge in the treatment of glioblastoma (GBM). Tumour reproductive cells (TRCs) have been implicated in the development of chemotherapy resistance. By culturing DBTRG cells in three-dimensional soft fibrin gels to enrich GBM TRCs and performing RNA-seq analysis, the expression of stanniocalcin-1 (STC), a gene encoding a secreted glycoprotein, was found to be upregulated in TRCs. Meanwhile, the viability of TMZ-treated TRC cells was significantly higher than that of TMZ-treated 2D cells. Analysis of clinical data from CGGA (Chinese Glioma Genome Atlas) database showed that high expression of STC1 was closely associated with poor prognosis, glioma grade and resistance to TMZ treatment, suggesting that STC1 may be involved in TMZ drug resistance. The expression of STC1 in tissues and cells was examined, as well as the effect of STC1 on GBM cell proliferation and TMZ-induced DNA damage. The results showed that overexpression of STC1 promoted and knockdown of STC1 inhibited TMZ-induced DNA damage. These results were validated in an intracranial tumour model. These data revealed that STC1 exerts regulatory functions on MGMT expression in GBM, and provides a rationale for targeting STC1 to overcome TMZ resistance.

Glioma lateralization: Focus on the anatomical localization and the distribution of molecular alterations (Review).

It is well known how the precise localization of glioblastoma multiforme (GBM) predicts the direction of tumor spread in the surrounding neuronal structures. The aim of the present review is to reveal the lateralization of GBM by evaluating the anatomical regions where it is frequently located as well as the main molecular alterations observed in different brain regions. According to the literature, the precise or most frequent lateralization of GBM has yet to be determined. However, it can be said that GBM is more frequently observed in the frontal lobe. Tractus and fascicles involved in GBM appear to be focused on the corticospinal tract, superior longitudinal I, II and III fascicles, arcuate fascicle long segment, frontal strait tract, and inferior fronto­occipital fasciculus. Considering the anatomical features of GBM and its brain involvement, it is logical that the main brain regions involved are the frontal­temporal­parietal­occipital lobes, respectively. Although tumor volumes are higher in the right hemisphere, it has been determined that the prognosis of patients diagnosed with cancer in the left hemisphere is worse, probably reflecting the anatomical distribution of some detrimental alterations such as TP53 mutations, PTEN loss, EGFR amplification, and MGMT promoter methylation. There are theories stating that the right hemisphere is less exposed to external influences in its development as it is responsible for the functions necessary for survival while tumors in the left hemisphere may be more aggressive. To shed light on specific anatomical and molecular features of GBM in different brain regions, the present review article is aimed at describing the main lateralization pathways as well as gene mutations or epigenetic modifications associated with the development of brain tumors.

Integrative single-cell and bulk transcriptome analyses identify a distinct pro-tumor macrophage signature that has a major prognostic impact on glioblastomas.

Glioblastoma (GBM) is a highly heterogeneous disease with poor clinical outcomes. To comprehensively dissect the molecular landscape of GBM and heterogeneous macrophage clusters in the progression of GBM, this study integrates single-cell and bulk transcriptome data to recognize a distinct pro-tumor macrophage cluster significantly associated with the prognosis of GBM and develop a GBM prognostic signature to facilitate prior subtypes. Leveraging glioma single-cell sequencing data, we identified a novel pro-tumor macrophage subgroup, marked by S100A9, which might interact with endothelial cells to facilitate tumor progression via angiogenesis. To further benefit clinical application, a prognostic signature was established with the genes associated with pro-tumor macrophages. Patients classified within the high-risk group characterized with enrichment in functions related to tumor progression, including epithelial-mesenchymal transition and hypoxia, displays elevated mutations in the TERT promoter region, reduced methylation in the MGMT promoter region, poorer prognoses, and diminished responses to temozolomide therapy, thus effectively discriminating between the prognostic outcomes of GBM patients. Our research sheds light on the intricate microenvironment of gliomas and identifies potential molecular targets for the development of novel therapeutic approaches.

[Patients with long-term survival in malignant gliomas after photodynamic therapy]. / Patsienty s dlitel'noi vyzhivaemost'yu pri zlokachestvennykh gliomakh posle fotodinamicheskoi terapii.

OBJECTIVE: Analysis of long-lived patients from the group of patients with glioblastomas after using photodynamic therapy in the structure of their complex treatment in order to assess the influence of various factors on their life expectancy. MATERIAL AND METHODS: In total, a single-center, retrospective categorical study analyzed the long-term results of treatment of 63 patients with glioblastoma in the structure of complex treatment including photodynamic therapy. Clinical factors (age, sex, number of cases, preoperative Karnofsky index, location and size of the tumor, radicality of the operation), histological (nuclear polymorphism, mitosis, vascular proliferation, necrosis), immunohistochemical (Ki-67, p53 index) molecular-genetic factors (expression of VEGF, MGMT, IDH, CD34), amount of radiation and chemotherapy were analyzed. RESULTS: In the entire group of patients, there was a direct correlation of life expectancy with MGMT status, IDH status, the number of courses of chemotherapy, the age of the patient, and the severity of the first surgical intervention. CONCLUSION: Clinical features such as age at diagnosis and extent of surgical resection and amount of chemotherapy have predictive value in assessing their effect on life expectancy. Mutations in IDH and MGMT promoter methylation were the most important molecular factors determining long-term patient survival.

Preoperative prediction of MGMT promoter methylation in glioblastoma based on multiregional and multi-sequence MRI radiomics analysis.

O6-methylguanine-DNA methyltransferase (MGMT) has been demonstrated to be an important prognostic and predictive marker in glioblastoma (GBM). To establish a reliable radiomics model based on MRI data to predict the MGMT promoter methylation status of GBM. A total of 183 patients with glioblastoma were included in this retrospective study. The visually accessible Rembrandt images (VASARI) features were extracted for each patient, and a total of 14676 multi-region features were extracted from enhanced, necrotic, "non-enhanced, and edematous" areas on their multiparametric MRI. Twelve individual radiomics models were constructed based on the radiomics features from different subregions and different sequences. Four single-sequence models, three single-region models and the combined radiomics model combining all individual models were constructed. Finally, the predictive performance of adding clinical factors and VASARI characteristics was evaluated. The ComRad model combining all individual radiomics models exhibited the best performance in test set 1 and test set 2, with the area under the receiver operating characteristic curve (AUC) of 0.839 (0.709-0.963) and 0.739 (0.581-0.897), respectively. The results indicated that the radiomics model combining multi-region and multi-parametric MRI features has exhibited promising performance in predicting MGMT methylation status in GBM. The Modeling scheme that combining all individual radiomics models showed best performance among all constructed moels.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Unveiling the role of TAGLN2 in glioblastoma: From proneural-mesenchymal transition to Temozolomide resistance.

Glioblastoma (GBM) presents a daunting challenge due to its resistance to temozolomide (TMZ), a hurdle exacerbated by the proneural-to-mesenchymal transition (PMT) from a proneural (PN) to a mesenchymal (MES) phenotype. TAGLN2 is prominently expressed in GBM, particularly in the MES subtype compared to low-grade glioma (LGG) and the PN subtype. Our research reveals TAGLN2's involvement in PMT and TMZ resistance through a series of in vitro and in vivo experiments. TAGLN2 knockdown can restrain proliferation and invasion, trigger DNA damage and apoptosis, and heighten TMZ sensitivity in GBM cells. Conversely, elevating TAGLN2 levels amplifies resistance to TMZ in cellular and intracranial xenograft mouse models. We demonstrate the interaction relationship between TAGLN2 and ERK1/2 through co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrometry analysis. Knockdown of TAGLN2 results in a decrease in the expression of p-ERK1/2, whereas overexpression of TAGLN2 leads to an increase in p-ERK1/2 expression within the nucleus. Subsequently, the regulatory role of TAGLN2 in the expression and control of MGMT has been demonstrated. Finally, the regulation of TAGLN2 by NF-κB has been validated through chromatin immunoprecipitation and ChIP-PCR assays. In conclusion, our results confirm that TAGLN2 exerts its biological functions by interacting with the ERK/MGMT axis and being regulated by NF-κB, thereby facilitating the acquisition of promoting PMT and increased resistance to TMZ therapy in glioblastoma. These results provide valuable insights for the advancement of targeted therapeutic approaches to overcome TMZ resistance in clinical treatments.

Gender differences in gliomas: From epidemiological trends to changes at the hormonal and molecular levels.

Gender plays a crucial role in the occurrence and development of cancer, as well as in the metabolism of nutrients and energy. Men and women display significant differences in the incidence, prognosis, and treatment response across various types of cancer, including certain sex-specific tumors. It has been observed that male glioma patients have a higher incidence and worse prognosis than female patients, but there is currently a limited systematic evaluation of sex differences in gliomas. The purpose of this study is to provide an overview of the association between fluctuations in sex hormone levels and changes in their receptor expression with the incidence, progression, treatment, and prognosis of gliomas. Estrogen may have a protective effect on glioma patients, while exposure to androgens increases the risk of glioma. We also discussed the specific genetic and molecular differences between genders in terms of the malignant nature and prognosis of gliomas. Factors such as TP53, MGMT methylation status may play a crucial role. Therefore, it is essential to consider the gender of patients while treating glioma, particularly the differences at the hormonal and molecular levels. This approach can help in the adoption of an individualized treatment strategy.

Adaptive fine-tuning based transfer learning for the identification of MGMT promoter methylation status.

Background.Glioblastoma Multiforme (GBM) is an aggressive form of malignant brain tumor with a generally poor prognosis.O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been shown to be a predictive bio-marker for resistance to treatment of GBM, but it is invasive and time-consuming to determine methylation status. There has been effort to predict the MGMT methylation status through analyzing MRI scans using machine learning, which only requires pre-operative scans that are already part of standard-of-care for GBM patients.Purpose.To improve the performance of conventional transfer learning in the identification of MGMT promoter methylation status, we developed a 3D SpotTune network with adaptive fine-tuning capability. Using the pretrained weights of MedicalNet with the SpotTune network, we compared its performance with a randomly initialized network for different combinations of MR modalities.Methods.Using a ResNet50 as the base network, three categories of networks are created: (1) A 3D SpotTune network to process volumetric MR images, (2) a network with randomly initialized weights, and (3) a network pre-trained on MedicalNet. These three networks are trained and evaluated using a public GBM dataset provided by the University of Pennsylvania. The MRI scans from 240 patients are used, with 11 different modalities corresponding to a set of perfusion, diffusion, and structural scans. The performance is evaluated using 5-fold cross validation with a hold-out testing dataset.Results.The SpotTune network showed better performance than the randomly initialized network. The best performing SpotTune model achieved an area under the Receiver Operating Characteristic curve (AUC), average precision of the precision-recall curve (AP), sensitivity, and specificity values of 0.6604, 0.6179, 0.6667, and 0.6061 respectively.Conclusions.SpotTune enables transfer learning to be adaptive to individual patients, resulting in improved performance in predicting MGMT promoter methylation status in GBM using equivalent MRI modalities as compared to a randomly initialized network.

Dissecting the prognostic signature of patients with astrocytoma isocitrate dehydrogenase-mutant grade 4: a large multicenter, retrospective study.

BACKGROUND: The World Health Organization (WHO) 2021 classification of central nervous system (CNS) tumors classified astrocytoma isocitrate dehydrogenase-mutant (A IDHm) with either microvascular proliferation and/or necrosis or homozygous deletion of CDKN2A/B as CNS grade 4 (CNS WHO G4), introducing a distinct entity and posing new challenges to physicians for appropriate management and prognostication. PATIENTS AND METHODS: We retrospectively collected information about patients diagnosed with A IDHm CNS WHO G4 at three reference neuro-oncological Italian centers and correlated them with survival. RESULTS: A total of 133 patients were included. Patients were young (median age 41 years) and most received post-operative treatment including chemo-radiation (n = 101) and/or temozolomide maintenance (n = 112). With a median follow-up of 51 months, the median overall survival (mOS) was 31.2 months, with a 5-year survival probability of 26%. In the univariate analysis, complete resection (mOS: 40.2 versus 26.3 months, P = 0.03), methyl-guaninemethyltransferase (MGMT) promoter methylation (mOS: 40.7 versus 18 months, P = 0.0136), and absence of telomerase reverse transcriptase (TERT) promoter mutation (mOS: 40.7 versus 18 months, P = 0.0003) correlated with better prognosis. In the multivariate models, lack of TERT promoter mutation [hazard ratio (HR) 0.23, 95% confidence interval (CI) 0.07-0.82, P = 0.024] and MGMT methylation (HR 0.40, 95% CI 0.20-0.81, P = 0.01) remained associated with improved survival. CONCLUSIONS: This is the largest experience in Western countries exploring the prognostic signature of patients with A IDHm CNS G4. Our results show that MGMT promoter methylation and TERT promoter mutation may impact clinical outcomes. This may support physicians in prognostication, clinical management, and design of future studies of this distinct diagnostic entity.

A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria.

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory.

Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.

Enhancing Temozolomide (TMZ) chemosensitivity using CRISPR-dCas9-mediated downregulation of O6-methylguanine DNA methyltransferase (MGMT).

PURPOSE: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O6-methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O6 position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ. METHOD: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer. RESULT: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line. CONCLUSION: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells.

Temperature-Wise Calibration Increases the Accuracy of DNA Methylation Levels Determined by High-Resolution Melting (HRM).

High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT.

Evaluation of the clinical use of MGMT methylation in extracellular vesicle-based liquid biopsy as a tool for glioblastoma patient management.

Glioblastoma (GB) is a devastating tumor of the central nervous system characterized by a poor prognosis. One of the best-established predictive biomarker in IDH-wildtype GB is O6-methylguanine-DNA methyltransferase (MGMT) methylation (mMGMT), which is associated with improved treatment response and survival. However, current efforts to monitor GB patients through mMGMT detection have proven unsuccessful. Small extracellular vesicles (sEVs) hold potential as a key element that could revolutionize clinical practice by offering new possibilities for liquid biopsy. This study aimed to determine the utility of sEV-based liquid biopsy as a predictive biomarker and disease monitoring tool in patients with IDH-wildtype GB. Our findings show consistent results with tissue-based analysis, achieving a remarkable sensitivity of 85.7% for detecting mMGMT in liquid biopsy, the highest reported to date. Moreover, we suggested that liquid biopsy assessment of sEV-DNA could be a powerful tool for monitoring disease progression in IDH-wildtype GB patients. This study highlights the critical significance of overcoming molecular underdetection, which can lead to missed treatment opportunities and misdiagnoses, possibly resulting in ineffective therapies. The outcomes of our research significantly contribute to the field of sEV-DNA-based liquid biopsy, providing valuable insights into tumor tissue heterogeneity and establishing it as a promising tool for detecting GB biomarkers. These results have substantial implications for advancing predictive and therapeutic approaches in the context of GB and warrant further exploration and validation in clinical settings.

MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma.

Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.

Accurate and comprehensive evaluation of O6-methylguanine-DNA methyltransferase promoter methylation by nanopore sequencing.

AIMS: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability. METHODS: We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification. RESULTS: Nanopore sequencing displayed a strong correlation (R2 = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island. CONCLUSIONS: This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.