Christopher E. Duymich

Identifying aggressive prostate cancer foci using a DNA methylation classifier

BackgroundSlow-growing prostate cancer (PC) can be aggressive in a subset of cases. Therefore, prognostic tools to guide clinical decision-making and avoid overtreatment of indolent PC and undertreatment of aggressive disease are urgently needed. PC has a propensity to be multifocal with several different cancerous foci per gland.ResultsHere, we have taken advantage of the multifocal propensity of PC and categorized aggressiveness of individual PC foci based on DNA methylation patterns in primary PC foci and matched lymph node metastases. In a set of 14 patients, we demonstrate that over half of the cases have multiple epigenetically distinct subclones and determine the primary subclone from which the metastatic lesion(s) originated. Furthermore, we develop an aggressiveness classifier consisting of 25 DNA methylation probes to determine aggressive and non-aggressive subclones. Upon validation of the classifier in an independent cohort, the predicted aggressive tumors are significantly associated with the presence of lymph node metastases and invasive tumor stages.ConclusionsOverall, this study provides molecular-based support for determining PC aggressiveness with the potential to impact clinical decision-making, such as targeted biopsy approaches for early diagnosis and active surveillance, in addition to focal therapy.

Identification of DNA Methylation–Independent Epigenetic Events Underlying Clear Cell Renal Cell Carcinoma

Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation-independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954-64. ©2016 AACR.

Genetic and Epigenetic Alterations in Bladder Cancer

Bladder cancer is one of the most common cancers worldwide, with a high rate of recurrence and poor outcomes as a result of relapse. Bladder cancer patients require lifelong invasive monitoring and treatment, making bladder cancer one of the most expensive malignancies. Lines of evidence increasingly point to distinct genetic and epigenetic alteration patterns in bladder cancer, even between the different stages and grades of disease. In addition, genetic and epigenetic alterations have been demonstrated to play important roles during bladder tumorigenesis. This review will focus on bladder cancer-associated genomic and epigenomic alterations, which are common in bladder cancer and provide potential diagnostic markers and therapeutic targets for bladder cancer treatment.

MP98-02 A POTENTIAL ROLE OF ABERRANT DNA METHYLATION IN THE CHEMORESISTANCE IN BLADDER CANCER CELLS. DNA METHYLATION INHIBITORS COULD RE-SENSITIZE DRUG-RESISTANCE BLADDER CANCER CELLS.

INTRODUCTION AND OBJECTIVES: Aberrant DNA methylation is one of the well-known epigenetic changes in cancer, although its involvement in the chemoresistance remains to be elucidated. In this study, we aimed to unravel the roles played by DNA methylation in chemoresistance in bladder cancer (BCa). METHODS: We established gemcitabine (GEM)-resistant (T24RG and UMUC3-RG) or cisplatin (CDDP)-resistant (T24-RC and UNUC3-RC) BCa cell lines by continuously treating their parental cells. Genome-wide DNA methylation was assessed by Infinium HumanMethylation450 BeadChip (HM450). To assess the chromatin accessibilities, cells were treated with a CpG methyltransferase M.SssI, after which DNA methylation was analyzed by HM450. To evaluate whether treatment with epigenetic drugs could overcome the chemoresistance in BCa, cells were treated with a DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR) and/or a histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), after which they were treated with or without GEM or CDDP. RESULTS: HM450 assays revealed increased levels of methylation at a number of CpG sites in the resistant cells as compared the parental cells (730 sites in T24-RC and 3856 sites in UMUC3-RC, respectively), however, a number of CpG sites remained unmethylated but loss chromatin accessibility in the resistant cells (1391 sites in T24RG and 1322 sites in UMUC3-RG, respectively. In CDDP-resistant BCa cells, a combination treatment with 5-Aza-CdR and CDDP synergistically suppressed cell proliferation, suggesting that 5-Aza-CdR restored CDDP-sensitivity (Figure A, B). In contrast, 5-Aza-CdR didn’t show any growth inhibitory effects in GEM-resistant cells (Figure C, D). Moreover, a treatment with SAHA with or without 5-Aza-CdR also failed to restore GEM-resistance in BCa cells (Figure E, F). CONCLUSIONS: Our results suggest that epigenetic alteration may be one of key factors for drug resistance, drug resistance cells could be desensitized by DNA methylation inhibitors especially in the CDDP-resistance in BCa cells.

MP65-10 RECURRENT NON-MUSCLE INVASIVE UROTHELIAL CARCINOMA TUMORS HAVE COMMON ANCESTRAL CLONE

reviewed by 3 different expert uro-pathologists and kappa statistic for interobserver variability was calculated. RESULTS: Unsupervised clustering of data from RNA sequencing revealed classification of three robust-non-overlapping molecular subtypes of NMIBC termed Grade Related Index (GRI) 1, GRI2 and GRI3. GRI1 comprised of almost exclusively LG tumours, while GRI3 clustered with HG MIBC tumours. After assessment by expert pathologists, kappa for interobserver variability in 1973 WHO histological grading was 0.40 whereas it reached 0.78 for the 2004 classification. Most discrepant cases clustered in molecular subtype GRI2. GRI subclassification independently predicted disease progression in NMIBC (p1⁄40.004, gray test). FGFR3 mutations, FGFR3::TACC3 fusion events and Hedgehog were strongly enriched in GRI1. GRI3 disease was associated with a germ stem cell-like phenotype and upregulation in APOBEC3B. CONCLUSIONS: WT sequencing data delineated three molecular classes of NMIBC, and improved prediction of disease progression from NMIBC to MI compared to conventional histologic grading. WT analysis could be integrated to a new WHO classification.

Abstract B03: Identification of epigenetic regulated genes through simultaneous analysis of DNA methylation and chromatin structure in uncultured tumors

The contribution of promoter DNA methylation to the alteration of caner related gene expression has been well studied, however, these genes can also potentially be altered by chromatin accessibility without involvement of DNA methylation and can be epigenetically inherited. In this study, we used an assay developed in our laboratory (AcceSssIble) that can simultaneously interrogate DNA methylation and chromatin accessibility allowing us to investigate the epigenetic changes in uncultured clear cell renal cell carcinoma (ccRCC) tumors and normal tissue to uncover genes that contribute to ccRCC tumorigenesis. AcceSssIble is both cost-effective and easily analyzed using simple and straightforward computational analysis. Our study revealed significant changes to the epigenome of ccRCC, especially identifying epigenetically up- (160) or down-regulated genes (180), which are dependent on accessibility changes with (30%) or without (70%) DNA methylation involvement, and which were validated by a cross-correlation of the identified genes with RNA-seq and DNA methylation data from larger cohorts of ccRCC samples from The Cancer Genome Atlas (TCGA). In addition, our findings also revealed these sets of genes not found to be commonly mutated in ccRCC and undergo epigenetic changes at higher frequencies than common ccRCC mutations. In addition, pathway analysis suggests that genetic and epigenetic alterations are independent events, and one such example includes a set of changes in HIF1α signaling pathway genes that are independent of the Von Hippel-Lindau (VHL) status. This suggests a novel epigenetic basis for HIF1αs role during tumorigenesis that may be a common occurrence in ccRCC. Thus, the AcceSssIble analysis on ccRCC samples revealed novel candidates for epigenetic driver genes which were previously undetectable by widely used methylation studies. Overall, this study provides a novel approach that can help identify new epigenetic therapeutic targets and treatment strategies complementing current approaches based primarily on the genetic makeup of primary tumors. Citation Format: Elinne Becket, Sameer Chopra, Christopher Duymich, Lin J. Justin, Jueng Soo You, Kurinji Pandiyan, Peter W. Nichols, Kimberly D. Siegmund, Peter A. Jones, Gangning Liang. Identification of epigenetic regulated genes through simultaneous analysis of DNA methylation and chromatin structure in uncultured tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B03.

Unique Role for a DNA Methyltransferase Isoform in Lung Cancer.

Aberrant DNA methylation patterns are one of the most studied epigenetic components in cancer development. In certain cancers, altered expression of DNA methyltransferase (DNMT) family members could potentially cause this DNA methylation re-patterning. Specifically, DNMT3B has over thirty different isoforms with varied expression in tissues (Ostler et al. 2007). Recent studies have shown that DNMT3B is responsible for gene body methylation by recognizing the H3K36me3 modification found in gene bodies, especially gene body remethylation after treatment with a DNA methylation inhibitor (5-Aza-CdR) (Baubec et al., 2015, Yang et al., 2014). The methylated gene bodies showed positive correlations with gene expression and, additionally, hypomethylation of gene bodies could lead to down regulation of gene expression (Yang et al. 2014). Non-small cell lung cancer (NSCLC) is one such disease which has overexpression of DNMT3B, including a specific subfamily which lacks an N-terminal domain (ΔDNMT3B) (Wang et al. 2006). In this issue of EBioMedicine the work presented by Ma et al. (Ma et al. 2015), provides exciting developments for NSCLC with findings connecting the aberrant DNA methylation patterns during tumorigenesis with the predominantly expressed DNMT3B isoform: ΔDNMT3B4-del, a truncated DNMT3B isoform lacking exons 21 and/or 22 which contain the catalytic domain.

Abstract A1-05: Elucidation of epigenetic driver genes in clear cell renal cell carcinoma using a newly developed assay, AcceSssIble

Background: While many studies have uncovered genetic mutations that drive tumorigenesis, far fewer have described epigenetic changes, such as nucleosome positioning and DNA methylation, which lead to the development of cancer. Therefore, accurately mapping these changes between normal and tumor tissue will provide novel information to identify genes that undergo epigenetic changes that drive tumorigenesis (“epigenetic driver genes”). In this study, we used an assay developed in our laboratory to investigate the epigenetic changes between clear cell renal cell carcinoma (ccRCC, the most common subtype of renal carcinoma) tumors and normal tissue to uncover genes that contribute to ccRCC tumorigenesis. Methods: Current methods to investigate epigenomic changes in clinical samples are expensive and require abundant biological sample material for analysis. We have developed a novel assay (“Acce SssI ble”) to simultaneously determine DNA methylation and chromatin accessibility in clinical samples. It is rapid and cost-effective, only requiring 20 mg of tissue, the Infinium HumanMethylation450 BeadChip platform, and the CpG methyltransferase M.SssI. We used this method to measure the changes in DNA methylation and chromatin accessibility in 9 matched pairs of ccRCC tumors and adjacent normal tissue from different patients, and intersected this data with RNA-seq data of 72 matched ccRCC samples and DNA methylation data of 160 matched ccRCC samples from The Cancer Genome Atlas (TCGA). Genes that were revealed to have the most changes in chromatin structure and expression were then targeted by siRNA knockdown for functional validation in ccRCC. Results: From the Acce SssI ble assay on 9 pairs of ccRCC patient tumor/normal samples, we uncovered 438 genes whose promoters change in chromatin accessibility in at least 2 ccRCC samples, both dependent and independent of DNA methylation changes, and have an accompanying change in gene expression in TCGA RNA-seq data. The results produce a striking figure in which chromatin accessibility changes are inversely correlated with DNA methylation but directly correlated with gene expression changes. Interestingly, loss of (DNA methylation change-dependent) accessibility preferentially occurred within CpG islands, while gain of (DNA methylation change-dependent) accessibility was strongly biased towards non-CpG islands. Meanwhile, chromatin accessibility changes independent of DNA methylation changes do not show preference in CpG content. Furthermore, pathway analyses reveal involvement of HIF1α signaling, cAMP-mediated signaling, and G-protein Coupled Receptor Signaling in the development of ccRCC. Lastly, we performed siRNA knockdown experiments on several top genes most changing in expression and accessibility, which revealed two genes, encoding type IV collagen and an RNA-binding protein, whose knockdown resulted in a significant increase in proliferation in normal kidney epithelial cells. Conclusions: Our study revealed a vast number of chromatin accessibility and accompanying gene expression changes that occur in gene promoters in the development of ccRCC, both dependent and independent of DNA methylation changes. Each individual tumor has a unique profile of epigenetic alterations. Moreover, almost none of the genes that were found to undergo epigenetic and resulting gene expression changes overlap with TCGA9s findings of commonly mutated genes in ccRCC. Overall, these studies represent novel approaches that can help identify new therapeutic target genes and treatment strategies for ccRCC, including personalized approaches. Citation Format: Elinne Coral Becket, Christopher Duymich, Yin-Wei Chang, Kurinji Pandiyan, Peter Nichols, Peter Jones, Inderbir Gill, Gangning Liang. Elucidation of epigenetic driver genes in clear cell renal cell carcinoma using a newly developed assay, AcceSssIble. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-05.

Abstract LB-130: Simultaneous evaluation of chromatin accessibility and DNA methylation in clear cell renal cell carcinoma by a newly developed assay, AcceSssIble

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Epigenetic changes, such as nucleosome positioning and DNA methylation, control gene expression and are altered in cancer. Therefore, accurately mapping these changes between normal and tumor tissue will provide novel information for identification of epigenetic driver genes, biomarker discovery, and anti-tumor therapy development for clear cell renal cell carcinoma (ccRCC), the most common subtype of renal carcinoma. Current methods are expensive and require a considerable amount of biological tissue. Methods: We have developed a novel assay (AcceSssIble) to determine DNA methylation and nucleosome occupancy in a single experiment. It is rapid and cost-effective, requiring the Infinium HumanMethylation450 BeadChip platform and the CpG methyltransferase M.SssI. Preliminary studies using fresh and frozen kidney tumors show that the epigenetic profile is maintained in frozen tissues, making this method suitable to interrogate archived clinical samples. Results: We have used AcceSssIble to assay the epigenomes of 5 fresh ccRCC tumors and their adjacent normal tissue, and have uncovered 257 genes that are associated with changes in chromatin accessibility in at least 4 ccRCC samples; 155 of these genes also show a change in endogenous DNA methylation levels in at least 4 tumors. These chromatin accessibility and DNA methylation changes occurred in the promoter regions, open reading frames, and the 3’ untranslated regions (UTRs) of the genes. Cross-referencing our results to the expression data of 72 matched ccRCC samples from The Cancer Genome Atlas (TCGA), we discovered that 29% (74/257) of these genes exhibited a significant change in gene expression, over half of which had been previously implicated in various cancers, including ccRCC. Furthermore, we observed chromatin accessibility changes in intergenic regions, including several which contain known transcription factor binding sites and ncRNA promoter regions. These sites might serve as potential enhancers for neighboring genes important to the development of ccRCC. Conclusions: In these studies, we were able to identify epigenetic changes in ccRCC samples associated with known oncogenes and tumor suppressor genes, as well as uncover novel targets. These targets, associated both within genes as well as in intergenic regions, undergo changes in chromatin structure and may be important in the development of ccRCC. Linking these findings to gene expression data revealed functionally relevant targets for ccRCC treatment. Expansion of these studies into a larger number of clinical samples across tumor grades will allow us to uncover biomarkers that can be used for personalized medicine. Citation Format: Elinne Becket, Christopher Duymich, Yin-Wei Chang, Kurinji Pandiyan, Peter A. Jones, Gangning Liang. Simultaneous evaluation of chromatin accessibility and DNA methylation in clear cell renal cell carcinoma by a newly developed assay, AcceSssIble. [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 LB-130. doi:10.1158/1538-7445.AM2014-LB-130

Abstract A11: Role of chromatin remodeler mutations in urothelial cell carcinoma of the bladder

Traditionally cancer is viewed as a disease driven by the accumulation of genetic alterations; nowadays this view has been expanded to include epigenetic alterations. Recent developments such as exome-sequencing have enabled researchers to analyze the entire exome of cancer cell lines and tissue samples to determine possible genetic mutations causing tumor onset/progression, leading to improved disease prognosis and treatment therapies. These studies have uncovered that genes encoding components of the epigenetic machinery are frequently mutated in urothelial cell carcinoma (UCC) of the bladder and many other tumor types. The effects of these mutations on the epigenetic landscape, e.g. DNA methylation, histone modification, and nucleosome positioning are as yet unknown. Given that the disruption of normal epigenetic patterns contributes to carcinogenesis, it is critical to study the effect of genetic alterations on the epigenome, since the latter are reversible by pharmacological intervention. In this study, we sequenced the exomes of 15 bladder cancer cell lines and compared them to 17 additional cell lines analyzed by the Cancer Cell Line Encyclopedia (CCLE). We have identified that epigenetic regulators in UCC of the bladder are commonly mutated, confirming other reports pertaining to UCC. The most frequently mutated genes include chromatin regulators such as KDM6A, ARID1A, HDAC4, and CREBBP. In addition to the discovery of genetic modifications in UCC cell lines, we studied the potential impact of these mutations on DNA methylation and nucleosome positioning by performing the Infinium Human Methylation 450K Array and the novel AcceSssIble assay, which is also based on the 450K array; comparing M.SssI CpG methyltransferase treated chromatin samples against controls, leading to the identification of nucleosome occupied CpG sites. To identify possible correlations between chromatin regulator mutations and DNA methylation patterns, we compared UCC cell lines bearing a specific chromatin regulator mutation to cell lines without the mutation and searched for significantly differentially methylated CpG sites. We found that UCC cell lines with mutations in ARID1A and HDAC4 show significant DNA methylation changes compared to cell lines not carrying the mutation. Our results show a possible direct impact of genetic mutations leading to epigenetic changes that may contribute to the tumor phenotype and might lead to novel therapeutic targets in UCC. Further studies will determine the effects of these specific chromatin regulator mutations on their target gene expression, DNA methylation and nucleosome occupancy. Loss and gain of function studies of these chromatin regulators will further our understanding of their roles in UCC tumor progression. The target genes of these studies will be further interrogated with commonly used epigenetic drugs such as 5-Azacitidine and HDAC inhibitors. Citation Format: Christopher E. Duymich, Jessica Charlet, Gangning Liang, Peter A. Jones. Role of chromatin remodeler mutations in urothelial cell carcinoma of the bladder. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A11.