H e m a t o lo g y E d u c a t io n - European Hematology Association

was thought that they are linked to a pure erythrocytosis
phenotype. However, exon 12 JAK2 mutation
patients develop thrombosis complications, secondary
myelofibrosis, and leukemia similarly to those harboring
JAK2 V617F. 30
Thrombopoietin receptor mutations
Approximately 8–10% of ET and PMF patients that
do not harbor JAK2 V617F carry mutations in the TpoR.
Fascinatingly, most mutations seem to concern
W515, 31,32 a tryptophan residue located in a cytosolic
juxtamembrane amphipathic sequence (RWQFP (Figure
3)), that maintains the receptor inactive in the absence
of Tpo. 33 Mutations of W515 to Leu, Lys, Ala, Arg have
been reported, and in patients it was shown that TpoR
W515 mutants occur at the HSC stage. 34 That many
mutations at W515 activate the receptor indicate that it
is the loss of Trp515 and not the acquisition of various
residues that leads to activation. In bone marrow
murine reconstitution experiments, TpoR W515L and
TpoR W515A were shown to induce a rapid, fatal MPN
with myelofibrosis, a phenotype that is much more
severe than that induced by JAK2 V617F. 32,35 In fact,
TpoR W515 mutants are probably the fastest myelofibrosis-inducing
oncogenes known. Using mass spectrometry,
it was shown that two cytosolic TpoR
residues, Y592 and Y626, were found to be phosphorylated
in cells transformed by TpoR W515A, and that
these two residues play opposite roles in transformation.
35 The Y592F mutation leads to hyperactivation of
JAK2 by TpoR W515A, while the Y626F mutation (or
Y112F counting only intracellular residues), in contrast,
abolished phenotype, without inhibiting JAK2 activation.
35 These results indicated that myelofibrosis induction
requires pathways downstream of Y626, and
indeed proteomics approaches showed that TpoR
W515A induces excessive MAP-kinase Erk1,2 and
STAT3 signaling. 35 A small molecule inhibitor of signaling
by TpoR Y626 could be a new approach in the treatment
of MPNs with mutated TpoR.
Familial and sporadic ET patients have been found to
harbor with low frequency the transmembrane S505N
activating mutation, 36–38 which leads to constitutive activation
of TpoR. Another TpoR mutation, located in the
extracellular juxtamembrane region at a position symmetrical
to W515, namely T487A, was shown to induce
a myeloproliferative phenotype in vivo, and has been
detected in a non-Down syndrome childhood acute
megakaryoblastic leukemia. 39 Overall, it is clear that the
juxtamembrane and transmembrane regions of TpoR
are true switch regions that can induce receptor activation
(Figure 3).
At present, it is unknown why TpoR mutants induce
a more severe and rapid phenotype when compared
with JAK2 V617F. Signaling by TpoR W515A may differ
from Tpo-activated TpoR, especially at the HSC level,
where normal TpoR is responsible for maintaining quiescence,
an effect opposite to its role in megakaryocyte
proliferation and differentiation. 40–42 Thus, signaling or
conformation by TpoR might not be similar in HSCs
and megakaryocytes. Further experiments with knockin
mice will be interesting to understand the precise role
London, United Kingdom, June 9-12, 2011
of TpoR W515A and of megakaryocytes in the development
of myelofibrosis.
Down-modulation of TpoR in MPN patient
megakaryocytes and platelets
A feature of many MPN patients is down-modulation
of TpoR cell surface and total levels in megakaryocytes
and platelets, without down modulation of TpoR
mRNA 8 (Figure 4). Examination of TpoR metabolism
pointed to defective maturation, accumulation of glycosylation-immature
(Endoglycosidase H-sensitive) TpoR, 43
characteristic of endoplasmic reticulum retention or
Figure 3. Several activating mutations in the thrombopoietin
receptor (c-MPL, TpoR) have been identified in myeloproliferative
disorders. Mutations cluster around the juxtamembrane
and transmembrane domains. Most frequently
W515 of the cytosolic juxtamembrane domain is
mutated to Lys, Leu or Ala. W515 is part of a short amphipathic
region that normally prevents self-activation of the
receptor. Mutation of transmembrane S505 to Asn is also
found in familial and sporadic MPNs. Mutation of a residue
symmetrically located on the extracellular juxtamembrane
region to W515, T487 to Ala also leads to constitutive activation
of TpoR and induces a MPD in mouse reconstitution
experiments. Furthermore, the human TpoR can be activated
by small molecules that bind to the extracellular juxtamembrane
region, further supporting the notion that this
region can switch on receptor signaling. These mutations,
especially W515K/L/A lead to constitutive activation of
the receptor, activation of JAK2 and phosphorylation of
Y626 of the cytosolic domain, which is required for induction
of MPN and myelofibrosis in vivo.
Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) | 247 |