Methionine restriction of glioma does not induce MGMT and greatly improves temozolomide efficacy in an orthotopic nude-mouse model: A potential curable approach to a clinically-incurable disease.

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. METHODS: We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. RESULTS: MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. CONCLUSION: These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

MGMT promoter methylation status prediction using MRI scans? An extensive experimental evaluation of deep learning models.

The number of studies on deep learning for medical diagnosis is expanding, and these systems are often claimed to outperform clinicians. However, only a few systems have shown medical efficacy. From this perspective, we examine a wide range of deep learning algorithms for the assessment of glioblastoma - a common brain tumor in older adults that is lethal. Surgery, chemotherapy, and radiation are the standard treatments for glioblastoma patients. The methylation status of the MGMT promoter, a specific genetic sequence found in the tumor, affects chemotherapy's effectiveness. MGMT promoter methylation improves chemotherapy response and survival in several cancers. MGMT promoter methylation is determined by a tumor tissue biopsy, which is then genetically tested. This lengthy and invasive procedure increases the risk of infection and other complications. Thus, researchers have used deep learning models to examine the tumor from brain MRI scans to determine the MGMT promoter's methylation state. We employ deep learning models and one of the largest public MRI datasets of 585 participants to predict the methylation status of the MGMT promoter in glioblastoma tumors using MRI scans. We test these models using Grad-CAM, occlusion sensitivity, feature visualizations, and training loss landscapes. Our results show no correlation between these two, indicating that external cohort data should be used to verify these models' performance to assure the accuracy and reliability of deep learning systems in cancer diagnosis.

Modulating MGMT expression through interfering with cell signaling pathways.

Guanine O6-alkylating agents are widely used as first-line chemotherapeutic drugs due to their ability to induce cytotoxic DNA damage. However, a major hurdle in their effectiveness is the emergence of chemoresistance, largely attributed to the DNA repair pathway mediated by O6-methylguanine-DNA methyltransferase (MGMT). MGMT plays an important role in removing the alkyl groups from lethal O6-alkylguanine (O6-AlkylG) adducts formed by chemotherapeutic alkylating agents. By doing so, MGMT enables tumor cells to evade apoptosis and develop drug resistance toward DNA alkylating agents. Although covalent inhibitors of MGMT, such as O6-benzylguanine (O6-BG) and O6-(4-bromothenyl)guanine (O6-4-BTG or lomeguatrib), have been explored in clinical settings, their utility is limited due to severe delayed hematological toxicity observed in most patients when combined with alkylating agents. Therefore, there is an urgent need to identify new targets and unravel the underlying molecular mechanisms and to develop alternative therapeutic strategies that can overcome MGMT-mediated tumor resistance. In this context, the regulation of MGMT expression via interfering the specific cell signaling pathways (e.g., Wnt/ß-catenin, NF-κB, Hedgehog, PI3K/AKT/mTOR, JAK/STAT) emerges as a promising strategy for overcoming tumor resistance, and ultimately enhancing the efficacy of DNA alkylating agents in chemotherapy.

Magnolol and Temozolomide exhibit a synergistic anti-glioma activity through MGMT inhibition.

Temozolomide (TMZ) is the leading chemotherapeutic agent used for glioma therapy due to its good oral absorption and blood-brain barrier permeability. However, its anti-glioma efficacy may be limited due to its adverse effects and resistance development. O6-Methylguanine-DNA-methyltransferase (MGMT), an enzyme associated with TMZ resistance, is activated via the NF-κB pathway, which is found to be upregulated in glioma. TMZ also upregulates NF-κB signaling like many other alkylating agents. Magnolol (MGN), a natural anti-cancer agent, has been reported to inhibit NF-κB signaling in multiple myeloma, cholangiocarcinoma, and hepatocellular carcinoma. MGN has already shown promising results in anti-glioma therapy. However, the synergistic action of TMZ and MGN has not been explored. Therefore, we investigated the effect of TMZ and MGN treatment in glioma and observed their synergistic pro-apoptotic action in both in vitro and in vivo glioma models. To explore the mechanism of this synergistic action, we found that MGN inhibits MGMT enzyme both in vitro and in vivo glioma. Next, we established the link between NF-κB signaling and MGN-induced MGMT inhibition in glioma. MGN inhibits the phosphorylation of p65, a subunit of NF-κB, and its nuclear translocation to block NF-κB pathway activation in glioma. MGN-induced NF-κB inhibition results in the transcriptional inhibition of MGMT in glioma. TMZ and MGN combinatorial treatment also impedes p65 nuclear translocation to inhibit MGMT in glioma. We observed a similar effect of TMZ and MGN treatment in the rodent glioma model. Thus, we concluded that MGN potentiates TMZ-induced apoptosis in glioma by inhibiting NF-κB pathway-mediated MGMT activation.

A Comprehensive Review of miRNAs and Their Epigenetic Effects in Glioblastoma.

Glioblastoma is the most aggressive form of brain tumor originating from glial cells with a maximum life expectancy of 14.6 months. Despite the establishment of multiple promising therapies, the clinical outcome of glioblastoma patients is abysmal. Drug resistance has been identified as a major factor contributing to the failure of current multimodal therapy. Epigenetic modification, especially DNA methylation has been identified as a major regulatory mechanism behind glioblastoma progression. In addition, miRNAs, a class of non-coding RNA, have been found to play a role in the regulation as well as in the diagnosis of glioblastoma. The relationship between epigenetics, drug resistance, and glioblastoma progression has been clearly demonstrated. MGMT hypermethylation, leading to a lack of MGMT expression, is associated with a cytotoxic effect of TMZ in GBM, while resistance to TMZ frequently appears in MGMT non-methylated GBM. In this review, we will elaborate on known miRNAs linked to glioblastoma; their distinctive oncogenic or tumor suppressor roles; and how epigenetic modification of miRNAs, particularly via methylation, leads to their upregulation or downregulation in glioblastoma. Moreover, we will try to identify those miRNAs that might be potential regulators of MGMT expression and their role as predictors of tumor response to temozolomide treatment. Although we do not impact clinical data and survival, we open possible experimental approaches to treat GBM, although they should be further validated with clinically oriented studies.

The Impact of O6-Methylguanine-DNA Methyltransferase (MGMT) Promoter Methylation on the Outcomes of Patients with Leiomyosarcoma Treated with Dacarbazine.

Dacarbazine is an important drug in the therapeutic landscape of leiomyosarcoma (LMS). Alkylating agents are subjected to resistance mechanisms based on anti-apoptotic pathways and repair mechanisms, including the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). In this retrospective study, the methylation status of the MGMT promoter in histological tumor samples from patients with LMS, dacarbazine-based regimens-treated, was measured and correlated with clinical outcomes aimed at optimizing the use of dacarbazine in soft tissue sarcomas. The patients with unmethylated MGMT had better outcomes than those with methylated MGMT. Patients without MGMT methylation had better Progression Free Survival (PFS) when aged ≥62 years compared to those aged <62 years, while PFS of patients with methylated MGMT was less favorable independently of age (p = 0.0054). The patients without a methylated MGMT gene had higher Disease control rate (DCR). These results are not in agreement with the role of the methylated MGMT gene in other tumors, and with this study, we demonstrated the correlation between methylated MGMT and poor prognosis; despite that, sample smallness, heterogeneity of LMS and of treatment history could be selection bias. Predictive markers of response to chemotherapies in sarcomas remain an unmet need.

Metronomic Temozolomide in Heavily Pretreated Patients With Recurrent Isocitrate Dehydrogenase Wild-type Glioblastoma: A Large Real-Life Mono-Institutional Study.

AIMS: Glioblastoma (GBM) is the most common primary malignant brain tumour in adults and frequently relapses. The aim of this study was to assess the efficacy and safety of metronomic temozolomide (TMZ) in the recurrent GBM population. MATERIALS AND METHODS: All patients treated at our centre between September 2013 and March 2021 were retrospectively reviewed. The main inclusion criteria were first-line therapy with the Stupp protocol, relapse after the first or subsequent line of therapy, treatment with a metronomic TMZ schedule (50 mg/m2 continuously) and histological diagnosis of isocitrate dehydrogenase wild-type GBM according to World Health Organization 2016 classification. RESULTS: In total, 120 patients were enrolled. The median follow-up was 15.6 months, the median age was 59 years, Eastern Cooperative Oncology Group performance status (ECOG-PS) was 0-2 in 107 patients (89%). O6-methylguanine-DNA-methyltransferase (MGMT) was methylated in 66 of 105 (62%) evaluable patients. The median number of prior lines of treatment was 2 (range 1-7). Three (2%) patients showed a partial response; 48 (40%) had stable disease; 69 (57%) had progressive disease. The median overall survival from the start of metronomic TMZ was 5.4 months (95% confidence interval 4.3-6.4), whereas the median progression-free survival (PFS) was 2.6 months (95% confidence interval 2.3-2.8). At univariate analysis, MGMT methylated and unmethylated patients had a median PFS of 2.9 and 2.1 months (P = 0.001) and a median overall survival of 5.6 and 4.4 months (P = 0.03), respectively. At multivariate analysis, the absence of MGMT methylation (hazard ratio = 2.3, 95% confidence interval 1.3-3.9, P = 0.004) and ECOG-PS ≤ 2 (hazard ratio = 0.5, 95% confidence interval 0.3-0.9, P = 0.017) remained significantly associated with PFS, whereas ECOG-PS ≤ 2 (hazard ratio = 0.4, 95% confidence interval 0.3-07, P = 0.001) was the only factor associated with overall survival. The most common grade 3-4 toxicities were haematological (lymphopenia 10%, thrombocytopenia 3%). CONCLUSIONS: Rechallenge with metronomic TMZ is a well-tolerated option for recurrent GBM, even in pretreated patients. Patients with methylated MGMT disease and good ECOG-PS seem to benefit the most from this treatment.

Prospective Evaluation of MGMT-Promoter Methylation Status and Correlations with Outcomes to Temozolomide-Based Chemotherapy in Well-Differentiated Neuroendocrine Tumors.

Temozolomide (TEM) as a single agent or in combination with capecitabine (CAPTEM) is active in well-differentiated advanced neuroendocrine tumors (NETs) of gastro-entero-pancreatic and thoracic origin. The predictive role of MGMT-promoter methylation in this setting is controversial. We sought to prospectively evaluate the MGMT-promoter methylation status ability to predict outcomes to TEM-based chemotherapy in patients with NET. A single-center, prospective, observational study has been conducted at the ENETS Center-of-Excellence Outpatient Clinic of the IRCCS Policlinico Sant'Orsola-Malpighi in Bologna, Italy. Patients with advanced, gastro-entero-pancreatic or lung well-differentiated NETs candidate to TEM-based chemotherapy and with available tumor samples for MGMT-promoter methylation assessment were included. The MGMT-promoter methylation status was analyzed by using pyrosequencing. The primary endpoint was progression-free survival (PFS) by the MGMT-promoter methylation status. Secondary endpoints included overall survival (OS), objective response rate (ORR), disease control rate (DCR), and safety. Survival outcomes were compared by restricted mean survival time (RMST) difference. Of 26 screened patients, 22 were finally enrolled in the study. The most frequent NET primary sites were the pancreas (64%) and the lung (23%). MGMT promoter was methylated in five tumors (23%). At a median follow-up time of 47.2 months (95%CI 29.3-89.7), the median PFS was 32.8 months (95%CI 17.2-NA), while the median OS was not reached. Patients in the methylated MGMT group, when compared to those in the unmethylated MGMT group, had longer PFS (median not reached [95%CI NA-NA] vs. 30.2 months [95%CI 15.2-NA], respectively; RMST p = 0.005) and OS (median not reached [95%CI NA-NA] vs. not reached [40.1-NA], respectively; RMST p = 0.019). After adjusting for confounding factors, the MGMT-promoter methylation status was independently associated to the PFS. Numerically higher ORR (60% vs. 24%; p = 0.274) and DCR (100% vs. 88%; p = 1.00) were observed in the methylated vs. unmethylated MGMT group. TEM-based chemotherapy was well-tolerated (adverse events grade ≥3 < 10%). In this prospective study, MGMT-promoter methylation predicted better outcomes to TEM-based chemotherapy in patients with NET.

Fused deep learning paradigm for the prediction of o6-methylguanine-DNA methyltransferase genotype in glioblastoma patients: A neuro-oncological investigation.

BACKGROUND: The O6-methylguanine-DNA methyltransferase (MGMT) is a deoxyribonucleic acid (DNA) repairing enzyme that has been established as an essential clinical brain tumor biomarker for Glioblastoma Multiforme (GBM). Knowing the status of MGMT methylation biomarkers using multi-parametric MRI (mp-MRI) helps neuro-oncologists to analyze GBM and its treatment plan. METHOD: The hand-crafted radiomics feature extraction of GBM's subregions, such as edema(ED), tumor core (TC), and enhancing tumor (ET) in the machine learning (ML) framework, was investigated using support vector machine(SVM), K-Nearest Neighbours (KNN), random forest (RF), LightGBM, and extreme gradient boosting (XGB). For tissue-level analysis of the promotor genes in GBM, we used the deep residual neural network (ResNet-18) with 3D architecture, followed by EfficientNet-based investigation for variants as B0 and B1. Lastly, we analyzed the fused deep learning (FDL) framework that combines ML and DL frameworks. RESULT: Structural mp-MRI consisting of T1, T2, FLAIR, and T1GD having a size of 400 and 185 patients, respectively, for discovery and replication cohorts. Using the CV protocol in the ResNet-3D framework, MGMT methylation status prediction in mp-MRI gave the AUC of 0.753 (p < 0.0001) and 0.72 (p < 0.0001) for the discovery and replication cohort, respectively. We presented that the FDL is ∼7% superior to solo DL and ∼15% to solo ML. CONCLUSION: The proposed study aims to provide solutions for building an efficient predictive model of MGMT for GBM patients using deep radiomics features obtained from mp-MRI with the end-to-end ResNet-18 3D and FDL imaging signatures.

BAG1, MGMT, FOXO1, and DNAJA1 as potential drug targets for radiosensitizing cancer cell lines.

BACKGROUND AND PURPOSE: Activation of some signaling pathways can promote cell survival and have a negative impact on tumor response to radiotherapy. Here, the role of differences in expression levels of genes related to the poly(ADP-ribose) polymerase-1 (PARP-1), heat shock protein 90 (Hsp90), B-cell lymphoma 2 (Bcl-2), and phosphoinositide 3-kinase (PI3K) pathways in the survival or death of cells following X-ray exposure was investigated. METHODS: Eight human cell cultures (MCF-7 and MDA-MB-231: breast cancers; MCF-12A: apparently normal breast; A549: lung cancer; L132: normal lung; G28, G44 and G112: glial cancers) were irradiated with X-rays. The colony-forming and real-time PCR based on a custom human pathway RT2 Profiler PCR Array assays were used to evaluate cell survival and gene expression, respectively. RESULTS: The surviving fractions at 2 Gy for the cell lines, in order of increasing radioresistance, were found to be as follows: MCF-7 (0.200 ± 0.011), G44 (0.277 ± 0.065), L132 (0.367 ± 0.023), MDA-MB-231 (0.391 ± 0.057), G112 (0.397 ± 0.113), A549 (0.490 ± 0.048), MCF-12A (0.526 ± 0.004), and G28 (0.633 ± 0.094). The rank order of radioresistance at 6 Gy was: MCF-7 < L132 < G44 < MDA-MB-231 < A549 < G28 < G112 < MCF-12A. PCR array data analysis revealed that several genes were differentially expressed between irradiated and unirradiated cell cultures. The following genes, with fold changes: BCL2A1 (21.91), TP53 (8743.75), RAD51 (11.66), FOX1 (65.86), TCP1 (141.32), DNAJB1 (3283.64), RAD51 (51.52), and HSPE1 (12887.29) were highly overexpressed, and BAX (-127.21), FOX1 (-81.79), PDPK1 (-1241.78), BRCA1 (-8.70), MLH1 (-12143.95), BCL2 (-18.69), CCND1 (-46475.98), and GJA1 (-2832.70) were highly underexpressed in the MDA-MB-231, MCF-7, MCF-12A, A549, L132, G28, G44, and G112 cell lines, respectively. The radioresistance in the malignant A549 and G28 cells was linked to upregulation in the apoptotic, DNA repair, PI3K, and Hsp90 pathway genes BAG1, MGMT, FOXO1, and DNAJA1, respectively, and inhibition of these genes resulted in significant radiosensitization. CONCLUSIONS: Targeting BAG1, MGMT, FOXO1, and DNAJA1 with specific inhibitors might effectively sensitize radioresistant tumors to radiotherapy.

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.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy.

BACKGROUND: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. METHODS: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. RESULTS: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. CONCLUSIONS: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Synergy between TMZ and individualized multimodal immunotherapy to improve overall survival of IDH1 wild-type MGMT promoter-unmethylated GBM patients.

The prognosis of IDH1 wild-type MGMT promoter-unmethylated GBM patients remains poor. Addition of Temozolomide (TMZ) to first-line local treatment shifted the median overall survival (OS) from 11.8 to 12.6 months. We retrospectively analyzed the value of individualized multimodal immunotherapy (IMI) to improve OS in these patients. All adults meeting the criteria and treated 06/2015-06/2021 were selected. Thirty-two patients (12f, 20m) had a median age of 47 y (range 18-69) and a KPI of 70 (50-100). Extent of resection was complete (11),

Phase I/II trial of meclofenamate in progressive MGMT-methylated glioblastoma under temozolomide second-line therapy-the MecMeth/NOA-24 trial.

BACKGROUND: Glioblastoma is the most frequent and malignant primary brain tumor. Even in the subgroup with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and favorable response to first-line therapy, survival after relapse is short (12 months). Standard therapy for recurrent MGMT-methylated glioblastoma is not standardized and may consist of re-resection, re-irradiation, and chemotherapy with temozolomide (TMZ), lomustine (CCNU), or a combination thereof. Preclinical results show that meclofenamate (MFA), originally developed as a nonsteroidal anti-inflammatory drug (NSAID) and registered in the USA, sensitizes glioblastoma cells to temozolomide-induced toxicity via inhibition of gap junction-mediated intercellular cytosolic traffic and demolishment of tumor microtube (TM)-based network morphology. METHODS: In this study, combined MFA/TMZ therapy will be administered (orally) in patients with first relapse of MGMT-methylated glioblastoma. A phase I component (6-12 patients, 2 dose levels of MFA + standard dose TMZ) evaluates safety and feasibility and determines the dose for the randomized phase II component (2 × 30 patients) with progression-free survival as the primary endpoint. DISCUSSION: This study is set up to assess toxicity and first indications of efficacy of MFA repurposed in the setting of a very difficult-to-treat recurrent tumor. The trial is a logical next step after the identification of the role of resistance-providing TMs in glioblastoma, and results will be crucial for further trials targeting TMs. In case of favorable results, MFA may constitute the first clinically feasible TM-targeted drug and therefore might bridge the idea of a TM-targeted therapeutic approach from basic insights into clinical reality. TRIAL REGISTRATION: EudraCT 2021-000708-39 . Registered on 08 February 2021.

DNA methyltransferase inhibitors combination therapy for the treatment of solid tumor: mechanism and clinical application.

DNA methylation, an epigenetic modification, regulates gene transcription and maintains genome stability. DNA methyltransferase (DNMT) inhibitors can activate silenced genes at low doses and cause cytotoxicity at high doses. The ability of DNMT inhibitors to reverse epimutations is the basis of their use in novel strategies for cancer therapy. In this review, we examined the literature on DNA methyltransferase inhibitors. We summarized the mechanisms underlying combination therapy using DNMT inhibitors and clinical trials based on combining hypomethylation agents with other chemotherapeutic drugs. We also discussed the efficacy of such compounds as antitumor agents, the need to optimize treatment schedules and the regimens for maximal biologic effectiveness. Notably, the combination of DNMT inhibitors and chemotherapy and/or immune checkpoint inhibitors may provide helpful insights into the development of efficient therapeutic approaches.

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.

Clinical Trial of MGMT(P140K) Gene Therapy in the Treatment of Pediatric Patients with Brain Tumors.

Gene transfer targeting hematopoietic stem cells (HSC) in children has shown sustained therapeutic benefit in the treatment of genetic diseases affecting the immune system, most notably in severe combined immunodeficiencies affecting T-cell function. The HSC compartment has also been successfully targeted using gene transfer in children with genetic diseases affecting the central nervous system, such as metachromatic leukodystrophy and adrenoleukodystrophy. HSCs are also a target for genetic modification in strategies aiming to confer drug resistance to chemotherapy agents so as to reduce off-target toxicity, and to allow for chemotherapy dose escalation with the possibility of enhanced therapeutic benefit. In a trial of this strategy in adult glioma patients, significant engraftment of gene-modified HSCs expressing a mutant of the DNA repair protein O6-methyl-guanine-methyl-transferase (MGMT(P140K)) showed potential in conferring drug resistance against the combined effect of O6-benzylguanine (O6BG)/temozolomide (TMZ) chemotherapy. The aim was to test the safety and feasibility of this approach in children with poor prognosis brain tumors. In this Phase I trial, seven patients received gene-modified HSC following myelo-suppressive conditioning, but with only transient low-level engraftment of MGMT(P140K) gene-modified cells detectable in four patients. All patients received O6BG/TMZ chemotherapy following infusion of gene-modified cells, with five patients eligible for chemotherapy dose escalation, though in the absence of demonstrable transgene-mediated chemoprotection. Since all gene-modified cell products met the criteria for release and assays for engraftment potential met expected outcome measures, inadequate cell dose, conditioning chemotherapy, and/or underlying bone-marrow function may have contributed to the lack of sustained engraftment of gene-modified cells. We were able to demonstrate safe conduct of a technically complex Phase I study encompassing manufacture of the gene therapy vector, genetically modified cells, and a drug product specifically for the trial in compliance with both local and national regulatory requirements.

Epi-drugs in combination with immunotherapy: a new avenue to improve anticancer efficacy.

Immune checkpoint factors, such as programmed cell death protein-1/2 (PD-1, PD-2) or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) receptors, are targets for monoclonal antibodies (MAbs) developed for cancer immunotherapy. Indeed, modulating immune inhibitory pathways has been considered an important breakthrough in cancer treatment. Although immune checkpoint blockade therapy used to treat malignant diseases has provided promising results, both solid and haematological malignancies develop mechanisms that enable themselves to evade the host immune system. To overcome some major limitations and ensure safety in patients, recent strategies have shown that combining epigenetic modulators, such as inhibitors of histone deacetylases (HDACi) or DNA methyltransferases (DNMTi), with immunotherapeutics can be useful. Preclinical data generated using mouse models strongly support the feasibility and effectiveness of the proposed approaches. Indeed, co-treatment with pan- or class I-selective HDACi or DNMTi improved beneficial outcomes in both in vitro and in vivo studies. Based on the evidence of a pivotal role for HDACi and DNMTi in modulating various components belonging to the immune system, recent clinical trials have shown that both HDACi and DNMTi strongly augmented response to anti-PD-1 immunotherapy in different tumour types. This review describes the current strategies to increase immunotherapy responses, the effects of HDACi and DNMTi on immune modulation, and the advantages of combinatorial therapy over single-drug treatment.

Epigenetics in radiotherapy: where are we heading?

Radiotherapy is an important component of anti-cancer treatment. However, not all cancer patients respond to radiotherapy, and with current knowledge clinicians are unable to predict which patients are at high risk of recurrence after radiotherapy. There is therefore an urgent need for biomarkers to guide clinical decision-making. Although the importance of epigenetic alterations is widely accepted, their application as biomarkers in radiotherapy has not been studied extensively. In addition, it has been suggested that radiotherapy itself introduces epigenetic alterations. As epigenetic alterations can potentially be reversed by drug treatment, they are interesting candidate targets for anticancer therapy or radiotherapy sensitizers. The application of demethylating drugs or histone deacetylase inhibitors to sensitize patients for radiotherapy has been studied in vitro, in vivo as well as in clinical trials with promising results. This review describes the current knowledge on epigenetics in radiotherapy.