This open-label, single-arm, phase II study combined enzastaurin with temozolomide plus radiation therapy (RT) to treat glioblastoma multiforme (GBM) and gliosarcoma. Adults with newly diagnosed disease and Karnofsky performance status (KPS) ≥ 60 were enrolled. Treatment was started within 5 weeks after surgical diagnosis. RT consisted of 60 Gy over 6 weeks. Temozolomide was given at 75 mg/m2 daily during RT and then adjuvantly at 200 mg/m2 daily for 5 days, followed by a 23-day break. Enzastaurin was given once daily during RT and in the adjuvant period at 250 mg/day. Cycles were 28 days. The primary end point was overall survival (OS). Progression-free survival (PFS), toxicity, and correlations between efficacy and molecular markers analyzed from tumor tissue samples were also evaluated. A prospectively planned analysis compared OS and PFS of the current trial with outcomes from 3 historical phase II trials that combined novel agents with temozolomide plus RT in patients with GBM or gliosarcoma. Sixty-six patients were enrolled. The treatment regimen was well tolerated. OS (median, 74 weeks) and PFS (median, 36 weeks) results from the current trial were comparable to those from a prior phase II study using erlotininb and were significantly better than those from 2 other previous studies that used thalidomide or cis-retinoic acid, all in combination with temozolomide plus RT. A positive correlation between O-6-methylguanine-DNA methyltransferase promoter methylation and OS was observed. Adjusting for age and KPS, no other biomarker was associated with survival outcome. Correlation of relevant biomarkers with OS may be useful in future trials.

Purpose

To address the association between sequence variants within the MGMT promoter-enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy.

Experimental Design

SNPs identified through sequencing a 1.9kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide was assessed.

Results

The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20–41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to temozolamide was strongly correlated with levels of MGMT methylation and expression.

Conclusions

These studies provide strong evidence that the A allele of a MGMT promoter-enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, temozolamide treatment may benefit a subset of lung cancer patients methylated for MGMT.

HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using RT-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrantly activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a PI3K inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with TRAIL resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets, and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.

The haploid human genome contains approximately 29 million CpGs that exist in a methylated, hydroxymethylated or unmethylated state, collectively referred to as the DNA methylome. The methylation status of cytosines in CpGs and occasionally in non-CpG cytosines influences protein–DNA interactions, gene expression, and chromatin structure and stability. The degree of DNA methylation at particular loci may be heritable transgenerationally and may be altered by environmental exposures and diet, potentially contributing to the development of human diseases. For the vast majority of normal and disease methylomes however, less than 1% of the CpGs have been assessed, revealing the formative stage of methylation mapping techniques. Thus, there is significant discovery potential in new genome-scale platforms applied to methylome mapping, particularly oligonucleotide arrays and the transformative technology of next-generation sequencing. Here, we outline the currently used methylation detection reagents and their application to microarray and sequencing platforms. A comparison of the emerging methods is presented, highlighting their degrees of technical complexity, methylome coverage and precision in resolving methylation. Because there are hundreds of unique methylomes to map within one individual and interindividual variation is likely to be significant, international coordination is essential to standardize methylome platforms and to create a full repository of methylome maps from tissues and unique cell types.

Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5′ CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.

Much recent effort has focused on identifying and characterizing cellular markers that distinguish tumor propagating cells (TPCs) from more differentiated progeny. We report here an unusual promoter DNA methylation pattern for one such marker, the cell surface antigen CD133 (Prominin 1). This protein has been extensively used to enrich putative cancer propagating stem-like cell populations in epithelial tumors, and especially, glioblastomas. We find that, within individual cell lines of cultured colon cancers and glioblastomas, the promoter CpG island of CD133 is DNA methylated, primarily, in cells with absent or low expression of the marker protein whereas lack of such methylation is evident in purely CD133+ cells. Differential histone modification marks of active versus repressed genes accompany these DNA methylation changes. This heterogeneous CpG island DNA methylation status in the tumors is unusual in that other DNA hypermethylated genes tested in such cultures preserve their methylation patterns between separated CD133+ and CD133− cell populations. Furthermore, the CD133 DNA methylation seems to constitute an abnormal promoter signature since it is not found in normal brain and colon but only in cultured and primary tumors. Thus, the DNA methylation is imposed on the transition between the active versus repressed transcription state for CD133 only in tumors. Our findings provide additional insight for the dynamics of aberrant DNA methylation associated with aberrant gene silencing in human tumors.

Background

Hypertonic saline (HTS) reduces the severity of lung injury in ischemia-reperfusion, endotoxin-induced and ventilation-induced lung injury. However, the potential for HTS to modulate the resolution of lung injury is not known. We investigated the potential for hypertonic saline to modulate the evolution and resolution of oleic acid induced lung injury.

Methods

Adult male Sprague Dawley rats were used in all experiments. Series 1 examined the potential for HTS to reduce the severity of evolving oleic acid (OA) induced acute lung injury. Following intravenous OA administration, animals were randomized to receive isotonic (Control, n = 12) or hypertonic saline (HTS, n = 12), and the extent of lung injury assessed after 6 hours. Series 2 examined the potential for HTS to enhance the resolution of oleic acid (OA) induced acute lung injury. Following intravenous OA administration, animals were randomized to receive isotonic (Control, n = 6) or hypertonic saline (HTS, n = 6), and the extent of lung injury assessed after 6 hours.

Results

In Series I, HTS significantly reduced bronchoalveolar lavage (BAL) neutrophil count compared to Control [61.5 ± 9.08 versus 102.6 ± 11.89 × 103 cells.ml-1]. However, there were no between group differences with regard to: A-a O2 gradient [11.9 ± 0.5 vs. 12.0 ± 0.5 KPa]; arterial PO2; static lung compliance, or histologic injury. In contrast, in Series 2, hypertonic saline significantly reduced histologic injury and reduced BAL neutrophil count [24.5 ± 5.9 versus 46.8 ± 4.4 × 103 cells.ml-1], and interleukin-6 levels [681.9 ± 190.4 versus 1365.7 ± 246.8 pg.ml-1].

Conclusion

These findings demonstrate, for the first time, the potential for HTS to reduce pulmonary inflammation and enhance the resolution of oleic acid induced lung injury.

Background

Meningiomas are common brain tumors that are classified into three World Health Organization grades (benign, atypical and malignant) and are molecularly ill-defined tumors. The purpose of this study was identify molecular signatures unique to the different grades of meningiomas and to unravel underlying molecular mechanisms driving meningioma tumorigenesis.

Results

We have used a combination of gene expression microarrays and array comparative genomic hybridization (aCGH) to show that meningiomas of all three grades fall into two main molecular groups designated 'low-proliferative' and 'high-proliferative' meningiomas. While all benign meningiomas fall into the low-proliferative group and all malignant meningiomas fall into the high-proliferative group, atypical meningiomas distribute into either one of these groups. High-proliferative atypical meningiomas had an elevated median MIB-1 labeling index and a greater frequency of copy number aberrations (CNAs) compared to low-proliferative atypical meningiomas. Additionally, losses on chromosome 6q, 9p, 13 and 14 were found exclusively in the high-proliferative meningiomas. We have identified genes that distinguish benign low-proliferative meningiomas from malignant high-proliferative meningiomas and have found that gain of cell-proliferation markers and loss of components of the transforming growth factor-beta signaling pathway were the major molecular mechanisms that distinguish these two groups.

Conclusion

Collectively, our data suggests that atypical meningiomas are not a molecularly distinct group but are similar to either benign or malignant meningiomas. It is anticipated that identified molecular and CNA markers will potentially be more accurate prognostic markers of meningiomas.

A single method that detects multiple common cancer types at an early stage would have the biggest payoff for cancer control, say Brena and colleagues.

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.

Background

Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.

Methods

We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting.

Results

Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100β-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins.

Conclusion

microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.