MGMT Promoter Region Research Articles

Longitudinal multimodal profiling of IDH-wildtype glioblastoma reveals the molecular evolution and cellular phenotypes underlying prognostically different treatment responses.

Despite recent advances in the biology of IDH-wildtype glioblastoma, it remains a devastating disease with median survival of less than 2years. However, the molecular underpinnings of the heterogeneous response to the current standard-of-care treatment regimen consisting of maximal safe resection, adjuvant radiation, and chemotherapy with temozolomide remain unknown. Comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent glioblastoma specimens from 106 patients was performed to investigate the molecular evolution and cellular phenotypes underlying differential treatment responses. While TERT promoter mutation and CDKN2A homozygous deletion were early events during gliomagenesis shared by initial and recurrent tumors, most other recurrent genetic alterations (eg, EGFR, PTEN, and NF1) were commonly private to initial or recurrent tumors indicating acquisition later during clonal evolution. Furthermore, glioblastomas exhibited heterogeneous epigenomic evolution with subsets becoming more globally hypermethylated, hypomethylated, or remaining stable. Glioblastoma that underwent sarcomatous transformation had shorter interval to recurrence and were significantly enriched in NF1, TP53, and RB1 alterations and the mesenchymal epigenetic class. Patients who developed somatic hypermutation following temozolomide treatment had significantly longer interval to disease recurrence and prolonged overall survival, and increased methylation at 4 specific CpG sites in the promoter region of MGMT was significantly associated with this development of hypermutation. Finally, an epigenomic evolution signature incorporating change in DNA methylation levels across 347 critical CpG sites was developed that significantly correlated with clinical outcomes. Glioblastoma undergoes heterogeneous genetic, epigenetic, and cellular evolution that underlies prognostically different treatment responses.

PATH-30. CLINICAL OUTCOMES AND PREDICTIVE BIOMARKERS FOR IDH-WILDTYPE GLIOBLASTOMAS DEVELOPING HYPERMUTATION FOLLOWING TEMOZOLOMIDE TREATMENT

Abstract Current standard of care for IDH-wildtype glioblastoma (GBM) includes maximal safe resection, adjuvant radiation, and chemotherapy with the alkylating agent temozolomide. A subset of gliomas treated with temozolomide develop somatic hypermutation with a predominance of C>T transitions due to the alkylation effects on DNA. However, the frequency of occurrence, clinical ramifications, and predictive biomarkers for developing temozolomide-induced hypermutation (TMZ-HM) in GBM are unknown. Here we performed comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent GBM from 106 patients. Seven patients (7%) developed hypermutation at time of first recurrence, while an additional five patients (5%) developed hypermutation at a subsequent recurrence. Patients who developed TMZ-HM had a significantly longer interval between initial surgery and first recurrence (27.8 vs. 9.9 months, p<0.001). In contrast to IDH-mutant gliomas where TMZ-HM is associated with poor prognosis, those patients with IDH-wildtype GBM who developed TMZ-HM had significantly longer overall survival from both initial surgery (67.9 vs. 20.3 months, p<0.001) and from time of recurrence after development of hypermutation (30.9 vs. 9.5 months, p=0.001). There was no difference in oncogenic alteration frequency in initial GBM that subsequently developed TMZ-HM. However, 4 specific CpG sites out of 12 total interrogated sites in the promoter region of MGMT (a DNA repair enzyme) and 5 specific CpG sites in the promoter region of KCNQ1DN (a negative regulator of c-Myc) were significantly more hypermethylated in those GBM that developed TMZ-HM. We then generated a weighted risk score across these CpG sites that can be used to prospectively identify GBM likely to develop TMZ-HM associated with favorable survival. In summary, patients with GBM that develop TMZ-HM follow a more favorable clinical course, and specific MGMT and KCNQ1DN promoter hypermethylation patterns could serve as a biomarker to identify these patients who may potentially benefit from extended alkylating chemotherapy treatment.

CTNI-27. SYSTEMIC DELIVERY OF LOW ENERGY RADIOFREQUENCY ELECTROMAGNETIC FIELDS FOR THE TREATMENT OF PATIENTS WITH PRIMARY BRAIN TUMORS

Abstract BACKGROUND Tumor Treating Fields plus maintenance temozolomide (mTMZ) showed a survival advantage in the first-line glioblastoma (GBM) treatment. The survival benefit remains limited in patients with unmethylated MGMT gene promoter. The AutEMsys device emits low-energy amplitude modulated systemic electromagnetic fields (EMF) at patient specific frequencies. It is an outpatient procedure that can be integrated with mTMZ. METHODS This study involved a prospective compassionate access program targeting adult GBM patients using an intrabuccal EMF exposure via AutEMsys in addition to chemoRT+mTMZ. The program explored the effects on survival and patients’ reported quality-of-life (QOL) by EORTC-QL-C30. Patients underwent a 90-minute session of EMF exposure therapy repeated every 2 weeks until progression, deterioration in QOL or death. All patients had pathological specimens for diagnosis, IDH mutation status through NGS techniques, and MGMT promoter region methylation status. RESULTS 31 participants were enrolled, including 29 with GBM and 2 with diffuse midline glioma. 14 patients (45%) received EMF+chemoRT followed by EMF+mTMZ and 17 patients (55%) received EMF+mTMZ (post chemoRT). Survival time was measured from the start of mTMZ to the date of the last follow-up. The median overall survival (mOS) for all 31 patients was 26.0 months (95% CI: 14.4 – 37.2 months). Specifically for 21 patients with unmethylated MGMT, the mOS was 25.0 months (95% CI: 15.9 – 34.1 months). Similarly, for the 28 patients with IDH wildtype status, the mOS remained 26.0 months (95% CI: 9.6 – 42.4 months). The addition of AutEMsys with chemoRT+mTMZ demonstrated an improved role functioning in 79% of patients with 25% of these with >10-point score. CONCLUSION The AutEMsys device has shown promising initial results when added to chemoRT+mTMZ treatment of GBM. These findings warrant further prospective investigation and validation in a multicenter clinical trial setting.

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.

DCas9/CRISPR-based methylation of O-6-methylguanine-DNA methyltransferase enhances chemosensitivity to temozolomide in malignant glioma

BackgroundMalignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ.MethodsVia lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis.ResultsWe used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects.ConclusiondCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications.

10015-NI-1 PREDICTION OF MGMT PROMOTOR METHYLATION STATUS IN GLIOBLASTOMA BY CONTRAST-ENHANCED T1-WEIGHTED IMAGE

Abstract PURPOSE The study aims to explore MRI phenotypes that predict the methylation status of the promoter region of MGMT (pMGMT) in glioblastoma. MATERIALS AND METHODS 202 histologically and molecularly confirmed glioblastoma patients (pMGMT methylated: 103, unmethylated: 99) at the Kansai Network for Molecular Diagnosis of Central Nervous Tumors (KNBTG) were used as a test cohort. 104 patients from the Cancer Imaging Archive (TCIA) / Cancer Genome Atlas (TCGA) dataset (pMGMT methylated: 103, unmethylated: 99) were used as a validation cohort. "Thickened structure" was defined when the solid component of the tumor had either a circumferential extension or occupied more than 50% of the entire volume of the tumor. "Methylated contrast phenotype" was defined by imaging findings with one of the following three features: indistinct enhancing circumferential border, heterogenous enhancement, and nodular enhancement. The inter-rater agreement between three independent blinded raters was first evaluated, and then the relationship between the image findings and pMGMT methylation status was investigated. RESULTS Fleiss's Kappa coefficient for "Thickened structure" was 0.68 for the KNBTG cohort and 0.56 for the TCIA cohort and for " Methylated contrast phenotype", 0.32 and 0.35, respectively. The odds ratio of "Methylated contrast phenotype" detecting pMGMT hypermethylation was 2.4 (p = 0.003) for the KNBTG cohort. The odds ratio of "Methylated contrast phenotype" associated with pMGMT methylation was 4.5 (p = 0.001) for those restricted to tumors presenting a "Thickened structure". The odds ratio for pMGMT methylation detected by "Methylated contrast phenotype" was 3.4 (p = 0.005) for the TCIA cohort. It was 7.1 (p = 0.009) when restricting to tumors presenting a "Thickened structure". CONCLUSION Glioblastoma showing both a "Thickened structure" structure and "Methylated contrast phenotype" is associated with pMGMT methylation, which may be a clinically useful imaging feature.

Analysis of DNA methylation profiles confirms a high proportion of immune cells in glioblastoma samples

Glioblastomas are malignant tumors that belong to the central nervous system and are challenging to diagnose due to their significant intratumoral heterogeneity, which makes molecular testing and diagnosis confirmation particularly difficult. In addition to identifying typical genetic mutations such as IDH, H3F3A G34 and K27, WHO recommendations emphasize the importance of analyzing the tumor epigenome to define its class based on DNA methylation patterns and methylation status of specific genomic regions, particularly the MGMT promoter region. Based on our clinical experience, molecular genetic studies sometimes yield contradictory results due to the heterogeneous cellular composition of glioblastomas. In this study, we present a series of observations made on 35 glioblastoma samples in which we compare the morphological features and the results of cell type detection by deconvolution method based on total DNA methylation profiles. Our results suggest that samples of mesenchymal class glioblastomas may contain over 50 % non-tumor immune cells, which should be considered in genetic testing of these tumors.

Curcumol reverses temozolomide resistance in glioma cells by regulating the UTX/MGMT axis

To explore the mechanism through which curcumol reverses primary drug resistance in glioma cells. The inhibitory effect of 10, 20, and 40 μg/mL curcumol were observed in human glioma cell lines A172 and U251. UTX-overexpressing glioma cells constructed by lentiviral transfection were treated with curcumol (40 μg/mL), temozolomide (TMZ; 10 μg/mL), or both, and the changes in cell viability, clone formation capacity and apoptosis were assessed using MTT assay, cell clone formation experiment, and flow cytometry; UTX activity in the cells was determined using a UTX detection kit, and the enrichment of UTX and H3K27me3 in the MGMT promoter region was detected with ChiP-qPCR. The protein expressions in glioma cells were detected using Western blotting and immunohistochemistry. In a nude mouse model bearing glioma xenografts, the effects of curcumol (20 mg/kg), TMZ (20 mg/kg) and their combination on tumor growth and expressions of UTX, H3K27me3 and MGMT were evaluated. Curcumol significantly inhibited the proliferation (P<0.05) and promoted apoptosis of cultured glioma cells (P<0.01). Curcumol, but not TMZ, produced significant inhibitory effect on tumor growth in the tumor-bearing mice (P<0.01). Curcumol significantly inhibited UTX activity and increased the expression level of H3K27me3 protein in the glioma cells. UTX overexpression obviously decreased H3K27me3 protein expression and reversed the effects of curcumol on glioma cell proliferation and apoptosis (P<0.01). Curcumol reduced the enrichment of UTX and H3K27me3 in the MGMT promoter region (P<0.05) and decreased MGMT protein expression, which was reversed by UTX overexpression. In both the in vivo and in vitro experiments, curcumol combined with TMZ significantly increased H3K27me3 protein expression in the glioma cells, reduced the expression of its downstream target gene MGMT, and enhanced TMZ sensitivity of the glioma cells. Curcumol can enhance glioma cell sensitivity to TMZ by regulating the UTX/MGMT axis.

MGMT Gene rs1625649 Polymorphism in Iranian Patients with Brain Glioblastoma: A Case Control Study.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor with poor prognosis and high potential of dispersion to other brain tissues in adult. Effective and modern choices of treatment including chemotherapy with alkylating agents marginally extend survival of GBM. However, alkylating agents can lead to highly harmful mismatch during DNA replication causing apoptosis and cell death. Accordingly, O6-Methylguanine-DNA methyltransferase (MGMT) removes alkyl adducts, thereby causing resistance to alkylating drugs. Single-Nucleotide Polymorphisms (SNPs) in MGMT promoter region may play a role in the regulation of MGMT expression and prediction of glioma development risk. In order to evaluate the clinical significance of rs1625649 SNP in the MGMT promoter region of glioblastoma, genomic DNA from a series of 54 patients with GBM and 50 healthy individuals in Iranian population were collected for tetra ARMS PCR amplification. None of the "A" or "C" alleles were associated with tumor occurrence, the "AA" genotype was more frequent in healthy subjects, and the "AC" genotype was 4.6 times more common in patients with GBM. The longest survival time was observed in the "CC" genotype; however, this difference was not statistically significant. On the other hand, homozygous rs1625649 (AA genotype) was significantly associated with a better survival than the cases with heterozygous rs1625649 (CA genotype) or wild type rs1625649 (CC genotype), predicting better response to temozolomide-based chemotherapy.

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma.

Temozolomide (TMZ) resistance has become an important obstacle affecting its therapeutic benefits. O6-methylguanine DNA methyltransferase (MGMT) is primarily responsible for the TMZ resistance in Glioblastoma multiforme (GBM) patients. In addition, active DNA damage repair pathways can also lead to TMZ resistance. Here, we reported a novel small-molecule inhibitor EPIC-0412 that improved the therapeutic efficacy of TMZ by inhibiting the DNA damage repair pathway and MGMT in GBM via epigenetic pathways. The small-molecule compound EPIC-0412 was obtained through high-throughput screening. RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), and chromatin immunoprecipitation (ChIP) assays were used to verify the effect of EPIC-0412. Co-immunoprecipitation (Co-IP) was used to elucidate the interactions of transcription factors at the MGMT promoter region. Animal experiments using a mouse model were performed to verify the efficacy of EPIC-0412 in sensitizing GBM cells to TMZ. EPIC-0412 physically interrupts the binding of HOTAIR and EZH2, leading to the upregulation of CDKN1A and BBC3, causing cell cycle arrest and apoptosis in GBM cells. EPIC-0412 inhibits DNA damage response in GBM cells through the p21-E2F1 DNA damage repair axis. EPIC-0412 epigenetically silences MGMT through its interaction with the ATF3-p-p65-HADC1 axis at the MGMT promoter region. The application of EPIC-0412 restored the TMZ sensitivity in GBM in vivo experiments. This study discovered a small-molecule inhibitor EPIC-0412, which enhanced the chemotherapeutic effect of TMZ by acting on the p21-E2F1 DNA damage repair axis and ATF3-p-p65-MGMT axis, providing evidence for combining epigenetic drugs to increase the sensitization toward TMZ in GBM patients.

Evaluation of Methylation Panel in the Promoter Region of p16INK4a , RASSF1A, and MGMT as a Biomarker in Sputum for Lung Cancer.

Lung cancer is the most common cause of cancer death in the world. Effective early detection and appropriate medications can help treat this deadly cancer. Therefore, early detection of lung cancer is of utmost importance, especially in screening high-risk populations (such as smokers) with an urgent need to identify new biomarkers. The present study aimed to demonstrate the potential of using the panel of DNA methylation as a biomarker for the early diagnosis of lung cancer from sputum samples. The methylated promoter of p16INK4a , RASSF1A, and MGMT genes was estimated by the methylation-specific polymerase chain reaction in a sample of 84 lung cancer patients (65 smokers and 19 non-smokers). Based on the results, p16INK4a promoter methylation was significantly associated with smoking habit and lung cancer progression in terms of histological grading and patient staging. The sensitivity and specificity of the p16INK4a gene as a biomarker for lung cancer were 71% and 90%, respectively. The methylated promoter of RASSF1A was less sensitive (48%) as a biomarker for lung cancer with 83%. The results demonstrated a strong association between promoter methylation of RASSF1A and late stages of lung cancer (P=0.0007). The sensitivity of the MGMT gene as a biomarker for lung cancer was 61% with high specificity (92%), compared to other candidate genes in this study. The epigenetic alteration in the promoter region of p16INK4a , RASSF1A, and MGMT genes is highly associated with cancer cell development. It is suggested that the use of these candidate biomarkers can be used as an adjunct to computed tomography screening to diagnose patients at high risk for lung cancer after validation.

Combining MGMT promoter pyrosequencing and protein expression to optimize prognosis stratification in glioblastoma.

Pyrosequencing (PSQ) represents the golden standard for MGMT promoter status determination. Binary interpretation of results based on the threshold from the average of several CpGs tested would neglect the existence of the “gray zone”. How to define the gray zone and reclassify patients in this subgroup remains to be elucidated. A consecutive cohort of 312 primary glioblastoma patients were enrolled. CpGs 74‐81 in the promoter region of MGMT were tested by PSQ and the protein expression was assessed by immunohistochemistry (IHC). Receiver operating characteristic curves were constructed to calculate the area under the curves (AUC). Kaplan‐Meier plots were used to estimate the survival rate of patients compared by the log‐rank test. The optimal threshold of each individual CpG differed from 5% to 11%. Patients could be separated into the hypomethylated subgroup (all CpGs tested below the corresponding optimal thresholds, n = 126, 40.4%), hypermethylated subgroup (all CpGs tested above the corresponding optimal thresholds, n = 108, 34.6%), and the gray zone subgroup (remaining patients, n = 78, 25.0%). Patients in the gray zone harbored an intermediate prognosis. The IHC score instead of the average methylation levels could successfully predict the prognosis for the gray zone (AUC for overall survival, 0.653 and 0.519, respectively). Combining PSQ and IHC significantly improved the efficiency of survival prediction (AUC: 0.662, 0.648, and 0.720 for PSQ, IHC, and combined, respectively). Immunohistochemistry is a robust method to predict prognosis for patients in the gray zone defined by PSQ. Combining PSQ and IHC could significantly improve the predictive ability for clinical outcomes.

The number of methylated CpG sites within the MGMT promoter region linearly correlates with outcome in glioblastoma receiving alkylating agents

MGMT-promoter methylation is associated with favorable outcome in glioblastoma. The aim of this study was to determine whether the absolute number of methylated Cytosine-Guanine-dinucleotide-(CpG-)sites within the DMR-2 island of the MGMT-promoter may correlate with outcome in a qualitative or quantitative fashion. In a cohort of newly diagnosed glioblastoma patients treated with stereotactic biopsy or open tumor resection plus concomitant chemoradiotherapy, we assessed MGMT-promoter methylation by methylation-specific polymerase-chain-reaction (MSP). Methylation of the CpG-sites 74–98 within the MGMT-promoter region was additionally analysed by Sanger sequencing, and the total number of methylated CpG-sites was correlated with outcome using proportional hazards models. 215 patients with glioblastoma were identified and stratified per MSP (positive: 53%, negative: 47%). Among MSP-positive tumors, hierarchical clustering identified three subgroups with different methylation rates (median: 80% vs. 52% vs. 47%), indicating a site-dependent methylation propagation. The methylation status of a given CpG-site indicated a neighborhood-dependent methylation propagation. Survival was linearly associated with the cumulative number of methylated CpG-sites. This was particularly true in patients who received at least one adjuvant cycle of temozolomide. Notably, all CpG-sites analyzed contributed similarly to effect size; this enabled a further predictive substratification of MSP-positive tumors with median OS ranging from as low as 17.1 months (< 18 methylated CpG-sites) to as high as 26.2 months (≥ 18 methylated CpG-sites) in the overall cohort. All in all, total number of methylated CpG-sites may correlate with outcome in a linear fashion. Such analysis may therefore add further predictive value to conventional methods of determining the MGMT-promoter status.

New molecular prognostic factors of adult diffuse lower-grade gliomas in post-2016 molecular era: a retrospective analysis from single center

Background and objective Several studies have examined the prognostic significance of IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation in lower-grade gliomas but most of these used the 2007 fourth edition of the WHO classification. We evaluate prognostic significance of these indicators in the 2016 WHO updated fourth edition of CNS tumor classification. Methods A total of 180 intracranial glioma patients diagnosed according WHO 2016 edition between December 2016 and December 2018 Jinling Hospital (Nanjing, China) were reviewed retrospectively. We performed survival analysis on 109 patients with complete molecular pathology and follow-up data. Results Histologically, 52 were diagnosed as astrocytoma (WHO grade II and III), 17 as oligodendrogliomas (WHO grade II and III) and 40 as GBMs. At last follow-up, 50.5% patients had experienced tumor progression and 34.9% had died. Among grade II and III cases 36.2% experienced tumor progression and 27.5% died. In univariate Kaplan–Meier analysis, multifocal tumor, EGFR mutation or amplification, PIK3CA mutation and IDHwt/TERTpwt group were associated with shorter PFS (p < 0.001, p = 0.003, p = 0.005, p < 0.001, respectively) and OS (p = 0.010, p = 0.020, p = 0.018, p < 0.001, respectively) as were older age (≥55 years), multifocal tumor, IDH1/2 wild type, 1p/19q non-codeletion and negative methylation in the MGMT promoter region. A Cox proportional hazards model was created demonstrating that single tumor (HR = 0.180, p = 0.04), MGMTp methylation (HR = 0.095, p = 0.003) and chemoradiotherapy (HR = 0.006, p = 0.002) were independent prognostic factors for OS. Conclusions Beyond histological classification as well as IDH1/2 mutation, 1p/19q codeletion status, we could incorporate IDH1/2mt combined with TERTpmt, EGFR mutation or amplification and PIK3CA mutation into the diagnostic criteria for DLGGs to supplement WHO 2016 pathological criteria.

TBIO-21. Lnc-TALC PROMOTES O6-METHYLGUANINE-DNA METHYLTRANSFERASE EXPRESSION VIA REGULATING THE C-MET PATHWAY BY COMPETITIVELY BINDING WITH miR-20b-3p

Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.

The distribution of isocitrate dehydrogenase mutations, O6-methylguanine-DNA methyltransferase promoter methylation, and 1p/19q codeletion in different glioma subtypes and their correlation with glioma prognosis in Taiwanese population: A single center study

The molecular characteristics of glioma patients, such as 1p/19q codeletion, MGMT promoter region methylation, and IDH1/IDH2 mutations, carry important information for tumor classification, biological behavior, and the prediction of patient survival. We sought to address the clinical characteristics of brain tumor patients in the Taiwanese population. Our aim was to compare the H&E-based classification and the 2016 WHO classification in glioma patients. 192 patients were recruited in this study including glioblastomas (GBMs) and lower-grade gliomas (LGGs). We performed assays to determine the statuses of 1p/19q codeletion, MGMT promoter methylation, and IDH mutations in tumors and assessed their associations with survival time. We also compared the difference between H&E and WHO 2016 classification. Using the H&E classification, the overall survival time (OST) was associated with IDH status in astrocytoma and oligoastrocytoma patients but not in oligodendroglioma and GBM patients. However, OST showed no significant correlation with MGMT promoter methylation in all the groups. Furthermore, no significant association was observed between 1p/19q codeletion and OST in GBM patients. Using the WHO 2016 classification, our results indicated that astrocytoma and oligodendroglioma patients with mutant IDH showed a significantly prolonged OST than patients with wild-type IDH. However, only oligodendroglioma patients with MGMT hypermethylation demonstrated statistically significant difference. Prognostic factors, including the age at diagnosis, IDH mutations, MGMT promoter methylation, and 1p/19q codeletion, could independently predict patient survival. Based on the WHO 2016 classification of glioma, the new histological groups provide a better and objective method to categorize glioma patients and predict patient survival.

Correlation of the quantitative level of MGMT promoter methylation and overall survival in primary diagnosed glioblastomas using the quantitative MethyQESD method

AimsO(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation is a high predictive factor for therapy results of temozolomide in patients with glioma. The objective of this work was to analyse the impact of MGMT...

TMIC-14. PD-L1 EXPRESSION IS NEGATIVELY CORRELATED TO OUTCOMES IN PATIENTS WITH MGMT METHYLATED PROMOTERS IN GBM

Abstract BACKGROUND Glioblastoma is a high-grade glioma and is considered the most aggressive primary brain tumor with a median overall survival of 11–15 months. Methylation of the promoter of the MGMT gene, which encodes a DNA repair protein, has been shown to be a good prognostic indicator for GBM survival. It is known that GBM has elevated expression of several immune checkpoint molecules that allow the tumor to evade anti-tumor immune response. Therefore, it is important to elucidate connections between prognostic factors like methylation status and immunologic markers in the tumor microenvironment (TME) that might lead to immunosuppression. METHODS Patients with resected GBM tumor samples were identified by pathology reports as having tumors with either methylated or unmethylated MGMT promoters. Corresponding patient tumor biopsy samples were embedded in paraffin, sectioned, and then stained using specific antibodies for known immune checkpoint molecules: PD-1, PD-L1, LAG-3, TIM-3, CSF1R, and IDO-1. RESULTS In our cohort of 34 patients, 17 had methylated MGMT promoter regions and 17 did not. We found no statistically significant difference between methylated and unmethylated MGMT promoter status for immune checkpoint expression. However, we did find a statistically significant negative correlation (P&lt; 0.05) between the degree of PD-L1 expression in the TME and the progression free survival of patients with the MGMT promoter methylation phenotype. CONCLUSION Our study examined the relationship between immune checkpoint markers and methylation status of MGMT promoters in GBM. While there was no statistically significant difference between these two groups within the immunological framework of this study, there was a statistically significant negative correlation showing that increased PD-L1 expression in patients with methylated MGMT promoter tumors correlated to a worse progression free survival. Future studies are indicated to investigate the correlation between PD-L1 expression in relation to GBM treatment prognosis and its dependence on MGMT promoter methylation.

MGMT-Deficiency Is a Biomarker to Guide Treatment of Solid Tumors with Temozolomide and ATR Inhibitors

Temozolomide (TMZ) is an alkylating agent that methylates guanine bases at the O6 position (O6meG). O6meG-DNA methyltransferase (MGMT) is a suicide enzyme that repairs O6meG lesions by direct reversal. The promoter region of MGMT is methylated in various types of solid tumors, including glioblastoma (GBM), colorectal cancer, non-small and small-cell lung cancer, gastric cancer, and pancreatic cancer. Numerous previous studies have demonstrated that TMZ is more effective in treating tumors deficient in MGMT. However, clinical use of TMZ has been largely limited to GBM. Thus, the goal of this study was to determine what drugs are synergistic with TMZ as a novel strategy to increase the efficacy of TMZ for a wider array of tumor types. We assessed in vitro and in vivo synergistic interactions with a variety of DNA repair inhibitors and alkylating agents, using a collection MGMT-proficient and -deficient cell line models across various tumor types. We found that TMZ, unlike other alkylators tested, specifically induces growth delay, DNA damage, and G2/M-phase cell cycle arrest in MGMT-deficient cells, but not in MGMT-proficient cells. Because the protein ATR activates the G2/M checkpoint in response to DNA damage, we hypothesized and verified that TMZ activates ATR more specifically in MGMT-deficient cells. Next, we hypothesized that ATR inhibitors (ATRi's) would increase sensitivity to TMZ. In vitro, VX-970, an ATRi currently in clinical trials, increased sensitivity to TMZ by over 1000-fold in MGMT-deficient cells, but not in MGMT-proficient cells. Depletion of MGMT with a competitive inhibitor (O6-benzylguanine) conferred exquisite sensitivity to combined therapy with ATRi and TMZ in multiple MGMT-proficient cell lines, including U2OS and HeLa. In vivo, we demonstrated that combined treatment with TMZ and ATRi inhibited tumor growth in comparison to either agent alone, in both MGMT-deficient LN229 cells (GBM) and an MGMT-deficient, GBM PDX model. ATRi’s also appear to have promising activity as radiosensitizers, which prompted us to test whether we could detect their effects in combination with ionizing radiation (IR) and TMZ, specifically in MGMT-deficient cells. Indeed, we were able detect significant efficacy with this triple combination in our cell line models using a range of IR doses. Our data suggest that MGMT status can be utilized as a critical biomarker to select tumors for treatment with ATRi-based combinations. As TMZ and RT currently are standard of care for GBM, and nearly 50% of tumors harbor MGMT-deficiency, these results form the basis for clinical trials testing the integration of ATRi’s into novel chemoradiation strategies in the future. In particular, we propose that the combination of ATRi’s with RT, during both the concurrent and adjuvant TMZ phases of treatment, will have great potential. Additional studies are currently underway in our laboratory to further validate the efficacy of this “triple strategy” in GBM.

Lnc-TALC promotes O6-methylguanine-DNA methyltransferase expression via regulating the c-Met pathway by competitively binding with miR-20b-3p

Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ-resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.