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G. Zardo - Identification of novel genetic and epigenetic targets in Leukemia by genome wide approaches
These comparisons have provided the following
results:
i) In HL-60, FAB M2, 249 out of 1188 (20.9%) analyzable
loci were found to be hypermethylated when
compared to the normal “master DNA methylation
profile”. For 149 out of 249 (59.8%) hypermethylated
loci, it was possible to assign the chromosomal
localization and corresponding DNA sequence.
ii) In NB-4 FAB M3 PML/RAR+, 386 out of 1188
(32.5%) analyzable loci had a hypermethylated status
when compared to the normal “master DNA methylation
profile”. For 220 out of 386 (57%) hypermethylated
loci, it was possible to assign the chromosomal
localization and corresponding DNA sequence.
iii) The cross-comparison between the DNA methylation
profiles of HL-60 FAB M2 and NB-4 FAB M3
PML/RAR+ showed common and diverse targets of
hypermethylation. 183 hypermetylated genomic loci
out of 1188 total loci (15.4%) were shared by the
two different cell lines. 63 out of 249 (25.3%) loci
were hypermethylated in HL-60 but not in the NB-4
cell line. 196 out of 386 (50.8%) loci were hypermethylated
in NB-4 but not in the HL-60 cell line.
iv) Retinoic acid in combination with chemotherapy
is the frontline treatment for the therapy of acute
promyelocitic leukemia (APL). APL is characterized
by the t(15;17) that generates the oncogenic fusion
protein PML/RAR that holds epigenetic activity.
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NB-4 is a human PML/RAR+ cell line considered to
be an in vitro model for the study of APL. With the
aim to identify targets of aberrant DNA methylation
that might contribute to the onset of APL in
humans, we treated the NB-4 cell line with retinoic
acid to determine if the differentiation process
induced by this drug might be associated to the
demethylation of specific loci aberrantly methylated.
We compared the RLGS DNA methylation profiles
of control NB-4 and NB-4 cells treated for 72 hours
with 1µM retinoic acid. In NB-4, PML/RAR+ cells,
retinoic acid induced the demethylation of 10
genomic loci (0.84%) out of 1188 analyzed. The
chromosomal localization and DNA sequence was
assigned to 5 out of 10 loci. The remaining 5 loci
will be identified using the “in silico” procedure
described in the main proposal.
In conclusion, our data show that a portion of DNA
methylation targets are shared by leukemic cells of
different FAB and carrying fusion proteins, but
above all, show specific DNA methylation signatures
depending on the stage of differentiation block and
presence of fusion proteins. Moreover, our data
prove that the in vitro treatment with retinoic acid of
NB-4 PML/RAR+ cells leads to the demethylation
of aberrantly methylated genomic loci that might be
relevant for the induction of the differentiation
process and to prevail over the differentiation block.