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

12 th Congress of the European Hematology Association
patients (7 male, 7 female, median age 68), thirteen ET-JAKV617Fpos
patients (6 male, 7 female, median age 72) and seven ET-JAK2V617Fneg
patients (3 male, 4 female, median age 66) were included in the study.
Samples of normal bone marrow were obtained from five healthy
donors and one sample was from a non-Hodkgin's lymphoma patient
with no lymphoma involvement of the marrow. Real-time PCR analysis
for ACE, AGT, AT1R1, REN genes and internal housekeeping gene
GAPDH was performed using Real Time PCR System. To compensate
for inter-PCR variations, normalisation of target genes (ACE, AGT,
AT1R1, REN) with an endogenous control (GAPDH) was performed.
JAK2 V617F mutational status was determined by the allele-specific
PCR. Results. ACE expression in BM of PV and ET patients was downregulated
to 2-20% of the donor BM values with no statistically significant
difference in expression between patients. AGT expression was
significantly higher in PV and in ET JAK2V617F pos comparing to ET
JAK2V617F neg patients. Renin has shown similar pattern of expression
as AGT: it was significantly higher in PV and in ET JAK2V617F pos
patients comparing to ET JAK2V617F neg patients. AT1R gene was significantly
higher expressed in PV patients in comparison to both ET subgroups,
JAK2V617F positive or negative patients. ET patients did not differ
for AT1R expression by their JAK2 mutational status. Conclusions.
Our findings indicate up-regulation of AGT, AT1R and REN genes and
down-regulation of ACE gene expression in clonal hematopoiesis of PV
and ET. Different expression pattern of major RAS components in BM
of PV and ET compared to normal BM is clearly related to the existence
of JAK2V617F mutation and less to the PV or ET disease phenotype.
However, interesting exception is excessive expression of AT1R in PV
that was not observed in ET irrespective of JAK2 mutation. This latest
observation provides ground for the future experimental and clinical
studies for the use of AT1R blockers in improving clinical management
of JAK2V617Fpos PV.
0237
THE MYELOPROLIFERATIVE DISEASE JAK2 V617F MUTANT CANNOT BE REGULATED BY
SOCS PROTEINS
M. Hookham, 1 J. Elliott, 1 Y. Suessmuth, 1 A. Ward, 2 J. Staerk, 3
W. Vainchenker, 4 M. Percy, 5 M.F. McMullin, 5 S. Constantinescu, 3
J. Johnston1
1 2 Queens University Belfast, BELFAST, United Kingdom; Deakin University,
BURWOOD, Australia; 3Ludwig Institute for Cancer Research, BRUSSELS,
Belgium; 4INSERUM Institut Gustave Roussy, PARIS, France; 5Haematology, Belfast City Hospital, BELFAST, United Kingdom
Background. Many myeloproliferative disorders including Polycythaemia
Vera (PV) are associated with a valine to phenylalanine
(V617F) somatic mutation in the pseudokinase domain of JAK2, which
leads to constitutive kinase activity. This JAK2 mutant requires a cognate
receptor such as the Epo receptor (EpoR) for transformation to
occur. Suppressor of cytokine signalling (SOCS) proteins are known to
strongly negative regulate erythropoietin (Epo) signalling through interaction
with both the EpoR and JAK2. Aims. The aim of this study was
to determine to role of SOCS3 in the regulation of JAK2 V617F. Methods.
Expressing the EpoR and JAK2 WT or JAK2 V617F in Ba/F3 cells that
also express SOCS under the control of the Tet-off system as well as
transient expression of the above proteins in 293T we also investigated
the expression of SOCS-3 in leukocytes derived from PV patients and
healthy donors. Results. We found that SOCS3 could not negatively regulate
the mutant JAK2. Furthermore we found that the activation of
JAK2 V617F can be enhanced in the presence of SOCS3 and that normal
SOCS turnover is inhibited, causing an accumulation of both proteins.
Conclusions. The presence of SOCS3 strongly inhibits cell proliferation
in the presence of wild-type JAK2, but SOCS could not inhibit
proliferation when co-expressed with JAK2 V617F. Moreover, leukocytes
derived from PV patients express SOCS3 at higher levels than
those derived from healthy donors. These findings suggest that the JAK2
V617F mutant can counteract normal SOCS3 regulation and may exploit
this to enhance the myeloproliferative disease.
0238
DETECTION OF ACTIVATED STAT5 IN THE CYTOPLASM OF NEOPLASTIC CELLS
IN VARIOUS MYELOID NEOPLASMS
K. Sonneck, 1 R. Fritz, 1 L. Müllauer, 1 K.V. Gleixner, 1 M. Mayerhofer, 1
S. Florian, 1 M. Kerenyi, 2 W.R. Sperr, 1 C. Sillaber, 1 R. Moriggl, 3 P. Valent1 1 Medical University of Vienna, VIENNA; 2 University of Vienna, IMP, VIEN-
NA; 3 Ludwig Boltzmann Institute of Cancer Res, VIENNA, Austria
86 | haematologica/the hematology journal | 2007; 92(s1)
Background. The signal transducer and activator of transcription 5
(STAT5) has recently been implicated as essential pro-oncogenic factor
in the pathogenesis of myeloid leukemias in mice (Cancer Cell
2005;7:87-99). More recently, STAT5 activation has also been described
to occur in human leukemias. However, so far, little is known about the
expression of activated/tyrosine phosphorylated STAT5 (pSTAT5) in
various myeloid neoplasms and about the distribution of pSTAT5 in the
cellular compartments of the normal and leukemic bone marrow (bm).
Methods. We have examined the expression of pSTAT5 in the bm in
patients with acute myeloid leukemia (AML, FAB M0, n=3, M1, n=6,
M2, n=4, M3, n=5, M4, n=5, M5, n=4, M6, n=5, M7, n=4), chronic
myeloid leukemia (CML, chronic phase, n=4, accelerated phase, n=5,
blast phase, n=5), and systemic mastocytosis (SM, n=30), as well as in
the normal bm (n=5). Expression of pSTAT5 was determined on paraffin-embedded
bm sections by immunohistochemistry using the
pSTAT5-specific antibody AX1. Results. In the normal bm, the antibody
AX1 was found to react with megakaryocytes and immature myeloid
progenitor cells, whereas erythroid cells and mature granulocytic cells
did not stain positive for AX1. In patients with AML and CML, the distribution
of pSTAT5 showed a similar pattern. In fact, pSTAT5 was
found to be expressed in leukemic blast cells without differences among
FAB types as well as megakaryocytic cells, but not in erythroid cells. In
patients with SM, neoplastic mast cells were found to be immunoreactive
for pSTAT5. Interestingly, in all patients and all cells examined,
pSTAT5 was found to be localized in the cytoplasm rather than in the
nucleus. The cytoplasmic distribution of pSTAT5 in neoplastic cells was
confirmed by immunocytochemical staining experiments performed on
primary isolated neoplastic cells (AML, CML, mastocytosis) and respective
cell lines (U937, KG1, K562, KU812, HMC-1). In each case, the reactivity
of neoplastic cells with the AX1 antibody was abrogated by preincubation
of the antibody with a pSTAT5-specific blocking peptide.
Moreover, the expression of cytoplasmic pSTAT5 in the leukemic cell
lines was demonstrable by flow cytometry. To study the molecular
mechanisms underlying STAT5-activation in neoplastic cells, Ba/F3 cells
with doxycycline-inducible expression of disease-specific oncoproteins,
namely BCR/ABL (CML) and KIT-D816V (SM) were employed. Induction
of these oncoproteins in Ba/F3 cells resulted in massive activation
of pSTAT5 and DNA binding activity as shown by EMSA and supershift
assays. Summary. Our data show that neoplastic cells in AML, CML,
and SM express cytoplasmic pSTAT5, and that disease-related oncoproteins
contribute to STAT5-activation. The particular cytoplasmic localization
of pSTAT5 in neoplastic cells suggests that apart from its function
as a transcription factor, pSTAT5 may have an additional role as a
cytoplasmic regulator in these malignancies.
0239
NF-E2 OVEREXPRESSION DELAYS ERYTHROID DIFFERENTIATION AND INCREASES
ERYTHROCYTE PRODUCTION
A.S. Magin, H. Pahl, M. Mutschler, M. Buerge, B. Will, H. L. Pahl
University Hospital Freiburg, FREIBURG, Germany
Background. A point mutation in the Jak2 kinase, Jak2V617F, is found
in up to 95% of patients with Polycythemia vera (PV). Jak2V617F leads
to constitutive kinase activation and in a mouse model recapitulates
many disease features including erythrocytosis. However, the molecular
mechanism by which the mutation exerts its effect has not been
delineated. The transcription factor Nuclear Factor-Erythroid 2 (NF-E2)
is overexpressed in the vast majority of PV patients. In murine cells, NF-
E2 overexpression leads to EPO-independent growth, a hallmark of PV.
Moreover, ectopic NF-E2 expression can reprogram myeloid cells
towards erythroid maturation. Aims. We therefore hypothesized that
Jak2V617F-induced erythrocytosis is mediated by NF-E2 overexpression.
Consequently, we investigated the effect of NF-E2 overexpression
in healthy peripheral blood CD34 + cells. Methods. Peripheral blood
CD34 + progenitor cells were purified from healthy donors using antibody-based
magnetic bead separation. Cells were retrovirally transduced
with a NF-E2 cDNA in conjunction with a GFP marker. GFPexpressing
cells were sorted and assayed for colony formation in methyl
cellulose, or maintained in liquid culture medium promoting erythroid
differentiation. Erythroid maturation was assessed by CD36 and
CD235a (Glycophorin A) staining and FACS analysis as well as Wright-
Giemsa staining of cytospins. Results. NF-E2 overexpression drastically
altered erythroid colony formation. While empty vector transduced cells
mainly formed CFU-E (70%±20%) and to a small degree BFU-E (30%
±20%), NF-E2 overexpressing cells formed almost exclusively BFU-E
(80%±5%). In addition, NF-E2 overexpressing BFU-E were larger and
more dispersed than control BFU-E. Concurrently, the absolute number