Davide Sciuscio

Extent and Patterns of MGMT Promoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Epigenetic Deregulation of DNA Repair and Its Potential for Therapy

Epigenetic silencing of essential components of DNA repair pathways is a common event in many tumor types, and comprise O6-methylguanine-DNA methyltransferase (MGMT), human mut L homolog 1 (hMLH1), Werner syndrome gene (WRN), breast cancer susceptibility gene 1 (BRCA1), and genes of the Fanconi anemia pathway. Most interestingly, some of these alterations become the Achilles heel of the affected tumors upon treatment with certain classes of anticancer agents. That is, patients whose tumors carry such defects can be stratified for respective therapy rendering some classic DNA damaging agents, such as alkylators or DNA crosslinking agents, into “targeted therapies.” Here we review some of the affected repair pathways that, when inactivated, sensitize the tumors to specific drugs and are thus exploitable for individualized therapy. (Clin Cancer Res 2009;15(16):5026–31)

Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma

BackgroundHOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma.ResultsWe observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively.ConclusionsBased on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Epigenetics and Brain Cancer

Epigenetic alterations have been recognized as important mechanisms in neoplastic transformation, malignant progression of cancer, and response to therapy. Epigenetic modifications include DNA methylation and posttranslational modifications of histone proteins that influence the chromatin structure. Moreover, with the identification of the RNA interference machinery, a new layer of gene regulation has been added to the definition. The coordinated interaction of these processes regulates gene expression activity. The disruption of these mechanisms of control is involved in a wide variety of pathologies, including but not restricted to cancer. Although epigenetic changes are somatically inheritable, they are reversible and hence may represent actionable targets for novel therapies.

Abstract 4031: MGMT methylation based outcome prediction is associated with two CpG regions separated by a prediction minimum centred at the initiation start site

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Promoter methylation of the repair gene MGMT is a predictive factor for benefit from alkylating agent therapy in glioblastoma patients. High through-put platforms for genome wide DNA methylation analysis have allowed establishing the methylome of large series of patient samples that has identified a glioma CpG methylator phenotype (G-CIMP). However, classification of samples as being methylated or unmethylated for a given gene is not obvious, since the relationship between CpG-methylation at a given site and the extent of the overall CpG island methylation, and the effect on gene silencing is strongly dependent on the location. For the MGMT gene 176 CpGs are interrogated on the 450K chip of which 18 are located in the CpG island associated with the promoter region. Correlation of MGMT methylation at these 18 CpG sites in 63 glioblastoma and 5 non tumoral brain tissues and methylation prediction established previously by methylation specific PCR (MSP) indicated 2 regions with highest association. The two areas were separated by a prediction minimum centered at the initiation start site. The correlation of MGMT expression with the methylation status of CpGs followed the same pattern with the strongest negative correlation reaching –0.5 at the 2 peaks. Subsequently we tested the predictive value of the 18 CpGs for outcome in these patients treated within clinical trials with temozolomide containing chemo-radiotherapy. Most intriguingly, correlation with gene expression, survival analyses and association with MSP displayed almost superposable images of CpGs, at the previously identified CpG regions! A model for MGMT methylation classification based on stepwise logistic regression was constructed with a kappa value close to 0.85. The model was validated in a published external data set of homogeneously treated patient cohort with anaplastic glioma for which methylation data and survival information was available, and for which MGMT methylation was reported as prognostic factor. The model was subsequently applied to the methylation data available from the The Cancer Genome Atlas (TCGA) glioblastoma data-set comprising 241 samples. Classification of the samples for their MGMT methylation status based on the predictor revealed that MGMT is frequently methylated in glioblastoma with G-CIMP (simulated p=0.001 chi-square, Monte-Carlo method), but is not a G-CIMP gene. Taken together, the location of methylated CpGs within the promoter is crucial for prediction of MGMT silencing and subsequent impact on outcome. Hence, for efficient mining of large scale methylation data we need models to identify regions of CpGs with a high impact on gene silencing or other features relevant for the understanding of tumor biology and effect on response to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4031. doi:1538-7445.AM2012-4031

Abstract 4927: MGMT promoter methylation is enriched in glioblastoma derived spheres as compared to respective original tumor tissue and is associated with loss of expression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Epigenetic silencing of the O6-methylaguanine DNA methyl transferase (MGMT) gene by promoter hypermethylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Quantitative methylation specific PCR (Q-MSP) suggests that not all cells in a glioblastoma exhibiting MGMT methylation carry a methylated MGMT allele, even after adjustment for tumor cell content. This raises the question of the clinically relevant methylation threshold for predicting response to therapy for treatment stratification. There are several potential reasons: heterogeneity of glioblastoma; contaminating normal tissue; a heterogenous methylation pattern not correctly detectable by the assay, or presence of MGMT methylation in distinct subpopulations of cells, namely tumor initiating cells. In order to address the question we investigated the degree and pattern of MGMT promoter methylation in 10 paired samples of glioblastoma and spheres derived thereof using Q-MSP and methylation specific clone sequencing. We observed that in MGMT methylated glioblastoma, the percentage of methylated alleles ranged from 10 to 90%. In contrast, in most of the respective glioblastoma derived spheres, cultured under stem cell conditions, methylated alleles were highly enriched (10/10 clones), even if both MGMT-alleles were retained. The methylation pattern was conserved in the spheres, even when passing them in nude mice. Most importantly, this hypermethylation was associated with complete loss of MGMT expression. For one case we observed only 50% methylated clones in the sphere fraction, associated with low expression of MGMT. Taken together, our data suggest that in MGMT methylated glioblastoma, cells with MGMT promoter hypermethylation are enriched in the sphere fraction which supposedly comprises cancer initiating cells. Thus, even a low percentage of MGMT methylation in a glioblastoma could be predictive marker for a benefit from alkylating agent therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4927.