Platinum-derived drugs such as cisplatin and carboplatin are among the most commonly used cancer chemotherapy drugs, but very few specific molecular and cellular markers predicting differential sensitivity to these agents in a given tumor type have been clearly identified. Epigenetic gene silencing is increasingly being recognized as a factor conferring distinct tumoral drug sensitivity, so we have used a comprehensive DNA methylation microarray platform to interrogate the widely characterized NCI60 panel of human cancer cell lines with respect to CpG methylation status and cisplatin/carboplatin sensitivity. Using this approach, we have found promoter CpG island hypermethylation-associated silencing of the putative DNA/RNA helicase Schlafen-11 (SLFN11) to be associated with increased resistance to platinum compounds. We have also experimentally validated these findings in vitro. In this setting, we also identified the BRCA1 interacting DHX9 RNA helicase (also known as RHA) as a protein partner for SLFN11, suggesting a mechanistic pathway for the observed chemoresistance effect. Most importantly, we have been able to extend these findings clinically, following the observation that those patients with ovarian and non-small cell lung cancer carrying SLFN11 hypermethylation had a poor response to both cisplatin and carboplatin treatments. Overall, these results identify SLFN11 epigenetic inactivation as a predictor of resistance to platinum drugs in human cancer.
A unique resource for systems pharmacology and genomic studies is the NCI-60 cancer cell line panel, which provides data for the largest publicly available library of compounds with cytotoxic activity (~21,000 compounds), including 108 FDA-approved and 70 clinical trial drugs as well as genomic data, including whole-exome sequencing, gene and microRNA transcripts, DNA copy number, and protein levels. Here we provide the first readily usable genome-wide DNA methylation database for the NCI-60, including 485,577 probes from the Infinium HumanMethylation450k BeadChip array, which yielded DNA methylation signatures for 17,559 genes integrated into our open access CellMiner version 2.0 (https://discover.nci.nih.gov/cellminer). Among new insights, transcript versus DNA methylation correlations revealed the epithelial/mesenchymal gene functional category as being influenced most heavily by methylation. DNA methylation and copy number integration with transcript levels yielded an assessment of their relative influence for 15,798 genes, including tumor suppressor, mitochondrial, and mismatch repair genes. Four forms of molecular data were combined, providing rationale for microsatellite instability for 8 out of the 9 cell lines in which it occurred. Individual cell line analyses showed global methylome patterns with overall methylation levels ranging from 17 to 84%. A six-gene model including PARP1, EP300, KDM5C, SMARCB1 and UHRF1 matched this pattern. Additionally, promoter methylation of two translationally relevant genes Schlafen 11 (SLFN11) and methylguanine methyltransferase (MGMT) served as indicators of therapeutic resistance or susceptibility, respectively. Overall, our database provides a resource of pharmacological data that can reinforce known therapeutic strategies and identify novel drugs and drug targets across multiple cancer types.
Promoter CpG island hypermethylation of tumor suppressor genes is an epigenetic hallmark of human cancer commonly associated with nucleosome occupancy and the transcriptional silencing of the neighboring gene. Nucleosomes can determine the underlying DNA methylation status. Herein, we show that the opposite is also true: DNA methylation can determine nucleosome positioning. Using a cancer model and digital nucleosome positioning techniques, we demonstrate that the induction of DNA hypomethylation events by genetic (DNMT1/DNMT3B deficient cells) or drug (a DNA demethylating agent) approaches is associated with the eviction of nucleosomes from previously hypermethylated CpG islands of tumor suppressor genes. Most importantly, the establishment of a stable cell line that restores DNMT1/DNMT3B deficiency shows that nucleosomes reoccupy their positions in de novo methylated CpG islands. Finally, we extend these results to the genomic level, combining a DNA methylation microarray and the nucleosome positioning technique. Using this global approach, we observe the dependency of nucleosome occupancy upon the DNA methylation status. Thus, our results suggest that there is a close association between hypermethylated CpG islands and the presence of nucleosomes, such that each of these epigenetic mechanisms can determine the recruitment of the other.
<p>Methylation gene probe selection and correlation to expression level.</p>
<p>Linear regression analysis of 15,798 genes with with transcript expression, DNA methylation, and DNA copy number data available.</p>
<p>128,394 probes used for the determination of gene methylation levels.</p>
<p>Legends for Supplemental Figures S1-S2.</p>
The National Cancer Institute Antitumor Cell line panel NCI60 is a very frequently used collection of cancer cell lines that has been largely characterized in different biological aspects. Data from proteomic and RNA expression analyses of this panel is already available to the scientific community. Also, the IC50 data for more than 100,000 different compound treatments is accessible in the developmental therapeutic program of NCI (dtp). However, very little has been explored about the epigenetic component of this panel. We have performed a thorough correlational study combining the available molecular information of NCI60, the data of our 450K NCI60 dataset and the IC50 data from the dtp to discover candidate genes for new biomarkers of resistance to antitumor agents. Finding new biomarkers to determine the most probable response to the therapy seems to be a key stage in the development of personalized medicine. Due to its stability and how easy it is to detect its level of methylation, DNA methylation changes appear to be a perfect candidate mark for the construction of this new generation of biomarkers. In this study we obtained multiples candidate genes whose methylation correlated to resistance to different drugs. In order to perform validation analyses we chose a gene that showed a strong positive correlation with the resistance to several DNA damage agents (DDA).Using overexpression and silencing assays we were able to demonstrate that the expression of this gene has a profound influence on the cell sensitivity to the DDA. Furthermore, through gene expression validation assays, such as 5-azacytidine treatments, we demonstrated that the promotor methylation of this gene is associated to its non-expression. In addition, we observed that this gene methylation correlates with a lower overall survival in an ovarian cancer cohort treated with platinum agents. Little is known about our candidate gene but it seems to be related to the DNA repair machine and also to have a function in the cell cycle control. These features could give an explanation to the importance of its presence in the response to DDA treatment. Our studies show that the analysis of DNA methylation changes in cancer cells in combination with other molecular alterations can be used in correlational studies to find trustworthy biomarkers for antitumor drug resistance. Citation Format: Vanesa Nogales, Catia Moutinho, Anna Martinez-Cardús, Sudhir Varma, J. Keith Killian, William C. Reinhold, Paul S. Meltzer, Yves Pommier, Manel Esteller. Discovery of biomarkers for antitumor drug resistance using 450K methylation data of NCI60. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1366. doi:10.1158/1538-7445.AM2014-1366
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