Effects of 6‑Aminonicotinic Acid Esters on the Reprogrammed Epigenetic State of Distant Metastatic Pancreatic Carcinoma

In the search for alternatives to 6-aminonicotinamide (6AN), a series of 6-aminonicotinic acid esters were designed and synthesized as precursors of 6-amino-NADP+, a potent inhibitor of 6-phosphogluconate dehydrogenase (6PGD). Like 6AN, some of these esters were found to reverse the loss of histone 3 lysine 9 trimethylation (H3K9me3) in patient-derived pancreatic ductal adenocarcinoma (PDAC) distant metastasis (A38-5). Among them, 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 6-aminonicotinate (5i) showed more potent antiproliferative activity than 6AN. Metabolite analysis revealed that compound 5i produced a marked increase in metabolites upstream of 6PGD, indicating intracellular inhibition of 6PGD by 6-amino-NADP+ derived from compound 5i through 6-aminonicotinic acid (6ANA) via the Preiss–Handler pathway. Despite the more potent pharmacological effects shown by compound 5i in A38-5, compound 5i was found to be substantially less toxic to primary hippocampal rat neurons compared to 6AN, indicating its therapeutic potential in targeting distant metastatic cells

DNA methylation of cord blood cell types: Applications for mixed cell birth studies

<p>Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test <i>P</i> < 10⁻⁸). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at <i>P</i> < 10⁻⁸) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.</p

Examples of CHARM results for two of the regions showing greatest DNAm differences between MDD cases and controls.

<p>The plots show percent methylation versus genomic location with each point representing the methylation level of an individual sample for a given probe. The curve represents averaged smoothed percent methylation values. The locations of CpG dinucleotides are indicated with black tickmarks on the X-axis. CpG density was calculated across the region using a standard density estimator and is represented by the smoothed black line. The location of the CpG island is denoted on the X-axis as an orange line. Gene annotation is indicated, showing <i>LASS2</i> in (a) and <i>PRIMA1</i> in (b). The thin outer grey line represents the transcript, while the thin inner lines represent a coding region. Filled in grey boxes represent exons.</p

Immunohistochemical pattern of AChE in frontal cortex.

<p>(A) In controls there is diffuse and intense pattern of immunoreactivity involving mainly the neuropil. (B) In MDD subjects, though variable, immunostaining was reduced. Both 200×. (C) In controls, there is virtually no perikaryal staining. (D) The latter contrasts with the pattern observed in some areas in MDD subjects, in which groups of pyramidal neurons display intense perikaryal staining, suggesting redistribution of the enzyme to the cell body. The red circles highlight examples. Both 640×.</p

Stanley Medical Research Institute MDD and control brain samples.

<p>Stanley Medical Research Institute MDD and control brain samples.</p

<i>PRIMA1</i> DNAm by diagnosis, and by covariate status^a.

a<p>Dx = diagnosis; PMI = postmortem interval; DNAm = DNA methylation; p-values<0.05 are italicized for clarity.</p

Results of bisulfite pyrosequencing for validation of CHARM in brain samples.

<p>Regions in or near four genes showed differences that remained statistically significant after correction for having tested 17 genes: (a) <i>LASS2</i>, (b) <i>CPSF3</i>, (c) <i>ZNF263</i>, (d) <i>PRIMA1</i>. The grey bars represent values from control brain sample DNA, while the black bars represent those from MDD brain samples. The Y-axis is percent DNA methylation, while the X-axis shows distance along the chromosome for each CpG dinucleotide assayed. One asterisk indicates a difference between MDD and control of p<0.05. Two asterisks indicates p<0.0029 (a correction for 17 regions tested). Three asterisks indicates p<0.00054 (a correction for 92 CpGs tested).</p

A Selective Phenelzine Analogue Inhibitor of Histone Demethylase LSD1

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor <i>in vitro</i> and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1–/– cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease

A Selective Phenelzine Analogue Inhibitor of Histone Demethylase LSD1

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor <i>in vitro</i> and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1–/– cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease