Dissecting the role of aberrant DNA methylation in human leukaemia

Abstract

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL fusion oncogene. However, many molecular mechanisms of the disease progression still remain poorly understood. A growing body of evidence suggests that the epigenetic abnormalities are involved in tyrosine kinase resistance in CML, leading to leukaemic clone escape and disease propagation. Here we show that, by applying cellular reprogramming to primary CML cells, aberrant DNA methylation contributes to the disease evolution. Importantly, using a BCR-ABL inducible murine model, we demonstrate that a single oncogenic lesion triggers DNA methylation changes, which in turn act as a precipitating event in leukaemia progression.

Figure 1. Reprogramming erases aberrant DNA methylation patterns in LiPS cells

A) DNA methylation profile of K562 and KBM7 cell lines, primary patient-derived CML cells, their corresponding LiPS clones, as well as human ES cells, CD34⁺-iPS and primary CD34⁺ cells from healthy donors. Each column of the heat map represents the average over two biological replicates. The heat map visualizes DNA methylation levels, with red color indicating low levels of DNA methylation and blue color indicating high levels. White color indicates DNA regions not covered by RRBS analysis. For each genomic region type, shown are the 2% of regions that scored highest according to a ranking score in differential analysis comparing the group of K562, KBM7, and primary CML samples to the group including all derived iPS cell lines (see also the Methods section). B) DNA methylation profile of SALL4 gene locus, hypermethylated in all the leukemia cells and demethylated after reprogramming (upper panel). DNA methylation profiles of HOXA5 and BRCA1 gene loci are shown in the middle and lower panels, respectively. Each row represents a sample, while each column represents a CpG dinucleotide. Blue color indicates high DNA methylation levels, and red color indicates low methylation levels. CpG islands are annotated with green bars. Mean promoter and gene methylation levels are indicated at the right.

Figure 2. Characterization of Differentially Methylated Regions distinguishing CML cells from their reprogrammed counterparts

A) Scatterplot comparing mean promoter methylation in CML cells with their derived LiPS cell lines. The blue color gradient depicts point density. The 500 promoters with highest combined ranking of absolute/relative differential methylation and statistical significance are depicted in red. B–C) P-values for significant Gene Ontology terms enriched among the 500 most highly ranking hypomethylated (red) and hypermethylated (green) gene promoters in reprogrammed cells relative to CML cells. D) Overlap with genomic and epigenomic features in an EpiExplorer analysis. The 500 most highly ranking differentially methylated tiling regions (blue) are compared to a background of 72,469 of 5kb tiling regions covered by RRBS (red). The ChIP-seq peak tracks are from the ENCODE project for the GM12878, HMEC, NHLF, NHEK, HUVEC and H1 ES cell lines, chromatin state segmentations are based on chromHMM and the gene, gene promoter (TSS-5kb to TSS+1kb), CpG islands, repeat and conserved locus annotation are from the UCSC Genome Browser. Overlaps of at least 1bp are shown as percentage of all regions.

Figure 3. Reprogramming restores myeloid differentiation decreasing the malignancy of leukemia cells

A) CD45⁺ CD15⁺ cells derived from LiPS 1 clone revealing a typical morphology of terminally differentiated monocytes (center) and macrophages (right) (Scale bars 20 µm). B) Survival curve of NSG mice transplanted with K562 cells (n=3), LiPS-derived CD45⁺ cells (n=4) and CD45+ LiPS3 cells (n=4). (*P<0.05; **P<0.01 and ***P<0.001; Mantel-Cox Survival Test) C) FACS analysis showing engraftment of K562 cells, LiPS-derived CD45⁺ cells and a negative control in NSG mice. Indicated are the percentages of CD45⁺ cells. D) Primary cells from two CML patients acquiring typical morphology of terminally differentiated monocytes after AZA treatment. E) FACS analysis showing high expression of myeloid differentiation marker CD11b and CD14 in primary CML cells after AZA treatment.

Figure 4. BCR-ABL activation induces aberrant DNA methylation pattern in murine HSCs

A) FACS analysis of BCR-ABL transgenic murine bone marrow mononuclear cells in which the BCR-ABL oncogene is not expressed (“control”), is expressed (“leukemic”) or repressed after previous expression (“rescued”). Leukemic mice showed moderate expansion of the HSCs population cKit⁺ Sca1⁺ and a strong expansion of the Mac1⁺ Gr1⁺ myeloid population compared to control and rescued animals. B) DNA methylation analysis of CpG islands in the Lin⁻ cKit⁺ Sca1⁺ population of BCR-ABL transgenic mice with different status of oncogene expression. Zoomed-in region shows example of CpG islands differentially methylated between leukemic, control and rescue mice. Each row represents the average of two biological replicates. Each column represents a complete CpG island. Blue: high methylation, Red: low methylation. White color indicates DNA regions not covered by RRBS analysis. C) Heatmap visualizing genes and promoters hypermethylated in leukemic mice versus control and rescued ones. D) High-resolution view of DNA methylation of the Hoxb1 gene locus, which was hypermethylated in leukemic mice as compared to the control mice. Each row represents the average of two biological replicates; each column represents a single CpG dinucleotide.

Figure 5. 5-Azacytidine reduces the oncogenic potential of BCR-ABL expressing cells

A) Peripheral Blood FACS analysis of BCR-ABL transgenic mice expressing BCR-ABL shows strong reduction of the Mac1⁺ and Gr1⁺ myeloid population in mice treated with Imatinib, AZA or combination of the two drugs. B) Survival curve of B6.SJL-Ptprc^a Pepc^b/BoyJ (pep boys) congenic recipient mice transplanted with BCR-ABL leukemic bone marrow shows prolonged survival rate upon AZA treatment as compared to Imatinib group and untreated mice. Between 8–10 mice (diagram of treatment shown in Supplementary Figure 5B) were used per each group.