Adoptive cell therapy for high grade gliomas using simultaneous temozolomide and intracranial mgmt-modified γδ t cells following standard post-resection chemotherapy and radiotherapy: current strategy and future directions.

Cellular therapies, including chimeric antigen receptor T cell therapies (CAR-T), while generally successful in hematologic malignancies, face substantial challenges against solid tumors such as glioblastoma (GBM) due to rapid growth, antigen heterogeneity, and inadequate depth of response to cytoreductive and immune therapies, We have previously shown that GBM constitutively express stress associated NKG2D ligands (NKG2DL) recognized by gamma delta (γδ) T cells, a minor lymphocyte subset that innately recognize target molecules via the γδ T cell receptor (TCR), NKG2D, and multiple other mechanisms. Given that NKG2DL expression is often insufficient on GBM cells to elicit a meaningful response to γδ T cell immunotherapy, we then demonstrated that NKG2DL expression can be transiently upregulated by activation of the DNA damage response (DDR) pathway using alkylating agents such as Temozolomide (TMZ). TMZ, however, is also toxic to γδ T cells. Using a p140K/MGMT lentivector, which confers resistance to TMZ by expression of O(6)-methylguanine-DNA-methyltransferase (MGMT), we genetically engineered γδ T cells that maintain full effector function in the presence of therapeutic doses of TMZ. We then validated a therapeutic system that we termed Drug Resistance Immunotherapy (DRI) that combines a standard regimen of TMZ concomitantly with simultaneous intracranial infusion of TMZ-resistant γδ T cells in a first-in-human Phase I clinical trial (NCT04165941). This manuscript will discuss DRI as a rational therapeutic approach to newly diagnosed GBM and the importance of repeated administration of DRI in combination with the standard-of-care Stupp regimen in patients with stable minimal residual disease.

A modified nucleoside O6-methyl-2'-deoxyguanosine-5'-triphosphate exhibits anti-glioblastoma activity in a caspase-independent manner.

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2'-deoxyguanosine-5'-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

OBJECTIVE: Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. METHODS: CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). RESULTS: Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. CONCLUSIONS: Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

Protocatechuic aldehyde acts synergistically with dacarbazine to augment DNA double-strand breaks and promote apoptosis in cutaneous melanoma cells.

BACKGROUND: Despite rapid developments in immunotherapy and targeted therapy, dacarbazine (DTIC)-based chemotherapy still has been placed at the first-line for advanced melanoma patients who are after failure of immunotherapy or targeted therapy. However, the limited response rate and survival benefit challenge the DTIC-based chemotherapy for advanced melanoma patients. METHODS: Two melanoma cell lines, A375 and SK-MEL-28 were cultured with PA and DTIC over a range of concentrations for 72 h and the cell viabilities were detected by CCK8 assay. The Bliss model and ZIP model were used for calculating the synergistic effect of PA and DTIC. DNA double-strand breaks in the two cell lines were examined by the Comet assay, and cell apoptosis was analyzed by flow cytometry. The short hairpin RNA (shRNA)-mediated knockdown, Real-time polymerase chain reaction (RT-PCR) and Western blot were performed for molecular analysis. RESULTS: In the present study, we report that Protocatechuic aldehyde (PA) synergistically enhances the cytotoxicity of DTIC to two melanoma cell lines, A375 and SK-MEL-28. The combination of PA and DTIC augments DNA double-strand breaks and increases cell apoptosis. Further mechanism study reveals that PA destabilizes MGMT protein (O-6-Methylguanine-DNA Methyltransferase) through the ubiquitin-proteasome process and directly repairs DTIC-induced genetic lesions. Knockdown of MGMT compromises the synergistic effect between PA and DTIC. CONCLUSION: Our study demonstrates that the bioactive compound, Protocatechuic aldehyde, synergistically promotes the cytotoxicity of DTIC to melanoma cells through destabilization of MGMT protein. It could be a potential candidate for melanoma chemotherapy.

Brain Tumor Radiogenomic Classification of O6-Methylguanine-DNA Methyltransferase Promoter Methylation in Malignant Gliomas-Based Transfer Learning.

Artificial Intelligence (AI) is the subject of a challenge and attention in the field of oncology and raises many promises for preventive diagnosis, but also fears, some of which are based on highly speculative visions for the classification and detection of tumors. A brain tumor that is malignant is a life-threatening disorder. Glioblastoma is the most prevalent kind of adult brain cancer and the 1 with the poorest prognosis, with a median survival time of less than a year. The presence of O6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation, a particular genetic sequence seen in tumors, has been proven to be a positive prognostic indicator and a significant predictor of recurrence.This strong revival of interest in AI is modeled in particular to major technological advances which have significantly increased the performance of the predicted model for medical decision support. Establishing reliable forecasts remains a significant challenge for electronic health records (EHRs). By enhancing clinical practice, precision medicine promises to improve healthcare delivery. The goal is to produce improved prognosis, diagnosis, and therapy through evidence-based sub stratification of patients, transforming established clinical pathways to optimize care for each patient's individual requirements. The abundance of today's healthcare data, dubbed "big data," provides great resources for new knowledge discovery, potentially advancing precision treatment. The latter necessitates multidisciplinary initiatives that will use the knowledge, skills, and medical data of newly established organizations with diverse backgrounds and expertise.The aim of this paper is to use magnetic resonance imaging (MRI) images to train and evaluate your model to detect the presence of MGMT promoter methylation in this competition to predict the genetic subtype of glioblastoma based transfer learning. Our objective is to emphasize the basic problems in the developing disciplines of radiomics and radiogenomics, as well as to illustrate the computational challenges from the perspective of big data analytics.

Temozolomide Resistance: A Multifarious Review on Mechanisms Beyond O-6-Methylguanine-DNA Methyltransferase.

BACKGROUND: Chemotherapy with the oral alkylating agent temozolomide still prevails as a linchpin in the therapeutic regimen of glioblastoma alongside radiotherapy. Because of the impoverished prognosis and sparse chemotherapeutic medicaments associated with glioblastoma, the burgeoning resistance to temozolomide has made the whole condition almost irremediable. OBJECTIVE: The present review highlights the possible mechanisms of drug resistance following chemotherapy with temozolomide. METHODS: The review summarizes the recent developments, as published in articles from Scopus, PubMed, and Web of Science search engines. DESCRIPTION: One of the prime resistance mediators, O-6-methylguanine-DNA methyltransferase, upon activation, removes temozolomide-induced methyl adducts bound to DNA and reinstates genomic integrity. In the bargain, neoteric advances in the conception of temozolomide resistance have opened the door to explore several potential mediators like indirect DNA repair systems, efflux mechanisms, epigenetic modulation, microenvironmental influences, and autophagy-apoptosis processes that constantly lead to the failure of chemotherapy. CONCLUSION: This review sheds light on recent discoveries, proposed theories, and clinical developments in the field of temozolomide resistance to summarize the complex and intriguing involvement of oncobiological pathways.

BET protein inhibition sensitizes glioblastoma cells to temozolomide treatment by attenuating MGMT expression.

Bromodomain and extra-terminal tail (BET) proteins have been identified as potential epigenetic targets in cancer, including glioblastoma. These epigenetic modifiers link the histone code to gene transcription that can be disrupted with small molecule BET inhibitors (BETi). With the aim of developing rational combination treatments for glioblastoma, we analyzed BETi-induced differential gene expression in glioblastoma derived-spheres, and identified 6 distinct response patterns. To uncover emerging actionable vulnerabilities that can be targeted with a second drug, we extracted the 169 significantly disturbed DNA Damage Response genes and inspected their response pattern. The most prominent candidate with consistent downregulation, was the O-6-methylguanine-DNA methyltransferase (MGMT) gene, a known resistance factor for alkylating agent therapy in glioblastoma. BETi not only reduced MGMT expression in GBM cells, but also inhibited its induction, typically observed upon temozolomide treatment. To determine the potential clinical relevance, we evaluated the specificity of the effect on MGMT expression and MGMT mediated treatment resistance to temozolomide. BETi-mediated attenuation of MGMT expression was associated with reduction of BRD4- and Pol II-binding at the MGMT promoter. On the functional level, we demonstrated that ectopic expression of MGMT under an unrelated promoter was not affected by BETi, while under the same conditions, pharmacologic inhibition of MGMT restored the sensitivity to temozolomide, reflected in an increased level of γ-H2AX, a proxy for DNA double-strand breaks. Importantly, expression of MSH6 and MSH2, which are required for sensitivity to unrepaired O6-methylguanine-lesions, was only briefly affected by BETi. Taken together, the addition of BET-inhibitors to the current standard of care, comprising temozolomide treatment, may sensitize the 50% of patients whose glioblastoma exert an unmethylated MGMT promoter.

Drug delivery in glioblastoma therapy: a review on nanoparticles targeting MGMT-mediated resistance.

INTRODUCTION: Glioblastoma multiforme (GBM) is the deadliest type of brain cancer with poor response to the available therapies, mainly due to intrinsic resistance mechanisms. Chemotherapy is based on alkylating agents, but DNA-repair mechanisms can revert this cytotoxic effect. O6-methylguanine-DNA methyltransferase (MGMT) protein is the primary mechanism for GBM resistance. Therefore, different strategies to suppress its activity have been explored. However, their clinical use has been hindered due to the high toxicity of MGMT inhibitors verified in clinical trials. AREAS COVERED: This review article aims to provide the current progress in the development of novel drug delivery systems (DDS) to overcome this resistance. Here, we also review the current knowledge on MGMT-mediated resistance and the clinical outcomes and potential risks of using MGMT inhibitors. EXPERT OPINION: To overcome therapeutic limitations, nano-based approaches have been proposed as a suitable solution to improve drug accumulation in the brain tumor tissue and decrease systemic toxicity. DDS to overcome MGMT-mediated resistance in GBM have been mostly developed to deliver MGMT inhibitors and for gene therapy to modulate MGMT gene expression.

Monocentric evaluation of Ki-67 labeling index in combination with a modified RPA score as a prognostic factor for survival in IDH-wildtype glioblastoma patients treated with radiochemotherapy.

PURPOSE: The prognosis for glioblastoma patients remains dismal despite intensive research on better treatment options. Molecular and immunohistochemical markers are increasingly being investigated as understanding of their role in disease progression grows. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been shown to have prognostic and therapeutic relevance for glioblastoma patients. Other markers implicated in tumor formation and/or malignancy are p53, Alpha thalassemia/mental retardation syndrome X-linked (ATRX), Epidermal Growth Factor Receptor splice variant III (EGFRvIII), and Ki-67, with loss of nuclear ATRX expression and lower Ki-67 index being associated with prolonged survival. For p53 and EGFRvIII the data are contradictory. Our aim was to investigate the markers mentioned above regarding progression-free (PFS) and overall survival (OS) to evaluate their viability as independent prognostic markers for our patient collective. METHODS: In this retrospective study, we collected data on patients undergoing radiotherapy due to isocitrate dehydrogenase (IDH) wildtype glioblastoma at a single university hospital between 2014 and 2020. RESULTS: Our findings confirm Ki-67 labeling index ≤ 20% as an independent prognostic factor for prolonged PFS as well as MGMT promoter methylation for both prolonged PFS and OS, in consideration of age and Eastern Cooperative Oncology Group (ECOG) status, chemotherapy treatment, and total radiation dose for PFS as well as additionally sex, resection status, and receipt of treatment for progression or recurrence for OS. Additionally, Ki-67 labeling index ≤ 20% showed a significant correlation with prolonged OS in univariate analysis. Modification of the recursive partitioning analysis (RPA) score to include Ki-67 labeling index resulted in a classification with the possible ability to distinguish long-term-survivors from patients with unfavorable prognosis. CONCLUSION: MGMT promoter methylation and Ki-67 labeling index were independent predictors of survival in our collective. We see further studies pooling patient collectives to reach larger patient numbers concerning Ki-67 labeling index as being warranted.

Temozolomide Followed by Combination With Low-Dose Ipilimumab and Nivolumab in Patients With Microsatellite-Stable, O6-Methylguanine-DNA Methyltransferase-Silenced Metastatic Colorectal Cancer: The MAYA Trial.

PURPOSE: This is a multicenter, single-arm phase II trial evaluating the efficacy and safety of an immune-sensitizing strategy with temozolomide priming followed by a combination of low-dose ipilimumab and nivolumab in patients with microsatellite-stable (MSS) and O6-methylguanine-DNA methyltransferase (MGMT)-silenced metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Patients with pretreated mCRC were centrally prescreened for MSS status and MGMT silencing (ie, lack of MGMT expression by immunohistochemistry plus MGMT methylation by pyrosequencing). Eligible patients received two priming cycles of oral temozolomide 150 mg/sqm once daily, days 1-5, once every 4 weeks (first treatment part) followed, in absence of progression, by its combination with ipilimumab 1 mg/kg once every 8 weeks and nivolumab 480 mg once every 4 weeks (second treatment part). The primary end point was the 8-month progression-free survival (PFS) rate calculated from enrollment in patients who started the second treatment part, with ≥ 4 out of 27 subjects progression-free by the 8-month time point as decision rule. RESULTS: Among 716 prescreened patients, 204 (29%) were molecularly eligible and 135 started the first treatment part. Among these, 102 (76%) were discontinued because of death or disease progression on temozolomide priming, whereas 33 patients (24%) who achieved disease control started the second treatment part and represented the final study population. After a median follow-up of 23.1 months (interquartile range, 14.9-24.6 months), 8-month PFS rate was 36%. Median PFS and overall survival were 7.0 and 18.4 months, respectively, and overall response rate was 45%. Grade 3-4 immune-related adverse events were skin rash (6%), colitis (3%), and hypophysitis (3%). No unexpected adverse events or treatment-related deaths were reported. CONCLUSION: The MAYA study provided proof-of-concept that a sequence of temozolomide priming followed by a combination of low-dose ipilimumab and nivolumab may induce durable clinical benefit in MSS and MGMT-silenced mCRC.

Dramatic clinical response in the treatment of small cell glioblastoma multiforme.

WHAT IS KNOWN AND OBJECTIVE: Small cell glioblastoma (scGBM) is a rare subtype of primary glioblastoma, which typically behave more aggressively compared with classical glioblastoma (GBMs). They are generally associated with poor responses to treatment, and optimal treatment is not known. CASE SUMMARY: We present the case of a 51-year-old woman with scGBM with O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation, demonstrating an unexpected dramatic clinical response to chemoradiotherapy. WHAT IS NEW AND CONCLUSION: This case highlights that treatment with temozolomide-based chemoradiotherapy is justified in patients with scGBM, despite their poor prognosis. MGMT methylation may be associated with clinical responses.

The Role of Delayed Radiotherapy Initiation in Patients with Newly Diagnosed Glioblastoma with Residual Tumor Mass.

OBJECTIVE: Treatment for newly diagnosed isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) includes maximum safe resection, followed by adjuvant radio(chemo)therapy (RCx) with temozolomide. There is evidence that it is safe for GBM patients to prolong time to irradiation over 4 weeks after surgery. This study aimed at evaluating whether this applies to GBM patients with different levels of residual tumor volume (RV). METHODS: Medical records of all patients with newly diagnosed GBM at our department between 2014 and 2018 were reviewed. Patients who received adjuvant radio (chemo) therapy, aged older than 18 years, and with adequate perioperative imaging were included. Initial and residual tumor volumes were determined. Time to irradiation was dichotomized into two groups (≤28 and >28 days). Univariate analysis with Kaplan-Meier estimate and log-rank test was performed. Survival prediction and multivariate analysis were performed employing Cox proportional hazard regression. RESULTS: One hundred and twelve patients were included. Adjuvant treatment regimen, extent of resection, residual tumor volume, and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation were statistically significant factors for overall survival (OS). Time to irradiation had no impact on progression-free survival (p = 0.946) or OS (p = 0.757). When stratified for different thresholds of residual tumor volume, survival predication via Cox regression favored time to irradiation below 28 days for patients with residual tumor volume above 2 mL, but statistical significance was not reached. CONCLUSION: Time to irradiation had no significant influence on OS of the entire cohort. Nevertheless, a statistically nonsignificant survival prolongation could be observed in patients with residual tumor volume > 2 mL when admitted to radiotherapy within 28 days after surgery.

Joint application of biochemical markers and imaging techniques in the accurate and early detection of glioblastoma.

Glioblastoma is a primary brain tumor with the most metastatic effect in adults. Despite the wide range of multidimensional treatments, tumor heterogeneity is one of the main causes of tumor spread and gives great complexity to diagnostic and therapeutic methods. Therefore, featuring noble noninvasive prognostic methods that are focused on glioblastoma heterogeneity is perceived as an urgent need. Imaging neuro-oncological biomarkers including MGMT (O6-methylguanine-DNA methyltransferase) promoter methylation status, tumor grade along with other tumor characteristics and demographic features (e.g., age) are commonly referred to during diagnostic, therapeutic and prognostic processes. Therefore, the use of new noninvasive prognostic methods focused on glioblastoma heterogeneity is considered an urgent need. Some neuronal biomarkers, including the promoter methylation status of the promoter MGMT, the characteristics and grade of the tumor, along with the patient's demographics (such as age and sex) are involved in diagnosis, treatment, and prognosis. Among the wide array of imaging techniques, magnetic resonance imaging combined with the more physiologically detailed technique of H-magnetic resonance spectroscopy can be useful in diagnosing neurological cancer patients. In addition, intracranial tumor qualitative analysis and sometimes tumor biopsies help in accurate diagnosis. This review summarizes the evidence for biochemical biomarkers being a reliable biomarker in the early detection and disease management in GBM. Moreover, we highlight the correlation between Imaging techniques and biochemical biomarkers and ask whether they can be combined.

The influence of methylation status on a stochastic model of MGMT dynamics in glioblastoma: Phenotypic selection can occur with and without a downshift in promoter methylation status.

Glioblastoma originates in the brain and is one of the most aggressive cancer types. Glioblastoma represents 15% of all brain tumours, with a median survival of 15 months. Although the current standard of care for such a tumour (the Stupp protocol) has shown positive results for the prognosis of patients, O-6-methylguanine-DNA methyltransferase (MGMT) driven drug resistance has been an issue of increasing concern and hence requires innovative approaches. In addition to the well established drug resistance factors such as tumour location and blood brain barriers, it is also important to understand how the genetic and epigenetic dynamics of the glioblastoma cells can play a role. One important aspect of this is the study of methylation status of MGMT following administration of temozolomide. In this paper, we extend our previously published model that simulated MGMT expression in glioblastoma cells to incorporate the promoter methylation status of MGMT. This methylation status has clinical significance and is used as a marker for patient outcomes. Using this model, we investigate the causative relationship between temozolomide treatment and the methylation status of the MGMT promoter in a population of cells. In addition by constraining the model to relevant biological data using Approximate Bayesian Computation, we were able to identify parameter regimes that yield different possible modes of resistances, namely, phenotypic selection of MGMT, a downshift in the methylation status of the MGMT promoter or both simultaneously. We analysed each of the parameter sets associated with the different modes of resistance, presenting representative solutions as well as discovering some similarities between them as well as unique requirements for each of them. Finally, we used them to devise optimal strategies for inhibiting MGMT expression with the aim of minimising live glioblastoma cell numbers.

O6-Methylguanine-DNA methyltransferase (MGMT): A drugable target in lung cancer?

This manuscript addresses the role of O6-methylguanine-DNA methyltransferase (MGMT) as a biomarker in the oncogenesis of cancer and the opportunity of turning this gene into a drugable target in neuroendocrine tumours of the lung. Studies in brain tumours conclude that MGMT promoter methylation is considered a strong predictive factor for a favourable outcome for treatment with temozolomide, e.g. alkylating agent. We conducted a systematic review of MGMT in non-small cell lung cancer (NSCLC), small-cell lung cancer (SCLC) and other pulmonary neuroendocrine tumours (NETs) to evaluate whether MGMT is a prognostic and/or predictive factor to select patients with lung cancer who can benefit from treatment with temozolomide. In NSCLC MGMT promoter methylation is not a prognostic and predictive factor, hence temozolomide has no place. In SCLC and NET patients with a MGMT promoter methylation benefit of temozolomide has to be confirmed.Temozolomide can be considered a 'personalized' treatment if the predictive role of MGMT is further confirmed.