Genomic impact of transient low-dose decitabine treatment on primary AML cells

Abstract

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

Figure 1

In vitro expansion of human AML cells. (A) Growth curves (top panels) and morphology (bottom panels) of primary AML cells plated on different stromal cells for 3 weeks. All cells were grown in the presence of human IL3, IL6, SCF, TPO, and FLT3 ligand. (B) Metaphase fluorescence in situ hybridization (FISH) for MLL rearrangement on UPN 410324 after 1 week of stromal coculture with a dual-color break apart probe, showing 1 normal 11q23 locus (yellow, arrow) and a typical rearrangement (separate red and green), as well as an extra 3′ MLL signal (red). This pattern was seen in 100 of 100 cells, and the identical rearrangement pattern was seen at the time of diagnosis. (C) Identification of AML-specific mutations in 2 primary AML samples. Shown are the variant allele frequencies at day 0 (black) and after 7 days of culture on HS27 stroma (red). (D) Engraftment of UPN 476081 at 16 weeks in the bone marrow of NSG mice after 2 weeks of growth on HS27 (right), compared with 476081 after overnight culture (left).

Figure 2

Characterization of UPN 476081. Cells from 476081 were grown on stroma for 4 days, followed by a 3-day treatment with cytarabine (AraC) or decitabine (DAC). Drug was administered daily. After 3 days of treatment, the number of viable cells (A), annexin-positive cells (B), and total 5-methycytosine content by LC-MS/MS (C) was determined. (D-E) cell cycle profiles were determined after overnight EdU incorporation and DNA content was measured by FxCycle Violet DNA dye. Shown are representative flow plots (D) and distributions of cell cycle phase after drug treatment (E). All experiments were performed in triplicate; error bars represent 95% confidence intervals; asterisk indicates a significant change from mock treatment. (F) CD45^dim/SS^low blast gate (top panel) and expression of CD33 and CD14 antigens (bottom panel) of cells from 476081 after drug treatment. (G) Heatmap showing mRNA fold change using the nanostring platform of drug treated cells relative to mock. Up-regulated genes are shown in red; down-regulated genes in green. Experiments were performed in triplicate; all data were normalized to GAPDH expression levels.

Figure 3

Characteristics of primary de novo AML cells. Cells were grown on stroma in the absence of drug for 4 days, followed by a 3-day treatment with either 100nM AraC or 100nM DAC. (A) Fold change of cells during the 3 days of drug treatment. (B) Fraction of cells that excluded 7-AAD, relative to the mock treated samples. (C) LC-MS/MS determination of 5-methylcytosine; all values are shown relative to the percentage methylcytosine in the untreated samples. (D) Measurement of H19 mRNA using the nanostring platform; fold change is shown relative to the mock treated cells. Data points in panels A through C represent the mean of technical replicates (n = 3). mRNA measurements were performed in duplicate. Error bars represent 95% confidence intervals.

Figure 4

Distribution of methylation values for a representative primary AML sample (721214) relative to gene and CpG island annotations, and expression. (A) Methylation value distributions for CpGs in promoters (green), gene bodies (orange), 3′ untranslated regions (purple); the distribution for all genic CpGs is shown in black. (B) Methylation value distributions for CpGs in islands (green), within 2 kbp of islands (orange), 2 to 4 kbp from islands (purple), and outside of islands (black). (C) Methylation value distributions for CpGs in promoters of genes stratified by array-based expression quartile. (D) Methylation value distributions for all genic CpGs stratified by array-based expression quartile.

Figure 5

Effects of short-term DAC treatment on primary AML samples. (A) Distribution of methylation values for all interrogated CpGs from 8 primary AML samples treated with DAC (blue), cytarabine (red), or DMSO (black). (B) Change in methylation values on treatment with DAC, versus methylation in mock-treated samples, for a representative primary AML sample (721214). (C) Hierarchical clustering of methylation values of 8 primary AML samples treated with DAC, AraC, or vehicle using the 1000 most variable CpGs across all arrays.

Figure 6

Global and focal patterns of DAC-induced hypomethylation. Distribution of methylation changes after short-term DAC treatment as a function of methylation level (binned by β value on x-axis) in the mock-treated sample at CpGs in promoters, gene bodies, and with respect to CpG island annotation. A representative primary AML sample (721214) is shown.

Figure 7

DAC-induced changes in expression and methylation. (A-B) Change in log2 expression versus change in mean methylation value at promoter-associated CpGs between DAC and mock-treated samples. The mean methylation value was calculated using all promoter CpGs annotated for each RefSeq transcript. A representative case (721214, panel A) is shown, as well as the case with profound DAC-induced hypomethylation (775109, panel B). (C) Heatmap representation showing probes with consistent expression changes (fold-change > 1.5 and FDR < 0.05) across all 18 AML samples. (D-E) Change in log2 expression versus change in mean methylation value at promoter-associated CpGs between decitabine and mock-treated samples at a selected group of transcripts. (D) Black points represent 7 genes (TKTL1, H19, COL14A1, PGF, DAZL, PNMA5, and AB128832) up-regulated by DAC treatment (each point represents a single transcript in a single AML sample). Also shown are CDKN2B and CDH1, 2 genes commonly reported to be regulated by methylation in AML cells. (E) Points represent individual transcripts (SCARB1, RSRC1, CYTH4, WDR87, SIPA1L3, MEGF6, CCDC62, ELF2, NCL, and SNORA75) with the promoter CpGs that have largest DAC-induced change in methylation.