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

The optimal treatment for patients with low-grade glioma (LGG) WHO grade II remains controversial. Overall survival ranges from 2 to over 15 years depending on molecular and clinical factors. Hence, risk-adjusted treatments are required for optimizing outcome and quality of life. We aim at identifying mechanisms and associated molecular markers predictive for benefit from radiotherapy (RT) or temozolomide (TMZ) in LGG patients treated in the randomized phase III trial EORTC 22033. As candidate biomarkers for these genotoxic treatments, we considered the DNA methylome of 410 DNA damage response (DDR) genes. We first identified 62 functionally relevant CpG sites located in the promoters of 24 DDR genes, using the LGG data from The Cancer Genome Atlas. Then we tested their association with outcome [progression-free survival (PFS)] depending on treatment in 120 LGG patients of EORTC 22033, whose tumors were mutant for isocitrate dehydrogenase 1 or 2 (IDHmt), the molecular hallmark of LGG. The results suggested that seven CpGs of four DDR genes may be predictive for longer PFS in one of the treatment arms that comprised MGMT, MLH3, RAD21, and SMC4. Most interestingly, the two CpGs identified for MGMT are the same, previously selected for the MGMT-STP27 score that is used to determine the methylation status of the MGMT gene. This score was higher in the LGG with 1p/19q codeletion, in this and other independent LGG datasets. It was predictive for PFS in the TMZ, but not in the RT arm of EORTC 22033. The results support the hypothesis that a high score predicts benefit from TMZ treatment for patients with IDHmt LGG, regardless of the 1p/19q status. This MGMT methylation score may identify patients who benefit from first-line treatment with TMZ, to defer RT for long-term preservation of cognitive function and quality of life.

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short-term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30-37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30-min preincubation of isolated mitochondria at 25-40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short-term high temperature events associated with global warming.

Purpose: The methylation status of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is predictive for benefit from temozolomide in glioblastoma (GBM). A clinically optimized cutoff was sought allowing patient selection for therapy without temozolomide, while avoiding to withhold it from patients who may potentially benefit. Experimental Design: Quantitative MGMT methylation-specific PCR data were obtained for newly diagnosed patients with GBM screened or treated with standard radiotherapy and temozolomide in four randomized trials. The pooled dataset was randomly split into a training and test dataset. The unsupervised cutoff was obtained at a 50% probability to be (un)methylated. ROC analysis identified an optimal cutoff supervised by overall survival (OS). Results: For 4,041 patients valid MGMT results were obtained, whereof 1,725 were randomized. The unsupervised cutoff in the training dataset was 1.27 (log2[1,000 × (MGMT+1)/ACTB]), separating unmethylated and methylated patients. The optimal supervised cutoff for unmethylated patients was −0.28 (AUC = 0.61), classifying “truly unmethylated” (≤−0.28) and “gray zone” patients (>−0.28, ≤1.27), the latter comprising approximately 10% of cases. In contrast, for patients with MGMT methylation (>1.27) more methylation was not related to better outcome. Both methylated and gray zone patients performed significantly better for OS than truly unmethylated patients [HR = 0.35, 95% confidence interval (CI), 0.27–0.45, P < 0.0001; HR = 0.58, 95% CI, 0.43–0.78, P < 0.001], validated in the test dataset. The MGMT assay was highly reproducible upon retesting of 218 paired samples (R2 = 0.94). Conclusions: Low MGMT methylation (gray zone) may confer some sensitivity to temozolomide treatment, hence the lower safety margin should be considered for selecting patients with unmethylated GBM into trials omitting temozolomide.

Purpose: EORTC 26082 assessed the activity of temsirolimus in patients with newly diagnosed glioblastoma harboring an unmethylated O6 methylguanine-DNA-methyltransferase (MGMT) promoter. Experimental Design: Patients (n = 257) fulfilling eligibility criteria underwent central MGMT testing. Patients with MGMT unmethylated glioblastoma (n = 111) were randomized 1:1 between standard chemo-radiotherapy with temozolomide or radiotherapy plus weekly temsirolimus (25 mg). Primary endpoint was overall survival at 12 months (OS12). A positive signal was considered >38 patients alive at 12 months in the per protocol population. A noncomparative reference arm of 54 patients evaluated the assumptions on OS12 in a standard-treated cohort of patients. Prespecified post hoc analyses of markers reflecting target activation were performed. Results: Both therapies were administered per protocol with a median of 13 cycles of maintenance temsirolimus. Median age was 55 and 58 years in the temsirolimus and standard arms, the WHO performance status 0 or 1 for most patients (95.5%). In the per protocol population, 38 of 54 patients treated with temsirolimus reached OS12. The actuarial 1-year survival was 72.2% [95% confidence interval (CI), 58.2–82.2] in the temozolomide arm and 69.6% (95% CI, 55.8–79.9) in the temsirolimus arm [hazard ratio (HR) 1.16; 95% CI, 0.77–1.76; P = 0.47]. In multivariable prognostic analyses of clinical and molecular factors, phosphorylation of mTORSer2448 in tumor tissue (HR 0.13; 95% CI, 0.04–0.47; P = 0.002), detected in 37.6%, was associated with benefit from temsirolimus. Conclusions: Temsirolimus was not superior to temozolomide in patients with an unmethylated MGMT promoter. Phosphorylation of mTORSer2448 in the pretreatment tumor tissue may define a subgroup benefitting from mTOR inhibition. Clin Cancer Res; 22(19); 4797–806. ©2016 AACR.

Purpose The clinical consequences of PMS2 germline mutations are poorly understood compared with other Lynch-associated mismatch repair gene (MMR) mutations. The aim of this European cohort study was to define the cancer risk faced by PMS2 mutation carriers. Methods Data were collected from 98 PMS2 families ascertained from family cancer clinics that included a total of 2,548 family members and 377 proven mutation carriers. To adjust for potential ascertainment bias, a modified segregation analysis model was used to calculate colorectal cancer (CRC) and endometrial cancer (EC) risks. Standardized incidence ratios (SIRs) were calculated to estimate risks for other Lynch syndrome–associated cancers. Results The cumulative risk (CR) of CRC for male mutation carriers by age 70 years was 19%. The CR among female carriers was 11% for CRC and 12% for EC. The mean age of CRC development was 52 years, and there was a significant difference in mean age of CRC between the probands (mean, 47 years; range, 26 to 68 years) and other family members with a PMS2 mutation (mean, 58 years; range, 31 to 86 years; P < .001). Significant SIRs were observed for cancers of the small bowel, ovaries, breast, and renal pelvis. Conclusion CRC and EC risks were found to be markedly lower than those previously reported for the other MMR. However, these risks embody the isolated risk of carrying a PMS2 mutation, and it should be noted that we observed a substantial variation in cancer phenotype within and between families, suggesting the influence of genetic modifiers and lifestyle factors on cancer risks.