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Understanding oncogenic epigenetic mechanisms in brain tumors is crucial for improved diagnosis and treatment. Recently DNA methylation has proven to be powerful for brain tumor characterization and diagnostic classification. To evaluate tumor type specific features, we compared atypical teratoid/rhabdoid tumors (AT/RTs), medulloblastomas (MBs), and choroid plexus tumors with each other by integrating DNA methylation (507 samples), gene expression (120 samples), and transcription factor (TF) -binding data. Different tumor entities were used to find unique changes affecting each of the entities and further to identify functions driven by these changes. Our results provide insight on how the aberrant DNA methylation induces oncogenesis of AT/RTs. These tumors are known for their aggressiveness and exceptionally low mutation rates. Our results suggest that in AT/RT, elevated DNA methylation masks the binding sites of TFs such as NEUROD1, ASCL1 and MYCN driving neural development. DNA methylation in AT/RTs is also associated with reduced gene expression for specific neural regulators such as NEUROG1 and NEUROD2. For MBs, DNA methylation patterns predict a more advanced differentiation state. In MB, we found masked TF binding sites for TFs such as REST and ZEB1 that normally inhibit neural differentiation. We then wanted to further characterize DNA methylation and compared these tumors to pluripotent stem cells (PSCs) and normal fetal brain samples. As a result, we were able to find two different regulatory programs in AT/RTs: One in which DNA methylation is similar to PSCs and which harbors mostly neural TF binding sites. Second program has AT/RT-specific DNA methylation, and these sites are uniquely associated with polycomb repressive complex 2 members. However, this second program also covers neural TF binding sites and is likely to have relevance in oncogenic regulation.

Fast progress of the next generation sequencing (NGS) technology has allowed global transcriptional profiling and genome-wide mapping of transcription factor binding sites in various cellular contexts. However, limited number of replicates and high amount of data processing may weaken the significance of the findings. Comparative analyses of independent data sets acquired in the different laboratories would greatly increase the validity of the data. Runx2 is the key transcription factor regulating osteoblast differentiation and bone formation. We performed a comparative analysis of three published Runx2 data sets of chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis in osteoblasts from mouse and human origin. Moreover, we assessed the similarity of the corresponding transcription data of these studies available online. The ChIP-seq data analysis confirmed general features of Runx2 binding, including location at genic vs intergenic regions and abundant Runx2 binding on promoters of the highly expressed genes. We also found high frequency of Runx2 DNA binding without a consensus Runx2 motif at the binding site. Importantly, mouse and human Runx2 showed moderately similar binding patterns in terms of peak-associated closest genes and their associated genomic ontology (GO) pathways. Accordingly, the gene expression profiles were highly similar and osteoblastic phenotype was prominent in the differentiated stage in both species. In conclusion, ChIP-seq method shows good reproducibility in the context of mature osteoblasts, and mouse and human osteoblast models resemble each other closely in Runx2 binding and in gene expression profiles, supporting the use of these models as adequate tools in studying osteoblast differentiation.

Background Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs) and circulating tumor DNAs (ctDNA). Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches. Methods We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments. Results Mutations in TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II) and late stage (III and IV) cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177) of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that are consistent with cancer progression or response to EGFR drug treatment. Conclusions This study demonstrates that ctDNA mutation rates in the key tumor-associated genes are clinical parameters relevant to smoking status and mortality. Mutations in ctDNA may serve as an early detection tool for cancer. This study quantitatively confirms the hypothesis that ctDNAs in circulation is the result of dissemination of aggressive tumor clones and survival of resistant clones. This study supports the use of ctDNA profiling as a less-invasive approach to monitor cancer progression and selection of appropriate drugs during cancer evolution. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0468-1) contains supplementary material, which is available to authorized users.

The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.

DNA methylation has proven to be powerful for brain tumor characterization and diagnostic classification. To obtain information about the oncogenic role of DNA methylation, we analyzed medulloblastoma, choroid plexus, and atypical teratoid/rhabdoid tumors (AT/RTs) with public data from 450K-methylation arrays (N=584) and gene-expression arrays (N=110). In addition, two AT/RTs, five choroid plexus tumors and three medulloblastomas were analyzed by using reduced representation bisulfite sequencing, exome sequencing, and RNA-sequencing of matched samples. Only few somatic alterations in addition to SMARCB1 deletion were present in our AT/RTs. DNA methylation analysis generated 2325-5739 and 17175-25187 differentially methylated regions (DMRs) between tumor types in 450K array and RRBS sequencing data, respectively. AT/RTs harbored generally higher DNA methylation levels than the other tumor types. Next, DNA methylation differences were integrated with gene expression data. Surprisingly, only eight genes showed cancer-specific association between differential DNA methylation and an opposite expression change at promoter or linked enhancer in both public and in-house data. There were 44 cancer-specific genes with expression-methylation association when DNA methylation analysis was extended to genomic neighborhoods. To gain information about changes in epigenetic regulation between tumor types, we studied which previously experimentally validated transcription factor (TF) binding sites are enriched in cancer specific DMRs. Several TFs known to promote neural development, such as NEUROG2 and NEUROD1, were enriched in regions hypermethylated in AT/RT, whereas TFs, such as SMAD2, involved in the inhibition of neural development were associated with regions hypermethylated in medulloblastoma. This suggests that DNA methylation is regulating especially the target sites for neural regulators in AT/RT tumors, thus inhibiting neural development. Expression differences did not explain the predicted decreased activity of most of these neural TFs. Low number of genes with cancer-specific expression and methylation change is at least partly explained by the different gene expression patterns in medulloblastomas and choroid plexus tumors, thus providing different references for comparison. Also differences in the measurement techniques contribute to this. Taken together, these results suggest that DNA methylation has a role as an epigenetic regulator for the oncogenesis of AT/RTs. Citation Format: Kirsi Johanna Granberg, Joonas Tuominen, Kristiina Nordfors, Meeri Pekkarinen, Ville Kytölä, Sergei Häyrynen, Ebrahim Afyounian, Olli Lohi, Pauli Helen, Juha Kesseli, Joonas Haapasalo, Hannu Haapasalo, Matti Nykter. DNA methylation analysis reveals epigenetic regulation of neural differentiation in AT/RTs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-173.

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses. publishedVersion

Background: Cancers related to tobacco use and African-American ancestry are under-characterized by genomics. This gap in precision oncology research represents a major challenge in the health disparities in the United States. Methods: The Precision Oncology trial at the Wake Forest Baptist Comprehensive Cancer Center enrolled 431 cancer patients from March 2015 to May 2016. The composition of these patients consists of a high representation of tobacco-related cancers (e.g., lung, colorectal, and bladder) and African-American ancestry (13.5%). Tumors were sequenced to identify mutations to gain insight into genetic alterations associated with smoking and/or African-American ancestry. Results: Tobacco-related cancers exhibit a high mutational load. These tumors are characterized by high-frequency mutations in TP53, DNA damage repair genes (BRCA2 and ATM), and chromatin remodeling genes (the lysine methyltransferases KMT2D or MLL2, and KMT2C or MLL3). These tobacco-related cancers also exhibit augmented tumor heterogeneities. Smoking related genetic mutations were validated by The Cancer Genome Atlas dataset that includes 2,821 cases with known smoking status. The Wake Forest and The Cancer Genome Atlas cohorts (431 and 7,991 cases, respectively) revealed a significantly increased mutation rate in the TP53 gene in the African-American subgroup studied. Both cohorts also revealed 5 genes (e.g. CDK8) significantly amplified in the African-American population. Conclusions: These results provide strong evidence that tobacco is a major cause of genomic instability and heterogeneity in cancer. TP53 mutations and key oncogene amplifications emerge as key factors contributing to cancer outcome disparities among different racial/ethnic groups.

Colorectal cancer (CRC) accounts for high mortality. So far, there is lack of markers capable of predicting which patients are at risk of aggressive course of the disease. Protein phosphatase‐2A (PP2A) inhibitor proteins have recently gained interest as markers of more aggressive disease in certain cancers. Here, we report the role of PP2A inhibitor PME‐1 in CRC. PME‐1 expression was assessed from a rectal cancer patient cohort by immunohistochemistry, and correlations were performed for various clinicopathological variables and patient survival. Rectal cancer patients with higher cytoplasmic PME‐1 protein expression (above median) had less recurrences (P = 0.003, n = 195) and better disease‐free survival (DFS) than the patients with low cytoplasmic PME‐1 protein expression (below median). Analysis of PPME‐1 mRNA expression from TCGA dataset of colon and rectal adenocarcinoma (COADREAD) patient cohort confirmed high PPME1 expression as an independent protective factor predicting favorable overall survival (OS) (P = 0.005, n = 396) compared to patients with low PPME1 expression. CRC cell lines were used to study the effect of PME‐1 knockdown by siRNA on cell survival. Contrary to other cancer types, PME‐1 inhibition in CRC cell lines did not reduce the viability of cells or the expression of active phosphorylated AKT and ERK proteins. In conclusion, PME‐1 expression predicts for a favorable outcome of CRC patients. The unexpected role of PME‐1 in CRC in contrast with the oncogenic role of PP2A inhibitor proteins in other malignancies warrants further studies of cancer‐specific function for each of these proteins.