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

0414
COMPARATIVE ANALYSIS OF THE CONSTITUTIVE ACTIVE JAK2V617F, JAK2T875N AND
MPLW515L ALLELES IN A MURINE BONE MARROW TRANSPLANT MODEL OF
MYELOPROLIFERATIVE DISEASE
G. Wernig, T. Mercher, Y. Pikman, R.L. Levine, D.G. Gilliland
HMS, Brigham & Women's Hospital, BOSTON, USA
Present in the 95% of polycythemia vera (PV) patients and in 50-60%
of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis
(IMF), the JAK2V617F mutation, resulting in constitutive activation
of the JAK/STAT pathway, is the most frequent genetic event in
myeloproliferative disease (MPD). Little is know about additional mutations
which contribute to JAK2V617F negative MPD. Very recently, two
additional mutations were identified in JAK2V617F – patients:
JAK2T857N, detected in the human AMKL cell line CHRF-288-11 and
the somatic mutation in the thrombopoietin receptor, MPLW515L,
detected in ~10% of patients with JAK2V617F – MF. JAK2V617F,
JAK2T875N and MPLW515L were analysed in murine models of bone
marrow transplantation and sufficiency for the development of MPD has
been shown for each of them. Comparison of these three models provides
insight into the JAK2-positive and JAK2 – MPDs that may inform
therapeutic strategies. Furthermore, developing murine models of disease
for each of these alleles may provide novel insights into phenotypic
variance among the MPDs. The JAK2V617F mouse model is characterized
by a striking, longterm erythrocytosis, a strain dependent degree
of leukocytosis and the development of bone marrow and splenic reticulin
fibrosis. Despite the presence of megakaryocytic hyperplasia, there
is impaired megakaryocytic polyploidization, and platelet counts are not
augmented. JAK2T875N constitutively activates downstream effectors
including STAT5 in hematopoietic cells in vitro. In a murine bone marrow
transplantation model JAK2T875N, although the T875N substitution
is localized in a different domain of the JAK2 kinase, results in a phenocopy
of JAK2V617F disease, causing a MPD with features of AMKL
including megakaryocytic hyperplasia in spleen, a polyploidization
defect and increased reticulin fibrosis in bone marrow and spleen, with
normal platelet and leukocyte counts. Similarly, the MPLW515L mutation
also transformed hematopoietic cell lines to cytokine independent
growth through activation of the JAK/STAT pathway. in vivo expression
of MPLW515L, caused a rapidly fatal, fully penetrant MPD that was
characterized by a marked leukocytosis, splenomegaly, bone marrow
reticulin fibrosis with a strain specific disease latency. However, - in contrast
to both JAK2 mutations - there was megakaryocyte hyperplasia
with normal megakaryoctye ploidy and marked thrombocytosis in the
3-4 million/uL range that was accompanied by thrombotic complications.
Our data demonstrate important phenotypic differences between
JAK2V617F, JAK2T875N and MPLW515L induced MPDs. Most striking
was the effect on the megakaryocyte lineage. JAK2V617F and
JAK2T875N induced megakaryocytic hyperplasia with a block in
megakaryocyte maturation and lack of thrombocytosis, whereas
MPLW515L enhanced megakarypoiesis resulting in severe thrombocytosis.
Furthermore, marked leukocytosis developed in MPLW515L but
not in JAK2V617F and JAK2T875N expressing C57Bl/6 mice. These findings
indicate that the three mutations have different impacts on proliferation
and/or survival of various hematopoietic progenitors. Since all
three mutations signal through the JAK/STAT pathway, these data suggest
that the MPLW515L allele activates transcriptional programs not
activated by JAK2 mutations that confer the ability for megakaryocytic
development and thrombocytosis.
12 th Congress of the European Hematology Association
0415
EXPRESSION AND EFFECTS OF NUCLEAR FACTOR-ERYTHROID DERIVED 2 (NF-E2) IN
MURINE AND HUMAN HEMATOPOIESIS
B. Will, 1 M. Mutschler, 2 C. Scholl, 3 U. Steidl, 4 E. Levantini, 4
C.S. Huettner, 5 D.G. Tenen, 4 H.L. Pahl6 1 Harvard Medical School, BOSTON, USA; 2 University of Freiburg,
FREIBURG, Germany; 3 Brigham and Women's Hospital, BOSTON, USA;
4 Harvard Institutes of Medicine, BOSTON, USA; 5 Medical College of Wisconsin,
MILWAUKEE, USA; 6 University Hospital of Freiburg, FREIBURG, Germany
The basic region-leucine zipper transcription factor NF-E2 plays an
important role in terminal erythroid and megakaryocytic maturation
and regulates the differentiation of erythroblasts and megakaryocytes.
NF-E2 knock out mice die at birth due to lack of platelets, but only display
mild defects in the erythroid lineage. NF-E2 expression has been
reported in murine cKit+/Sca-1+/Lin- (KSL) cells. However, its specific
role in stem cells and early progenitors remains unclear. NF-E2 is overexpressed
in patients with Polycythemia vera (PV). Several studies have
demonstrated that the degree of NF-E2 overexpression in individual PV
patients correlates with the proportion of Jak2V617F'positive granulocytes
and the ability to form endogenous erythropoietin-independent
colonies in vitro. Interestingly, the Jak2V617F activating mutation is present
in uncommitted progenitor cells (Lin-/CD34 + /CD38 – /CD90 + ) and
results in enhanced erythroid differentiation capacity. However, the role
of NF-E2 in stem and progenitor cells and its potential cooperation with
Jak2V617F in PV is unclear. In an approach to clarify the function of NF-
E2 in normal hematopoiesis and PV we investigated nf-e2 mRNA expression
in human and murine hematopoietic cells at different developmental
stages as well as the effects of NF-E2 overexpression in murine models.
Nf-e2 expression was evaluated in FACS sorted human and murine
stem and progenitor populations by qRT-PCR. A lentiviral construct (NF-
E2-IRES-GFP) was used to transduce KSL and cKit+/Sca-1-/Lin- cells,
which were subsequently assayed for colony formation in methylcellulose.
A tetracycline-inducible mouse model (TET-OFF) was generated by
introducing an nf-e2 transgene under control of the tetracycline responsive
element and subsequent crossing to a CD34-promoter-tTA transgenic
strain. Nf-e2 displayed a similar differentiation-specific expression
pattern in murine and human hematopoiesis: nf-e2 mRNA was detected
in LT-HSCs and ST-HSCs. During lineage commitment nf-e2 was
downregulated in both CMPs and GMPs but was again upregulated in
MEPs. In murine cells, highest nf-e2 expression levels were found in
proerythroblasts followed by a consecutive decrease during maturation
to orthochromatic erythroblasts. Enforced expression of NF-E2 following
lentiviral transduction of murine KSL and cKit+/Sca-1-/Lin- progenitor
cells caused markedly decreased BFU-E and CFU-E colony formation
in the presence of erythropoietin. In the tetracycline-inducible mouse
model NF-E2 overexpression in KSL and cKit+/Sca-1-/Lin- cells similarly
led to impaired erythroid colony formation in vitro. Lentiviral NF-E2
overexpression did not appear to affect maturation of committed erythroid
progenitor cells in in vitro differentiation assays of murine Ter119spleen
and fetal liver (E14.5) cells. In these assays, Jak2V617F likewise
showed no effect. Our study shows that NF-E2 expression is regulated
in a biphasic manner in both murine and human erythropoiesis. Forces
expression of NF-E2 in various human and murine stem- and progenitor
cells decreases erythroid colony cloning efficiency.
haematologica/the hematology journal | 2007; 92(s1) | 153