Abstract
Background: 5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome wide assays for 5-hmC determination are needed as many of the ...techniques commonly used to assay 5-methylcytosine (5-mC), including conventional methyl-sensitive restriction digest and bisulfite sequencing, are incapable of distinguishing between 5-mC and 5-hmC.
Results: Glycosylation of 5-hmC residues by beta-Glucosyl Transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. We used this premise to modify the HELP-tagging assay to identify both 5-mC and 5-hmC loci in the genome. The HELP tagging assay uses massive parallel sequencing to analyze the cytosine methylation status of 2.1 million CpGs in the genome. Libraries were generated from genomic DNA digested with HpaII, MspI and β-GT +MspI. Comparison of HpaII and MspI digested samples led to determination of 5-mC loci, while comparison of β-GT +MspI with MspI digested samples identified 5-hmC residues. This modified “HELP-GT” assay allowed multiplexing of 8 libraries per sequencing lane to generate an average of 6-10 million HpaII/MspI reads per sample with an average depth of coverage between 6-11x for each CCGG site. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and at the locus specific level by qRT-PCR of 5-hmC pulldown DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. This assay was then used to analyze 5-hmC profiles of two pancreatic cancer cell lines (Pa03C and Pa04C) that were compared with pancreatic control cells (HPNE). Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells with significantly increased 5-hmC at Promoters, Gene bodies and Transcription factor binding sites. 5-hmC was also increased at many oncogenic promoters such as GATA6 in pancreatic cancer and correlated with its overexpression. 5-hmC profiles were also able to distinguish between cancer and control cells with greater discrimination (unsupervised hierarchical clustering) when compared to 5-mC patterns.
Conclusions: The HELP-GT assay allows a high resolution, simultaneous determination of 5-hmC and 5-mC loci from small amounts of DNA with the utilisation of modest sequencing resources. This assay is able to provide single base pair resolution analysis of over 1 million sites in the human genome with the use of 1μg of genomic DNA. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis.
Citation Format: Sanchari Bhattacharyya, Yiting Yu, Masako Suzuki, Nathaniel Cambpell, Jozef Mazdo, Aparna Vasantkumar, Tushar D. Bhagat, Sangeeta Nischal, Ulrich Steidl, Lucy Godley, Anirban Maitra, John M. Greally, Amit Verma. Genome wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2013-4231
Abstract
Myelodysplastic syndromes (MDS) are hematologic malignancies characterized by hematopoietic stem cell dysfunction that leads to ineffective hematopoiesis and cytopenias. Even though a third ...of MDS cases transform to leukemia, most of the mortality in MDS is due to low blood counts that occur because of failure of hematopoiesis. Development of effective treatments for MDS have been impeded by a limited understanding of the molecular pathways that suppress hematopoietic stem cells. We previously demonstrated that the myelosuppressive cytokine TGF-β mediated SMAD2 activation can inhibit stem and progenitor cells in MDS. We demonstrated that SMAD7, a negative regulator of TGF-β receptor-I kinase, is markedly reduced in MDS, and can lead to ineffective hematopoiesis by overactivation of Smad2/3 mediated TGF-β signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3′UTR of the gene and found a highly conserved binding site for microRNA-21. Strikingly, we observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age matched controls. miR-21 was shown to directly bind to the 3′UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-β signaling in a TGF-β overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Even though miR-21 was found to be elevated in MDS, the mechanisms of its elevation in MDS as well as in other cancers are not well elucidated. The miR-21 gene locus has been shown to contain upstream STAT3 binding sites. Furthermore, STAT3 has also been shown to directly lead to miR-21 upregulation in myeloma and other immune cells. Genomic profiling of highly purified hematopoietic stem cells in MDS by us demonstrated that STAT3 is selectively upregulated in these cells. STAT3 overexpression was validated in an expanded set of 183 MDS CD34+ cells as well. These results suggest that a STAT3-miR-21 pathway leads to enhancement of TGF-β signaling, thus leading to ineffective hematopoiesis in MDS.
Citation Format: Tushar D. Bhagat, Li Zhou, Lubomir Sokol, Ioannis Mantzaris, Sanchari Bhattacharyya, Shanisha A.K. Gordon, Yiting Yu, Krishna Gundabolu, Sangeeta Nischal, Rahul Polineni, Carolina Schinke, Grigorios Chrysofakis, Amittha Wickrema, Andrea Pellagatti, Jacqueline Boultwood, Ulrich Steidl, Gang Liu, Alan F. List, Markus Bitzer, Amit Verma. MicroRNA mediated regulation of TGF-β signaling leads to stem cell alterations in myelodysplastic syndromes. 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 5224. doi:10.1158/1538-7445.AM2014-5224
Abstract 5008
Cure rates of acute leukemia remain poor emphasizing the need to for novel therapies. All trans retinoic acid (ATRA) is an effective therapeutic agent in a subtype of acute myeloid ...leukemia and relieves transcriptional repression induced by the PML-RAR oncoprotein by binding to the retinoic acid receptor. Even though ATRA is effective, the treatment course is characterized by a high rate of toxicity and ATRA resistance is seen in some cases of acute promyelocytic leukemia. In an attempt to improve outcomes, we devised a methodology for creation of boronic acid and other newer retinoic acid analogues. Our lead compound, MA-21 was generated by replacing the terminal carboxyl group of ATRA with a boronic ester using Wittig reactions. Computation modeling revealed that MA-21 can fit in the RARa pocket and can form increased covalent bonding with cysteine residues within the receptor. As opposed to other synthetic retinoids, the addition of a boron atom resulted in significantly enhanced cytotoxicity in leukemic cell lines, even those that were resistant to ATRA. MA-21 at 1uM dose led to significant reduction in proliferation of ATRA sensitive NB4 APL cell line (1.8 fold decrease after 96hrs, p= 0.028) as well as in ATRA resistant cell lines NB4.007/6 (3.3 fold decrease after 72hrs and 2.1 decrease after 96hrs, p values of 0.018 and 0.046) and NB4.306 (2.6 fold decrease after 96hrs, p= 0.032). MA-21 was able to induce these effects by inducing significant G2/M cell cycle arrest and not by increased apoptosis or cellular differentiation. Cell cycle was assessed by Flow Cytometry after 96hrs of incubation and showed a significant increase in G2/M percentage in the ATRA sensitive and resistant cell lines compared to DMSO (NB4 cell line- 1.35 fold increase, p= 0.035; NB4.007/6- 1.35 fold increase, p= 0.015 and NB4.306- 2 fold increase, p= 0.023). Thus, we demonstrate novel synthetic methodology to synthesize boron containing novel retinoids and demonstrate the potential of these compounds as therapeutic agents in resistant leukemias. Display omitted
No relevant conflicts of interest to declare.
Abstract 784
Gene expression is a tightly regulated process and is influenced by aberrant epigenetic changes that can lead to carcinogenesis. We used the HELP (HpaII tiny fragment Enrichment by ...Ligation-mediated PCR) assay to perform an unbiased genome-wide analysis of DNA methylation in 11 MGUS, 16 newly diagnosed myeloma, 17 relapsed myeloma, and 8 healthy control samples. The HELP assay uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling, and co-hybridization to quantitatively determine individual promoter methylation of 25,626 loci. The methylome analysis was performed using CD 138+ sorted bone marrow plasma cells in all cases. We observed extensive DNA methylation changes in myeloma that were seen even in MGUS cases when compared to normal plasma cells. Unsupervised clustering of all samples showed that MGUS samples were distinct but epigenetically closer to normal plasma cells than to cases of newly diagnosed or relapsed Myeloma. MGUS cases were characterized predominantly by aberrant hypomethylation, with 456 hypomethylated probes versus 130 hypermethylated probes (cutoff criteria were defined as a difference of means > 1.5 and significance of this difference with p < 0.001, Figure 1) that affected pathways regulated by NF-kb transcription factor. Untreated newly diagnosed myeloma samples were also predominantly hypomethylated (461 hypomethylated probes, 83 hypermethylated probes) when compared to controls. In addition to NF-kB, the MAP kinase and PI3 kinase regulated pathways were affected by hypomethylated genes. In contrast, cases of relapsed myeloma showed predominantly hypermethylated loci (221 hypomethylated, 560 hypermethylated probes) when compared to controls and involved the TNF and retinoblastoma pathways. A large number of important genes including the tumor suppressors CDKN2A and CDKN2B were aberrantly hypermethylated in this cohort.
Display omitted
Analysis of our differentially methylated targets using the Molecular Signatures Database (MSigDB, Tamayo, et al. 2005, PNAS 102, 15545–15550), showed significant overlap for hypomethylated genes in MGUS and new multiple myeloma (MM). These gene sets contain genes with promoter regions around transcription start site containing motifs which match annotation for transcription factors SP1 and TCF3 as well as enrichment for genes with gene ontology annotations for plasma membrane proteins. Hypermethylated genes in new MM show significant overlap with genes in the neighborhood of RAD23A, CTBP1, G22P1 and SMC1, suggesting impairment of DNA repair, as well as enrichment for genes associated with apoptosis and programmed cell death.
In conclusion, genome-wide DNA methylation analysis is able to clearly differentiate between normal bone marrow plasma cells, MGUS as well as new MM and relapsed MM cells. Correlation of significantly differentially methylated genes with published gene sets reveals enrichment for genes involved in DNA repair, cell-cell signaling, cell death, apoptosis and cell cycle regulation.
Mehta:Celgene: Consultancy, Speakers Bureau; Takeda/Millennium: Speakers Bureau; Onyx: Research Funding. Singhal:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding; Celgene: Speakers Bureau.
Abstract 627
Myeloproliferative diseases (MPD) are clonal hematologic disorders that present with increased numbers of functional, mature, terminally differentiated myeloid elements. Even though ...genetic, biochemical and functional studies have provided important insights into the pathogenesis of MPDs, the role of epigenetic changes in disease pathobiology is not well elucidated. We performed the HELP assay to study genome-wide methylation patterns in cases of Polycythemia Vera (PV), Essential Thrombocytosis (ET) and Idiopathic Myelofibrosis (IMF) and compared it with normal matched controls. The HELP assay uses differential methylation specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG methylation of 25636 loci. Analysis of 26 MPD neutrophil samples comprising 9 cases of ET, 6 cases of PV and 11 Cases of IMF was performed and compared to normal healthy controls. Unsupervised clustering based on global methylation profiles showed that IMF cases formed a distinct epigenetic cluster, while PV and ET cases were more similar to the normal controls. Further analysis of epigenetic differences between these groups showed that PV and ET samples were characterized by aberrant hypermethylation when compared to controls and had 143 genes that were uniformly hypermethylated. These genes are involved in pathways regulated by the NF-Kb and HNF-4alpha transcription factors. IMF on the other hand was characterized by both aberrantly hyper (n=162) and hypomethylated (n=95) loci when compared to controls. The pathways affected by hypomethylated genes were involved in cytokine cell signaling and MAP kinases. We subsequently validated these observations in an independent set of 8 IMF cases compared to 8 age matched controls. Methylation profiles obtained from whole blood by the HELP assay were able to clearly separate IMF cases from controls demonstrating the validity of our observations. These changes were quantified on a whole genome level by the Luminometric methylation assay (LUMA) that also revealed significantly more hypomethylation in IMF when compared to PV and ET cases (49% hypomethylation in IMF vs. 38% in PV/ET, p<0.05). Selected changes were validated by Mass Array sequencing.
We next wanted to determine the epigenomic effects of Jak2V617F mutation and compared methylation profiles of MPD cases with and without the mutation. We observed that cases with Jak2 mutation had a higher number of differentially hypomethylated loci. This striking difference was seen by the LUMA assay also with 67% hypomethylation seen in mutant cases when compared to 48% in those without the mutation (p=0.02). This observation was validated in vitro in a cell line (FDCP) that expressed wither WT or mutant Jak2 kinase. We observed that expression of the mutant kinase led to global hypomethylation. This observation builds on recent data demonstrating nuclear binding of the mutant Jak2 kinase and its effects on the histone epigenetic machinery.
In conclusion, we report that MPDs are characterized by various novel epigenetic alterations that affect important functional pathways and IMF is grossly epigenetically distinct from ET and PV. The Jak2 mutation also affects the methylome and leads to global hypomethylation that potentially contributes to the genomic instability and disease pathobiology.
No relevant conflicts of interest to declare.
Abstract 3813
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-b is a hematopoietic inhibitory cytokine that has been indirectly ...linked to the pathogenesis of some subsets of MDS. We have shown that smad2, a component of the TGF- signaling pathway, is constitutively activated and upregulated in MDS progenitors (Blood, 112(8):3434; 2008). Since there is conflicting data about upregulation of TGF- b levels in MDS, we next sought to determine the molecular basis of TGF- b pathway activation in this disease. We observed that smad-7, a negative regulator of TGF- b receptor-I kinase, is markedly down regulated in MDS and leads overactivation of the receptor and subsequent smad2 phosphorylation / activation in this disease (Cancer Res, 71(3):955–63). In the present study we wanted to determine the cause of smad7 reduction in MDS. Since microRNA dysregulation has been reported in many malignancies, we explored the 3’UTR of the smad7 gene for putative microRNA binding sites and observed predicted mir-21 and mir-15/16 binding sites that were conserved across species. mir-21 was found to be elevated in a microarray screen in MDS (British J. Haem. 153(1):24–32) and was subsequently found by us to be significantly elevated by qPCR in MDS marrow samples when compared with age matched controls (TTest, N=11 in each group, P Value= 0.02). Luciferase reporters containing wild type and mutant 3’ UTR of the smad7 gene were then used to determine whether mir21 was able to directly bind to the predicted sequence in the gene. Enforced expression of mir-21 was able to inhibit the wild type smad7 reporter expression and a mutation of 4 complementary residues in the 3’UTR led to abrogation of this effect, thus demonstrating direct effects of mir-21 on smad7 gene. To test the role of mir-21 in regulating TGF-b signaling in vivo, we used chemically modified, locked nucleic acid (LNA) inhibitors of mir-21. These were used in a TGF-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with the mir-21 inhibitor led to significant increases in RBC counts when compared to placebo (P value<0.01, T test) and led to increase in smad7 expression and decrease in smad2 phosphorylation in bone marrow progenitors of the treated mice. Finally, mir-21 inhibitor treatment led to increases in erythroid and myeloid colony formation from human MDS bone marrow stem cells, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate that mir-21 mediated reduction in smad-7 contributes to ineffective hematopoiesis in MDS by activating TGF-beta signaling in bone marrow progenitors. Most importantly, these studies illustrate the therapeutic potential of mir-21 inhibitors in this disease.
List:Celgene: Consultancy.
In this paper, we show how mobile drawing methodologies can bring the dynamic, relational and non-representational qualities of landscape encounters to the foreground. The research paper discusses a ...mobile drawing project that took place in the Kathmandu Valley, Nepal. The project entitled 'Taxi Guff-Gaff' invited participants to undertake a collaborative drawing and conversational journey. Mobile drawing together on a bumpy taxi journey required artist participants to move together and literally 'pay attention to the moment at hand'. In so doing it produced imagery that foregrounds the inherent dynamic quality of all our landscape encounters. We propose that mobile drawing offers an immersive way to relate to the urban landscape and each other and can open up spaces of landscape research that centre on speculative forms of thinking, being, drawing and conversation.
Pediatric optic neuritis (ON) is a rare disease that has not been well characterized. The Pediatric ON Prospective Outcomes Study (PON1) was the first prospective study to our knowledge aiming to ...evaluate visual acuity (VA) outcomes, including VA, recurrence risk, and final diagnosis 2 years after enrollment.
Nonrandomized observational study at 23 pediatric ophthalmology or neuro-ophthalmology clinics in the United States and Canada.
A total of 28 (64%) of 44 children initially enrolled in PON1 (age 3-<16 years) who completed their 2-year study visit.
Participants were treated at the investigator's discretion.
Age-normal monocular high-contrast VA (HCVA). Secondary outcomes included low-contrast VA (LCVA), neuroimaging findings, and final diagnoses.
A total of 28 participants completed the 2-year outcome with a median enrollment age of 10.3 years (range, 5-15); 46% were female, and 68% had unilateral ON at presentation. Final 2-year diagnoses included isolated ON (n = 11, 39%), myelin oligodendrocyte glycoprotein-associated demyelination (n = 8, 29%), multiple sclerosis (MS) (n = 4,14%), neuromyelitis optica spectrum disease (NMOSD) (n = 3, 11%), and acute disseminated encephalomyelitis (n = 2, 7%). Two participants (7%; 95% confidence interval CI, 1-24) had subsequent recurrent ON (plus 1 participant who did not complete the 2-year visit); all had MS. Two other participants (7%) had a new episode in their unaffected eye. Mean presenting HCVA was 0.81 logarithm of the minimum angle of resolution (logMAR) (∼20/125), improving to 0.14 logMAR (∼20/25
) at 6 months, 0.12 logMAR (∼20/25
) at 1 year, and 0.11 logMAR (20/25
) at 2 years (95% CI, -0.08 to 0.3 20/20
-20/40
). Twenty-four participants (79%) had age-normal VA at 2 years (95% CI, 60-90); 21 participants (66%) had 20/20 vision or better. The 6 participants without age-normal VA had 2-year diagnoses of NMOSD (n = 2 participants, 3 eyes), MS (n = 2 participants, 2 eyes), and isolated ON (n = 2 participants, 3 eyes). Mean presenting LCVA was 1.45 logMAR (∼20/500
), improving to 0.78 logMAR (∼20/125
) at 6 months, 0.69 logMAR (∼20/100
) at 1 year, and 0.68 logMAR (∼20/100
) at 2 years (95% CI, 0.48-0.88 20/50
-20/150
).
Despite poor VA at presentation, most children had marked improvement in VA by 6 months that was maintained over 2 years. Associated neurologic autoimmune diagnoses were common. Additional episodes of ON occurred in 5 (18%) of the participants (3 relapses and 2 new episodes).
