The Thrombopoietin/MPL Pathway in Hematopoiesis and
Fu-Sheng Chou and James C. Mulloy*
Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center,
Cincinnati, Ohio 45229
Hematopoietic stem cells (HSC) comprise a small percentage of total hematopoietic cells. Their ability to self-renewal is key to the continuous
replenishment of the hematopoietic system with newly formed functional blood cell types while maintaining their multipotential capacity.
primitive stage, and with the knowledge applied, will potentially lead to improved clinical transplantation outcomes. In this review, we will
summarize the current understanding of the role of the thrombopoietin/MPL signaling pathway in HSC maintenance during adult and fetal
hematopoiesis. We will also speculate on the downstream key players in the pathway based on published data, and summarize the role of this
pathway in leukemia. J. Cell. Biochem. 112: 1491–1498, 2011.
? 2011 Wiley-Liss, Inc.
KEY WORDS: THPO; MPL; HSC; HEMATOPOIETIC DEVELOPMENT; LEUKEMIA
multiple lineages that carry out their various functions of supplying
oxygen, defending against microorganism insults, and maintaining
homeostasis. The late-stage functional cells have a limited lifespan,
so the system relies on primitive stem and progenitor cells to
continuously proliferate and differentiate in order to replace the lost
cells. It is generally accepted that HSC have the ability to self-renew,
meaning that upon cell division, at least one daughter cell inherits
the characteristics of the mother cell and retains ‘‘stemness.’’
Furthermore, HSC are predominantly quiescent and reside in
osteoblastic niches near the trabecular bones, protecting them from
extrinsic insults. HSC have been further categorized into long- and
short-term HSC based on cell surface immunophenotypic markers
and functional correlates in serial transplantation assays [Wilson
et al., 2009].
The maintenance of the HSC pool is critical to support a healthy
organism. Loss of HSC from cell death (e.g., irradiation) or from
intrinsic or extrinsic stimuli that disturb their ability to self-renew
will cause inevitable bone marrow failure, leading to pancytopenia,
and eventual death. Therefore, understanding the signals that are
essential for HSC homeostasis is of significant interest to the field of
hematology and stem cell biology. In addition to self-renewing
proliferation, cellular viability may also be involved in determining
the HSC pool size. Circulating cytokine signals as well as paracrine
he hematopoietic system consists of hematopoietic stem cells
(HSC), hematopoietic progenitors, and differentiated cells of
cytokine signals from the bone marrow niche microenvironment,
3, and angiopoietin have been found to participate in HSC
homeostasis. In this review, we will focus on the effect of the
thrombopoietin (THPO)/MPL signaling pathway on HSC biology.
DISCOVERY OF THE THPO/MPL PATHWAY
Two decades ago, v-mpl was identified as a viral oncogene
responsible for transforming myeloproliferative leukemia virus
(MPLV)-infected hematopoietic progenitors [Souyri et al., 1990]. v-
mpl is an envelope protein that shares striking structural similarities
with members of the cytokine receptor superfamily. Two years
later, molecular cloning identified the human homolog, MPL, as a
hematopoietic growth factor receptor [Vigon et al., 1992]. Two
isoforms (MPL-P and MPL-K) were identified that differed at their
30ends due to alternative splicing, leading to distinct cytoplasmic
domains (Fig. 1). Later findings suggested that the two isoforms
are co-expressed, with MPL-P being the dominant form [Horikawa
et al., 1997]. MPL-P contains the intracellular domain that is
highly homologous to v-mpl in sequence, suggesting MPL-P is the
isoform involved in signal transduction [Vigon et al., 1992]. Similar
to other members of the type I cytokine receptor family, MPL
contains the characteristic extracellular domain structure that
Journal of Cellular
Journal of Cellular Biochemistry 112:1491–1498 (2011)
*Correspondence to: James C. Mulloy, PhD, Cincinnati Children’s Hospital Medical Center, Division of Experimental
Hematology and Cancer Biology, Leukemia Biology Program, 3333 Burnet Ave., Mail Location 7013, TCHRF 7529,
Cincinnati, Ohio, USA. E-mail: James.Mulloy@cchmc.org
Received 21 February 2011; Accepted 22 February 2011 ? DOI 10.1002/jcb.23089 ? ? 2011 Wiley-Liss, Inc.
Published online 25 February 2011 in Wiley Online Library (wileyonlinelibrary.com).
includes two pairs of cysteine residues and a WSXWS motif for
directing proper folding, with highest homology to interleukin-3
and erythropoietin receptors [Vigon et al., 1992; Alexander and
Dunn, 1995]. However, MPL is unique among family members in
that it contains two of these motifs[Alexander and Dunn, 1995]. The
cytoplasmic domain of MPL contains the Box 1 and Box 2
membrane-proximal motifs that are essential for binding of the
Janus kinase 2 (JAK2) protein to initiate signaling (Fig. 1) [Gurney
et al., 1995].
Expression of MPL is largely confined to tissues that support
hematopoiesis, including bone marrow, spleen, and fetal liver.
MPL has been found on immature human CD34þCD38?stem
and progenitor cells, megakaryocyte progenitors and platelets,
and was recently shown to be expressed primarily in the most
primitive CD34þCD38?CD90þCD45RA?cell compartment [Goar-
don et al., 2011]. Initial characterization of MPL-deficient mice
found severe thrombocytopenia but relatively normallevels ofother
hematological cell types [Gurney et al., 1994]. Subsequently,
multiple groups identified THPO as the primary ligand for the MPL
receptor [Lok et al., 1994; Wendling et al., 1994]. Upon THPO
binding, MPL receptors undergo homodimerization to initiate
intracellular signaling, including activation of the JAK2/signal
transducers and activators of transcription (STAT) pathway (Fig. 2)
[Ezumi et al., 1995]. THPO/MPL signal transduction was shown to
play critical roles in thrombopoiesis, from ex vivo megakaryocyte
progenitor expansion and differentiation to in vivo platelet
production in BALB/c mice [Kaushansky et al., 1994; Lok et al.,
THE ROLE OF THPO/MPL SIGNALING IN
MURINE ADULT HEMATOPOIESIS
In addition to its role in thrombopoiesis, THPO was also found to
play a role in expanding erythroid and granulocytic–monocytic
progenitors [Kaushansky et al., 1996]. In addition, ex vivo
hematopoietic progenitor studies of MPL?/?mice revealed a more
than50%decrease intotal colony-forming unitcells (CFU-C) aswell
assignificantreductionsinmyeloid, erythroid,megakaryocytic, and
blast CFU-C [Alexander et al., 1996]. Similar findings were observed
in cells derived from THPO?/?mice [Carver-Moore et al., 1996].
Surprisingly, no reduction in peripheral blood counts other than
platelet number was observed, implying that maturation of
progenitors into differentiated and functional blood cells remained
largely unaffected [Alexander et al., 1996]. These data indicate a
potential defect in the multipotent cell compartment or even in the
stem cell compartment due to loss of THPO/MPL signaling.
Subsequently, ex vivo studies successfully defined the role of THPO
in expanding or maintaining the pool of transplantable HSC, further
establishing the responsiveness of cells in the primitive hemato-
poietic compartment tothis cytokine signaling pathway [Matsunaga
et al., 1998; Yagi et al., 1999].
To further characterize HSC defects in MPL?/?mice, Kimura et al.
first performed in vivo CFU-spleen (CFU-S) assays by injecting bone
marrow cells derived from MPLþ/þor MPL?/?(on the 129/Sv
background) mice into MPLþ/þor MPL?/?recipients, followed by
irradiation of the recipient mice at 11Gy [Kimura et al., 1998]. The
results showed a significant loss of CFU-S from MPL?/?donor cells.
Fig. 1.Schematic representation of human MPL-K, MPL-P, and v-mpl.
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Interestingly, MPLþ/þdonor cells gave rise to equal frequency of
CFU-S in both MPLþ/þand MPL?/?recipients, suggesting that the
CFU-S defect is cell-intrinsic. Subsequently, a long-term repopulat-
ing cell assay was performed using increasing numbers of test cells
from MPLþ/þand MPL?/?mice to compete with a fixed number of
MPLþ/þcompetitor cells [Kimura et al., 1998]. Analyses of relative
engraftment in the primary transplants at 9–10 months as well as in
the secondary and tertiary transplants at 3-month time points
all showed MPL?/?test cells significantly lost competitiveness,
indicating a repopulating defect of MPL?/?long-term HSC.
Alternatively, this pronounced phenotype could also result from
the total absence of long-term repopulating cells in the MPL?/?
In vivo studies on THPO?/?mice also led to similar conclusions.
One elegant experiment demonstrated that transplantation of
normal bone marrow cells into lethally irradiated THPO?/?recipient
mice resulted in a 10- to 20-fold reduction in the expansion of
transplantable HSC when compared to the THPOþ/þrecipient group,
as 7.5?106donor cells from the THPO?/?primary transplant
is required to reconstitute and rescue 80% of lethally irradiated
recipient mice in the secondary transplantation while 3?105cells
fromtheTHPOþ/þprimaryrecipient micewas sufficient torescueall
irradiated secondary recipient mice [Fox et al., 2002]. Exogenous
administration of a physiological dose of THPO to the THPO?/?
primary recipient mice completely rescued the phenotype. One
puzzling finding from this same report was that short-term
repopulating ability (5 weeks post-transplantation) of normal bone
marrow cells was also compromised in THPO?/?recipient mice,
leading to significantly reduced rescue of the lethally irradiated
recipient mice. Intriguingly, exogenous THPO administration did
not reverse this phenotype. Although a detailed progenitor analysis
was not done, this phenotype may be due to ineffective production
of sufficient numbers of megakaryocytic progenitors from the
transplanted cells, even with THPO rescue. Further examination
of why THPO?/?recipient mice failed to support hematopoietic
was more than a 10-fold decrease in Lin?Sca-1þKitþand Lin?Sca-
1þKitþFlt3?cells in the THPO?/?recipient mice 3 weeks after
transplantation when compared to their wild-type counterpart,
suggesting that the THPO/MPL pathway is required to maintain this
compartment in the engrafted marrow [Qian et al., 2007]. It is also
have given rise to these cells in the absence of THPO immediately
THE THPO/MPL PATHWAY IN PRIMATE
The study of the influence of the THPO/MPL pathway on the
maintenance of HSC has been extended to non-human primates
and humans. THPO injection rescued irradiation-induced loss of
multilineage hematopoietic progenitors in primates, including
GEMM-CFU, GM-CFU, BFU-E, and MEG-CFU [Farese et al.,
1996]. This was associated with faster recovery of peripheral blood
counts following irradiation. Human CD34þCD38?hematopoietic
cells derived from umbilical cord blood can be further separated
into MPLþand MPL?cells using an anti-MPL antibody against
the surface receptors. CD34þCD38?MPLþcells, but not CD34þ
CD38?MPL?cells, were able to efficiently engraft transplanted
human fetal bone marrow fragments in immunodeficient mice
[Solar et al., 1998].
Fig. 2. THPO/MPL downstream signaling pathways and proposed regulatory roles in self-renewal.
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Interestingly, a MPL point mutation in the extracellular ligand-
binding domain was identified in a rare human disease called
congenital amegakaryocytic thrombocytopenia (CAMT) [Ballmaier
et al., 2001; Steinberg et al., 2007]. Patients with CAMT present with
isolated thrombocytopenia in infancy, and develop pancytopenia
later in their childhood. Progenitor assays using bone marrow
mononuclear cells showed a significant reduction in clonogenic
cells not only in the megakaryocytic lineage but also in myeloid and
erythroid lineages when compared to healthy donors. Due to its
phenotypic similarities with the findings of thrombocytopenia and
the stem cell defect in MPL?/?mice, the authors proposed that both
thrombocytopenia and pancytopenia observed in CAMT patients
result from MPL loss-of-function mutations.
Moreover, a retrospective clinical study on patients undergoing
bone marrow transplantation found two independent factors that
predict poor transplantation prognosis—one is post-transplantation
platelet engraftment of <150,000/ml; the other is development of
idiopathic secondary transplant thrombocytopenia (ISPT) [Ninan
lower dose of CD34þcells/kg upon transplant, and the latter with a
low dose of CD34þCD38?cells/kg, suggesting defects in short- and
long-term engraftment, respectively. The utilization of thrombopoi-
esis to predict transplantation outcome implies that the THPO/MPL
signaling pathway has a significant influence on HSC homeostasis.
THE ROLE OF THPO/MPL SIGNALING DURING
Due to multi-lineage progenitor defects observed in MPL?/?and
THPO?/?mice, it is reasonable to hypothesize that the defects may
already be evident during embryonic development. In situ
hybridization revealed MPL mRNA expression in Aorta-Gonad-
AGM, MPL is exclusively expressed in the hematopoietic clusters
[Petit-Cocault et al., 2007]. It is possible that the earliest
hematopoietic cells that emerge from the aortic endothelium are
responsive to THPO signaling. A transplantation assay showed
defects in long-term repopulation and engraftment in secondary
recipients repopulated with MPL?/?E11.5 AGM cells, suggesting
loss of MPL-mediated signaling has a negative impact onthe quality
or quantity of the earliest transplantable hematopoietic cells. A
colony formation assay also showed reduced CFC, mostly due to a
reduction in BFU-E and CFU-Mk, in E11.5 MPL?/?AGM. A closer
look at the absolute number of CD45þKitþand CD34þKitþcells in
the E10.5 AGM showed a slight increase in MPL?/?AGM, but
no difference between E11.5 MPLþ/þand MPL?/?AGM cells,
suggesting a quantitative defect transiting from E10.5 to E11.5 in
the MPL?/?AGM [Fleury et al., 2010]. Interestingly, quantitative
PCR showed a slight, yet significant, reduction in the expression
levels of anti-apoptotic Bcl-2 and Bcl-xL [Fleury et al., 2010].
It is possible that the reduced expression of survival factors is
responsible for the observed defect of MPL?/?AGM cells in the
transplantation assay. Whether it is responsible for the observed
defects in AGM remain to be further investigated. Moreover,
reductions in the mRNA expression of Meis1 and HoxB4, homeobox
proteins that are involved in leukemia formation, were detected in
identification of the key downstream factors should take advantage
oftheinducible murinegeneticmodels tobestexamine theeffectsat
a specific developmental stage.
An early study separated fetal liver AA4þSca1þkitþcells into
MPLþand MPL?populations, and found that AA4þSca1þkitþMPLþ
cells were capable of engrafting lethally irradiated mice but
AA4þSca1þkitþMPL?cells were not in a competitive repopulation
assay [Solar et al., 1998]. Surprisingly, initial analysis of E12.5 fetal
liver hematopoietic progenitor cells revealed no differences in the
frequency of progenitors at various developmental stages between
MPLþ/þand MPL?/?embryos [Alexander et al., 1996]. However,
a recent time course study was performed on fetal liver lin?AA4þ
Sca1þ(highly enriched in HSC), lin?CD34þKitþ[enriched in long-
term hematopoietic reconstituting cells, (LTR)], and hematopoietic
progenitors, where the respective functional readouts for each cell
type were secondary transplantation (primary transplantation ?20
weeks, secondary transplantation of 7 weeks), primary transplanta-
tion at week 20, and colony formation [Petit-Cocault et al., 2007].
Fetal liver cellularity was also examined. Results showed that total
cellularity was low in E10.5 MPL?/?fetal liver when compared to
the MPLþ/þcounterpart, yet no difference in cellularity was
observed at E12.5 and E14.5, suggesting a delayed migration of
cellularity between E10.5 and E12.5 may accountfor reduced CFC in
MPL?/?at E10.5 but the lack of differences between the two groups
at E12.5. Surprisingly, CFC frequencies of all types were reduced
again in E14.5 MPL?/?fetal liver, possibly indicating the existence
that led to reduced progenitor output. Indeed, higher numbers of
E14.5 fetal liver cells, but not E12.5, were required to establish
engraftment in recipient mice at week 20, suggesting decreased
donor cells were required from MPL?/?fetal liver to establish
engraftment in the secondary recipients, further indicating reduced
signaling negatively impacts on the pool size of early hematopoietic
cells, predisposing to a defect in sustaining hematopoiesis later in
Notably, another study comparing E14.5 wild-type and THPO?/?
fetal liver cells did not find any repopulating defects [Qian et al.,
2007]. In this study, 2?104Lin?ScaþKitþCD34?Flt3?(defined as
LT-HSC) cells were injected into wild-type recipient mice. This lack
of effect could possibly be due to the significantly higher number of
cells used in the study. Alternatively, because cells from THPO?/?
mice express MPL, it is also possible that the cells regain their
repopulating ability when injected into wild-type mice. Moreover,
the different combinations of immunophenotypic markers used
for defining the HSC also make the comparison and interpretation
cells [Fleury et al., 2010]. Future studies on the
SPECULATION ON THE ROLE OF THE THPO/MPL
PATHWAY IN PROMOTING HSC SURVIVAL
It has been proposed that apoptosis plays a role in the regulation of
the size of the HSC compartment. Overexpression of Bcl-2 in the
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hematopoietic system using the H-2Kbpromoter led to increased
numbers of Lin?KithighSca-1highThy1.1lowLT-HSC, with more cells
in the G0phase. This is associated with increased repopulating
potential in a competitive transplantation assay [Domen et al.,
2000]. Moreover, Bcl-2 overexpression under the control of Sca-1
transcriptional regulatory elements also led to increased numbers of
Sca-1þcells in the AGM, leading to enhanced long-term (4 months)
engraftment in a competitive transplantation assay when limiting
cell numbers (1?104cells) were injected into the recipient mice
[Orelio et al., 2004]. Interestingly, in a serial transplantation assay
using E12 AGM cells and E12 fetal liver cells, Bcl-2 overexpressing
cells engrafted 100% and 50% of the secondary recipients,
respectively (the primary recipient mice with 75–100% engraftment
of E12 cells were used as donors for secondary transplantation). In
contrast, none of the secondary recipients injected with wild-type
cells had any detectable engraftment. These data indicate that
apoptosis/cell survival plays a significant role in regulating the
quantity or quality of HSC both during development and in adult
Although THPO by itself does not support ex vivo growth of
cells [Sitnicka et al., 1996]. Furthermore, one experiment showed
that Bcl-2 overexpression (under the control of H-2K promoter) in
suggesting that the THPO/MPL pathway may have a survival-
promoting downstream component for maintaining the HSC pool
size [Qian et al., 2007]. It is therefore of interest to determine which
anti-apoptotic molecules and pathways are responsible for the
regulation of HSC homeostasis and whether they are regulated by
the THPO/MPL-mediated signaling cascades.
In the study of thrombopoiesis, Bcl-xL is a critical downstream
developing megakaryocytes [Kozuma et al., 2007; Sanz et al., 2001].
As Bcl-xL knock-out leads to embryonic lethality at E13, and since
Bcl-xL, but not Bcl-2, is expressed in human CD34þCD38?cells, it is
possible that the THPO/MPL/Bcl-xL pathway may be functionally
important in these more primitive cells [Motoyama et al., 1995;
Park et al., 1995]. It is worth noting, however, that data from both
mouse and human studies indicate that Mcl-1 functionally plays
a critical role in promoting survival of the early compartment
(Lin-Sca-1þKitþin mice and CD34þCD38?in human cells)
[Opferman et al., 2005; Campbell et al., 2010]. Whether the
THPO/MPL pathway regulates Mcl-1 protein levels in cells included
in these populations, and whether Bcl-xL and Mcl-1 play non-
THE THPO/MPL PATHWAY SUPPORTS HSC IN
THEIR QUIESCENT STATE AND INTERACTION
WITH THE OSTEOBLASTIC NICHE
In addition to survival, THPO/MPL signaling has also been shown to
regulate the quiescent state of Lin?ScaþKitþCD34?Flt3?cells in the
THPO?/?genetic mouse model [Qian et al., 2007]. This is associated
with reduced expression of p57KIP2 in Lin?ScaþKitþCD34?Flt3?
and Lin?ScaþKitþCD34þFlt3?cells as well as decreased expression
of p19INK4D and p21CIP1 in the Lin?ScaþKitþFlt3?population
in THPO?/?mice, suggesting that THPO/MPL signaling may be
involved in the regulation of cell cycle progression. Another study
showed that MPL-expressing hematopoietic cells in the bone
marrow are in close proximity to THPO-producing osteoblastic
niche cells, suggesting that the THPO/MPL signal is highly enriched
in that microenvironment to maintain HSC in their quiescent state
[Yoshihara et al., 2007]. Interestingly, as mentioned above, Bcl-2
ectopic expression in the HSC compartment also led to an increased
percentage of cells in the G0 phase, raising the question as to
whether THPO-mediated quiescence is a consequence of improved
HOMEOBOX GENES AS DOWNSTREAM
EFFECTORS OF THE THPO/MPL PATHWAY IN
Homeobox (Hox) transcription factors have been implicated in HSC
self-renewal. In addition to downregulation of HoxB4 in MPL?/?
E10.5 AGM cells, as mentioned above, HoxB4, HoxA5, HoxA9, and
HoxA10, which were highly expressed in Lin?ScaþKitþCD34?Flt3?
long-term repopulating cells in wild-type mice, were detected at
very low levels in the equivalent population in THPO?/?mice [Qian
cell line found that THPO signaling stimulates expression of HoxB4
via the p38 mitogen-activated protein kinase (MAPK) pathway
[Kirito et al., 2003]. Moreover, THPO stimulation also promotes
Meis1 mRNA expression as well as nuclear translocation of HoxA9
without affecting its total protein levels in UT-7/THPO and Sca-
1þKitþGr-1?cells [Kirito et al., 2004]. Perhaps the next important
question is whether any (or all) of the Hox proteins play a key role in
determining the cell fate in response to THPO stimulation. It would
also be critical to directly examine whether THPO/MPL signaling
plays arole in determiningsymmetric versus asymmetric division as
well as polarity formation during development and in adult bone
CROSSTALK BETWEEN THE THPO/MPL
PATHWAY AND RUNX1
Runx1, a master transcription factor in hematopoiesis, was also
found to be significantly downregulated in MPL?/?AGM when
compared to wild-type AGM cells [Petit-Cocault et al., 2007]. It is
not known whether THPO/MPL signaling directly regulates Runx1
or whether these results may be due to comparison of different cell
population as a consequence of loss of THPO/MPL signaling. Runx1
endothelium during embryonic development [Chen et al., 2009].
Intriguingly, no obvious defect in HSC emergence was described in
MPL?/?embryos, and the AGM cellularity (CD45þc-Kithigh) at E10.5
was paradoxically higher than that in WT embryos [Petit-Cocault
et al., 2007; Fleury et al., 2010]. Moreover, Runx1 is viewed as a
differentiation promoter that guides HSC emergence from hemo-
genicendothelium, yetTHPO/MPLsignaling seems tobe responsible
for the maintenance of HSC (and long-term repopulating
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hematopoietic cells) by promoting cell survival and/or maintaining
quiescence. In addition, Runx1 inducible knock-out in the adult
hematopoietic system showed no effect on HSC or increased
HSC activity, while THPO or MPL knock-out is associated with
continuous loss of HSC phenotypes [Ichikawa et al., 2004]. Notably,
the technical aspects of the studies may need to taken into
consideration, as Cre recombinase inducible knock-out was used in
the Runx1 study but germline knock-out was used in the THPO/MPL
studies. Nonetheless, similar to MPL knock-out, Runx1 knock-out in
adult also leads to insufficient platelet production [Growney et al.,
2005]. On the other hand, expression of a Runx1 dominant-negative
mutant in Lin?Sca-1þKitþcells, or in embryonic stem cells induced
for hematopoietic cell development, led to increased expression
of MPL, suggesting that endogenous Runx1 plays a role in the
repression of the MPL gene in these early hematopoietic cells [Satoh
et al., 2008]. The authors further proposed that the repressive effect
results from recruitment of mSin3A, a co-repressor, by Runx1 to the
promoter region of the MPL gene, although the chromatin
immunoprecipitation assay was performed on Lin?Sca-1þcells, a
different population than the cells in which the phenotypes
were shown. Overall, the significance of Runx1 downregulation
in MPL?/?mice and the reciprocal regulation between MPL and
Runx1 remain to be further investigated.
THE ROLE OF THPO/MPL IN LEUKEMOGENESIS
When human MPL was cloned, it was known that v-mpl was the
truncated form of the cytokine receptor lacking the extracellular
ligand-binding domains. As v-mpl is by itself sufficient to promote
transformation of murine bone marrow cells, and given that the
THPO/MPL pathway is active in the HSC compartment, it is
reasonable to postulate that activation of the THPO/MPL pathway
may play a role in leukemogenesis. An early study found that MPL
mRNA expression is detectable by Northern blotting in 26 out of 50
cases of acute myeloid leukemia (AML). Subsequent in vitro assay
showed that blast cells from 22 out of the 26 MPLþ cases responded
to exogenous THPO for proliferation [Matsumura et al., 1995].
Except for French-American-British M7 (megakaryoblastic) AML
that all express MPL, MPL expression does not correlate with FAB
classification (M0–M6). Another study showed 47% (53 out of 114)
ofAMLand14% (2out of14)of acutelymphoblastic leukemia(ALL)
expressed surface MPL (detected by biotinylated THPO). Interest-
ingly, both ALL cases showed in vitro proliferative response to THPO
[Takeshita et al., 1998]. Yet another study showed that, although 37
out 60 ALL cases expressed surface MPL, none of them responded to
11 AML samples stimulated with THPO responded with either
improved survival or proliferation. Serum THPO levels were
significantly lower in patients with MPL-expressing AML when
compared to AML without MPL expression or ALL, implicating
uptake of THPO by the blast cells in the former group [Corazza et al.,
MPL mutation was detected in 5–9% of JAK2V617F-negative
myelofibrosis with myeloid metaplasia (MMM) cases and in 1% of
essential thrombocythemia (ET), two classes of myeloproliferative
disorders [Pikman et al., 2006]. Although patients with MMM or ET
may evolve into myelodysplastic syndrome or AML, MPL mutations
were notfound inthe lattertwo secondary diseases [Pardananiet al.,
2006]. The most common mutation was W515L or W515K, which
resides in the transmembrane domain (Fig. 1). Expression of
MPLW515L in 32D, UT7, or Ba/F3 cells led to cytokine-independent
growth. This was associated with constitutive phosphorylation of
JAK2, STAT3, STAT5, AKT, and ERK, suggesting constitutive
activation of the mutant receptors. Transplantation of MPLW515L-
overexpressing bone marrow cells, but not MPLwildtype-overexpres-
sing cells, into lethally irradiated recipient mice leads to develop-
ment of myeloproliferative disorders with full penetrance, further
verifying the disease-causing role of the mutation [Pikman et al.,
2006]. Indeed, MPLW515L mutation was found in three out of 12
acute megakaryoblastic leukemia that are either associated with
trisomy 21 or t(9;22), further implying a role of such mutation in
pathogenesis [Hussein et al., 2009]. On the other hand, while other
mutations of MPL in the transmembrane domain have also been
identified, in vitro and in vivo assays failed to show their growth-
promoting role [Chaligne et al., 2008].
Given the role of the THPO/MPL pathway in maintaining the
repopulating ability of normal HSC, it would be interesting to
investigate whether the THPO/MPL pathway plays a role in the
regulation of leukemia initiating cells and whether blocking the
pathway would aid in combating disease relapse. Delivery of a MPL
inhibitor would be relatively straightforward because of surface
expression of the receptors. However, potential adverse effects such
as thrombocytopenia or premature bone marrow failure may hinder
its clinical use. Recently, one intriguing report showed that
eltrombopag, a non-peptide MPL agonist licensed for use in the
treatment of chronic idiopathic thrombocytopenic purpura, para-
doxically decreased ex vivo growth of primary AML blasts while
recombinant THPO did not [Kalotaand Gewirtz, 2010]. This could be
due to overwhelming stress induction following receptor activation
by eltrombopag in blast cells, or due to aberrant structure or
function of the MPL protein in malignancy, leading to a cytotoxic
effect upon eltrombopag binding. In any case, this report
demonstrates the potential use of targeting this pathway as a novel
therapeutic strategy in leukemia treatment, and merits further
Since its discovery 20 years ago, researchers have been rigorously
is now clear that this pathway is not only critical for thrombopoiesis
but also for the regulation of HSC. However, how the THPO/MPL
pathway affects HSC homeostasis and at what stage of differentia-
tionthepathwayplaysacritical roleis stillnotfully understood.It is
also an open question as to how to validate the germline THPO and
MPL knock-out models in the study of adult HSC without
conditionally targeted alleles. A tissue-specific inducible mouse
genetic model system may solve this issue. With the advancement of
scientific and imaging tools and the expansion of our knowledge
regarding HSC biology in general, future studies should focus on a
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closer look at the effect of the pathway on self-renewing cell
divisions at a single-cell level, as well as its downstream effector
molecules that direct polarity establishment and niche interaction.
Moreover, as there is clearly a survival-promoting component of the
THPO/MPL pathway in the long-term repopulating HSC, a closer
look at how the pro-apoptotic and anti-apoptotic signals achieve
their balance and what molecules are involved will allow a more
thorough understanding of how the HSC pool size is maintained by
the THPO/MPL pathway. Equally important is how we translate our
understanding of the pathway in normal HSC into leukemia stem
cells. So far, most of the clinical studies are merely correlative, and
the sample sizes are too small. Although some ex vivo functional
experiments were performed to demonstrate the responsiveness of
blast cells to THPO stimulation, a more relevant biological system is
needed to facilitate our understanding of the influence of this
pathway on disease pathogenesis. The current biggest obstacle is the
lack of an inducible knock-out mouse model to study the effect of
the THPO/MPL pathway in leukemia formation and progression.
Moreover, the development of xenotransplantation models in
immunodeficient mice will allow us to study the molecular effect of
the THPO/MPL pathway on patient-derived blast cells and leukemia
repopulating cells, and to test investigational inhibitors in a pre-
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