methylguanine-DNA Methyltransferase Expression Research Articles

Identification of MGMT Downregulation Induced by miRNA in Glioblastoma and Possible Effect on Temozolomide Sensitivity.

Glioblastoma multiforme (GBM) remains one of the tumors with the worst prognosis. In recent years, a better overall survival (OS) has been described in cases subjected to Gross Total Resection (GTR) that were presenting hypermethylation of Methylguanine-DNA methyltransferase (MGMT) promoter. Recently, also the expression of specific miRNAs involved in MGMT silencing has been related to survival. In this study, we evaluate MGMT expression by immunohistochemistry (IHC), MGMT promoter methylation and miRNA expression in 112 GBMs and correlate the data to patients' clinical outcomes. Statistical analyses demonstrate a significant association between positive MGMT IHC and the expression of miR-181c, miR-195, miR-648 and miR-767.3p between unmethylated cases and the low expression of miR-181d and miR-648 and between methylated cases and the low expression of miR-196b. Addressing the concerns of clinical associations, a better OS has been described in presence of negative MGMT IHC, in methylated patients and in the cases with miR-21, miR-196b overexpression or miR-767.3 downregulation. In addition, a better progression-free survival (PFS) is associated with MGMT methylation and GTR but not with MGMT IHC and miRNA expression. In conclusion, our data reinforce the clinical relevance of miRNA expression as an additional marker to predict efficacy of chemoradiation in GBM.

Givinostat Inhibition of Sp1-dependent MGMT Expression Sensitizes Glioma Stem Cells to Temozolomide.

Givinostat is a pan-histone deacetylase (HDAC) inhibitor that has demonstrated excellent tolerability as well as efficacy in patients with polycythemia vera. Accumulating in vitro and in vivo evidence suggests givinostat is also promising as a therapeutic agent targeting glioma stem cells (GSCs), the cancer stem cells of glioblastoma (GBM) considered responsible for its intractable nature. However, it remains to be shown how givinostat impacts the therapeutic effects of temozolomide, a DNA-alkylating agent and the key component of GBM treatment given not only during postoperative radiotherapy but also thereafter as maintenance chemotherapy. The effects of givinostat and knockdown of O6-methylguanine-DNA methyltransferase (MGMT) or Sp1 on the mRNA and protein expression of relevant genes in human GSC lines were examined by RT-PCR and western blot analyses. The dye exclusion method was used to evaluate cell viability. Givinostat enhanced the cytotoxic activity of temozolomide in GSC lines expressing MGMT, in which the MGMT expression was shown to contribute to their temozolomide resistance. Givinostat inhibited MGMT expression in GSCs and, in parallel, the expression of Sp1, a transcription factor involved in the control of MGMT promoter activity. Knockdown experiments demonstrated Sp1 expression was indeed required for MGMT expression in GSCs. Givinostat, in addition to its own cytotoxic activity, sensitizes GSCs to temozolomide by inhibiting Sp1-dependent MGMT expression in GSCs. Combining givinostat with temozolomide could therefore be a rational therapeutic strategy to effectively eliminate GSCs and thus help overcome the therapy resistance of GBM.

P15.02 Loss of Williams Syndrome Transcription Factor (WSTF) leads to improved temozolomide sensitivity in human glioblastoma cells irrespective of MGMT expression

Abstract BACKGROUND The prognosis for newly diagnosed adult glioblastoma multiforme (GBM) patients is poor even after standard therapy with a median survival of approximately 14–15 months. The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) efficiently counteracts formation of the most lethal DNA adducts by temozolomide (TMZ) chemotherapy, and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has previously been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling together with SMARCA5 in the WICH complex) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its ability to regulate the DDR in GBMs, is so far not known. In this study, we investigated whether loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. MATERIAL AND METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. For each cell line, three knockout clones were chosen for further investigation. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding WSTF-expressing control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p <0.0001, U-251: Clone 7/35.1/70.2 vs control: p <0.0001/p= 0.0107/p= 0.0119). Furthermore, two out of three clones of T98G and U-251 (T98G Clone 13 and 128 vs control, p <0.0001, U-251 Clone 7 vs control, p= 0.0062; clone 70.2, p= 0.0052) showed significantly reduced plating efficiency compared to control cells. CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.

TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT.

BackgroundOur previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.MethodsThe distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.ResultsOverexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.ConclusionsOur results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.

Profound, durable and MGMT-independent sensitivity of glioblastoma cells to cyclin-dependent kinase inhibition.

TG02 is a novel cyclin-dependent kinase (CDK) inhibitor and thought to act mainly via CDK-9 inhibition-dependent depletion of short-lived oncoproteins such as MCL-1 or c-MYC. We studied the activity of TG02 in 9 human long-term glioma cell lines (LTC) and 5 glioma-initiating cell lines (GIC) using various cell death assays in vitro and in the LN-229 LTC and ZH-161 GIC models in vivo. TG02 exhibits strong anti-tumor cell activity with EC50 concentrations in the nanomolar range. Median survival in the LN-229 and ZH-161 models was moderately prolonged by TG02. Neither constitutive CDK levels nor those of MCL-1 or c-MYC correlated with sensitivity to TG02. Cdk-9 or cdk-5 gene silencing alone did not fully reproduce the effects of TG02. C-myc gene silencing inhibited cell growth, but did not modulate TG02 activity. Electron microscopy revealed cell death to be essentially apoptotic. High concentrations of TG02 induced annexin V binding and minor caspase 3 cleavage, but the pan-caspase inhibitor, zVAD-fmk, or BCL-2 or MCL-1 gene transfer only moderately attenuated TG02-induced cell death, and caspase inhibition did not prevent loss of MCL-1 or c-MYC. TG02 activity was independent of O6 -methylguanine DNA methyltransferase expression. Repetitive exposure to TG02 did not generate an acquired TG02 resistance phenotype, but accumulation of MCL-1, loss of c-MYC, or senescence. TG02 is a highly potent apoptosis-inducing agent in glioma cells in vitro. Caspase inhibition does not rescue TG02-treated cells and repetitive exposure fails to confer acquired resistance, supporting the clinical evaluation of TG02 in glioblastoma.

Resveratrol restores sensitivity of glioma cells to temozolamide through inhibiting the activation of Wnt signaling pathway.

Malignant gliomas are aggressive primary neoplasms that originate in the glial cells of the brain or the spine with notable resistance to standard treatment options. We carried out the study with the aim to shed light on the sensitization of resveratrol to temozolomide (TMZ) against glioma through the Wnt signaling pathway. Initially, glioma cell lines with strong resistance to TMZ were selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the glioma cells were subjected to resveratrol, TMZ, Wnt signaling pathway inhibitors, and activators. Cell survival rate and inhibitory concentration at half maximum value were detected by MTT, apoptosis by flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, in vitro proliferation by hanging drop method and β-catenin translocation into nuclei by TOP/FOP-FLASH assay. The expressions of the Wnt signaling pathway-related and apoptosis-related factors were determined by western blot analysis. Nude mice with glioma xenograft were established to detect tumorigenic ability. Glioma cell lines T98G and U138 which were highly resistant to TMZ were selected for subsequent experiments. Resveratrol increased the efficacy of TMZ by restraining cell proliferation, tumor growth, and promoting cell apoptosis in glioma cells. Resveratrol inhibited Wnt2 and β-catenin expressions yet elevated GSK-3β expression. Moreover, the Wnt signaling pathway participates in the sensitivity enhancing of resveratrol to TMZ via regulating O 6 -methylguanine-DNA methyltransferase (MGMT) expression. Resveratrol sensitized TMZ-induced glioma cell apoptosis by repressing the activation of the Wnt signaling pathway and downregulating MGMT expression, which may confer new thoughts to the chemotherapy of glioma.

Analysis of the correlation of MGMT and XRCC1 gene expression with the pathogenesis of glioma and its mechanism analysis

Objective To investigate the relationship between the expression of O6- methyl guanine DNA methyltransferase (MGMT), X-ray repair cross complementation gene 1 (XRCC1) and the incidence of glioma. Methods From February 2015 to September 2017, 53 glioma patients (glioma group) in our hospital were enrolled in the study. 50 patients with hypertensive intracerebral hemorrhage were selected as control A group, and 106 healthy volunteers as control B group. Immunohistochemical staining was used to detect the expression of MGMT and XRCC1 in brain tissue of glioma group and control A group, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect the polymorphism of MGMT and XRCC1 gene in glioma group and control B group. Results The positive expression rates of MGMT and XRCC1 in the tissues of brain glioma were 47.17% and 39.62%, respectively, which were significantly higher than those in the control A group (P 0.05); There was no correlation between the expression of MGMT and XRCC1 in glioma tissues (rs=0.162, P>0.05); The proportion of XRCC1 genotype AG+ GG in brain glioma group was 58.49%, which was significantly higher than that of control B group (P<0.05); The proportion of MGMT genotype LP+ PP in brain glioma group was 28.30%, which was significantly higher than that of control B group (P<0.05). Conclusions MGMT and XRCC1 are increased significantly in glioma brain tissues, but not correlated with pathological grades; The polymorphism of MGMT and XRCC1 genes may be related to the susceptibility of gliomas. Key words: DNA modification methylases/GE; X-ray repair cross complementing protein 1/GE; Glioma/GE; Polymorphism, genetic

Abstract 4831: DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide

Abstract Introduction: O6 methylguanine DNA methyltransferase (MGMT) repairs the DNA damage caused by alkylating agents [temozolomide (TMZ)] leading to chemoresistence. Members of the aldehyde dehydrogenase (ALDH) family of isoenzymes serve as markers of cancer stem cells and contribute to chemotherapy resistance. Disulfiram (DSF) inhibits MGMT through ubiquitin mediated degradation; it is also a specific inhibitor of ALDH. We therefore, hypothesized that DSF through MGMT and ALDH inhibition decreases stemness and sensitizes GBM cells to TMZ. Methods: Normal astrocytes as well as established MGMT expressing GBM cell lines (LN18, T98G, U138, U118) and MGMT expressing (unmethylated) patient derived glioblastoma cells (ANII 7730 and ANII 7754) were treated with DSF/Cu +/- TMZ in various doses and combinations. We have also evaluated MGMT, ALDH levels and Sox2 expression. Cell viability, apoptotic assay and caspase 3/7 assay were used to evaluate inhibitory effect of various treatment combinations. Results: DSF and TMZ have a minimal effect on normal astrocyte growth. DSF alone inhibited MGMT in established MGMT expressing glioblastoma cells (5 to 10 µM). DSF alone was unable to inhibit MGMT in patient derived brain tumor cells (up to 20µM). Addition of copper (&amp;lt;/=1µM) significantly inhibited MGMT (~ 90%) in patient derived GBM cells which correlated with significant growth inhibition even when DSF was used at low concentrations (&amp;lt;/=1µM). This suggests that addition of copper to DSF may reduce any potential, dose dependent DSF related neurotoxicity. Temozolomide alone did not inhibit MGMT in established cell lines nor patient derived brain tumor cell lines (up to 1000µM). DSF+Cu further sensitized patient derived GBM cells to TMZ and significantly inhibited GBM cell growth (~ 95%) and did not inhibit normal astrocyte growth. Further DSF+Cu and DSF+Cu+TMZ combinations caused significant apoptotic cell death and significantly increased caspase 3/7 in patient derived unmethylated GBM cells. Similarly, combination of DSF+Cu and TMZ+DSF+Cu caused significant drop in ALDH activity in patient derived brain tumor cells compared to untreated controls and single agents. DSF induced ALDH and MGMT inhibition correlated with decrease in SOX2. MGMT expression canceled by CRISPR/Cas9 led to significant decrease in SOX2 expression in primary cell cultures. Conclusions: Our findings suggest that DSF/Cu treatment, a dual MGMT and ALDH inhibitor, suppresses stemness. Furthermore, our results confirm that combination of TMZ and DSF/Cu significantly inhibited glioblastoma cell growth compared to TMZ alone and untreated controls. Citation Format: George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah Donohoe, Dmitry Bosenko, Santhi D. Konduri. DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4831.

Transcriptional control of O6 -methylguanine DNA methyltransferase expression and temozolomide resistance in glioblastoma.

O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma-initiating cells (GIC) with stem-like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real-time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF-κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S-to-M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF-κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF-κB by the small molecule inhibitor, BAY 11-7082, or siRNA-mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over-expression of MGMT which results from increased activity of the NF-κB pathway in this cell culture model of glioma stem-like cells. Read the Editorial Highlight for this article on page 688.

Panobinostat and its combination with 3-deazaneplanocin-a induce apoptosis and inhibit In vitro tumorigenesis and metastasis in GOS-3 glioblastoma cell lines

Aim: Glioblastoma is the most malignant primary brain tumor. The treatment consists of surgery, with or without radiotherapy, and temozolomide, with a life expectancy of 12–15 months after diagnosis. Glioblastoma is resistant to conventional antitumor therapies. In this work, we present a preliminary in vitro study of two epigenetic drugs against GOS-3 glioblastoma cells. Methods: We used (1) panobinostat, a histone deacetylase inhibitor, and (2) 3-deazaneplanocin-A (DZ-Nep), an inhibitor of enhancer of zeste homolog 2 (EZH2) (enzyme of the polycomb repressor complex 2, polycomb group of proteins that trimethylate lysine 27 of histone 3-H3K27 me3-), as treatments that might modulate the PI3K pathway, affected in GOS-3 cells due to PTEN haploinsufficiency. The glioblastoma cell line GOS-3 was exposed to DZ-Nep and panobinostat treatments, separately and in combination, over a period of 2 days, after which cell migration, clonogenicity, and molecular expression characterization assays were performed. Results: Panobinostat alone or the combination of panobinostat plus DZ-Nep inhibited clonogenicity, metastasis, angiogenesis, epithelial–mesenchymal transition, and entry in the S phase of the cell cycle and induced apoptosis in GOS-3 glioblastoma cells. On the contrary, DZ-Nep inhibited cell migration (single treatment) and O(6)-methylguanine-DNA methyltransferase expression (DZ-Nep or double treatment). Conclusion: Panobinostat alone or the combination of panobinostat and DZ-Nep induce apoptosis and inhibit in vitro tumorigenesis and metastasis in GOS-3 glioblastoma cell lines.

Abstract 2035: MGMT inhibition leads to CDK4/6 inhibition and enhances palbociclib and abemaciclib activity in breast cancer

Abstract Background: MGMT (O6 methylguanine DNA methyltransferase), a DNA repair protein leading to chemotherapy resistance, increasingly studied for its cell cycle regulatory functions, also known to control ER expression and function, is overexpressed in a majority of cancers, including breast cancer. MGMT inhibition has been reported to restore ER function and sensitivity to hormonal therapy in tamoxifen resistant breast cancer. CDK4/6 is a cell cycle regulator targeted by a new class of drugs in the treatment of breast cancer in patients who had progressed during prior endocrine therapy. We investigated a potential correlative role between MGMT and CDK4/6 expression/activity. In this therapeutic context MGMT inhibition would have the dual role of increasing/restoring effect of endocrine therapy and facilitate activity of CDK4/6 inhibitors (Palbociclib and Abemaciclib). Methods: We have tested the effect of Antabuse (disulfiram, DSF), as an MGMT inhibitor, at nontoxic doses, on the expression of CDK4/6, or in combination with Palbociclib (PB) or Abemaciclib (LY2835219 - LY) on ER+ breast cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to PB or/and LY and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + LY and moderate synergism for DSF + PB. DSF, alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY), significantly inhibits expression of MGMT, CDK4/6, ERα and aldehyde dehydrogenase activity - all involved in breast cancer cell cycle proliferation and tumorigenesis. Furthermore, PB and LY dose dependently decreased MGMT and CDK4 expression in breast cancer cells and significantly accumulated breast cancer cells in G1 phase of the cell cycle. DSF, alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY) caused significant apoptosis in breast cancer cells. DSF inhibited colony formation which was further enhanced by addition of PB/LY (DSF ± PB/LY). Similarly, DSF alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY) decreased the metastatic potential of breast cancer cells. Conclusions: Our findings suggest that DSF as an MGMT inhibitor significantly enhances the antitumor effect of CDK4/6 inhibitors (PB or LY) in ER+ breast cancer. Citation Format: George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah L. Donohoe, Maxwell Albiero, Tarun Jella, Olivia Fukui, Cameron Piron, Santhi D. Konduri. MGMT inhibition leads to CDK4/6 inhibition and enhances palbociclib and abemaciclib activity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2017-2035

Abstract 285: Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including breast cancer. MGMT is a DNA repair protein leading to chemo resistance. MGMT expression directly correlates with ER expression and tamoxifen resistance. Aldehyde Dehydrogenase (ALDH) activity, as a progenitor/stem cell marker, while it has been reported to inversely correlate with MGMT expression in other cancers, has also been linked to chemotherapy and radiation resistance. Methods: We have tested the effect of Antabuse (disulfiram, DSF), as a dual MGMT and ALDH inhibitor, at nontoxic doses, in combination with Temozolomide (TMZ) or/and Cyclophosphamide (CP) on ER+ breast cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to TMZ or/and CP and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + CP and moderate synergism for DSF + TMZ. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP), significantly inhibits expression of MGMT, aldehyde dehydrogenase, ERα, E2F and BIRC5 gene (survivin) - all involved in breast cancer tumorigenesis. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) caused significant apoptosis in breast cancer cells. In a dose dependent manner, DSF inhibited colony formation, effect which was further enhanced by addition of TMZ/CP (DSF ± TMZ/CP). Similarly, DSF alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) decreased the metastatic potential of breast cancer cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and ALDH significantly enhances the antitumor effect of alkylators as CP and TMZ in ER+ breast cancer Citation Format: George C. Bobustuc, Deborah Donohoe, Alisher Holmuhamedov, Srivenugopal Kalkunte, Santhi D. Konduri. Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 285.

MGMT expression predicts response to temozolomide in pancreatic neuroendocrine tumors.

Temozolomide (TEM) showed encouraging results in well-differentiated pancreatic neuroendocrine tumors (WDPNETs). Low O(6)-methylguanine-DNA methyltransferase (MGMT) expression and MGMT promoter methylation within tumors correlate with a better outcome under TEM-based chemotherapy in glioblastoma. We aimed to assess whether MGMT expression and MGMT promoter methylation could help predict the efficacy of TEM-based chemotherapy in patients with WDPNET. Consecutive patients with progressive WDPNET and/or liver involvement over 50% who received TEM between 2006 and 2012 were retrospectively studied. Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines. Nuclear expression of MGMT was assessed by immunochemistry (H-score, 0-300) and MGMT promoter methylation by pyrosequencing. Forty-three patients (21 men, 58years (27-84)) with grade 1 WDPNET (n=6) or 2 (n=36) were analyzed. Objective response, stable disease, and progression rates were seen in 17 patients (39.5%), 18 patients (41.9%), and 8 patients (18.6%), respectively. Low MGMT expression (≤50) was associated with radiological objective response (P=0.04) and better progression-free survival (PFS) (HR=0.35 (0.15-0.81), P=0.01). Disease control rate at 18months of treatment remained satisfying with an MGMT score up to 100 (74%) but dropped with a higher expression. High MGMT promoter methylation was associated with a low MGMT expression and longer PFS (HR=0.37 (0.29-1.08), P=0.05). Low MGMT score (≤50) appears to predict an objective tumor response, whereas an intermediate MGMT score (50-100) seems to be associated with prolonged stable disease.

MGMT in colorectal cancer: a promising component of personalized treatment.

The identification of new, effective drugs is a pressing need in colorectal cancer (CRC) rescue therapy. Data examining O (6)-methylguanine-DNA-methyl transferase (MGMT) and its predictive role in temozolomide (TMZ) treatment in CRC are scarce. In this study, the effect of MGMT status on the cytotoxic sensitivity caused by TMZ was analyzed using cytology proliferation assays in colon cancer cell lines. MGMT protein expression was assessed with immunohistochemistry in 385 patients. Concordance between primary and metastatic sites and the role of MGMT status on survival were statistically analyzed. TMZ sensitivity was significantly affected by the level of MGMT protein expression. Of 385 cases, 13 (3.4%) demonstrated loss of MGMT expression. However, low MGMT expression levels were significantly more common in signet ring cell carcinomas (p = 0.011). In 111 of 385 cases, the overall concordance of MGMT status between primary tumor and metastatic sites was 66.67% (κ = 0.271, p < 0.001). The median progression-free survival was significantly different between groups with low or high MGMT expression for the irinotecan-based regimen (p = 0.025), but MGMT protein expression was not observed to be a prognostic factor. In conclusion, MGMT was an important in vitro predictor of TMZ activity in CRC. The rate of MGMT protein loss was low in metastatic CRC patients from China, and MGMT might be more commonly lost in signet ring cell carcinoma. The MGMT status at primary and metastatic sites was consistent, but the power of concordance was poor. Further study into these topics is warranted.

Sensitivity Analysis of the MGMT-STP27 Model and Impact of Genetic and Epigenetic Context to Predict the MGMT Methylation Status in Gliomas and Other Tumors

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Our model MGMT-STP27 allows prediction of the methylation status of the MGMT promoter using data from the Illumina's Human Methylation BeadChips (HM-27K and HM-450K) that is publically available for many cancer data sets. Here, we investigate the impact of the context of genetic and epigenetic alterations and tumor type on the classification and report on technical aspects, such as robustness of cutoff definition and preprocessing of the data. The association between gene copy number variation, predicted MGMT methylation, and MGMT expression revealed agene dosage effect on MGMT expression in lower grade glioma (World Health Organization gradeII/III) that in contrast to glioblastoma usually carry two copies of chromosome 10 on which MGMT resides (10q26.3). This implies some MGMT expression, potentially conferring residual repair function blunting the therapeutic effect of alkylating agents. A sensitivity analyses corroborated the performance of the original cutoff for various optimization criteria and for most data preprocessing methods. Finally, we propose an R package mgmtstp27 that allows prediction of the methylation status of the MGMT promoter and calculation of appropriate confidence and/or prediction intervals. Overall, MGMT-STP27 is a robust model for MGMT classification that is independent of tumor type and is adapted for single sample prediction.

The tumoral A genotype of the MGMT rs34180180 single-nucleotide polymorphism in aggressive gliomas is associated with shorter patients' survival.

Malignant gliomas are the most common primary brain tumors. Grade III and IV gliomas harboring wild-type IDH1/2 are the most aggressive. In addition to surgery and radiotherapy, concomitant and adjuvant chemotherapy with temozolomide (TMZ) significantly improves overall survival (OS). The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter is predictive of TMZ response and a prognostic marker of cancer outcome. However, the promoter regions the methylation of which correlates best with survival in aggressive glioma and whether the promoter methylation status predictive value could be refined or improved by other MGMT-associated molecular markers are not precisely known. In a cohort of 87 malignant gliomas treated with radiotherapy and TMZ-based chemotherapy, we retrospectively determined the MGMT promoter methylation status, genotyped single nucleotide polymorphisms (SNPs) in the promoter region and quantified MGMT mRNA expression level. Each of these variables was correlated with each other and with the patients' OS. We found that methylation of the CpG sites within MGMT exon 1 best correlated with OS and MGMT expression levels, and confirmed MGMT methylation as a stronger independent prognostic factor compared to MGMT transcription levels. Our main finding is that the presence of only the A allele at the rs34180180 SNP in the tumor was significantly associated with shorter OS, independently of the MGMT methylation status. In conclusion, in the clinic, rs34180180 SNP genotyping could improve the prognostic value of the MGMT promoter methylation assay in patients with aggressive glioma treated with TMZ.

Expression profiling of O(6) methylguanine-DNA-methyl transferase in prolactinomas: a correlative study of promoter methylation and pathological features in 136 cases.

BackgroundLow-level expression of O6 methylguanine-DNA-methyl transferase (MGMT) prolactinomas has been noted previously in case reports, although what modulates MGMT expression remains unclear. This study therefore aimed to delineate the factors regulating MGMT expression in prolactinomas.MethodsWe retrospectively reviewed 136 prolactinoma patients who were treated in our center between January 2000 and September 2013. Expression of MGMT, Ki-67, and p53 protein were examined by immunohistochemical staining, and MGMT promoter methylation evaluated with methylation-specific PCR.ResultsMGMT immunopositivity was <25 % in 106/136 tumor specimens (77.94 %). MGMT immunoexpression was positively correlated with age (r = 0.251, p = 0.003), but inversely correlated with p53 staining (r = −0.153, p = 0.021). Moreover, reduced MGMT expression was more frequent in atypical prolactinomas (p = 0.044). Methylated MGMT promoter was confirmed in 10/46 specimens (21.7 %), all of which had low level or absent MGMT staining. Both p53 protein (r = −0.33, p = 0.025) and promoter methylation (r = −0.331, p = 0.025) were negatively associated with MGMT expression. Multivariate logistic analysis indicated that age (odds ratio [OR] = 1.127. 95 % confidence interval [CI] 1.027–1.236, p = 0.012) and p53 (OR = 0.116. 95 % CI 0.018–0.761, p = 0.025) staining were independent determents of MGMT expression.ConclusionsThe majority of prolactinomas, especially atypical prolactinomas, showed low-level or no MGMT immunoexpression, providing a rationale for the utility of temozolomide as an alternative to managing prolactinomas. In summary, epigenetic and transcriptional regulation are involved in silencing MGMT expression.

Abstract 3493: Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including pancreatic cancer. MGMT has been the focus of significant research for its role in the repair of DNA damage caused by chemotherapeutic agents. Aldehyde Dehydrogenase as a progenitor/stem cell marker has been linked to chemotherapy and radiation resistance. Methods: We tested the administration of Antabuse (disulfiram, DSF) at nontoxic doses in combination with gemcitabine (GEM) and Abraxane (ABX) on pancreatic cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases pancreatic cancer cell growth in a dose-dependent manner. DSF further sensitizes pancreatic cancer cells to GEM and ABX and significantly inhibits pancreatic cancer growth without causing unwanted side effects to the normal pancreatic cells. Dose effect and isobologram studies confirm synergistic activity of DSF and ABX combination and moderate synergism for DSF and GEM combination. DSF, either alone or in combination with GEM (DSF ± GEM) and/or ABX (DSF ± ABX), significantly inhibits expression of MGMT, aldehyde dehydrogenase, antigen Ki-67 and BIRC5 gene (survivin) - all involved in pancreatic cancer tumorigenesis. DSF+/-GEM+/-ABX induces the expression of p21Cip1, a cell cycle inhibitor. We show an inverse correlation between aldehyde dehydrogenase activity and MGMT expression, where MGMT expression is high, aldehyde dehydrogenase activity is low and where aldehyde dehydrogenase activity is high, MGMT levels were low. The ALDH3A1 expression is significantly higher in highly metastatic cancer cells (L3.6pl) compared to other metastatic cancer cells (PANC1, Mia PaCa2, ASPC1). We further report that in our model DSF at very low doses (achievable in human serum with standard DSF clinical dosing) effectively inhibits both aldehyde dehydrogenase and MGMT in pancreatic cancer cells. DSF also induces caspase activation indicative of mitochondrial induced apoptosis in these cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and aldehyde dehydrogenase potentially provides a novel therapeutic approach to inhibiting pancreatic cancer growth/metastasis and synergistically enhances GEM and ABX activity. Citation Format: George C. Bobustuc, Alisher Holmuhamedov, Kalkunte S. Srivenugopal, Jacob C. Frick, JAMES L. WEESE, Santhi D. Konduri. Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2015-3493

Abstract 4458: The HIV-derived protein Vpr52-96 has anti-glioma activity in vitro and in vivo

Abstract Background: Patients with actively replicating human immunodeficiency virus (HIV) are highly radiosensitive and exhibit adverse reactions even at low irradiation doses. A major underlying cause is high intra- and extracellular levels of a virus-encoded peptide termed viral protein R (Vpr). As Vpr efficiently crosses the blood-brain barrier and accumulates in astrocytes, we examined whether it may be utilized as a drug in the treatment of glioblastoma multiforme (GBM). Methods: Four glioblastoma-derived cell lines with and without lentivirus-mediated Methylguanine-DNA methyltransferase (MGMT) overexpression (U251, U87, U251-MGMT, U87-MGMT) were exposed to Vpr, temozolomide (TMZ), conventional photon irradiation (2-6 Gy) or to combinations. Caspase assays were employed to detect apoptosis. Colony formation assays were used to analyse clonogenic survival. Dose escalation was performed using n = 12 NOD/SCID/γc−/− (NSG) mice in a classical 3+3 manner. This strain was also used as an orthotopic implant model of glioma using luciferase-expressing U87 cells (n = 18). Continuous Vpr administration was achieved using osmotic pumps, irradiation (3×5 Gy) was performed using a small-animal irradiation device and intracerebral tumor growth was monitored using repetitive contrast-enhanced micro CT scans. Results: Vpr showed high rates of acute toxicities with a LD50 of 4.0±1.1 μM for U251 and a LD50 of 15.7±7.5 μM for U87 cells. Caspase assays revealed Vpr-induced apoptosis in U251 but not in U87 cells. Vpr also efficiently inhibited clonogenic survival both in U251 and U87 cells and showed additive effects with irradiation. In contrast to TMZ, Vpr acted independently of the MGMT expression status. Dose escalation in mice (n = 12) was feasible and resulted in no evident renal or liver toxicity. Similar to irradiation with 3 × 5 Gy (n = 8), Vpr treatment of orthotopically implanted NSG mice (n = 5) delayed tumor growth and significantly (p = 0.041; log-rank test) prolonged overall survival compared to untreated animals (n = 5). Conclusion: The HIV-encoded peptide Vpr exhibits all properties of an effective chemotherapeutic drug and may be a useful agent in the treatment of GBM. Citation Format: Jens Kuebler, Stefanie Kirschner, Linda Hartmann, Grit Welzel, Maren Engelhardt, Carsten Herskind, Marlon R. Veldwijk, Christian Schultz, Manuela Felix, Gerhard Glatting, Patrick Maier, Frederik Wenz, Marc A. Brockmann, Frank A. Giordano. The HIV-derived protein Vpr52-96 has anti-glioma activity in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4458. doi:10.1158/1538-7445.AM2015-4458

Evaluation of extracellular matrix protein CCN1 as a prognostic factor for glioblastoma.

Recently, research efforts in identifying prognostic molecular biomarkers for malignant glioma have intensified. Cysteine-rich protein 61 (CCN1) is one of the CCN family of matricellular proteins that promotes cell growth and angiogenesis in cancers through its interaction with several integrins. In this study, we investigated the relationships among CCN1, O(6)-methylguanine-DNA methyltransferase expression, the tumor removal rate, and prognosis in 46 glioblastoma patients treated at the Okayama University Hospital. CCN1 expression was high in 31 (67%) of these patients. The median progression-free survival (PFS) and overall survival (OS) times of patients with high CCN1 expression was significantly shorter than those of patients with low CCN1 expression (p<0.005). In a multivariate Cox analysis, CCN1 proved to be an independent prognostic factor for patient survival [PFS, hazard ratio (HR)=3.53 (1.55-8.01), p=0.003 and OS, HR=3.05 (1.35-6.87), p=0.007]. Moreover, in the 31 patients who underwent gross total resection, the PFS and OS times of those with high CCN1 expression were significantly shorter than those with low CCN1 expression. It was concluded that CCN1 might emerge as a significant prognostic factor regarding the prognosis of glioblastoma patients.