MGMT Inhibitor Research Articles

Construction of a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity in human breast tissues

O6-methylguanine methyltransferase (MGMT) is responsible for dealkylation of naturally occurring O6-methylguanines, and it is closely related with DNA replication, transcription, and cancers. Herein, we develop a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity. When MGMT is present, the MGMT-catalyzed demethylation reaction initiates the cleavage of biotinylated dumbbell probes by PvuII restrictive enzyme, releasing two DNA fragments with 3′-OH end. The resultant DNA fragments can trigger terminal transferase (TdT)- and click chemistry-assisted isothermal amplification to obtain abundant G-rich sequences. The G-rich sequences can be captured by magnetic beads to produce a high chemiluminescence signal. This biosensor can greatly amplify the chemiluminescence signal, facilitating label-free and template-free measurement of MGMT. Especially, the introduction of dumbbell probe and PvuII enzyme can efficiently eliminate the false positive and improve the assay specificity. This biosensor possesses high sensitivity with a detection limit of 1.4 × 10−9 ng/μL, and it may accurately quantify the intracellular MGMT. Importantly, this biosensor can be used to screen the MGMT inhibitors and distinguish the MGMT level in breast tumor tissues and normal tissues, with great potential in drug discovery and cancer diagnosis.

Novel non-nucleoside MGMT inhibitors: potential in combined alkylating chemotherapy

Novel non-nucleoside MGMT inhibitors: potential in combined alkylating chemotherapy

Non-nucleoside O6-methylguanine-DNA methyltransferase inhibitors in murine spontaneous tumor experimental chemotherapy in vivo

Alkylating chemotherapy agents are well-established for inducing DNA lesions that result in apoptosis in cancer cells. However, the efficacy of these agents is often diminished due to the activity of the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which confers resistance to chemotherapy by catalyzing dealkylation reactions. Recent studies have identified novel non-nucleoside MGMT inhibitors with promising properties. In this study, we evaluated the effectiveness of these novel non-nucleoside MGMT inhibitors in combination with alkylating chemotherapy in vivo. Our experimental model involved ICR female mice that spontaneously developed malignant tumors. These mice were treated with a combination of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and new MGMT inhibitors. We analyzed tumor growth dynamics and observed the levels of MGMT and other proteins using western blot analysis. Our findings demonstrated that the addition of MGMT inhibitors significantly improved the tumor growth-inhibiting effects of the alkylating chemotherapy. Tumor growth was more effectively suppressed in the mice receiving the combination therapy compared to those receiving the alkylating agents alone. Additionally, MGMT levels were significantly reduced following the combined treatment. Furthermore, the active form of caspase 3 was detected in treated tumors, suggesting that the reduction in tumor growth may be mediated through an apoptotic pathway. These results underscore the potential for these novel MGMT inhibitors to enhance the efficacy of alkylating agents in cancer therapy, holding substantial promise for improving therapeutic outcomes against tumors that exhibit high MGMT activity.

A simple and rapid in vitro assay foridentification of direct inhibitors of O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that is overexpressed in certain tumors and is associated with resistance to the DNA alkylating agent temozolomide. MGMT inhibitors show potential in combating temozolomide resistance, but current assays for MGMT enzyme activity and inhibition, primarily oligonucleotide-based and fluorescent probe-based, are laborious and costly. The clinical relevance of temozolomide therapy calls for more convenient methodologies to study MGMT inhibition. Here, we extended the application of SNAP-Capture magnetic beads to develop a novel MGMT inhibition assay that demonstrated efficacy not only with known MGMT inhibitors, but also with the aldehyde dehydrogenase inhibitor, disulfiram. The assay uses standard fluorescence microscopy as a simple and reliable detection method, and is translationally applicable in drug discovery programs.

Abstract 4560: Assessing cancer drug combination efficacy across 900+ PRISM cell lines in a multiplexed screening assay

Abstract Combination therapies are crucial in cancer care, yet identifying which combination will benefit a specific patient is challenging. The vast array of clinical, investigational, and tool anticancer agents and diverse cancer contexts makes investigating many drug combinations over a large number of cell lines infeasible. Here, we describe a multiplexed screening approach using the PRISM assay, where drug combinations are screened on >900 DNA-barcoded cancer cell lines. We analyze cell viability data in drug combinations and single agents using established metrics to describe the combined effects such as additivity, synergy, and antagonism. Quantifying and classifying combined effects will help prioritize specific combinations for follow-up investigations and provide biological and mechanistic insights, thus enabling rationally designed combination therapies. We illustrate our methodology through data from three drug combinations that exemplify synergy and antagonism. In the first combination, temozolomide+O6-benzylguanine (alkylating agent + MGMT inhibitor), we found that cell lines expressing MGMT were sensitized to temozolomide in the presence of O6-benzylguanine, thus illustrating synergy. In the second combination, we screened two anti-apoptosis compounds A-1331852+AZD5991 (BCL-xL + MCL1 inhibitor) and found that BCL-xL and MCL1 inhibition were also synergistic. In the third combination, ML210+ferrostatin-1 (GPX4 inhibitor inducing ferroptosis + ferroptosis inhibitor), we found that ferrostatin-1 antagonized the effects of ML210. Our analysis also reveals the importance of appropriate dose selection and analytical metrics for reliably measuring combined effects. In conclusion, the PRISM platform's multiplexed cell line screening is a robust method for characterizing rationally designed drug combinations, offering a systematic approach to enhance the precision of combination therapy development in cancer care. Citation Format: Ashish Bino George, Shiker Nair, Mustafa Kocak, Ellen Nguyen, Alvin Kalathungal, Anthony Fazio, Cole Ponsi, Aydin Golabi, Melissa A. Ronan, Matthew G. Rees, Jennifer A. Roth. Assessing cancer drug combination efficacy across 900+ PRISM cell lines in a multiplexed screening assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4560.

Novel MGMT inhibitors increase the sensitivity of glioma MGMT-positive cells to treatment with alkylating agents in vitro

Novel MGMT inhibitors increase the sensitivity of glioma MGMT-positive cells to treatment with alkylating agents in vitro

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/μL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

MODL-02. DEVELOPMENT OF RODENT GLIOBLASTOMA MODELS OF MGMT-MEDIATED TEMOZOLOMIDE RESISTANCE

Abstract O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine DNA damage lesions generated by alkylators such as temozolomide (TMZ), the standard chemotherapeutic for glioblastoma (GBM) treatment. High MGMT expression leads to TMZ resistance and is correlated with lower survival, making it an important prognostic biomarker and potential therapeutic target. Xenograft models with human GBM cell lines require implantation into immunodeficient animals, precluding the study of MGMT-dependent alkylator sensitivity in the setting of an intact tumor immune microenvironment. In this study, we developed rat and mouse glioma models of MGMT overexpression and characterized response to alkylator therapy. RG2 rat glioma and SB28 mouse glioma cells, which have low endogenous MGMT levels, were transduced with cDNA expressing FLAG-tagged MGMT to generate an isogenic pair. RG2 MGMT+ and SB28 MGMT+ cells showed robust expression of MGMT, which was effectively abrogated by MGMT inhibitors. In vitro cell viability assays showed that MGMT overexpression resulted in increased resistance to TMZ compared to parental cells, and this resistance was reversed when cells were concurrently treated with the MGMT inhibitor lomeguatrib. To model MGMT overexpression in vivo, luciferase-expressing RG2 MGMT+ cells were implanted into the brains of syngeneic Fischer 344 rats. Rats were treated with TMZ at 50mg/kg daily x 5 days via intraperitoneal injection. Tumor growth was measured by bioluminescence imaging and rats were monitored for survival and toxicity. RG2 MGMT+ tumors were highly resistant to TMZ, as treatment did not improve survival or delay tumor growth. Overall, our newly developed isogenic rat and mouse glioma models recapitulate MGMT-dependent TMZ sensitivity and provide useful preclinical tools for studying MGMT as a biomarker and potential therapeutic target in GBM. In addition, these syngeneic models facilitate studies on the immune modulation of chemotherapeutic agents in the context of MGMT expression and allows for testing of chemoimmunotherapy combinations.

Epigenetic Activation of TUSC3 Sensitizes Glioblastoma to Temozolomide Independent of MGMT Promoter Methylation Status.

Temozolomide (TMZ) is an important first-line treatment for glioblastoma (GBM), but there are limitations to TMZ response in terms of durability and dependence on the promoter methylation status of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT-promoter-hypermethylated (MGMT-M) GBMs are more sensitive to TMZ than MGMT-promoter-hypomethylated (MGMT-UM) GBMs. Moreover, TMZ resistance is inevitable even in TMZ-sensitive MGMT-M GBMs. Hence, epigenetic reprogramming strategies are desperately needed in order to enhance TMZ response in both MGMT-M and MGMT-UM GBMs. In this study, we present novel evidence that the epigenetic reactivation of Tumor Suppressor Candidate 3 (TUSC3) can reprogram sensitivity of GBM stem cells (GSCs) to TMZ irrespective of MGMT promoter methylation status. Interrogation of TCGA patient GBM datasets confirmed TUSC3 promoter regulation of TUSC3 expression and also revealed a strong positive correlation between TUSC3 expression and GBM patient survival. Using a combination of loss-of-function, gain-of-function and rescue studies, we demonstrate that TUSC3 reactivation is associated with enhanced TMZ response in both MGMT-M and MGMT-UM GSCs. Further, we provide novel evidence that the demethylating agent 5-Azacitidine (5-Aza) reactivates TUSC3 expression in MGMT-M GSCs, whereas the combination of 5-Aza and MGMT inhibitor Lomeguatrib is necessary for TUSC3 reactivation in MGMT-UM GSCs. Lastly, we propose a pharmacological epigenetic reactivation strategy involving TUSC3 that leads to significantly prolonged survival in MGMT-M and MGMT-UM orthotopic GSCs models. Collectively, our findings provide a framework and rationale to further explore TUSC3-mediated epigenetic reprogramming strategies that could enhance TMZ sensitivity and outcomes in GBM. Mechanistic and translational evidence gained from such studies could contribute towards optimal design of impactful trials for MGMT-UM GBMs that currently do not have good treatment options.

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1β), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

A Biomimetic Nanomedicine Targets Orthotopic Glioblastoma by Combinatorial Co‐Delivery of Temozolomide and a Methylguanine‐DNA Methyltransferase Inhibitor

AbstractGlioblastoma (GBM) is the most aggressive intracranial tumor that occurs in the central nervous system (CNS) and has no effective treatment due to the fact of drug resistance. To combat drug resistance, TMZ is generally administrated in combination with other chemotherapeutic drugs. Unfortunately, drug combinations used to date only show modest improvements in anti‐tumor effects and have increased toxicity. Herein, an ApoE peptide decorated GBM cancer cell membrane camouflaged nanomedicine (AMNPs@TMZ+LM) to specifically co‐deliver TMZ and the MGMT inhibitor lomeguatrib (LM) for combinatorial GBM treatment is developed. Incorporation of LM not only effectively suppresses the repair of damaged DNA, but also inhibits tumor cell proliferation by inducing cell cycle arrest. Importantly, this biomimetic nanomedicine achieves high BBB penetration and enhances sensitivity to both TMZ‐resistant (U251‐TR) and GBM stem cells (GSCs). Notably, AMNPs@TMZ+LM results in significant inhibition of the growth of orthotopically implanted U251‐TR tumor in mice. In comparison, treatment with single drug loaded nanomedicines results in compromised anti‐GBM effects. Histological analyses and blood parameter studies show good biocompatibility of the nanomedicine. Hence, this biomimetic nanomedicine provides a potential new approach to overcome the limitations of current GBM chemotherapy regimens by co‐delivery of TMZ and the MGMT inhibitor LM.

TMOD-25. LATENT SOX9-POSITIVE CELLS BEHIND MYC-DRIVEN MEDULLOBLASTOMA RELAPSE

Abstract Tumor recurrence developing from therapy resistance, immune escape and metastasis is the leading cause of death in medulloblastoma, the most frequent malignant pediatric brain tumor. Amplification of MYC genes is the most common genetic alteration in Group 3 and Group 4 subgroups that constitute two thirds of medulloblastoma. SOX9 is a transcription factor present in stem cells in the normal brain but is limited to rare, quiescent cells in medulloblastoma patients with MYC gene amplifications. By studying paired primary-recurrent patient samples and patient-derived xenografts we here identified significant accumulation of SOX9-positive cells in Group 3 and Group 4 relapses. To follow relapse at the single cell level we developed an inducible dual Tet model of MYC-driven MB, where MYC was re-directed from the treatment-sensitive bulk cells to resistant, dormant SOX9-positive cells by doxycycline. In this model, distant recurrent tumors and spinal metastases developed. SOX9 promoted immune escape, DNA repair suppression and was essential for recurrence. Tumor cell dormancy was non-hierarchical, migratory and depended on MYC suppression by SOX9 to promote relapse. By using computational modeling and treatment we also showed how doxorubicin and MGMT inhibitors were specifically targeting recurrent cells that could be of potential use in future treatments for patients affected by these fatal relapses.

Lomeguatrib Increases the Radiosensitivity of MGMT Unmethylated Human Glioblastoma Multiforme Cell Lines.

Background: Treatment resistance of glioblastoma multiforme to chemo- and radiotherapy remains a challenge yet to overcome. In particular, the O6-methylguanine-DNA-methyltransferase (MGMT) promoter unmethylated patients have only little benefit from chemotherapy treatment using temozolomide since MGMT counteracts its therapeutic efficacy. Therefore, new treatment options in radiotherapy need to be developed to inhibit MGMT and increase radiotherapy response. Methods: Lomeguatrib, a highly specific MGMT inhibitor, was used to inactivate MGMT protein in vitro. Radiosensitivity of established human glioblastoma multiforme cell lines in combination with lomeguatrib was investigated using the clonogenic survival assay. Inhibition of MGMT was analyzed using Western Blot. Cell cycle distribution and apoptosis were investigated to determine the effects of lomeguatrib alone as well as in combination with ionizing radiation. Results: Lomeguatrib significantly decreased MGMT protein and reduced radiation-induced G2/M arrest. A radiosensitizing effect of lomeguatrib was observed when administered at 1 µM and increased radioresistance at 20 µM. Conclusion: Low concentrations of lomeguatrib elicit radiosensitization, while high concentrations mediate a radioprotective effect.

Regulation of temozolomide resistance in glioma cells via the RIP2/NF-κB/MGMT pathway.

BackgroundTemozolomide (TMZ) is a first‐line chemotherapy drug for the treatment of malignant glioma and resistance to it poses a major challenge. Receptor‐interacting protein 2 (RIP2) is associated with the malignant character of cancer cells. However, it remains unclear whether RIP2 is involved in TMZ resistance in glioma.MethodsRIP2 expression was inhibited in TMZ‐resistant glioma cells and normal glioma cells by using small interfering RNA (siRNA) against RIP2. Plasmid transfection method was used to overexpress RIP2. Cell counting kit‐8 assays were performed to evaluate cell viability. Western blotting or immunofluorescence was performed to determine RIP2, NF‐κB, and MGMT expression in cells. Flow cytometry was used to investigate cell apoptosis. TMZ‐resistant glioma xenograft models were established to evaluate the role of the RIP2/NF‐κB/MGMT signaling pathway in drug resistance.ResultsWe observed that RIP2 expression was upregulated in TMZ‐resistant glioma cells, whereas silencing of RIP2 expression enhanced cellular sensitivity to TMZ. Similarly, upon the induction of RIP2 overexpression, glioma cells developed resistance to TMZ. The molecular mechanism underlying the process indicated that RIP2 can activate the NF‐κB signaling pathway and upregulate the expression of O6‐methylguanine‐DNA methyltransferase (MGMT), following which the glioma cells develop drug resistance. In the TMZ‐resistant glioma xenograft model, treatment with JSH‐23 (an NF‐κB inhibitor) and lomeguatrib (an MGMT inhibitor) could enhance the sensitivity of the transplanted tumor to TMZ.ConclusionWe report that the RIP2/NF‐κB/MGMT signaling pathway is involved in the regulation of TMZ resistance. Interference with NF‐κB or MGMT activity could constitute a novel strategy for the treatment of RIP2‐positive TMZ‐resistant glioma.

The O6-methyguanine-DNA methyltransferase inhibitor O6-benzylguanine enhanced activity of temozolomide + irinotecan against models of high-risk neuroblastoma.

DNA-damaging chemotherapy is a major component of therapy for high-risk neuroblastoma, and patients often relapse with treatment-refractory disease. We hypothesized that DNA repair genes with increased expression in alkylating agent resistant models would provide therapeutic targets for enhancing chemotherapy. In-vitro cytotoxicity of alkylating agents for 12 patient-derived neuroblastoma cell lines was assayed using DIMSCAN, and mRNA expression of 57 DNA repair, three transporter, and two glutathione synthesis genes was assessed by TaqMan low-density array (TLDA) with further validation by qRT-PCR in 26 cell lines. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was upregulated in cell lines with greater melphalan and temozolomide (TMZ) resistance. MGMT expression also correlated significantly with resistance to TMZ+irinotecan (IRN) (in-vitro as the SN38 active metabolite). Forced overexpression of MGMT (lentiviral transduction) in MGMT non-expressing cell lines significantly increased TMZ+SN38 resistance. The MGMT inhibitor O6-benzylguanine (O6BG) enhanced TMZ+SN38 in-vitro cytotoxicity, H2AX phosphorylation, caspase-3 cleavage, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling. TMZ+IRN+O6BG delayed tumor growth and increased survival relative to TMZ+IRN in two of seven patient-derived xenografts established at time of death from progressive neuroblastoma. We demonstrated that high MGMT expression was associated with resistance to alkylating agents and TMZ+IRN in preclinical neuroblastoma models. The MGMT inhibitor O6BG enhanced the anticancer effect of TMZ+IRN in vitro and in vivo. These results support further preclinical studies exploring MGMT as a therapeutic target and biomarker of TMZ+IRN resistance in high-risk neuroblastoma.

CTNI-49. PHASE I STUDY OF MGMT-P140K TRANSFECTED HEMATOPOETIC PROGENITOR CELLS COMBINED WITH TMZ/O6BG DOSE ESCALATION FOR NEWLY DIAGNOSED, UNMETHYLATED GLIOBLASTOMA: TOLERANCE AND EVIDENCE OF SURVIVAL BENEFIT

Abstract INTRODUCTION GBM has median survival of 12 months despite current SOC therapy. The most important mechanism of TMZ resistance is the O6-methylguanine-DNA methyltransferase (MGMT) gene. The MGMT inhibitor O6-benzylguanine (BG) demonstrated efficacy in depleting MGMT but approach led to unacceptable bone marrow toxicity and has thus been abandoned. We hypothesized that chemoprotection of hematopoietic HPC with an MGMT mutant (MGMT-P140K) characterized by normal methyltransferase activity, coupled with low affinity for BG would maximize anti-tumor response while enabling patients to tolerate TMZ & BG dose escalation with minimal toxicity. A phase I trial was performed to test this hypothesis. METHODS 10 adults with newly diagnosed MGMT unmethylated, IDH-1 WT, GBM underwent standard surgery and radiation, followed by transplantation with autologous CD34+ HPC transfected with MGMT-P140K using a lentiviral vector. We tested tolerance and efficacy of three different paradigms for conditioning bone marrow and re-infusion of HPC. RESULTS Treatment was moderately toxic with 3/10 patients suffering grade 3–4 hematologic toxicity; no high grade non-hematologic toxicity was observed. Viral transduction rates ranged from 3–75% and were clearly improved in Arm III utilizing BCNU conditioning and intra-patient dose escalation of TMZ/O6GB. In patients tolerating 3 cycles or more, P140K-MGMT gene markings in peripheral blood and bone marrow cells increased 3-26-fold with only mild (Grade 2–3) myelosuppression consistent with chemo-protection as hypothesized. Median PFS and OS was 24 and 33 months respectfully, and three patients in Arm III were progression free at 36 months with one progression free at 48 months. OS exceeded RPA & Nomogram predicted survival by 3.6-fold, suggesting clinical benefit. Viral insertion site analysis demonstrate no clonal dominance. CONCLUSIONS P140K-MGMT transfected HPC enables TMZ/ BG dose escalation with acceptable toxicity and increased survival in a small cohort of selected patients. A U-01 funded phase II study is ongoing at UH and NIH.

Current Translational Insights into MGMT Methylation Regulating Temozolomide Sensitivity and Resistance in Glioblastoma Multiforme

Background:Temozolomide is used as frontline chemotherapy in the management of glioblastoma multiforme (GBM); however, its clinical utility is limited by the occurrence of significant resistance, majorly caused due to direct DNA repair. O6- methylguanine-DNA-methyltransferase (MGMT), a DNA repair protein, mediates this direct repair pathway and reverses the activity of temozolomide.Methods:We characterize and underscore the functional relevance and molecular aspects of MGMT in the development of sensitivity/resistance to temozolomide treatment. We review early translational, as well as clinical, evidence for the role of MGMT in mediating temozolomide resistance in vitro in cell lines, in vivo in small animals as well as in GBM patients.Results:Various approaches have been delineated to mitigate MGMT-induced temozolomide resistance. The most promising means in discovery biology appears to be the co-administration of MGMT inhibitors such as O6 benzyl guanine or lomeguatrib. Surprisingly, the validation of these pharmacologic inhibitors to assess the reversal of chemoresistance by appropriately designed safety and efficacy trials in combination with temozolomide is yet to be demonstrated.Conclusions:Taken together, given the regulation of temozolomide resistance by MGMT, intermediate and late discovery groups may focus their efforts on pharmacologic inhibition of MGMT, singly or in combination with radiotherapy or immunotherapy, to combat temozolomide resistance in GBM patients. In addition, one may speculate that the combined clinical use of temozolomide with a drug regulator-approved MGMT inhibitor as well as an immune checkpoint inhibitor such as nivolumab may prove beneficial. Future studies may also investigate any inter-ethnic variability in population pharmacogenetics of MGMT and pharmacometric approaches to optimize cancer precision medicine.

Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma

BackgroundTemozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system,...

Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT.

DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is O6-methylguanine (m6G), which base pairs with thymine during replication to cause GC → AT mutations. The gpt delta C57BL/6J mouse strain of Nohmi et al. (Mol. Mutagen 1996, 28, 465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from gpt delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by N-methyl-N-nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental N-nitrosamines, such as N-nitrosodimethylamine and therapeutic agents such as Temozolomide. The DNA repair protein MGMT (O6-methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of Temozolomide in cancer therapy. The gpt delta MEFs were treated with MNU to establish dose-dependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC → AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using O6-methyl-d3-guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation frequency seen in the same cells. This work establishes gpt delta MEFs as a versatile tool for probing mutagenesis by environmental and therapeutic agents and as a cell culture model in which chemical genetics can be used to determine the impact of DNA repair on biological responses to DNA damaging agents.

EXTH-50. THIOREDOXIN REDUCTASE1 AND MGMT SYNTHETIC LETHALITY ENHANCES CYTOTOXICITY OF PRIMA-1MET(APR-246) AND AURANOFIN IN GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM), the most common and advanced primary brain malignancy in adults remains an incurable disease, despite aggressive treatment with surgery, radiation therapy and chemoradiation using the alkylating agent, Temozolomide (TMZ). PRIMA-1MET (APR-246), a small molecule designed to restore mutant (mut)p53 function has been shown to affect cellular redox status through targeting thioredoxin reductase 1 (TrxR1) in wild-type (wt)p53 cancer cells. We have recently shown that PRIMA-1MET exerts cytotoxic effects status preferentially in GBM cell lines expressing low levels of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), known for its role in resistance to TMZ. We hypothesized that PRIMA-1MET mediates its growth inhibitory effects by modulating the redox balance and investigated the potential relationship between MGMT, redox balance and TrxR1. We show that PRIMA-1MET decreased TrxR1 expression levels preferentially in MGMT-low expressing isogenic GBM cell lines. Using pharmacological agents that modulate reactive oxygen species (ROS) levels i.e., ROS scavenger, N-acetylcysteine and ROS inducer, L-Buthionine-Sulfoximine, we show that PRIMA-1MET exerts its growth-inhibitory effects through increased ROS. Strikingly, we identified a novel positive relationship between MGMT and TrxR1, wherein high MGMT expression is associated with high expression of TrxR1 and low levels of ROS. Treatment with the MGMT inhibitor, O6-Benzylguanine, or the TrxR1-targeting FDA-approved drug Auranofin validated our findings. Interestingly, the latter exerted significantly more pronounced cytotoxic effects compared to PRIMA-1MET in GBM cell lines. Additional studies are warranted to assess PRIMA-1MET in combination with TrxR1-targeting therapies and propose repurposing of Auranofin as a novel strategy to improve the dismal outcome of patients with GBM.