A WHIM satisfactorily addressed

From www.bloodjournal.org by guest on February 5, 2015. For personal use only.
The paper by Yao et al now establishes
Shh as an autocrine factor for MC motility,
fostering chemotactic movement toward the
PDGF-BB–releasing endothelium. PDGF-BB
in turn acts upstream of Shh in MCs during
this process, and both pathways feed into
the phosphatidylinositol 3-kinase (PI3K) and
extracellular signal-regulated kinase 1/2
(ERK1/2) cascades via speciﬁc kinase isoforms.1
Herein, Shh appears to play the role of a delayed
reinforcement of the PI3K and ERK1/2 signal,
possibly retaining pathway activity on an
elevated level when the PDGF-BB boost has
already vanished in the haze (see ﬁgure).
When putting these novel ﬁndings in the
context of published data, they intriguingly ﬁt
to the described arterializing role of Shh that
was suggested to act via upregulation of VEGF
in somites that in turn leads to Notch pathway
activity in the endothelium.4 Yao et al describe
that VEGF-induced PDGF-BB release from
the endothelium activates Shh expression
in MCs so that one could envision a selfsustaining circle leading to vascular stability
(see ﬁgure). Although this aspect was not
touched on by Yao et al, a maintenance function
in the vasculature might be suggestive, because
Shh is important for blood-brain barrier
integrity, which also relies on PDGFBB–mediated pericyte recruitment.8,9
Besides the insufﬁciently understood cellautonomous effects of Hh in the endothelium,
these ﬁndings raise a number of important
questions. For example, with which players
other then PDGF-BB, such as angiopoietin/
Tie, Dll4/Notch, and transforming growth
factor b, does Shh interact during MC
recruitment? Because the Wnt/b-catenin
pathway has been suggested to regulate
PDGF-BB in the endothelium, leading to MC
recruitment to tumor vessels,10 it remains to be
deciphered if this would contribute to the
PDGF-BB–driven function of Shh in MCs
observed by Yao et al. Furthermore, the
contribution of other sources of Shh (ie,
astrocytes in the brain) to MC recruitment
needs to be clariﬁed (see ﬁgure).
In conclusion, the study by Yao et al
has successfully exposed that autocrine
Shh signaling in MCs is crucial for their
recruitment to endothelial cells and
consequently for vessel stability. Future
studies are required to determine whether
targeting the Shh pathway in MCs might
have beneﬁcial effects in diseases involving
pathologically increased or decreased MC
2286
recruitment, such as in regenerative and tumor
angiogenesis.
Conﬂict-of-interest disclosure: The author declares
no competing ﬁnancial interests. n
REFERENCES
1. Yao Q , Renault M-A, Chapouly C, et al. Sonic
hedgehog mediates a novel pathway of PDGF-BBdependent vessel maturation. Blood. 2014;123(15):2429-2437.
2. N¨usslein-Volhard C, Wieschaus E. Mutations
affecting segment number and polarity in Drosophila.
Nature. 1980;287(5785):795-801.
3. Rowitch DH, S-Jacques B, Lee SM, Flax JD, Snyder
EY, McMahon AP. Sonic hedgehog regulates proliferation
and inhibits differentiation of CNS precursor cells.
J Neurosci. 1999;19(20):8954-8965.
and CNS immune quiescence. Science. 2011;334(6063):
1727-1731.
6. Renault M-A, Roncalli J, Tongers J, et al. Sonic
hedgehog induces angiogenesis via Rho kinase-dependent
signaling in endothelial cells. J Mol Cell Cardiol. 2010;
49(3):490-498.
7. Briscoe J, Th´erond PP. The mechanisms of
Hedgehog signalling and its roles in development and
disease. Nat Rev Mol Cell Biol. 2013;14(7):416-429.
8. Daneman R, Zhou L, Kebede AA, Barres BA.
Pericytes are required for blood-brain barrier integrity
during embryogenesis. Nature. 2010;468(7323):562-566.
9. Armulik A, Genov´e G, M¨ae M, et al. Pericytes
regulate the blood-brain barrier. Nature. 2010;468(7323):
557-561.
4. Nagase T, Nagase M, Machida M, Fujita T.
Hedgehog signalling in vascular development. Angiogenesis.
2008;11(1):71-77.
10. Reis M, Czupalla CJ, Ziegler N, et al. Endothelial
Wnt/b-catenin signaling inhibits glioma angiogenesis and
normalizes tumor blood vessels by inducing PDGF-B
expression. J Exp Med. 2012;209(9):1611-1627.
5. Alvarez JI, Dodelet-Devillers A, Kebir H, et al. The
Hedgehog pathway promotes blood-brain barrier integrity
© 2014 by The American Society of Hematology
l l l CLINICAL TRIALS & OBSERVATIONS
Comment on McDermott et al, page 2308
A----------------------------------------------------------------------------------------------------WHIM satisfactorily addressed
Hal E. Broxmeyer1
1
INDIANA UNIVERSITY SCHOOL OF MEDICINE
In this issue of Blood, McDermott et al present the intriguing, clinically relevant,
and perhaps unexpected ﬁndings for the efﬁcacy and safety of long-term
administration of low-dose plerixafor treatment of patients with warts,
hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome.1
W
HIM is a rare disorder of primary
immunodeﬁciency associated with
warts, hypogammaglobulinemia, retention of
neutrophils in the bone marrow, and recurring
bacterial infections. Until this study (Clinical
Trials.gov identiﬁer: NCT00967785),1 there
was no treatment that allowed for long-term
control of infections and warts in this
syndrome. The rationale for using plerixafor
(also known as AMD3100 and Mozobil) was
to take advantage of the peripheral blood
neutrophil and other leukocyte-mobilizing
capacity of AMD3100/plerixafor to increase
numbers of leukocytes in the circulation2 to
alleviate some symptoms of this disease. This
was a reasonable approach as a majority of
patients with WHIM have an autosomaldominant mutation of CXCR4, a chemokine
receptor, believed to increase intracellular
signaling that allows retention of neutrophils
and other leukocytes at their tissue sites. The
leukocyte-mobilizing effects of CXCR4
antagonism are relatively rapid, usually
within minutes to hours, and cessation of
administration of the antagonist is quickly
(usually within hours) followed by decreased
numbers of blood cells.2,3 Hence, maintaining
increased levels of circulating leukocytes would
require frequent dosing of patients with the
antagonist. Frequent dosing, however, has
implications for possible problems involving
desensitization of the receptor to the
antagonizing effector, and induction of serious
side effects and safety concerns. In fact,
CXCR4 is a coreceptor for HIV, and attempts
at continuing antagonism of CXCR4 by
AMD3100 to prevent or decrease HIV
infection were associated with severe side
effects.4 AMD3100 has been used successfully
to mobilize hematopoietic stem (HSC) and
progenitor (HPC) cells to the blood for
collection for use in hematopoietic cell
transplantation (HCT),3,5,6 but due to the rapid
and effective mobilization of HSCs/HPCs, and
its synergy in this effect with granulocyte
colony-stimulating factor, the AMD3100
BLOOD, 10 APRIL 2014 x VOLUME 123, NUMBER 15
From www.bloodjournal.org by guest on February 5, 2015. For personal use only.
Schematic of results from the McDermott et al study,1 and questions emanating from the results.
needed only to be administered short-term to
donors for efﬁcient mobilization of enough
cells for effective HCT.6
The strength of the article by McDermott
et al1 is that the 3 patients with WHIM assessed
in this study were given plerixafor at low dose
(twice a day by self-administration) for
6 months without side-effects (and with
greatly decreased infectious episodes) and,
in combination with imiquimod, improved
control of warts (see ﬁgure). In retrospect,
the design of this long-term study is quite
impressive; the effectiveness of long-term
low-dose administration of plerixafor was not
necessarily predictable, even though it followed
2 phase 1 clinical trials with plerixafor
administered for 1 to 2 weeks to patients
with WHIM.7,8
There are interesting consequences of
low-dose administration of plerixafor noted in
this article,1 including the slow reversion to
baseline levels of leukocytes after chronic
plerixafor low-dosing was discontinued, and
a thorough read of this clinical study is
warranted with the understanding that this
work is only a beginning to proving the clinical
efﬁcacy of plerixafor for treatment of some of
the health issues associated with WHIM. It
may not be a cure, but it clearly demonstrates
health beneﬁts for patients with WHIM
syndrome, and follow-up conﬁrmatory studies
are warranted.
BLOOD, 10 APRIL 2014 x VOLUME 123, NUMBER 15
This study1 brings up a number of
questions and possibilities (see ﬁgure) that
could be experimentally evaluated in context
of further clinical trials in this and other
disorders associated with decreased circulating
leukocytes, and in animal models of disease,
as AMD3100 works in mice, and higher
animals, as well as in humans. An intriguing
ﬁnding was the apparent lack of desensitization
of CXCR4 to the leukocyte-mobilizing
effects of plerixafor, which is interesting
because CXCR4 is a G-protein–linked
7-transmembrane spanning receptor that
manifests desensitization to a natural ligand,
stromal-derived factor-1 (SDF-1)/CXCL12.
It has been reported that rapid mobilization
of HPCs by AMD3100 in mice may be
mediated at least in part by CXCR4-dependent
release of SDF-1/CXCL12 from stromal cells
in the bone marrow.9 In this context, it
would be of interest to assess serum levels of
SDF-1/CXCL12 in patients being treated
with long-term low-dose plerixafor.
Perhaps it is CXCR4-dependent release of
SDF-1/CXCL12 that is in part responsible for
the increase in circulating leukocytes and their
sustained levels for a while after cessation of
plerixafor. It would also be of interest to see
whether, along with plerixafor-induced
increases in circulating leukocytes, there is also
an increase in phenotypically deﬁned and
functionally active subsets of long- and short-
term repopulating human HSCs and HPCs, as
well as other cells which may play a role in
regenerative medicine, such as endothelial
colony-forming cells (ECFCs) and
mesenchymal stem/stromal cells (MSCs).
Although mobilization of HSCs, HPCs,
ECFCs, and/or MSCs may not play a role in
helping to ameliorate some of the problems
inherent in patients with WHIM syndrome,
such information could be of importance for
other disease states, as endogenous tissue repair
from certain stress conditions may reﬂect the
enhanced circulation of these stem/progenitor
cell populations.
It is becoming clear that levels of circulating
blood cells may be under control of circadian
oscillations, as has recently been reported for
HSCs,10 and correct timing of mobilization of
such cells may greatly enhance the numbers of
cells mobilized. Hence, studies of modiﬁed
timing of low-dose plerixafor may increase
efﬁcacy. Whether clinical use of long-term
administration of low-dose plerixafor can be
adequately translated for treatment of other
disorders associated with low blood leukocyte
counts is of interest; this will require that
there are enough leukocytes available in bone
marrow and other tissue sites for potential
mobilization, and the leukocytes in these
patients would need to function normally,
as in the evaluated 3 patients with WHIM
syndrome. What the study by McDermott
et al1 (and those publications preceding this
work that established AMD3100/plerixafor
as a mobilizing agent) demonstrates is that
ingenuity, creativity, and ﬂexibility in
experimental design, starting at a basic science
level and preceding through preclinical
investigation, can lead to efﬁcacious and safe
manipulations that improve health care. This is
what the scientiﬁc process is about.
Conﬂict-of-interest disclosure: The author
declares no competing ﬁnancial interests. n
REFERENCES
1. McDermott DH, Liu Q, Velez D, et al. A phase 1
clinical trial of long-term, low-dose treatment of WHIM
syndrome with the CXCR4 antagonist plerixafor. Blood.
2014;123(15):2308-2316.
2. Hendrix CW, Flexner C, MacFarland RT, et al.
Pharmacokinetics and safety of AMD-3100, a novel
antagonist of the CXCR-4 chemokine receptor, in human
volunteers. Antimicrob Agents Chemother. 2000;44(6):
1667-1673.
3. Liles WC, Broxmeyer HE, Rodger E, et al.
Mobilization of hematopoietic progenitor cells in healthy
volunteers by AMD3100, a CXCR4 antagonist. Blood.
2003;102(8):2728-2730.
2287
From www.bloodjournal.org by guest on February 5, 2015. For personal use only.
4. Hendrix CW, Collier AC, Lederman MM, et al;
AMD3100 HIV Study Group. Safety, pharmacokinetics,
and antiviral activity of AMD3100, a selective CXCR4
receptor inhibitor, in HIV-1 infection. J Acquir Immune
Deﬁc Syndr. 2004;37(2):1253-1262.
5. Broxmeyer HE, Orschell CM, Clapp DW, et al. Rapid
mobilization of murine and human hematopoietic stem and
progenitor cells with AMD3100, a CXCR4 antagonist.
J Exp Med. 2005;201(8):1307-1318.
6. Broxmeyer HE. Preclinical experience with AMD3100
for mobilization of hematopoietic stem and progenitor
cells. In: Fruehauf S, Jens Zeller W, Calandra G, eds.
Novel Developments in Stem Cell Mobilization: Focus on
CXCR4. Part 1. Chapter 1. New York, NY: Springer;
2012:3-22.
7. Dale DC, Bolyard AA, Kelley ML, et al. The CXCR4
antagonist plerixafor is a potential therapy for
myelokathexis, WHIM syndrome. Blood. 2011;118(18):
4963-4966.
8. McDermott DH, Liu Q, Ulrick J, et al. The
CXCR4 antagonist plerixafor corrects panleukopenia in
patients with WHIM syndrome. Blood. 2011;118(18):
4957-4962.
9. Dar A, Schajnovitz A, Lapid K, et al. Rapid
mobilization of hematopoietic progenitors by AMD3100
and catecholamines is mediated by CXCR4-dependent
SDF-1 release from bone marrow stromal cells. Leukemia.
2011;25(8):1286-1296.
10. M´endez-Ferrer S, Lucas D, Battista M, Frenette PS.
Haematopoietic stem cell release is regulated by circadian
oscillations. Nature. 2008;452(7186):442-447.
© 2014 by The American Society of Hematology
l l l THROMBOSIS & HEMOSTASIS
Comment on Nogami et al, page 2420
FV
and APC resistance: the plot thickens
----------------------------------------------------------------------------------------------------Elisabetta Castoldi1
1
MAASTRICHT UNIVERSITY
In this issue of Blood, Nogami et al report on a novel factor V (FV) gene mutation
(FV Trp1920→Arg, FVNara) associated with activated protein C (APC) resistance
and a severe thrombotic phenotype in a young Japanese patient.1 Since the
affected amino acid residue is located in the light chain of FV, far from the known
APC-cleavage sites, this discovery may afford new insights into the molecular
mechanisms of APC resistance.
T
he serine-protease APC plays a major
anticoagulant role by proteolytically
inactivating factors Va (FVa) and VIIIa
(FVIIIa), the essential cofactors of the
prothrombinase and intrinsic tenase
complexes, respectively. Both reactions are
greatly stimulated by anionic phospholipids
and by the APC-cofactor protein S. APC
cleaves FVa at Arg306, Arg506, and Arg679,
all located in the heavy chain of the protein,
whereas the light-chain anchors FVa to the
membrane surface. On the other hand, APC
also cleaves the inactive precursor FV
(particularly at Arg506), converting it into a
Thrombin generation curves obtained in platelet-poor plasma from a normal control (A) and from the FVNara homozygous patient (B) in the absence and presence of APC. Plasma from the FVNara homozygous patient is completely
insensitive to the anticoagulant action of APC. Professional illustration by Marie Dauenheimer.
2288
still poorly deﬁned anticoagulant cofactor that
stimulates the inactivation of FVIIIa by the
APC/protein S complex. Therefore, FV(a)
is both a substrate and a cofactor of APC
(reviewed in Castoldi and Rosing2).
In 1993, a reduced anticoagulant response
of plasma to APC (APC resistance) was ﬁrst
described in a thrombophilic family3 and
quickly recognized as the most common
risk factor for venous thrombosis in the
Caucasian population. Soon afterward, the
FV Arg506→Gln (FVLeiden) mutation was
identiﬁed as the underlying genetic defect.4
This mutation eliminates the APC-cleavage
site at Arg506, thereby hampering not only
the APC-mediated inactivation of FVaLeiden
(which relies entirely on the protein S–dependent
cleavage at Arg306), but also the conversion
of FVLeiden into an APC cofactor for FVIIIa
inactivation. The FVLeiden mutation is present
in ;5% of Caucasians, but is virtually absent in
the indigenous populations of Africa, America,
Eastern Asia, and Australia. More recently,
other FV gene mutations associated with APC
resistance have been identiﬁed in various
populations, including Arg306→Thr
(FVCambridge), Arg306→Gly (FVHong Kong),
Ile359→Thr (FVLiverpool), and Glu666→Asp
(reviewed in Castoldi and Rosing2).
Remarkably, all of these variants predict
amino acid changes at or close to the
APC-cleavage sites in the heavy chain of FV(a),
and their mechanisms of action can be
rationalized in terms of reduced cleavage
at these sites.
The interesting study by Nogami et al
describes a 13-year-old Japanese boy who
developed recurrent venous thrombosis during
oral anticoagulant treatment.1 The patient
had reduced FV levels (40 IU/dL antigen,
10 IU/dL activity) and pronounced APC
resistance (see ﬁgure), prompting FV gene
sequencing. This revealed a novel homozygous
missense mutation (Trp1920→Arg, FVNara) in
the C1 domain of FV, which was not found in
50 healthy Japanese people. In line with the
observations made in the patient, recombinant
FVNara showed reduced expression in
conditioned media (;50% of wild-type FV)
and conferred APC resistance to reconstituted
FV-deﬁcient plasma. Moreover,
detailed characterization of the mutant in
model systems indicated that: (1) the
APC-mediated inactivation of FVaNara is
severely impaired and hardly sensitive to
stimulation by protein S; and (2) FVNara
BLOOD, 10 APRIL 2014 x VOLUME 123, NUMBER 15
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2014 123: 2286-2288
doi:10.1182/blood-2014-02-557579
A WHIM satisfactorily addressed
Hal E. Broxmeyer
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