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

12 th Congress of the European Hematology Association
sis during differentiation. Surprisingly, the repression of p19 induced a
decrease in MK differentiation. Indeed, the about 70% diminution in p19
expression leads to a 38.6%±17% decrease in CD41 high CD42 high MKs.
This decrease was not linked to a differentiation blockage but to a delay
in differentiation. The number of proplatelet forming MKs was diminished
when cells were transduced with shRNA p19. However, when
mature CD41 ++ CD42 ++ cells were analyzed, no difference in the number
of proplatelet forming MK cells was detected suggesting that p19 did not
play a direct role in platelet formation. Knowing that polyploidization
is linked to MK differentiation, we tried to dissect the role of p19 in the
arrest of endomitosis and in the regulation of MK maturation. To perform
such experiments, polyploid MKs were transduced with shRNA
p19 and 72 hours later, 22.3±6.6% increase in mean ploidy level was
observed. However, when each ploidy class was analyzed separately, a
slight decrease in CD42 expression in polyploid MKs was observed suggesting
that these cells will accelerate their maturation once the cell
cycle is achieved. To confirm our results, CD34 + cells were transduced
by a lentivirus encoding for p19 cDNA, a 59.7±6.8% decrease in mean
ploidy level was detected. Furthermore, in each ploidy class, an important
increase in CD41 and slight increase in CD42 expression is
observed. All together our results demonstrated that p19 has an important
role in the arrest of endomitosis allowing the acceleration of MK
maturation.
0150
THE LEUKEMOGENIC CALM/AF10 FUSION PROTEIN ALTERS THE LOCALIZATION OF THE
EPIGENETIC REPRESSOR IKAROS
A. Greif, 1 B. Tizazu, 1 A. Krause, 1 E. Kremmer, 2 S.K. Bohlander1 1 GSF / LMU, MUNICH; 2 GSF, MUNICH, Germany
The t(10;11)(p13;q14) translocation leads to the fusion of the CALM
and AF10 genes. This translocation can be found as the sole cytogenetic
abnormality in acute lymphoblastic leukemia, acute myeloid leukemia
and also in malignant lymphomas. The expression of CALM/AF10 in
primary murine bone marrow cells triggers the development of an
aggressive leukemia in a murine bone marrow transplantation model.
Here we show that AF10 interacts with the epigenetic repressor Ikaros
in yeast-two-hybrid assays. Interestingly, Ikaros is required for normal
development of lymphocytes and aberrant expression of Ikaros has been
found in leukemia. In a murine model, the expression of a dominant negative
isoform of Ikaros causes leukemias and lymphomas. The Ikaros
interaction domain of AF10 was mapped to the leucine zipper domain
of AF10, which is required for malignant transformation by both the
CALM/AF10 and the MLL/AF10 fusion protein. The interaction
between AF10 and Ikaros was confirmed by GST-pulldown and coimmunoprecipitation.
In contrast to AF10, CALM/AF10 alters the
nuclear localization of Ikaros. The transcriptional repressor activity of
Ikaros is reduced by AF10. These results suggest that CALM/AF10 might
have a dominant negative effect on Ikaros, and thereby block differentiation
of the leukaemia propagating cell in CALM/AF10 positive
leukemias.
Figure 1. In vivo localization in co-transfected NIH 3T3 fibroblasts (Confocal
Laser Scan).
0151
BRAIN-EXPRESSED X-LINKED-2 (BEX2): EPIGENETIC REGULATION OF A POTENTIAL
MARKER FOR ACUTE MYELOID LEUKEMIA WITH MIXED LINEAGE LEUKEMIA
REARRANGEMENTS
H. Quentmeier, C. Fischer, J. Reinhardt, M. Zaborski, H.G. Drexler
DSMZ, BRAUNSCHWEIG, Germany
54 | haematologica/the hematology journal | 2007; 92(s1)
In epigenetically regulated genes, methyl-CpG recognizing proteins
like MeCP2 may bind to methylated CpG-rich areas and contribute to
silencing of these genes by recruitment of histone deacetylases (HDAC)
and histone methyltransferases. Previously, we identified BEX2 as candidate
gene for the diagnosis of acute myeloid leukemia (AML) with
mixed lineage leukemia translocations (MLLmu), similarly as extensively
described for HOX gene expression in MLLmu acute lymphoblastic
leukemia. Human brain expressed X-linked (BEX) is a novel gene family
consisting of at least six family members. Human BEX1 and BEX2 are
highly expressed in various brain-derived tissues, show diverse expression
patterns in peripheral tissues like the liver or pancreas, but are not
expressed in hematopoietic tissues like spleen, thymus, lymph node or
peripheral blood lymphocytes. We show here that a strict correlation
exists between the methylation status of the BEX2 5´ CpG-rich area and
expression of BEX2 mRNA: BEX2-negative MLL wild-type (MLLwt) cell
lines showed hypermethylation, BEX2-positive MLL mutant (MLLmu)
cell lines showed hypomethylation of this specific CpG-rich area. Supporting
the view that the expression of BEX2 is epigenetically regulated,
we found that treatment of MLLwt cell lines with the demethylating
agent Aza-2´deoxycytidine (Aza) induced BEX2 expression in
MLLwt cell lines. Furthermore, treatment of MLLwt cell lines with the
HDAC inhibitor trichostatin provoked upregulation of BEX2 mRNA in
these cells, alone and in combination with the demethylating agent Aza.
Chromatin immunoprecipitation assays confirmed the specific binding
of acetylated histone H3 to the BEX2 5´region in BEX2-positive, but not
in BEX2-negative cells. Methylated CpG-rich areas may not only silence
genes by recruitment of HDAC and histone methyltransferases, but also
by preventing binding of specific transcription factors to their DNA target
regions. The CpG-rich area in the 5´region of BEX2 contains an
ARNT-1 binding site. Stimulation of cells with 3-methylcholantrene (3-
MC) leads to binding of the receptor AhR to ARNT-1, translocation of
the AhR/ARNT-1 complex from the cytoplasm into the nucleus, and
induction of ARNT-1 target genes like CYP1A1. A clearly positive effect
of 3-MC on BEX2 expression was detectable only, if MLLwt cells were
preincubated with Aza. These data suggest that BEX2 negativity in
MLLwt cell lines is the consequence of CpG hypermethylation, recruitment
of HDAC and prevention of ARNT-1 binding to its cognate binding
site.
0152
DOWN REGULATION OF DLX3 EXPRESSION IN MLL/AF4 CHILDHOOD LYMPHOBLASTIC
LEUKEMIAS IS MEDIATED BY PROMOTER REGION HYPERMETHYLATION
M. Campo Dell'Orto, 1 B. Banelli, 2 E. Giarin, 1 B. Accordi, 1 L. Trentin, 1
M. Romani, 2 G. Te Kronnie, 1 G. Basso1 1 2 University of Padua, PADUA; Laboratory of Experimental Oncology C,
GENOA, Italy
Background. Chromosomal translocations that inactivate or create new
fusion genes are a common hallmark of acute lymphoblastic leukemia
(ALL) and may serve as diagnostic and prognostic markers in subtypes
of ALL. Aberrant methylation of clustered cytosine-guanosine motifs
(CpG), especially in CpG islands located in gene promoter regions, is an
early and essential step in tumour development and methylation has
been proved to be a mechanism of gene silencing as common as the disruption
of tumour-suppressor genes by mutation or deletion. Moreover,
DNA hypermethylation and transcriptional silencing of genes involved
in tumor invasiveness, cell growth and apoptosis, may also influence
recurrence after treatment and overall survival. Inactivation of cancerrelated
genes by DNA methylation is a frequent event also in paediatric
and adult ALL and, by looking at differences in the methylation pattern
of multiple genes, specific risk groups among patients have been identified.
The DLX genes implicated in haematopoiesis and in a number of
other processes with highly dynamic spatio-temporal expression patterns
and well-defined CpG islands in their promoter regions, are attractive
targets for methylation studies in leukemia subtypes. In particular,
the down-regulation of DLX2, 3 and 4 had been described in a group of
paediatric B-ALL characterized by the t(4;11)(MLL-AF4) chromosomal
rearrangement and, in several cancer cell lines, is directly connected to
an increased resistance to apoptosis. The presence of extended CpG
islands at the 5’ end of DLX2, 3 and 4 genes, their possible role in resistance
to apoptosis, and the recent finding of DLX5 promoter methylation
as one of the epigenetic markers of chronic lymphoblastic leukemia,
prompted us to study the methylation and gene expression pattern of
DLX2, 3 and 4 in specific paediatric ALL subtypes. Aims. Our main purpose
was to understand if, in pediatric leukemias, the methylation of
DLX2, 3 and 4 genes could have a functional role in their gene and protein
expression and if differential methylation patterns were able to dis-