Low-dose 5-aza induces distinct genome-wide activation of p53 target genes and unique repression of pluripotency genes in NT2/D1 cells.

<p><b>A</b>, Venn diagrams of expression microarray data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone-0053003-g002" target="_blank">Figure 2</a> indicating a large overlap in the genes upregulated (left) and down regulated (middle) in NT2/D1 cells with 1 day 5-aza treatment (fold change >1.3) compared to 3 day 5-aza treatment (fold change >1.5). A Venn diagram (right) of microarray data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone-0053003-g002" target="_blank">Figure 2</a> showing a large degree of overlap in genes upregulated 1.5-fold or greater by both 3 day 5-aza and cisplatin treatments. Genes listed in each Venn diagram are altered with p<0.05. Of the overlap genes, 13 are known p53 target genes in NT2/D1 cells. <b>B</b>, Partitioning around mediods (PAM) analysis of 5-aza and cisplatin regulated genes in NT2/D1 cells. The 1130 genes changed 1.5-fold or greater with BH corrected p-value of <0.02 were subjected to PAM analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#s4" target="_blank">Methods</a>. The number of genes in each of the five clusters and the mean silhouette width (MSW) value for each cluster is indicated. Expression intensity values for representative genes in Cluster 2, Cluster 3, and Cluster 4 is provided on the left. Error bars are S.E.M. * = p<0.05; ** = p<0.005 compared to untreated controls.</p

DNMT3B knockdown inhibits low-dose 5-aza mediated genome-wide activation of p53 target genes and repression of pluripotency genes.

<p><b>A</b>, Summary of results from expression microarray data of NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. Knockdown of DNMT3B greatly reduced the number of genes altered with 5-aza treatment while DNMT3B knockdown alone results in minimal expression changes. The number of genes altered was based on the average of 3 biological replicates with 1.5-fold or greater change and a p value<0.01. <b>B</b>, Unsupervised hierarchical cluster analysis of the expression data in (A) was performed on the 541 genes altered more than 1.8-fold with p value<0.01 ANOVA BH corrected, between NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. Upregulated gene are red, downregulated genes are green. C, Partitioning around mediods (PAM) analysis of 1169 genes changed 1.5-fold or greater with BH corrected p-value of <0.01. Cluster 1 and Cluster 2 of 6 total clusters are shown. All clusters are provided in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone.0053003.s004" target="_blank">Fig S4</a>. The number of genes in the clusters and the mean silhouette width (MSW) value is indicated. Expression intensity values for representative genes in Cluster 1 (pluripotent genes) and Cluster 2 (p53 target genes) is provided on the right and additional prominent members are provided below each cluster. Error bars are S.E.M. * = p<0.05 compared to untreated control.</p

Confirmation of promoter DNA demethylation with low-dose 5-aza in NT2/D1 cells.

<p><b>A</b>, Representative bisulfite pyrosequencing tracing from NT2/D1 cells treated with vehicle control or 10 nM 5-aza for 3 days in the promoter region of SOX15. Shaded areas are CpG sites. <b>B</b>, Decrease in DNA methylation of the RIN1, SOX15 and TLR4 promoter with 3 day 10 nM 5-aza treatment of NT2/D1 cells as determined by bisulfite pyrosequencing. Average of biological triplicate determinations. Error bars are standard deviation. ** = p<0.005. SOX15 values represent the average methylation value across two CpG sites. RIN1 and TRL4 values represent the average methylation across three CpG sites. <b>C</b>, Increased gene expression of RIN1, SOX15 and TLR4 with 3 day 10 nM 5-aza treatment of NT2/D1 cells as determined by real-time PCR. Average of biological triplicate determinations. Error bars are standard deviation. * = p<0.05; ** = p<0.005.</p

Low-dose 5-aza and DNMT3B knockdown alters genome-wide promoter demethylation in NT2/D1-R1 cells.

<p><b>A</b>, Summary of results from Illumina 27K beadchip DNA promoter methylation analysis of NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. The number of promoter methylation alterations was based on the average of 3 replicates with 1.3-fold or greater change and a p value<0.05. <b>B</b>, Venn diagrams depicting degree of overlap in genes altered in expression or DNA promoter methylation in NT2/D1-R1 cells due to low dose 5-aza or DNMT3B knockdown. <b>Top</b>, High degree of overlap in genes in NT2/D1-R1 cells demethylated with low dose 5-aza and demethylated with DNMT3B knockdown (left). Little overlap in genes in NT2/D1-R1 cells undergoing increased DNA methylation with low dose 5-aza and increased DNA methylation with DNMT3B knockdown (right). The numbers represent methylation changes of 1.3-fold or greater with p<0.05. <b>Middle</b>, A moderate degree of overlap in genes in NT2/D1-R1 cells that underwent decreased DNA methylation and increased gene expression with 5-aza (left) and little overlap in genes in NT2/D1-R1 cells that underwent increased DNA methylation and increased gene expression with 5-aza. The numbers represent methylation changes of 1.3-fold or greater with p<0.05 and expression changes of 1.5-fold or greater with p<0.01. <b>Bottom</b>, A large degree of overlap in genes demethylated after 5-aza treatment of NT2/D1 and NT2/D1-R1 cells. The six overlapping genes are shown. The numbers represent methylation changes of 1.3-fold or greater with p<0.05. <b>C</b>, Unsupervised hierarchical clustering of the promoter DNA methylation of the 4 treatment arm values among just the 388 genes changed 1.3-fold or greater, p value<0.05 with 5-aza in the control cells depicting the large degree of overlap in genes undergoing demethylation with 5-aza and DNMT3B knockdown. Increased methylation = red, decreased methylation = green.</p

Low-dose 5-aza induces an early, robust, and unique reprogramming of gene expression in NT2/D1 cells.

<p><b>A</b>, Scatter plot of microarray gene expression differences in NT2/D1 cells. Each point represents the average of 3 biological replicates. Points above the diagonal line represent genes upregulated and points below the diagonal line repressed with 1 day 5-aza treatment (top), 3 day 5-aza treatment (middle), and cisplatin treatment (bottom), compared to control. The number of altered genes above the indicated thresholds were changed with p<0.05. <b>B</b>, Robust induction of gene expression occurs with low-dose 5-aza. Box-whisker plot of expression levels of genes (1296) changed among groups >1.2-fold and p<0.02 ANOVA, Benjamini Hochberg (BH) corrected indicated a upward shift in gene expression with 5-aza. <b>C</b>, Low dose 5-aza induced an early and unique program of gene expression compared to cisplatin. Unsupervised hierarchical cluster analysis of the expression profile of 5-aza and cisplatin altered genes was performed on the 898 genes (rows) altered between the samples (columns) more than 1.5-fold with p value<0.01 ANOVA, BH corrected. Upregulated gene are red, downregulated genes are green. Large overlap in 1 day and 3 day 5-aza treatments and distinct regulation compared to cisplatin is evident. Genes regulated in a similar manner by 5-aza and cisplatin are in brackets.</p

Knockdown of DNMT3B results in resistance to low-dose 5-aza downstream of DNA damage and p53 protein induction.

<p><b>A</b>, DNMT3B knockdown in NT2/D1-R1 cells leads to resistance to low-dose 5-aza. Indicated doses of 5-aza were added fresh each day for 3 days to exponentially growing cultures of NT2/D1-R1 lentiviral control cells and NT2/D1-R1 cells stably expressing a lentiviral shDNMT3B construct. Viable cell growth and survival were measured. Data was normalized to no drug treatment. Error bars (within symbols) are standard deviation. * p = <0.05 compared to untreated controls. <b>B</b>, Low dose 5-aza induces DNA damage in NT2/D1-R1 cells independent of apoptosis. Cells were pretreated with vehicle or 20 µM Z-VAD-FMK (z-VAD) and treated with 100 nM 5-aza or 2 µM cisplatin for 1 day prior to Western analysis. Accumulation of pH2AX is indicative of double strand breaks. The arrow labeled cPARP indicates cleaved PARP that is indicative of apoptosis. Densitometry of pH2AX normalized to actin is shown. <b>C</b>, DNMT3B knockdown in NT2/D1-R1 cells results in resistant to low-dose 5-aza induced cell death and G2 cell cycle arrest. Cells were treated with 10 nM 5-aza for 3 days and then assayed for cell cycle analysis. Substantial cells in subG1 are indicative of cell death. <b>D</b>, Knockdown of DNMT3B in NT2/D1-R1 cells inhibits low-dose 5-aza mediated cell death but not p53 protein induction or induction of DNA damage. NT2/D1-R1 cells with no lentivirus and cells treated with sh-control lentvirus and sh-DNMT3B lentivirus were treated with 10 nM 5-aza for 3 days, 1 µM cisplatin for 12 hours or the combination. Western analysis was performed for p53, PARP and pH2AX.</p

Comparison of treatment related changes in gene expression for responding versus non-responding patients (POST minus PRE).

<p>Immune therapy leads to activation of stimulatory pathways in responding subjects. Examples of activated pathways are shown. The CD28 co-stimulatory, nuclear factor of activated T-cell (NFAT) and HIFα pathways were relatively up-regulated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050221#pone-0050221-g004" target="_blank">Figure 4A–4C</a>) for responding subjects. Another key pathway (IPA) differentially regulated in R vs. NR PBLs was the Flt3 signaling pathway (fms-like tyrosine kinase receptor-3; CD135) in which genes were overall down-regulated in non-responding patients but up-regulated in responding patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050221#pone-0050221-g004" target="_blank">Figure 4D</a>). To determine whether the genes picked by ingenuity software are able to discriminate between responding and non-responding patients, we performed supervised (selected genes) clustering analysis of genes important for Flt3 signaling. This clearly grouped responders and non-responders in 2 distinct groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050221#pone-0050221-g004" target="_blank">Figure 4D</a>), strengthening the IPA-result. Supervised clusters of pathways in 4A–4C led to similar results (data not shown).</p

Serum cytokine (27 plex) assay results.

*<p>p<0.05.</p><p>Ten cytokines showed significant up-regulation in patients vs. healthy controls; 5 additional cytokines showed a clear increase in mRCC patients, but due to low sample size and high standard deviations these contrasts did not reach significance. IL-1b, IL-2, IL-5, IL-9, IL-10, IL-15 and IL-17 had detectable values in very few of the 16 tested samples and were thus excluded from the analysis and table.</p

Comparison of gene expression of PBLs of mRCC patients and healthy controls following normalization of microarray expression data. Gene Set Enrichment Analysis (GSEA) was performed for 17 mRCC patients PRE (yellow background, #10–26) vs. 9 healthy controls (grey background, #1–9).

<p>GSEA uses a Kolmogorov Smirnov statistic to explore unique gene signature of small groups of genes within a data set. Shown are representative pathways that were up-regulated in the mRCC samples (p<0.05).</p

Separate comparison of gene expression post-treatment versus gene expression pre-treatment for responding and non-responding patients. Ingenuity pathway analysis suggests differences in regulation of T-cell (<b>Figure 3A</b>) and B-cell (<b>Figure 3B</b>) receptor signaling.

<p>Most of the molecules in the B-cell as well as in the T-cell receptor pathway are down-regulated for non-responding patients upon immunotherapy (POST PRE). Red: up-regulation, Green: down-regulation.</p