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

0245
POTENT AND SELECTIVE INHIBITION OF EEC COLONY FORMATION IN JAK2V617F
POLYCYTHEMIA VERA AND THROMBOCYTHEMIA BY LOW DOSES OF ITF2357, A NEW
HISTONE DEACETYLASE INHIBITOR
V. Guerini, O. Spinelli, A. Salvi, G. Finazzi, T. Barbui, M. Introna,
J. Golay, A. Rambaldi
Ospedali Riuniti of Bergamo, BERGAMO, Italy
Background. A somatic point mutation in the JAK2 gene (JAK2V617F)
has been recently recognized as the key pathogenetic lesion in Polycythemia
Vera (PV) and Essential Thrombocythemia (ET). Histone-
Deacetylase inhibitors (HDACi) are known inducers of cell differentiation,
apoptosis and cell cycle arrest of neoplastic cells. ITF2357 is a new
HDACi (Italfarmaco, Milano, Italy) which, at low micro molar concentration
in vitro, inhibits the secretion of several cytokines such as IL-1, IL-
6, VEGF and IFN-γ and exerts a potent anti tumor activity against multiple
myeloma (MM) and acute myeloid leukemia cells (AML). For these
latter properties, this drug is currently tested in Phase I/II trials in AML
and MM patients. Aims. We investigated the ability of ITF2357 and the
prototypic HDAC inhibitor Suberoyl Anilide Hydroxamic Acid (SAHA),
to inhibit the spontaneous endogenous erythroid colony (EEC) growth of
hematopoietic stem cells obtained from patients with PV (all positive for
JAK2V617F), ET (JAK2V617F positive in 53%) and Idiopathic Erythrocytosis
(all negative for JAK2V617F ). Methods. The in vitro EEC assays was
performed using mononuclear cells (MNC) from peripheral blood samples
obtained from PV, ET and IE patients at the time of regular followup
visits in our clinic. The inhibitory activity played by ITF2357 on the
EEC colony growth was performed with or without the addition of
exogenous cytokines including GM-CSF, IL-6, G-CSF, IL-3, EPO and Stem
Cell Factor, in the presence of a log scale concentration of ITF2357 (from
0.001 to 0.75 mM). We also investigated the JAK2V617F at the single
colony level on colonies picked-up at the end of a 14 days EEC assay. The
molecular analysis of JAK2V617F was performed using allele specific
Polymerase Chain Reaction (PCR) on DNA extracted by a single colony.
Results. MNC obtained from IE or ET patients negative for JAK2V617F
neither exhibited spontaneous EEC formation nor EPO hypersensitivity
(from 0.1 UI/mL up to 10UI/mL). On the contrary, MNC from JAK2V617F
PV and ET patients invariably sustained the spontaneous EEC outgrowth
with a marked hypersensitivity to exogenous cytokines. ITF2357 induced
a 90% EEC inhibition in all JAK2V617F PV and ET patients at 0.01mM
concentration while SAHA displayed a similar inhibitory activity only
when used at 0.25 mM. When the JAK2V617F mutation analysis was
performed on single colonies obtained from PV and ET cells plated with
exogenous cytokines, the addition of ITF2357 allowed the reproducible
outgrowth of normal hematopoietic colonies ranging from 30 to 60% in
different experiments. Conclusions. ITF2357, at concentration easily
attained after oral administration, shows a potent inhibitory activity on
the autonomous proliferation of hematopoietic stem cells of PV and TE
carrying JAK2V617F mutation. The inhibitory effect of ITF2357 is remarkably
selective and more evident on JAK2V617F mutated hematopoietic
progenitors. This warrants evaluating the clinical activity of this molecule
in Phase II trials designed for patients with chronic myeloproliferative disorders
carrying the JAK2V617F mutation.
0246
A SMALL DELETION AT 20Q13.13 INDICATES NFATC2 AS A CANDIDATE GENE IN
ESSENTIAL THROMBOCYTHEMIA
L. Vieira, 1 A. Vaz, 1 P. Matos, 1 M. Nogueira, 1 B. Marques, 1
A.P. Ambrósio, 1 A.M. Pereira, 2 M.G. Da Silva, 3 P. Jordan1 1 Instituto Nacional de Saúde, LISBOA; 2 Hospital Garcia de Orta, ALMADA;
3 Instituto Português de Oncologia, LISBOA, Portugal
Essential thrombocythemia (ET) is a chronic myeloproliferative disorder
(MPD) characterized by megakaryocytic proliferation and an excessive
production of platelets. Approximately half of ET and most other
MPD patients show a somatic activating point mutation in JAK2
(JAK2V617F) which is believed to represent a secondary pathogenetic
event. Recent data from the literature showed that JAK2V617F is significantly
associated with deletion of the long arm of chromosome 20, suggesting
that a yet undefined molecular lesion on 20q precedes JAK2V617F
and is responsible for initiation of clonal hematopoiesis. We describe a
cytogenetic and molecular study of an acquired three-way translocation
t(X;20;16)(p11;q13;q23~24) identified in a JAK2V617F-positive ET
patient. Using fluorescence in situ hybridization with 27 different probes
mapping along the 20q12-20q13.2 region, we identified a 500-kilobase
deletion associated with a breakpoint at 20q13.13. Subsequent deletion
12 th Congress of the European Hematology Association
mapping using polymerase chain reaction (PCR) analysis confirmed deletion
of 3 genes: nuclear factor of activated T-cells cytoplasmic 2 (NFATC2),
SAL-like 4 (SALL4) and ATPase class II type 9A (ATP9A). The SALL4 gene
has recently been implicated in the pathogenesis of acute myeloid
leukemia, however, we have sequenced the SALL4 coding sequence and
flanking intronic regions and demonstrate that it is not mutated in 28
patients with ET. We further found that neither SALL4 nor ATP9A are
expressed in 2 JAK2V617F-positive cell lines (SET-2 and HEL) although
their expression was detected in 16 different normal tissue samples. In
contrast, NFATC2 was strongly expressed in SET-2, HEL and in 7 other
leukemic cell lines and normal leukocytes, making it the most likely candidate
gene from the deleted region. To further address the role of
NFATC2 in megakaryocyte proliferation, we simulated the effects of a
deletion by suppressing NFATC2 gene expression in SET-2 megakaryocytic
cells using small interfering RNAs. After 24 hours of transfection,
NFATC2 mRNA and protein levels were 48% reduced compared to SET-
2 cells transfected with a control siRNA. The specific suppression of
NFATC2 resulted in a 34% increase in cell number which was accompanied
by an increase in megakaryocyte cell size. Furthermore, the suppression
of NFATC2 correlated with an up-regulation of cyclin B1 and
cyclin E protein levels, indicating that the higher cell number was a consequence
of cell cycle progression. Since NFATC2 regulates the expression
of several hematopoietic cytokines and is expressed at high levels in
megakaryocytes, mutations that affect NFATC2 expression may have
important consequences in the control of megakaryopoiesis and consequently,
contribute to ET pathogenesis.
0247
EVIDENCE THAT OVERPRODUCTION OF CYTOKINES AND OTHER MOLECULES LINKED TO
JAK2 ACTIVATION CONTRIBUTES TO ABNORMAL HEMATOPOIESIS IN POLYCYTHEMIA
VERA
M. Boissinot, I. Corre, D. Pineau, S. Hermouet, Y. Jacques
Institut de Biologie, NANTES CEDEX, France
Background. Polycythemia vera (PV) is a clonal myeloproliferative disorder
arising from a multipotent progenitor and resulting in excessive erythropoiesis.
Activating mutations of JAK2, a key signalling molecule for
hematopoietic cytokines, are found in almost all PV patients. The V617F
mutation (JAK2-V617F) is found in >90% of PV; other JAK2 mutations
have been described in JAK2-V617F negative PV. However, there is evidence
that onset of clonality and/or disease phenotype may be due to
other molecular abnormalities. We previously described the partial
dependence of PV erythroid progenitors on interleukin-11 (IL-11), a
cytokine that activates JAK2, stimulates erythropoiesis and is overproduced
in PV, notably by bone marrow (BM) stromal cells (BMSC). Aims.
To identify other JAK2-activating molecules with altered production in
PV. Methods. We used cytokine antibody arrays (Raybiotech Inc.) to establish
the cytokine profile of serum from 20 PV patients and 27 patients
with second erythrocytosis (SE) and of BM plasma (21 PV, 21 SE). Molecules
of interest were then measured by ELISA in serum (32 PV, 33 SE) and
in BM plasma (27 PV, 26 SE). Correlations between cytokine levels, blood
cell counts and%JAK2-V617F, measured by quantitative allele-specific
PCR in blood granulocytes, were analysed using Pearson’s or Spearman’s
rank correlation tests. Results. In addition to IL-11, cytokine arrays revealed
two new molecules linked to JAK2 activation and present at high levels in
PV: tissue inhibitor of metalloproteases-1 (TIMP-1) and hepatocyte growth
factor (HGF). Overexpression of TIMP-1 in PV was confirmed by ELISA
in BM plasma (102 ng/ml vs 58 ng/ml in SE, p=0.013). Overexpression of
HGF was confirmed by ELISA in BM plasma (PV: 7929 pg/ml vs 4438
pg/mL in SE, p=0.01) and in serum (PV: 5176 pg/mL vs 1765 pg/ml in SE,
p