Genomic locations of elderly AML differential methylation

We were attentive to the importance of defining genomic regions where the methylation levels are unique. Large epigenetic changes that occur in specific regions of the genome might be of additional significance.

So, we analysed particular patterns in the distribution of the elderly AML DMRs across genomic positions. We made two main observations: 1) most chromosomes had comparable amounts of hyperMRs and hypoMRs, except for chromosomes 16 and 17 where the number of hypermethylations in samples of cluster of elderly AML was clearly high (Figure 4.5.4A) and 2) there was a tendency of the hyperMRs to be located in the tips of chromosomes (Figure 4.5.4B).

The higher methylation levels of the samples from the cluster of the elderly were more striking in chromosome 17. This was the chromosome with the highest number of hyperMRs (109 of the 979, 11%) and many of these regions were located in the q25.3 band (Figure 4.5.4B, box). This was an unusual concentration of hypermethylation on the tip of chr17 in which 35 hyperMRs (35/109, 32%, Table 4.5.1) were concentrated in about 2.4Mb (highest density area in the blue box of Figure 4.5.4B).

Within this area, there were 2 major regions strongly hypermethylated, the regions related to neighboring genes ACTG1/KIAA1447 and to RPTOR/CHMP6.

These were the 4 genes that had the highest number of hypermethylated regions from

the cluster of elderly associated to them. There were 9 hyperMRs per each pair of

genes (ACTG1/KIAA1447 and RPTOR/CHMP6) and each of these regions related at

least to 3CpGs.

Figure 4.5.4 Distribution of DMRs of the cluster of elderly AML across chromosomes.

(A) The bar graph reports the number of DMRs from the cluster of elderly vs cluster of young in each

chromosome (comparison of hierarchical clusters in Figure 4.5.1). These were classified as

hypermethylated (hyperMRs, in red) or hypomethylated (hypoMRs, in blue) according to the methylation

level in the cluster of elderly AML. (B) The graph shows the hyperMRs plotted according to their genomic

position from the start of the short arm to the end of the long arm (oriented left to right) of chromosomes

1 to 22. The density plots represent the number of hyperMRs calculated by partitioning the genome in

0.2Mb regions. Chromosome 17q25.3 band was highlighted with a blue box. The scale of genomic regions

is given in Mb. Red region of chromosome ideograms marks the centromeres.

Table 4.5.1 HyperMRs of the

When examined in detail different CpGs across this 2.4Mb region have different

factors that correlate with their methylation levels. Some CpGs seem strongly affected

by mutations and other clearly more related to patients’ age. As an example of this, we

show the regions of the 3 adjacent genes KIAA1447, ACTG1 and FSCN2 (Figure

4.5.5). Some CpGs were clearly different between the 2 hierarchical clusters

(examples in boxes A and B), while others were particularly hypomethylated in

DNMT3A/NPM1/FLT3 mutated samples (example in box C) and/or very strongly

hypermethylated in the IDH1/2 mutated patients (example in box D).

Figure 4.5.5 Distinct methylation profiles of elderly in the KIAA1447/ACTG1/FSCN2 region.

DNA-methylation from the CpGs in the tip of the long arm of chromosome 17 using beta-values scaled from unmethylated (-5, blue) to fully methylated (5, red). In the depiction the 273 AML samples from the two cohorts (TCGA and SAL) are in horizontal and positions of CpGs according to GRCh37/hg19 are in vertical. The CpG islands of the genomic region are marked with blue boxes. Patients are annotated according to the group of the 1 ^st division of hierarchical clustering (cluster of elderly, purple annotation, or young, orange annotation, Figure 4.5.1), age group according to patient chronological age (elderly for ≥65 years old, black annotated, or young for <65 years old, yellow annotation) and mutation information for 2 relevant high rate mutated groups (IDH1/2, red annotation, and triple mutated DNMT3A/NPM1/FLT3, blue annotation).

The particular hypermethylation pattern of the tip of chr17 overlapped several

genes that were part of endocytosis and phagocytosis processes (see gene

enrichment in Figure 4.5.3), with emphasis on functions in actin organization. The

ACTG1 codes for the gama-actin in the cellular cytoskeletons and is one of the genes

in the biological process enrichment phagocytosis of the hyperMRs ¹³⁶ . Gama-actin is

also important for cell adhesion, being involved with the cadherins/protocadherins and

catenins for the regulation of adherens junctions ¹³⁶ (families of proteins for which we

found many other DMRs). The third gene FSCN2 (Fascin Actin-Bundling Protein 2, Retinal) codes a protein with an actin-binding function which is expressed only in retina and blood ¹³⁶ .

Interestingly, in their study Bullinger and colleagues in 2010 ¹³⁷ connected the hypermethylation in this region of the chr17 long arm overlapping the neighboring genes KIAA1447 and ACTG1 to poor survival in AML (these confirmations are marked with * in Table 4.5.2B, next chapter). Not much is known about KIAA1447, an alias of BAHCC1 (BAH Domain And Coiled-Coil Containing 1). This is a gene encoding a protein with a chromatin binding domain, interacting with the chromatin silencing complex for transcription regulation ¹³⁶ .

Furthermore, BAIAP2 is the gene that encodes BAI1 Associated Protein 2, a protein that functions as an insulin receptor tyrosine kinase substrate in the brain ¹³⁶ . It interacts with CDC42, being necessary for the CDC42-mediated reorganization of the actin cytoskeleton and whose RNAi phenotype is a reduction of endocytosis.

Moreover, CHMP6 is also involved in endocytosis and is in this genomic region ¹³⁶ . CHMP6 is a probable core component of the endosomal sorting required for transport complex III which is involved in multivesicular bodies formation and sorting of endosomal cargo proteins ¹³⁶ .

Besides BAIAP2, in this region were two other genes of proteins involved in metabolism, namely in insulin response, RPTOR and FASN. The gene RPTOR codes for the protein Regulatory Associated Protein Of MTOR Complex 1 ¹³⁶ . RPTOR or RAPTOR is involved in the control of the mammalian target of rapamycin complex 1 (mTORC1) activity which regulates cell growth, survival, and autophagy/longevity in response to nutrient and hormonal signals ¹³⁶ . A DMR overlapping RPTOR was found in the most recent paper reporting DMRs with correlations to survival probability (using this TCGA methylation data) ¹³⁸ . They advanced this gene to have one of the 8 DMRs signature that could be used as a prognostic marker and have a crucial role in AML.

In turn, FASN codes for the Fatty acid synthase, which main function is to

catalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA and is a

downstream responder in the insulin signaling pathway ¹³⁶ .