The antileukemic activity of decitabine upon PML/RARA-negative AML blasts is supported by all-trans retinoic acid: in vitro and in vivo evidence for cooperation

Abstract

The prognosis of AML patients with adverse genetics, such as a complex, monosomal karyotype and TP53 lesions, is still dismal even with standard chemotherapy. DNA-hypomethylating agent monotherapy induces an encouraging response rate in these patients. When combined with decitabine (DAC), all-trans retinoic acid (ATRA) resulted in an improved response rate and longer overall survival in a randomized phase II trial (DECIDER; NCT00867672). The molecular mechanisms governing this in vivo synergism are unclear. We now demonstrate cooperative antileukemic effects of DAC and ATRA on AML cell lines U937 and MOLM-13. By RNA-sequencing, derepression of >1200 commonly regulated transcripts following the dual treatment was observed. Overall chromatin accessibility (interrogated by ATAC-seq) and, in particular, at motifs of retinoic acid response elements were affected by both single-agent DAC and ATRA, and enhanced by the dual treatment. Cooperativity regarding transcriptional induction and chromatin remodeling was demonstrated by interrogating the HIC1, CYP26A1, GBP4, and LYZ genes, in vivo gene derepression by expression studies on peripheral blood blasts from AML patients receiving DAC + ATRA. The two drugs also cooperated in derepression of transposable elements, more effectively in U937 (mutated TP53) than MOLM-13 (intact TP53), resulting in a "viral mimicry" response. In conclusion, we demonstrate that in vitro and in vivo, the antileukemic and gene-derepressive epigenetic activity of DAC is enhanced by ATRA.

Fig. 1. Decitabine and all-trans retinoic acid exhibit cooperative effects on inhibition of proliferation and reduction of viability in AML cell lines.

A Treatment scheme. B Baseline-corrected proliferation of OCI-AML3, MOLM-13, MV4-11, U937, THP1, and HL-60 cells treated with DAC (100 nM) and ATRA (1 µM) after 120 h. Experiments were conducted in technical triplicates. C Proliferation and viability of U937 and MOLM-13 cells were measured 72 and 120 h after the first dose of Decitabine, untreated or treated with DMSO, DAC (U937: 200 nM; MOLM-13: 100 nM), ATRA (1 µM) or DAC + ATRA in combination. Three independent experiments, each with three independent technical replicates, were conducted. Standard deviation is shown as error bars. D Caspase-3/7 activity in U937 (left panel) and MOLM-13 cells (right panel) displayed as fold-change relative to a DMSO-treated control at 72 and 120 h. Six (72 h) and nine (120 h) independent experiments, each with three independent technical replicates, were conducted, respectively. Standard deviation is shown as error bars. Statistical significance was tested for by unpaired t-test with a threshold of p < 0.05.

Fig. 2. Decitabine and all-trans retinoic acid cooperate in inducing transcriptome changes in AML cells.

A Global expression changes in U937 and MOLM-13 were measured by RNA-seq (FDR < 0.01). Cells were treated with DAC, ATRA or both in combination, and harvested 72 h after the first dose of Decitabine. Bar and Venn diagrams depict the numbers of altered transcripts in comparison to untreated; upregulated transcripts are shown in red, downregulated in blue. B Comparison of significantly regulated genes in U937 and MOLM-13 treated with DAC + ATRA vs. untreated (determined by RNA-seq). C Heatmaps (unsupervised clustering) of the commonly regulated genes in U937 or MOLM-13, comparing untreated (untr.) to DAC + ATRA-treated cells. D GO analysis for the commonly up- or downregulated genes in U937 and MOLM-13 by DAC + ATRA.

Fig. 3. Cooperation between DAC and ATRA in modulating chromatin accessibility.

A Enrichment of accessible chromatin sites across the whole genome determined by ATAC-sequencing in U937 cells (technical triplicates) either untreated (untr.) or treated with DMSO, DAC, ATRA or DAC + ATRA at 72 h. Sequencing was performed with 40 million reads per sample (paired-ends). Mean alignment was at about 99%, with 51–63% uniquely mapped sequences. B Principal component (PC) analysis (PCA) of the assay for transposase-accessible chromatin sequencing (ATAC-seq) data of control (untreated, CNTL; n = 3), ATRA (n = 3), DAC (n = 3), and DAC + ATRA-treated (n = 3) samples. Chromatin accessibility of the 200,000 most variably accessible peaks was used for dimensionality reduction. C Heatmap of the 50,000 most variable differentially accessible regions (DARs) from the ATRA vs. untreated (CNTL), DAC vs. CNTL, and ATRA + DAC vs. CNTL comparison. Euclidean distance of the z-scaled chromatin accessibility of DARs in all samples was visualized. D Homer transcription factor motif enrichment of differentially accessible regions (adj. p-value < 0.05 and absolute log 2 fold-change >1), stratified in open and closed DARs. The top five most significantly enriched transcription factor motifs as well as all significantly enriched (adjusted p-value < 0.05) retinoic acid response (RAR) motifs are visualized. The union set of all peaks was used as a background for enrichment analyses.

Fig. 4. Transcriptional induction of HIC1 and CYP26A1 by DAC and ATRA is associated with changes in chromatin accessibility.

A, B mRNA expression (normalized read-counts) of HIC1 (A) and CYP26A1 (B) of both untreated U937 cells and U937 after treatment with either DAC, ATRA, or DAC + ATRA at 72 h was determined by RNA-seq (left panel). Chromatin accessibility of these cells and treatment modes was determined by ATAC-seq (upper right panel). Blue boxes below the ATAC-seq track represent significant effects. Annotations for POLR2A binding sites (green boxes), retinoic acid receptors RARA and RXRA (brown boxes) and CpG islands (black boxes) were extracted from ChiP-seq data accessed from ENCODE and GEO (right panel). CpG methylation data of U937 cells untreated or treated with DAC were determined by Infinium HumanMethylation450 BeadChip Assay [8] (lower right panel).

Fig. 5. Transcriptional induction of GBP4 and LYZ by DAC and ATRA is associated with changes in chromatin accessibility.

A, B mRNA expression (normalized read-counts) of GBP4 (A) and LYZ (B) of both untreated U937 cells and U937 after treatment with either DAC, ATRA, or DAC + ATRA at 72 h was determined by RNA-seq (left panel). Chromatin accessibility of these cells and treatment modes was determined by ATAC-seq (upper right panel). Blue boxes below the ATAC-seq track represent significant effects. Annotations for POLR2A binding sites (green boxes), retinoic acid receptors RARA and RXRA (brown boxes) and CpG islands (black boxes) were extracted from ChiP-seq data accessed from ENCODE and GEO (right panel). CpG methylation data of U937 cells untreated or treated with DAC were determined by Infinium HumanMethylation450 BeadChip Assay [8] (lower right panel).

Fig. 6. Global DNA demethylation by DAC is not enhanced by ATRA.

A U937 cells (technical triplicates) were treated with either DAC (lilac), ATRA (green), DAC + ATRA (orange) or left untreated (untr.; pink) and analyzed after 72 and 120 h. Global DNA methylation was acquired by Infinium HumanMethylation450 BeadChip Assay [8]. Methylation data was analyzed with RnBeads as described in Materials and Methods and Suppl. Methods. The heatmap displays the methylation percentiles (%) for all 24 samples analyzed. Unsupervised, hierarchical clustering showed subclustering in the DAC/DAC + ATRA arm for the two time points with an outlier in the 120 h group. B LINE-1 methylation of three selected CpGs was determined by MassArray in U937 cells (technical triplicates) treated with DAC, ATRA or DAC + ATRA at 72 h.

Fig. 7. Changes in primary blasts of AML patients treated with decitabine and all-trans retinoic acid.

A Venn-diagram of transcriptionally regulated genes in U937 and MOLM-13 cells (determined by RNA-seq) and AML patients (n = 6) treated with DAC + ATRA (primary AML blasts; expression arrays performed on day 0 and day 8, compared to untreated). B GO analysis of up- or downregulated genes, in U937 and MOLM-13, and primary AML blasts. C Scatter plots of HIC1, GBP4 and LYZ expression changes in patient samples (day 8 vs. day 0) having received DAC only (n = 9) or DAC + ATRA (n = 6). Fold-changes are displayed. Statistical significance was tested for by unpaired t-test with a threshold of p < 0,05 (ns, not significant). D Scatter plots displaying expression changes in CTSG, ELANE, IL7R and CXCR1 as described in Fig. 7C. E Scatter plot of MYC expression changes (downregulated by DAC + ATRA compared to DAC alone) as described in Fig. 7C. F Levels of fetal hemoglobin (HbF,%) in AML patients’ peripheral blood erythrocytes before treatment (baseline) and after 2 courses (p.c.2) of treatment with either DAC only (n = 6) or DAC + ATRA (n = 5) were determined by HPLC as described in Materials and Methods.

Fig. 8. Decitabine and ATRA cooperate in derepressing transposable elements in AML cells, activating dsRNA sensing.

A Global expression changes of TEs in U937 and MOLM-13 were measured by RNA-seq (FDR < 0.05). Cells were treated with DAC, ATRA or both in combination and harvested 72 h after first dose of Decitabine. Bar and Venn diagrams show the numbers of altered TEs for each treatment (DAC, ATRA, DAC + ATRA) in comparison to untreated; upregulated transcripts shown in red, downregulated in blue. B Comparison of regulated TEs in U937 and MOLM-13 treated with DAC + ATRA vs. untreated determined by RNA-seq. 102 out of 207 TE (49.3%) transcripts show concordance regarding induction vs. repression, as depicted in the lower graph. C Waterfall plots of the 10 most up- or downregulated TEs in U937 or MOLM-13 treated with DAC + ATRA. D Immunoblot results for RIG-I, MDA5 and MAVS in whole-cell lysates of U937 and MOLM-13 treated as indicated above. Beta-actin was used as loading control; the space between protein bands was cropped to conserve space. Results for the poly(I:C) sample in MOLM-13, repeated due to technical issues, were replaced.