12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
0635
DAP-KINASE HYPERMETHYLATION AND APOPTOSIS IN MYELODYSPLASTIC SYNDROMES
M.T. Voso, D. Gumiero, F. D'Alò, A. Scardocci, M. Greco, E. Fabiani,
A. Di Ruscio, F. Guidi, S. Rutella, S. Bellesi, L. Pagano, S. Hohaus,
G. Leone
Università Cattolica S. Cuore, ROMA, Italy
Background. Aberrant CpG island methylation in the context of the
promoter region of multiple genes, leading to their silencing, have gained
increasing recognition as important factors in development and progression
of myelodysplastic syndromes (MDS). Among genes regulated by
promoter hypermethylation, death-associated protein kinase (DAPkinase)
is a proapoptotic calcium/calmodulin-regulated serine/threonine
kinase that participates in several apoptotic pathways. Aims. We studied
the frequency of DAP-kinase promoter hypermethylation in a group
of 106 MDS patients and analyzed its effect on apoptotic cell levels in
their bone marrow samples. The contribution to apoptotis of DAPkinase
re-activation due to demethylating agents, as decitabine and azacitidine,
was studied in cell line models. Methods. DNA was extracted
from bone marrow mononuclear cells of MDS patients at the time of
diagnosis and during the disease follow-up. DAP-kinase promoter
methylation and expression were analyzed by methylation-specific PCR
and real time RT-PCR, respectively. CD34 + cells were isolated from bone
marrow samples by immunomagnetic beads. Apoptosis was evaluated
by 7-amino-actinomycin-D (7-AAD) incorporation and quantified by a
FACScan flow cytometer. Results. DAP-kinase was methylated in 37%
of MDS patients. No association with patients’ characteristics was
found. Sequential samples from 11 MDS patients were studied during
follow-up, at a median of 12.4 months (range 1.8-40.6 months) from initial
diagnosis. Eight patients, 7 of whom with disease progression, maintained
the same DAP-kinase unmethylated (n=6) or methylated (2
patients) status, while only 2 patients gained DAP-kinase hypermethylation,
and one patient became unmethylated. Freshly isolated CD34 +
cells from MDS samples showed a DAP-kinase methylation status similar
to the CD34 – cell fractions. Moreover, DAP-kinase hypermethylation
was associated to reduced mRNA expression in CD34 + cells, when compared
to unmethylated samples. Apoptosis was higher in bone marrow
samples from MDS patients compared to normal controls. In MDS
patients, the apoptosis rate increased in bone marrow mononuclear cells
and CD34 + cells of patients unmethylated for DAP-kinase, when compared
to methylated patients (1.8±0.5% vs 0.4±0.1% in MNC, p=0.068,
and 0.5±0.2% vs 0.08±0.04% in CD34 + cells, p=0.1, respectively). We
used the HL-60 cell line to study the effect of demethylating agents on
DAP-kinase function. Restoration of the unmethylated state of DAPkinase
occurred after 72-96 hours of exposure to 1 micromolar
decitabine, and was associated to re-expression of DAP-kinase transcripts.
Moreover, treatment was associated to cell growth inhibition and
significant increase of apoptotic cells. In particular, following 4 days of
treatment of HL60 cells with azacitidine, trichostatin A, or both drugs,
we found a cell growth inhibition of 65%, 32% and 82%, respectively,
compared to mock treated cells. Correspondingly, mean apoptosis was
2.2%±0.3% in the control, 11.3±2.8% in the presence of azacitidine
(p=0.02), 5.7%±2.3% in the presence of trichostatin A (p=0.13), while
the combination of the two drugs led to a synergistic effect, with apoptosis
increasing to 41.9%±9.8% (p=0.009). Summary and conclusions.
DAP-kinase promoter hypermethylation reduces apoptosis in MDS and
its re-expression may have an important role in the response to epigenetic
treatment.
238 | haematologica/the hematology journal | 2007; 92(s1)
0636
THE ORAL IRON CHELATOR ICL670 IS A POTENT INHIBITOR OF NF-ΚB AND THIS
ACTIVITY IS INDEPENDENT FROM IRON OVERLOAD IN MDS CELLS
D. Cilloni, V. Rosso, E. Messa, F. Messa, F. Arruga, I. Defilippi,
R. Catalano, S. Carturan, C. Bittoto, C. Boveri, P. Nicoli, E. Bracco,
G. Saglio
University of Turin, TURIN, Italy
Background. Patients affected by Myelodysplastic Syndromes (MDS)
undergo iron overload due to blood transfusions. Recently oral chelation
is under evaluation to reduce iron induced organ damage. It was reported
that iron chelation is able to reduce the Hb and platelets transfusion
requirement. Finally, it was reported that iron activates NF-κB through
TNF? release. Recently it was demonstrated that NF-κB is abnormally
activated not only in acute leukemias but also in MDS patients Aims. The
aim of the study was to evaluate the effects of the oral chelator ICL670
( Novartis) on NK-kB activity in order to identify a possible mechanism
responsible for the observed reduced transfusion requirements during
chelation therapy. Methods. After informed consent 20 BM samples were
collected from MDS patients. Eight were RA, 8 RAEB, and 4 AML secondary
to MDS (s-AML). 12 of the patients presented iron overload
(evaluated by SQUID biomagnetic liver susceptometry) and high ferritin
levels. The remaining 8 patients were collected at diagnosis before transfusions
and they presented normal liver iron concentration (LIC) and ferritin
levels. MNC cells were separated and incubated with 100 µm
ICL670 for 3 hrs. Moreover, K562 and HL60 cells were analyzed as control.
Incubated and control cells were evaluated for NF-κB activity using
both EMSA and ELISA method. Results. We detected an increased activation
of NF-κB as compared to healthy subjects in 4 out of 8 RA 6 out
of 8 RAEB, in all the cases of s-AML and in cell lines. No significant difference
was detected in NF-κB activity comparing patients with or without
iron overload (p=0,5). The levels of NF-κB activity increase during
disease progression being higher in RAEB and s-AML as compared to RA
(p=0,003). Regression analysis demonstrated a correlation between NFκB
activity and blast percentage (r=0,75) but no correlation between NFκB
and ferritin levels (r=0,5). Among patients with increased NF-κB
(n=14) the incubation with ICL670 induced a significant reduction of NFκB
activity (p=0,0002). No significant difference was detected in NF-κB
inhibition comparing patients with or without iron overload. In addition,
ICL670 also inhibits NF-κB activity in HL60 and K562 cells. Conclusions.
NF-κB is abnormally activated in MDS patients and this is not apparently
related to iron overload being present in many patients before transfusion
with normal ferritin levels and in cell lines. ICL670 acts as a potent
NF-κB inhibitor and this property could explain the activity on BM cells
which results in the improvement of the Hb and platelets levels. This latter
effect seems to be independent from the reduction of iron strorage
induced by oral chelation.