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

12 th Congress of the European Hematology Association
genetic mechanisms involved in the silencing of MHC2TA transcription
in leukaemia T-cells also to histone modifications at the CIITA-PIII region.
Using chromatin immunoprecipitation (ChIP) assays we found that the
level of acetylated histone H3 in CIITA-PIII chromatin in T-leukaemia
was strongly reduced when compared with CIITA-expressing T-lymphoma
cells. The opposite was noted for the triple-methylated lysine 27
modification in histone H3. This modification is associated with compact
chromatin and transcriptional silent genes. Subsequently, we also
established by ChIP that the enhancer of zeste homolog 2 (EZH2), which
has intrinsic histone methyltransferase activity, is recruited into CIITA-PIII
chromatin in T-leukemia cells. Together our data reveal that, in addition
to DNA methylation modifications, histone methylation modifications are
associated with transcriptional silencing of MHC2TA in T-leukaemia and
provide a link with components of the polycomb group family of proteins.
0930
UNCOVERING THE EPIGENETIC PATHOMECHANISM IN 13Q14
D. Mertens, 1 M. Ruppel, 2 A. Philippen, 3 C. Tschuch, 2 V. Fleig, 2
C. Mund, 2 F. Lyko, 2 B. Radlwimmer, 2 H. Döhner, 3 S. Stilgenbauer, 3
P. Lichter2 1 University Hospital Ulm, DKFZ Heidelberg, ULM; 2 DKFZ, HEIDELBERG;
3 University Hospital, ULM, Germany
Introduction. Deletions in chromosomal band 13q14.3 distal to RB1
occur in a variety of human neoplasms like B-cell chronic lymphocytic
leukaemia (CLL), indicating a tumor suppressor mechanism in this region.
Intriguingly, several characteristics of the region of interest point to an epigenetic
pathomechanism: i) candidate genes lack point mutations, yet ii)
these genes are downregulated in tumors, iii) the presence of large noncoding
RNA genes in 13q14.3 is reminiscent of imprinted regions where
only one gene copy is active. The data we show here led us to propose
a novel oncogenic mechanism where already in healthy tissue only one
gene copy is active while one gene copy is randomly chosen for silencing.
Loss of the single active copy is then sufficient for complete loss of
gene function in tumor cells. Currently we are trying to identify the (epi-
)genetic element that controls the whole locus. Aims. Identification of
the epigenetic regulatory mechanism localized in 13q14.3. Methods and
Results. We performed FISH analyses of hematopoietic and nonhematopoietic
cell lines to assess replication timing and chromatin packaging
of the critical region. In line with an imprinting mechanism, we find
that the two copies of the critical region replicate asynchronously and/or
show delayed chromatid segregation, suggesting differential chromatin
packaging of the two copies of 13q14.3. Next, we found by sequencing
of SNPs that 13q14.3 candidate genes are expressed from one copy only
in healthy probands. However, expression originated from either the
maternal or paternal copy, excluding an imprinting mechanism. In order
to identify the regulatory element, we performed DNA methylation
analyses and could show that one of the CpG islands of the region is
methylated. We could also show a functional interconnection of DNA
methylation and gene expression, as demethylating agents and histone
hyperacetylation induced biallelic expression. However, replication timing
was not affected. Currently we are employing array- and capillary
electrophoresis-based analysis of DNA-methylation (aPRIMES and bio-
COBRA) and chromatin-immunoprecipitation on arrayed CpG-libraries
(chIP on chip) with antibodies specific for histone modifications in order
to identify the epigenetic element regulating the critical region. Conclusions.
We propose that differential replication timing represents an early
epigenetic mark that distinguishes the two copies of 13q14.3, resulting
in differential chromatin packaging and monoallelic expression. This has
profound effects for the tumor suppressor mechanism localized in
13q14.3: Deletion of the single active copy of the region at 13q14.3,
which is detected in more than 50% of CLL tumors, will suffice for complete
loss of tumor suppressor function, as the remaining gene copies are
epigentically silenced. In addition, we are currently identifying the locus
control region that orchestrates gene expression in the critical region.
Thus, we provide a model for the pathomechanism of 13q14.3 in CLL
by the interaction of genetic lesions and epigenetic silencing.
0931
β-TRCP MEDIATES UBIQUITINATION AND DEGRADATION OF THE ERYTHROPOIETIN
RECEPTOR AND CONTROLS CELL PROLIFERATION
F. Verdier, L. Meyer, B. Deau, H. Forejtnikova, D. Dumenil,
F. Margottin-Goguet, C. Lacombe, P. Mayeux
Cochin Institute, PARIS, France
Control of intensity and duration of erythropoietin (Epo) signalling
348 | haematologica/the hematology journal | 2007; 92(s1)
is necessary to tightly regulate red blood cells production. After Epo
stimulation of erythroid cells, 2 types of signal are transduced via the
Epo receptor (Epo-R): positive signals involved in survival and proliferation,
and negative signals involved in signal arrest. We have recently
shown that the ubiquitin/ proteasome system plays a major role in the
control of Epo-R signalling duration and desensitisation processes.
Indeed, after Epo stimulation the Epo-R is ubiquitinated and its intracellular
part is degraded by the proteasome, preventing further signal
transduction. The remaining part of the receptor, together with associated
Epo is internalised and degraded by the lysosomes (Walrafen et al.
2005 Blood, 105, 600-608). Our aim was to identify the E3 ubiquitin ligase
involved in Epo-R ubiquitination. The Epo-R contains a putative β-
Trcp binding site in its intracellular domain. Interestingly, this putative
binding sequence is located in a region of the Epo-R that is deleted in
erythroid progenitors from patients with familial polycythemia. We
show that β-Trcp is responsible for Epo-R ubiquitination upon Epo
stimulation. After Epo stimulation, β-Trcp binds to the Epo-R and this
binding is dependent on Jak2 activation. Mutation of the Ser 462 residue
of the Epo-R, located in the consensus β-Trcp binding site abolished β-
Trcp binding, Epo-R ubiquitination and EpoR cleavage by the proteasome.
Activation of the mutated Epo-R is prolonged in comparaison
with Epo-R WT and BaF3 cells expressing this mutated receptor unable
to bind β-Trcp are hypersensitive to Epo. Whether the removal of the
β-Trcp binding site contributes to the hypersensitivity to Epo in familial
polycythemia is currently under study.
0932
IDENTIFICATION OF PROTEIN TYROSINE KINASES THAT CAN SUPPORT THE
PROLIFERATION AND SURVIVAL OF HEMATOPOIETIC CELLS
E. Lierman
K.U. Leuven, LEUVEN, Belgium
Introduction. Protein tyrosine kinases are an important family of signaling
proteins involved in the proliferation and survival of cells. They
are frequently activated in leukemia through mutation or their involvement
in chromosomal translocations often leading to fusion of kinase
domains with homodimerization domains present in the partner genes.
Examples of this are the BCR-ABL fusion in CML, the ETV6-PDGFRβ
fusion in CMML, and the NPM-ALK fusion in ALCL. Aims/Methods.
For many tyrosine kinases it remains unclear whether they can be activated
by enforced homodimerization and whether these activated
kinases would stimulate proliferation and survival pathways, leading to
transformation of hematopoietic cells. To test this we generated fusions
between the homodimerization domain of ETV6 and a variety of tyrosine
kinase domains, and determined if expression of these constructs
resulted in the transformation of Ba/F3 cells to IL3 independent growth.
Results. As a first method, we performed a retroviral insertional mutagenesis
screen for which we used a modified retroviral vector containing
an exon encoding the homodimerization domain of ETV6 followed
by an artificial splice donor site. When inserted in the host genome, this
vector would drive the expression of a fusion transcript consisting of
the exon encoding the homodimerization domain, and some exons of
the gene in which the retrovirus was inserted. Using this screen, we
obtained 423 independent Ba/F3 clones that were able to proliferate in
the absence of IL3. 271 of these clones contained insertions in 8 different
tyrosine kinase genes: Abl1, Fgfr1, Hck, Jak2, Lck, Mertk, Mst1r
and Tnk1. In all these cases, the fusion between the homodimerization
domain of ETV6 and the kinases were in-frame and the kinase domains
were completely included in the generated fusion genes. In the other
152 Ba/F3 clones, no meaningful fusion transcripts could be detected.
In a second method, we directly generated expression plasmids containing
fusions between the homodimerization domain of ETV6 and different
tyrosine kinase domains. So far, our results show that AXL,
EPHA2, EPHB4, FGFR1, KIT, SRC, SYK, TYRO3, YES1 and ZAP70 all
can be activated by homodimerization and support IL3-independent
growth of Ba/F3 cells. When analyzing the activation of downstream
signaling proteins such as PI3K, Akt, ERK, SRC kinases and STATs, significant
differences were observed between these different oncogenic
kinases. Conclusions. Our results identify a number of tyrosine kinase
proteins that can be activated by homodimerization and lead to stimulation
of proliferation and survival pathways in hematopoietic cells.
Some of these tyrosine kinase genes are novel candidate oncogenes.
The study of specific signaling pathways activated by these activated
tyrosine kinases may be usefull for the design of novel therapies.