Publications by authors named "Jozef Madzo"

34 Publications

and Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Cancer Res 2021 Oct 2;81(19):5089-5101. Epub 2021 Jul 2.

Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.

Somatic variants in and are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as , and , or with mutations affecting related signaling pathways such as and . Here, we show that and mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment and , whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of mimicked the effect of mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that and mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: and mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-3761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487956PMC
October 2021

Accelerated aging in normal breast tissue of women with breast cancer.

Breast Cancer Res 2021 05 22;23(1):58. Epub 2021 May 22.

Coriell Institute for Medical Research, 403 Haddon Ave., Camden, NJ, 08103, USA.

Background: DNA methylation alterations have similar patterns in normal aging tissue and in cancer. In this study, we investigated breast tissue-specific age-related DNA methylation alterations and used those methylation sites to identify individuals with outlier phenotypes. Outlier phenotype is identified by unsupervised anomaly detection algorithms and is defined by individuals who have normal tissue age-dependent DNA methylation levels that vary dramatically from the population mean.

Methods: We generated whole-genome DNA methylation profiles (GSE160233) on purified epithelial cells and used publicly available Infinium HumanMethylation 450K array datasets (TCGA, GSE88883, GSE69914, GSE101961, and GSE74214) for discovery and validation.

Results: We found that hypermethylation in normal breast tissue is the best predictor of hypermethylation in cancer. Using unsupervised anomaly detection approaches, we found that about 10% of the individuals (39/427) were outliers for DNA methylation from 6 DNA methylation datasets. We also found that there were significantly more outlier samples in normal-adjacent to cancer (24/139, 17.3%) than in normal samples (15/228, 5.2%). Additionally, we found significant differences between the predicted ages based on DNA methylation and the chronological ages among outliers and not-outliers. Additionally, we found that accelerated outliers (older predicted age) were more frequent in normal-adjacent to cancer (14/17, 82%) compared to normal samples from individuals without cancer (3/17, 18%). Furthermore, in matched samples, we found that the epigenome of the outliers in the pre-malignant tissue was as severely altered as in cancer.

Conclusions: A subset of patients with breast cancer has severely altered epigenomes which are characterized by accelerated aging in their normal-appearing tissue. In the future, these DNA methylation sites should be studied further such as in cell-free DNA to determine their potential use as biomarkers for early detection of malignant transformation and preventive intervention in breast cancer.
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http://dx.doi.org/10.1186/s13058-021-01434-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140515PMC
May 2021

The developmental origins of sex-biased expression in cardiac development.

Biol Sex Differ 2019 09 5;10(1):46. Epub 2019 Sep 5.

Fels Institute for Cancer Research, Lewis Katz School of Medicine, Temple University, 3400 N. Broad St, Philadelphia, PA, 19140, USA.

Background: Expression patterns between males and females vary in every adult tissue, even in organs with no conspicuous dimorphisms such as the heart. While studies of male and female differences have traditionally focused on the influence of sex hormones, these do not account for all the differences at the molecular and epigenetic levels. We previously reported that a substantial number of genes were differentially expressed in male and female mouse embryonic stem (ES) cells and revealed dose-dependent enhancer activity in response to Prdm14, a key pluripotency factor expressed more highly in female ES cells. In this work, we investigated the role of Prdm14 in establishing sex-specific gene expression networks. We surveyed the sex-specific landscape in early embryogenesis with special reference to cardiac development. We generated sex-specific co-expression networks from mouse ES cells, examined the presence of sex-specific chromatin domains, and analyzed previously published datasets from different developmental time points to characterize how sex-biased gene expression waxes and wanes to evaluate whether sex-biased networks are detectable throughout heart development.

Results: We performed ChIP-seq on male and female mouse ES cells to determine differences in chromatin status. Our study reveals sex-biased histone modifications, underscoring the potential for the sex chromosome complement to prime the genome differently in early development with consequences for later expression biases. Upon differentiation of ES cells to cardiac precursors, we found sex-biased expression of key transcription and epigenetic factors, some of which persisted from the undifferentiated state. Using network analyses, we also found that Prdm14 plays a prominent role in regulating a subset of dimorphic expression patterns. To determine whether sex-biased expression is present throughout cardiogenesis, we re-analyzed data from two published studies that sampled the transcriptomes of mouse hearts from 8.5 days post-coitum embryos to neonates and adults. We found sex-biased expression at every stage in heart development, and interestingly, identified a subset of genes that exhibit the same bias across multiple cardiogenic stages.

Conclusions: Overall, our results support the existence of sexually dimorphic gene expression profiles and regulatory networks at every stage of cardiac development, some of which may be established in early embryogenesis and epigenetically perpetuated.
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http://dx.doi.org/10.1186/s13293-019-0259-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727560PMC
September 2019

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cell 2018 11 25;175(5):1244-1258.e26. Epub 2018 Oct 25.

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
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http://dx.doi.org/10.1016/j.cell.2018.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247954PMC
November 2018

Poly(ADP-ribose) polymerase 1 is necessary for coactivating hypoxia-inducible factor-1-dependent gene expression by Epstein-Barr virus latent membrane protein 1.

PLoS Pathog 2018 11 5;14(11):e1007394. Epub 2018 Nov 5.

Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania, United States of America.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Poly(ADP-ribose) polymerase 1 (PARP1) regulates accessibility of chromatin, alters functions of transcriptional activators and repressors, and has been directly implicated in transcriptional activation. Previously we showed that LMP1 activates PARP1 and increases Poly(ADP-ribos)ylation (PARylation) through PARP1. Therefore, to identify targets of LMP1 that are regulated through PARP1, LMP1 was ectopically expressed in an EBV-negative Burkitt's lymphoma cell line. These LMP1-expressing cells were then treated with the PARP inhibitor olaparib and prepared for RNA sequencing. The LMP1/PARP targets identified through this RNA-seq experiment are largely involved in metabolism and signaling. Interestingly, Ingenuity Pathway Analysis of RNA-seq data suggests that hypoxia-inducible factor 1-alpha (HIF-1α) is an LMP1 target mediated through PARP1. PARP1 is acting as a coactivator of HIF-1α-dependent gene expression in B cells, and this co-activation is enhanced by LMP1-mediated activation of PARP1. HIF-1α forms a PARylated complex with PARP1 and both HIF-1α and PARP1 are present at promoter regions of HIF-1α downstream targets, leading to accumulation of positive histone marks at these regions. Complex formation, PARylation and binding of PARP1 and HIF-1α at promoter regions of HIF-1α downstream targets can all be attenuated by PARP1 inhibition, subsequently leading to a buildup of repressive histone marks and loss of positive histone marks. In addition, LMP1 switches cells to a glycolytic 'Warburg' metabolism, preferentially using aerobic glycolysis over mitochondrial respiration. Finally, LMP1+ cells are more sensitive to PARP1 inhibition and, therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies.
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http://dx.doi.org/10.1371/journal.ppat.1007394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237423PMC
November 2018

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

J Virol 2018 09 29;92(18). Epub 2018 Aug 29.

Fels Institute for Cancer Research & Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania, USA

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas. EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.
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http://dx.doi.org/10.1128/JVI.00755-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146685PMC
September 2018

TET1-Mediated Hypomethylation Activates Oncogenic Signaling in Triple-Negative Breast Cancer.

Cancer Res 2018 08 11;78(15):4126-4137. Epub 2018 Jun 11.

Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania.

Both gains and losses of DNA methylation are common in cancer, but the factors controlling this balance of methylation remain unclear. Triple-negative breast cancer (TNBC), a subtype that does not overexpress hormone receptors or HER2/NEU, is one of the most hypomethylated cancers observed. Here, we discovered that the TET1 DNA demethylase is specifically overexpressed in about 40% of patients with TNBC, where it is associated with hypomethylation of up to 10% of queried CpG sites and a worse overall survival. Through bioinformatic analyses in both breast and ovarian cancer cell line panels, we uncovered an intricate network connecting TET1 to hypomethylation and activation of cancer-specific oncogenic pathways, including PI3K, EGFR, and PDGF. TET1 expression correlated with sensitivity to drugs targeting the PI3K-mTOR pathway, and CRISPR-mediated deletion of TET1 in two independent TNBC cell lines resulted in reduced expression of PI3K pathway genes, upregulation of immune response genes, and substantially reduced cellular proliferation, suggesting dependence of oncogenic pathways on TET1 overexpression. Our work establishes TET1 as a potential oncogene that contributes to aberrant hypomethylation in cancer and suggests that TET1 could serve as a druggable target for therapeutic intervention. This study addresses a critical gap in knowledge of how and why methylation is prognostic in breast cancer and shows how this information can be used to stratify patients with TNBC for targeted therapy. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072603PMC
August 2018

Nerve Injury-Induced Chronic Pain Is Associated with Persistent DNA Methylation Reprogramming in Dorsal Root Ganglion.

J Neurosci 2018 07 6;38(27):6090-6101. Epub 2018 Jun 6.

Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania 19140 and

Nerve injury-induced hyperactivity of primary sensory neurons in the dorsal root ganglion (DRG) contributes to chronic pain development, but the underlying epigenetic mechanisms remain poorly understood. Here we determined genome-wide changes in DNA methylation in the nervous system in neuropathic pain. Spinal nerve ligation (SNL), but not paclitaxel treatment, in male Sprague Dawley rats induced a consistent low-level hypomethylation in the CpG sites in the DRG during the acute and chronic phases of neuropathic pain. DNA methylation remodeling in the DRG occurred early after SNL and persisted for at least 3 weeks. SNL caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands, in introns, intergenic regions, and repetitive sequences. In contrast, SNL caused more gains of methylation in the spinal cord and prefrontal cortex. The DNA methylation changes in the injured DRGs recapitulated developmental reprogramming at the neonatal stage. Methylation reprogramming was correlated with increased gene expression variability. A diet deficient in methyl donors induced hypomethylation and pain hypersensitivity. Intrathecal administration of the DNA methyltransferase inhibitor RG108 caused long-lasting pain hypersensitivity. DNA methylation reprogramming in the DRG thus contributes to nerve injury-induced chronic pain. Restoring DNA methylation may represent a new therapeutic approach to treat neuropathic pain. Epigenetic mechanisms are critically involved in the transition from acute to chronic pain after nerve injury. However, genome-wide changes in DNA methylation in the nervous system and their roles in neuropathic pain development remain unclear. Here we used digital restriction enzyme analysis of methylation to quantitatively determine genome-wide DNA methylation changes caused by nerve injury. We showed that nerve injury caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands in the dorsal root ganglion. Reducing DNA methylation induced pain hypersensitivity, whereas increasing DNA methylation attenuated neuropathic pain. These findings extend our understanding of the epigenetic mechanism of chronic neuropathic pain and suggest new strategies to treat nerve injury-induced chronic pain.
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http://dx.doi.org/10.1523/JNEUROSCI.2616-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031579PMC
July 2018

Demethylator phenotypes in acute myeloid leukemia.

Leukemia 2018 10 7;32(10):2178-2188. Epub 2018 Mar 7.

Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, 19140, USA.

Acute myeloid leukemia (AML) often harbors mutations in epigenetic regulators, and also has frequent DNA hypermethylation, including the presence of CpG island methylator phenotypes (CIMPs). Although global hypomethylation is well known in cancer, the question of whether distinct demethylator phenotypes (DMPs) exist remains unanswered. Using Illumina 450k arrays for 194 patients from The Cancer Genome Atlas, we identified two distinct DMPs by hierarchical clustering: DMP.1 and DMP.2. DMP.1 cases harbored mutations in NPM1 (94%), FLT3 (71%) and DNMT3A (61%). Surprisingly, only 40% of patients with DNMT3A mutations were DMP.1, which has implications for mechanisms of transformation by this mutation. In contrast, DMP.2 AML was comprised of patients with t(8;21), inv(16) or t(15;17), suggesting common methylation defects connect these disparate rearrangements. RNA-seq revealed upregulated genes functioning in immune response (DMP.1) and development (DMP.2). We confirmed these findings by integrating independent 450k data sets (236 additional cases), and found prognostic effects by DMP status, independent of age and cytogenetics. The existence of DMPs has implications for AML pathogenesis and may augment existing tools in risk stratification.
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http://dx.doi.org/10.1038/s41375-018-0084-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128790PMC
October 2018

Ezh2 phosphorylation state determines its capacity to maintain CD8 T memory precursors for antitumor immunity.

Nat Commun 2017 12 14;8(1):2125. Epub 2017 Dec 14.

Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA, 19140, USA.

Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8 T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.
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http://dx.doi.org/10.1038/s41467-017-02187-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730609PMC
December 2017

Digital Restriction Enzyme Analysis of Methylation (DREAM).

Methods Mol Biol 2018 ;1708:247-265

Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, 19140, USA.

Digital Restriction Enzyme Analysis of Methylation (DREAM) is a method for quantitative mapping of DNA methylation across genomes using next-generation sequencing (NGS) technology. The method is based on sequential cuts of genomic DNA with a pair of restriction enzymes (SmaI and XmaI) at CCCGGG target sites. Unmethylated sites are first digested with SmaI. This enzyme cuts the sites in the middle at CCC^GGG, leaving behind blunt ended fragments. CpG methylation completely blocks SmaI; therefore, only unmethylated sites are cleaved. The remaining methylated sites are digested with XmaI in the next step. This enzyme is not blocked by CpG methylation. It cuts the recognition site sideways at C^CCGGG forming 5'-CCGG overhangs. The sequential cuts thus create distinct methylation-specific signatures at the ends of restriction fragments: 5'-GGG for unmethylated CpG sites and 5'-CCGGG for methylated sites. The DNA fragments resulting from the digestions are ligated to NGS adapters. Sequencing libraries are prepared using hexanucleotide barcodes for sample identification. Individual libraries with distinct barcodes are pooled and sequenced using a paired ends protocol. The sequencing reads are aligned to the genome and mapped to unique CCCGGG target sites. Methylation at individual CpG sites is calculated as the ratio of sequencing reads with the methylated signature to the total number of reads mapping to the site. Sequencing of 25 million reads per sample typically yields 50,000 unique CpG sites covered with hundreds of reads enabling accurate determination of DNA methylation levels. DREAM does not require bisulfite conversion, has a very low background, and has high sensitivity to low levels of methylation. The method is simple, cost-effective, quantitative, highly reproducible, and can be applied to any species.
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http://dx.doi.org/10.1007/978-1-4939-7481-8_13DOI Listing
July 2018

Caloric restriction delays age-related methylation drift.

Nat Commun 2017 09 14;8(1):539. Epub 2017 Sep 14.

Fels Institute for Cancer Research & Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania, 19140, USA.

In mammals, caloric restriction consistently results in extended lifespan. Epigenetic information encoded by DNA methylation is tightly regulated, but shows a striking drift associated with age that includes both gains and losses of DNA methylation at various sites. Here, we report that epigenetic drift is conserved across species and the rate of drift correlates with lifespan when comparing mice, rhesus monkeys, and humans. Twenty-two to 30-year-old rhesus monkeys exposed to 30% caloric restriction since 7-14 years of age showed attenuation of age-related methylation drift compared to ad libitum-fed controls such that their blood methylation age appeared 7 years younger than their chronologic age. Even more pronounced effects were seen in 2.7-3.2-year-old mice exposed to 40% caloric restriction starting at 0.3 years of age. The effects of caloric restriction on DNA methylation were detectable across different tissues and correlated with gene expression. We propose that epigenetic drift is a determinant of lifespan in mammals.Caloric restriction has been shown to increase lifespan in mammals. Here, the authors provide evidence that age-related methylation drift correlates with lifespan and that caloric restriction in mice and rhesus monkeys results in attenuation of age-related methylation drift.
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http://dx.doi.org/10.1038/s41467-017-00607-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599616PMC
September 2017

Sex chromosomes drive gene expression and regulatory dimorphisms in mouse embryonic stem cells.

Biol Sex Differ 2017 08 17;8(1):28. Epub 2017 Aug 17.

Fels Institute for Cancer Research, Temple University School of Medicine, 3400 N. Broad St. PAHB Room 201, Philadelphia, PA, 19140, USA.

Background: Pre-implantation embryos exhibit sexual dimorphisms in both primates and rodents. To determine whether these differences reflected sex-biased expression patterns, we generated transcriptome profiles for six 40,XX, six 40,XY, and two 39,X mouse embryonic stem (ES) cells by RNA sequencing.

Results: We found hundreds of coding and non-coding RNAs that were differentially expressed between male and female cells. Surprisingly, the majority of these were autosomal and included RNA encoding transcription and epigenetic and chromatin remodeling factors. We showed differential Prdm14-responsive enhancer activity in male and female cells, correlating with the sex-specific levels of Prdm14 expression. This is the first time sex-specific enhancer activity in ES cells has been reported. Evaluation of X-linked gene expression patterns between our XX and XY lines revealed four distinct categories: (1) genes showing 2-fold greater expression in the female cells; (2) a set of genes with expression levels well above 2-fold in female cells; (3) genes with equivalent RNA levels in male and female cells; and strikingly, (4) a small number of genes with higher expression in the XY lines. Further evaluation of autosomal gene expression revealed differential expression of imprinted loci, despite appropriate parent-of-origin patterns. The 39,X lines aligned closely with the XY cells and provided insights into potential regulation of genes associated with Turner syndrome in humans. Moreover, inclusion of the 39,X lines permitted three-way comparisons, delineating X and Y chromosome-dependent patterns.

Conclusions: Overall, our results support the role of the sex chromosomes in establishing sex-specific networks early in embryonic development and provide insights into effects of sex chromosome aneuploidies originating at those stages.
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http://dx.doi.org/10.1186/s13293-017-0150-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561606PMC
August 2017

A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer.

Nucleic Acids Res 2017 Aug;45(14):8269-8281

Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA.

TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine (5hmC), resulting in regulation of DNA methylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation, but it also limits its ability to regulate genes outside of CGIs. Here, we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in embryonic stem cells (ESCs) but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues. Overexpression of TET1ALT shows production of 5hmC with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT is aberrantly activated in multiple cancer types including breast, uterine and glioblastoma, and TET1 activation is associated with a worse overall survival in breast, uterine and ovarian cancers. Our data suggest that the predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site-specific demethylation outside of CGIs.
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http://dx.doi.org/10.1093/nar/gkx435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737541PMC
August 2017

Transcriptional Selectivity of Epigenetic Therapy in Cancer.

Cancer Res 2017 01 22;77(2):470-481. Epub 2016 Nov 22.

Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, Pennsylvania.

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5243171PMC
January 2017

DREAM: A Simple Method for DNA Methylation Profiling by High-throughput Sequencing.

Methods Mol Biol 2016 ;1465:111-27

Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, 19140, USA.

The digital restriction enzyme analysis of methylation (DREAM) is a simple method for DNA methylation analysis at tens of thousands of CpG sites across the genome. The method creates specific signatures at unmethylated and methylated CpG sites by sequential digests of genomic DNA with restriction endonucleases SmaI and XmaI, respectively. Both enzymes have the same CCCGGG recognition site; however, they differ in their sensitivity to CpG methylation and their cutting pattern. SmaI cuts only unmethylated sites leaving blunt 5'-GGG ends. XmaI cuts remaining methylated CC(me)CGG sites leaving 5'-CCGGG ends. Restriction fragments with distinct signatures at their ends are ligated to Illumina sequencing adaptors with sample-specific barcodes. High-throughput sequencing of pooled libraries follows. Sequencing reads are mapped to the restriction sites in the reference genome, and signatures corresponding to methylation status of individual DNA molecules are resolved. Methylation levels at target CpG sites are calculated as the proportion of sequencing reads with the methylated signature to the total number of reads mapping to the particular restriction site. Aligning the reads to the reference genome of any species is straightforward, since the method does not rely on bisulfite conversion of DNA. Sequencing of 25 million reads per human DNA library yields over 50,000 unique CpG sites with high coverage enabling accurate determination of DNA methylation levels. DREAM has a background less than 1 % making it suitable for accurate detection of low methylation levels. In summary, the method is simple, robust, highly reproducible, and cost-effective.
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http://dx.doi.org/10.1007/978-1-4939-4011-0_10DOI Listing
January 2018

TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differentiating colonocytes and colon cancer.

Sci Rep 2015 Dec 3;5:17568. Epub 2015 Dec 3.

Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.

The formation of differentiated cell types from pluripotent progenitors involves epigenetic regulation of gene expression. DNA hydroxymethylation results from the enzymatic oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) 5-mC dioxygenase enzymes. Previous work has mapped changes in 5-mC during differentiation of intestinal stem cells. However, whether or not 5-hmC regulates colonocyte differentiation is unknown. Here we show that 5-hmC regulates gene expression during colonocyte differentiation and controls gene expression in human colon cancers. Genome-wide profiling of 5-hmC during in vitro colonic differentiation demonstrated that 5-hmC is gained at highly expressed and induced genes and is associated with intestinal transcription factor binding sites, including those for HNF4A and CDX2. TET1 induction occurred during differentiation, and TET1 knockdown altered gene expression and inhibited barrier formation of colonocytes. We find that the 5-hmC distribution in primary human colonocytes parallels the distribution found in differentiated cells in vitro, and that gene-specific 5-hmC changes in human colon cancers are directly correlated with changes in gene expression. Our results support a model in which 5-hmC regulates differentiation of adult human intestine and 5-hmC alterations contribute to the disrupted gene expression in colon cancer.
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http://dx.doi.org/10.1038/srep17568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668370PMC
December 2015

TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

Cancer Res 2015 Jul 13;75(14):2833-43. Epub 2015 May 13.

Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, Pennsylvania. Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas.

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506197PMC
July 2015

A phase I and pharmacodynamic study of the histone deacetylase inhibitor belinostat plus azacitidine in advanced myeloid neoplasia.

Invest New Drugs 2015 Apr 9;33(2):371-9. Epub 2014 Dec 9.

Department of Medicine, The University of Chicago, Chicago, IL, USA,

Background We hypothesized that targeting two mechanisms of epigenetic silencing would be additive or synergistic with regard to expression of specific target genes. The primary objective of the study was to establish the maximum tolerated dose (MTD) of belinostat in combination with a fixed dose of azacitidine (AZA). Methods In Part A of the study, patients received a fixed dose of AZA, with escalating doses of belinostat given on the same days 1-5, in a 28 day cycle. Part B was designed to evaluate the relative contribution of belinostat to the combination based on analysis of pharmacodynamic markers, and incorporated a design in which patients were randomized during cycle 1 to AZA alone, or the combination, at the maximally tolerated dose of belinostat. Results 56 patients with myeloid neoplasia were enrolled. Dose escalation was feasible in part A, up to 1000 mg/m(2) dose level of belinostat. In Part B, 18 patients were assessable for quantitative analysis of specific target genes. At day 5 of therapy, MDR1 was significantly up-regulated in the belinostat/AZA arm compared with AZA alone arm (p = 0.0023). There were 18 responses among the 56 patients. Conclusions The combination of belinostat and AZA is feasible and associated with clinical activity. The recommended phase II dose is 1000 mg/m(2) of belinostat plus 75 mg/m(2) of AZA on days 1-5, every 28 days. Upregulation in MDR1 was observed in the combination arm at day 5 compared with the AZA alone arm, suggesting a relative biologic contribution of belinostat to the combination.
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http://dx.doi.org/10.1007/s10637-014-0194-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390421PMC
April 2015

DNA hydroxymethylation profiling reveals that WT1 mutations result in loss of TET2 function in acute myeloid leukemia.

Cell Rep 2014 Dec 4;9(5):1841-1855. Epub 2014 Dec 4.

Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY 10065, USA; The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA; The Feil Family Brain and Mind Research Institute, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA. Electronic address:

Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation.
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http://dx.doi.org/10.1016/j.celrep.2014.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267494PMC
December 2014

TET1-mediated hydroxymethylation facilitates hypoxic gene induction in neuroblastoma.

Cell Rep 2014 Jun 15;7(5):1343-1352. Epub 2014 May 15.

Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, IL 60637, USA; Committee on Cancer Biology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

The ten-eleven-translocation 5-methylcytosine dioxygenase (TET) family of enzymes catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), a modified cytosine base that facilitates gene expression. Cells respond to hypoxia by inducing a transcriptional program regulated in part by oxygen-dependent dioxygenases that require Fe(II) and α-ketoglutarate. Given that the TET enzymes also require these cofactors, we hypothesized that the TETs regulate the hypoxia-induced transcriptional program. Here, we demonstrate that hypoxia increases global 5-hmC levels, with accumulation of 5-hmC density at canonical hypoxia response genes. A subset of 5-hmC gains colocalize with hypoxia response elements facilitating DNA demethylation and HIF binding. Hypoxia results in transcriptional activation of TET1, and full induction of hypoxia-responsive genes and global 5-hmC increases require TET1. Finally, we show that 5-hmC increases and TET1 upregulation in hypoxia are HIF-1 dependent. These findings establish TET1-mediated 5-hmC changes as an important epigenetic component of the hypoxic response.
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http://dx.doi.org/10.1016/j.celrep.2014.04.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516227PMC
June 2014

Hydroxymethylation at gene regulatory regions directs stem/early progenitor cell commitment during erythropoiesis.

Cell Rep 2014 Jan 27;6(1):231-244. Epub 2013 Dec 27.

Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.

Hematopoietic stem cell differentiation involves the silencing of self-renewal genes and induction of a specific transcriptional program. Identification of multiple covalent cytosine modifications raises the question of how these derivatized bases influence stem cell commitment. Using a replicative primary human hematopoietic stem/progenitor cell differentiation system, we demonstrate dynamic changes of 5-hydroxymethylcytosine (5-hmC) during stem cell commitment and differentiation to the erythroid lineage. Genomic loci that maintain or gain 5-hmC density throughout erythroid differentiation contain binding sites for erythroid transcription factors and several factors not previously recognized as erythroid-specific factors. The functional importance of 5-hmC was demonstrated by impaired erythroid differentiation, with augmentation of myeloid potential, and disrupted 5-hmC patterning in leukemia patient-derived CD34+ stem/early progenitor cells with TET methylcytosine dioxygenase 2 (TET2) mutations. Thus, chemical conjugation and affinity purification of 5-hmC-enriched sequences followed by sequencing serve as resources for deciphering functional implications for gene expression during stem cell commitment and differentiation along a particular lineage.
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http://dx.doi.org/10.1016/j.celrep.2013.11.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976649PMC
January 2014

Alterations of 5-hydroxymethylcytosine in human cancers.

Cancers (Basel) 2013 Jun 25;5(3):786-814. Epub 2013 Jun 25.

Section of Hematology/Oncology, Department of Medicine, The University of Chicago, 5841 S. Maryland Ave., MC 2115, Chicago, IL 60637, USA.

Prior to 2009, 5-methylcytosine (5-mC) was thought to be the only biologically significant cytosine modification in mammalian DNA. With the discovery of the TET enzymes, which convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), however, intense interest has emerged in determining the biological function of 5-hmC. Here, we review the techniques used to study 5-hmC and evidence that alterations to 5-hmC physiology play a functional role in the molecular pathogenesis of human cancers.
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http://dx.doi.org/10.3390/cancers5030786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795365PMC
June 2013

Perturbations of 5-hydroxymethylcytosine patterning in hematologic malignancies.

Semin Hematol 2013 Jan;50(1):61-9

Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, IL 60637-1470, USA.

The recent identification of covalent cytosine modifications derived from the metabolism of 5-methylcytosine (5-mC) and catalyzed by the TET proteins has facilitated molecular insight into a new subclass of acute myeloid leukemias (AMLs). TET2-mutant AMLs have the predicted hypermethylation phenotype expected given the inability of the mutant TET2 protein to convert 5-mC to 5-hydroxymethylcytosine (5-hmC). In addition, IDH1/2 mutations confer a gain-of-function, allowing the enzymes to process α-ketoglutarate to 2-hydroxyglutarate, which inhibits the TET proteins and ultimately induces the same hypermethylation phenotype. New techniques are being developed rapidly that have the unprecedented capacity to distinguish among the various covalent cytosine modifications now known to exist. Soon, these methods will be harnessed to yield a new level of insight into AMLs with altered distribution of 5-hmC, information that may allow new diagnostic and therapeutic approaches for patients with this subtype of AML.
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http://dx.doi.org/10.1053/j.seminhematol.2013.01.004DOI Listing
January 2013

Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.

Cancer Cell 2011 Jul 30;20(1):11-24. Epub 2011 Jun 30.

Department of Pathology, NYU Cancer Institute, New York University School of Medicine, NY 10016, USA.

Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2(+/-) mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.
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http://dx.doi.org/10.1016/j.ccr.2011.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194039PMC
July 2011

The identification of (ETV6)/RUNX1-regulated genes in lymphopoiesis using histone deacetylase inhibitors in ETV6/RUNX1-positive lymphoid leukemic cells.

Clin Cancer Res 2007 Mar 26;13(6):1726-35. Epub 2007 Feb 26.

Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology/Oncology, 2nd Medical School, Charles University Prague, Prague, Czech Republic.

Purpose: Chimeric transcription factor ETV6/RUNX1 (TEL/AML1) is believed to cause pathologic block in lymphoid cell development via interaction with corepressor complex and histone deacetylase. We wanted to show the regulatory effect of ETV6/RUNX1 and its reversibility by histone deacetylase inhibitors (HDACi), as well as to identify potential ETV6/RUNX1-regulated genes.

Experimental Design: We used luciferase assay to show the interaction of ETV6/RUNX1 protein, ETV6/RUNX1-regulated gene, and HDACi. To identify ETV6/RUNX1-regulated genes, we used expression profiling and HDACi in lymphoid cells. Next, using the flow cytometry and quantitative reverse transcription-PCR, we measured differentiation changes in gene and protein expression after HDACi treatment.

Results: Luciferase assay showed repression of granzyme B expression by ETV6/RUNX1 protein and the reversibility of this effect by HDACi. Proving this regulatory role of ETV6/RUNX1, we identified, using complex statistical analysis, 25 genes that are potentially regulated by ETV6/RUNX1 protein. In four selected genes with known role in the cell cycle regulation (JunD, ACK1, PDGFRB, and TCF4), we confirmed expression changes after HDACi by quantitative analysis. After HDACi treatment, ETV6/RUNX1-positive cells showed immunophenotype changes resembling differentiation process compared with other leukemic cells (BCR/ABL, ETV6/PDGFRB positive). Moreover, ETV6/RUNX1-positive leukemic cells accumulated in G(1)-G(0) phase after HDACi whereas other B-lineage leukemic cell lines showed rather unspecific changes including induction of apoptosis and decreased proliferation.

Conclusions: Presented data support the hypothesis that HDACi affect ETV6/RUNX1-positive cells via direct interaction with ETV6/RUNX1 protein and that treatment with HDACi may release aberrant transcription activity caused by ETV6/RUNX1 chimeric transcription factor.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-2569DOI Listing
March 2007
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