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Targeting the Hypoxia-Sensing Pathway in Clinical Hematology

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Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly heterogeneous in terms of microvasculature and thus oxygen concentration. The importance of hypoxia and HIFs in the hematopoietic environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoiesis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in hemodialysis patients. Pharmacological stabilization of HIF-1α protein with PHD inhibitors is also emerging as an important regulator of HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the bone marrow in vivo, thereby protecting them from high doses of irradiation and accelerating hematological recovery. Recent findings also show that stabilization of HIF-1α increases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerixafor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation. These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology.
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Catherine E. Forristal and Jean-Pierre Levesque
Targeting the Hypoxia-Sensing Pathway in Clinical Hematology
doi: 10.5966/sctm.2013-0134 originally published online December 26, 2013
2014, 3:135-140.Stem Cells Trans Med
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Perspectives
Targeting the Hypoxia-Sensing Pathway in Clinical Hematology
CATHERINE E. FORRISTAL,JEAN-PIERRE LEVESQUE
Stem Cell Biology Group, Mater Research InstituteUniversity of Queensland, Woolloongabba, Queensland, Australia
SUMMARY
Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain
(PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly
heterogeneou s in terms of microvasculature an d thus oxygen concentration. Th e importance of hypoxia and HIFs in the hematopoie tic
environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be
pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoi-
esis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in
hemodialysis patients. Pharmac ological stabilization o f HIF-1aprotein with PHD inhib itors is also emerging as an impo rtant regulator of
HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the
bone marrow in viv o, thereby protecting them from hi gh doses of irradiation and accel erating hematological recove ry. Recent findings
also show that stabilization of HIF-1aincreases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerix-
afor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation.
These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology. STEM CELLS TRANSLATIONAL
MEDICINE 2014;3:135140
INTRODUCTION
Maintenance of oxygen homeostasis is critical for the survival of
organisms. On exposure to hypoxic conditions, a cellular response
is mounted by hypoxia-inducible factors (HIFs). HIFs are a family of
three transcription factors composed of one of three oxygen-
sensitive asubunitsHIF-1a, HIF-2a, and HIF-3aand a constitu-
tively expressed bsubunit HIF-1b, also called aryl hydrocarbon
receptor nuclear translocator (ARNT). Once the HIF-a:ARNT com-
plex is formed, it translocates to the nucleus and activates the tran-
scription of genes containing hypoxia-responsive elements (HREs)
[1, 2]. Hematopoietic cells including hematopoietic stem cells
(HSCs) express HIF-1amRNA, which is expressed ubiquitously by
all cells. In hypoxic conditions with oxygen (O
2
) concentration be-
low 2%, HIF-aproteins are stabilized and complex with ARNT to
translocate to the nucleus and initiate transcription of HRE-
containing genes. In normoxic conditions or when O
2
concentra-
tion exceeds 2%, HIF-1aprotein is degraded within 5 minutes by
the proteasome [3], preventing the formation of the transcription
factor and its translocation to the nucleus. The sensitization of
HIF-aproteins to proteasomal degradation in the presence of
O
2
is mediated by three prolyl hydroxylase domain (PHD) enzymes
that hydroxylate two proline residues within the oxygen-degradation
domain of HIF-aproteins (Fig. 1A) [4, 5]. These hydroxylated proline
residues then bind the von Hippel-Lindau tumor-suppressor protein
to form an E3 ubiquitin ligase complex that ubiquinates and targets
HIF-aprotein to the proteasome (Fig. 1B) [6, 7]. PHD enzymes
are iron(II)-dependent and utilize 2-oxoglutarate and O
2
as sub-
strates to hydroxylate proline residues [8]. In cultured cells, PHDs
areinactivewhenO
2
is ,2% in the extracellular milieu, resulting
in HIF-aprotein stabilization.
As noted previously, the expression of HIF-asubunits is predom-
inantly regulated by PHD-mediated proline hydroxylation. There
are three well known PHD isoforms, called PHD1, PHD2, and
PHD3, and all are reported to hydroxylate HIF-asubunits [9]. They
are encoded by three distinct genes: Egln2 for PHD1, Egln1 for
PHD2, and Egln3 for PHD3. A fourth PHD enzyme is also thought
to be involved in regulating HIF-asubunits and has been reported
to play a potential role in erythropoiesis [10, 11].
ROLE OF HIFSINCONTROLLING HEMATOPOIETIC STEM AND
PROGENITOR CELLS
HIF Expression in Hematopoietic Stem and Progenitor Cells
The importance of HIFs in development and hematopoiesis has
been demonstrated by genetic deletion of ARNT, which abro-
gates the function of both HIF-1aand HIF-2a. In the developing
embryo, ARNT is essential for multilineage hematopoietic pro-
genitors, vasculogenesis, and angiogenesis [12, 13]. HIF-1a
mRNA is ubiquitously expressed [14]. In steady state, HIF-1apro-
tein is detected only in the endosteal region of the bone marrow
(BM)andinsomediscretecellsinthecentralBM[15].Conse-
quently, HIF-1aprotein is generally below detection in whole
BM lysates [15, 16]; however, when HSCs are mobilized in the pe-
ripheral blood by administering granulocyte colony-stimulating
factor (G-CSF) or cyclophosphamide, HIF-1aprotein is stabilized
and found throughout the BM cavity [15].
Correspondence: Jean-Pierre Levesque, Ph.D., Mater Research, Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland 4102, Australia.
Telephone: 61-7-3443-7571; E-Mail: jplevesque@mmri.mater.org.au Received July 26, 2013; accepted for publication September 18, 2013; first published
online in SCTM EXPRESS December 26, 2013. ©AlphaMed Press; http://dx.doi.org/10.5966/sctm.2013-0134
STEM CELLS TRANSLATIONAL MEDICINE 2014;3:135140 www.StemCellsTM.com ©AlphaMed Press 2014
P
ERSPECTIVES
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Unlike HIF-1a,HIF-2amRNA expression is restricted. HIF-2a
is expressed by vascular endothelium, hepatocytes, and inter-
stitial and glomerular cells of the kidney. In the BM, HIF-2a
mRNA is primarily expressed by hematopoietic lineage-negative
cells [14]. HIF-2amRNA is detected at very low levels in HSCs;
however, in these cells, HIF-2aprotein is mainly localized to
the cytoplasm [14], suggesting that it is not transcriptionally ac-
tive [17].
The expression profile of HIF-3ahas been largely uncharacter-
ized; however, in the BM, HIF-3ais most highly expressed in HSCs
and is expressed at low levels in more differentiated progeny [14].
The function of HIF-3ais unknown because, unlike HIF-1aand
HIF-2a, HIF-3adoes not contain a DNA-binding domain. Further-
more, HIF-3acontains many splice variants, the most studied of
which is known as inhibitory PAS domain, which functions as
a dominant-negative regulator of the other two HIFsmediated
gene induction [18, 19].
HIFs Regulate Proliferation and Self-Renewal of
Hematopoietic Stem and Progenitor Cells
The importance of hypoxia and HIF-1ain the proliferation and
self-renewal of HSCs is well established. Indeed, culture of hu-
man and mouse HSCs in hypoxic conditions results in an accumu-
lation in phase G
0
of the cell cycle as a result of HIF-1aprotein
stabilization, which enhances expression of p21
cip
,p27
Kip1
,and
p57
Kip2
and reduces mitochondrial oxidative phosphorylation
[20, 21]. This results in increased frequency of long-term repo-
pulating HSCs in hypoxic cultures compared with cultures in air
[22, 23]. Conversely, conditional deletion of the Hif1a gene in
hematopoietic cells results in exaggerated HSC proliferation
in vivo, with premature exhaustion of HSC self-renewal poten-
tial, loss of serial reconstitution potential, and increased sen-
sitivity to cytotoxics [14]. Pharmacological stabilization of HIF-
1aprotein with PHD inhibitors or genetic stabilization by con-
ditional deletion of the VHL gene increases HSC quiescence in
vivo [14, 16].
HIF-1aprotein is difficult to detect in the mouse BM in steady
state because only a very small proportion of the BM cavity is
hypoxic in these conditions [15, 16]; however, HIF-1aprotein
can be stabilized in murine HSCs in the BM by administering
small compounds (e.g., dimethyloxalylglycine or the isoquino-
line dipeptidyl derivative FG-4497; Fig. 2) that inhibit PHDs by
competing with their physiological substrate 2-oxoglutarate
[16]. This pharmacological stabilization of HIF-1aprotein in-
creases quiescence and decreases proliferation of HSCs in the
BM in vivo [16].
Hematopoietic progenitor cells alsorequire the hypoxia-sensing
pathway to regulate cell cycling. Pharmacological stabilization of
HIF-1aprotein with PHD inhibitors decreases hematopoietic pro-
genitor cell (HPC) proliferation in vivo [16]. Unexpectedly, condi-
tional deletion of the Egln1 gene (encoding PHD2) in cells
expressing CD68 results in an increase in proliferation of HPCs in
steady state in vivo in an HIF-1a- and SMAD7-dependent manner
[24]; however, without lineage-tracking experiments to determine
which cells have the active CD68 gene promoter to conditionally
delete the Egln1 gene, it is not possible to conclude whether the
proliferative effect of PHD2 deletion on HPCs was cell intrinsic or
extrinsically mediated by CD68
1
macrophages or other cells of
the BM stroma.
Figure 1. Regulation of HIF-aproteins. (A): Hydroxylation of two
distinct proline residues is catalyzed by PHDs. (B): Regulation of
the HIF-aprotein under hypoxic and normoxic conditions. PHD
inhibitors block HIF-aproline hydroxylation and subsequent ubiq-
uitination. HIF-aproteins are stabilized. Abbreviations: ARNT, aryl
hydrocarbon receptor nuclear translocator; ATM, ataxia telangiec-
tasia mutated; DMOG, dimethyloxalylglycine;HRE, hypoxia-responsive
elements; PHD, prolyl hydroxylase domain; pVHL, von Hippel-Lindau
protein.
Figure 2. Chemical structures of 2-oxoglutarate, the physiological
substrate of PHD enzymes, and PHD inhibitors DMOG, FG-4592,
and Amgen compound 12{1,1,2} [44]. Additional structures of PHD
inhibitors based on the dipeptidyl-quinolone backbone can be found
in [44]. The structure of FG-4592 was found on the website for Sell-
eckchem Inc. (Houston, TX, http://www.selleckchem.com). Ab-
breviation: DMOG, dimethyloxalylglycine; PHD, prolyl hydroxylase
domain.
136 HIF-Stabilizing Therapies in Hematology
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HIFs Regulate Erythropoiesis by Erythropoietin
Production in the Kidney
Erythropoietin(EPO) expressionis regulated primarily by thePHD2-
HIF-2aaxis. Renal EPO production is regulated primarily at the
transcriptional level and is markedly induced by anemia or global
hypoxia encountered at high altitudes. EPO has been shown to
be releasedby interstitialrenal cells in rodentsand by renal progen-
itor cells located in the renal inner medulla in humans [25]. HIF-2a,
expressed in the endothelial, interstitial, and glomerular cells of
the kidney and hepatocytes, plays an important role in EPO pro-
duction, whereas HIF-1adoes not appear to play a role [9, 26,
27]. The major role of PHD2 in controlling HIF-2aprotein and
EPO production in the kidney has been recently confirmed in
mice with conditional deletion of the Egln1 gene in kidneys
and macrophages. In the absence of PHD2, severe erythrocytosis
with a 10-fold increase of EPO concentration in kidney extracts
and blood plasma was observed in an HIF-2a-dependent manner
[28]. In support of this notion, positive selection of adaptive poly-
morphism in genes encoding for HIF-2aand PHD2 has been noted
in human populations living at high altitude, such as Tibetans and
Andeans [29, 30].
ARE HEMATOPOIETIC STEM AND PROGENITOR CELL NICHES IN THE
BM HYPOXIC?
The BM is highly heterogeneous in terms of microvasculature
[3137]. Consequently, HSCs are exposed to varying levels of ox-
ygen perfusion depending on their location in the BM. Serially
reconstituting, quiescent HSCs tend to reside in areas with very
low blood perfusion, whereas more proliferative HSCs with
a lower reconstitution potential tend to reside in areas that
are more perfused by blood [38]. Furthermore, quiescent HSCs
are more frequent in the endosteal region of the BM, at two cell
diameters, or on average 10 mm, from the interface with the
compact bone [36, 39, 40]. This endosteal region is thought to
be hypoxic because of binding of pimonidazole [14, 15], a com-
pound that covalently binds to protein adducts when O
2
concen-
tration is below 10 mmHg or when the oxidative state is low;
however, most HSCs reside in proximity to endothelial sinuses,
even in the endosteal region of the BM. Moreover, different
cells can demonstrate differing pimonidazole binding regardless
of direct oxygen concentration, depending on their metabolic
profile. Indeed, two recent reports indicate that HSCs and HPCs
can bind pimonidazole independently of their location in the BM
[36, 37]. Consequently, HSCs may be more correctly referred to
as low oxidative phosphorylation cellsrather than hypoxic
cells,and the so-called osteoblastic niche should rather be re-
ferredtoasanosteovascularniche [37]. Nevertheless, a pro-
portion of phenotypic HSCs reside immediately adjacent to
endothelial cells forming blood sinuses and vessels and are away
from the endosteum [41, 42].
Because of this heterogeneity of the HSC microenvironment,
not all HSCs and progenitor cells are exposed to the same oxi-
dative conditions in steady state. Approximately 50%60% of
long-term reconstituting HSCs defined by the lineage-negative
Kit
1
Sca1
1
CD48
2
CD150
1
phenotype, and approximately
20%40% of more heterogeneous lineage-negative Kit
1
Sca1
1
cells reside in poorly perfused, low-oxidative regions of
the BM in steady state, as demonstrated by Hoechst 33342 in
vivo perfusion and pimonidazole staining [14, 38]. Interestingly
these proportions coincide with the proportion of quiescent
HSPCs in the BM: Approximately 60% of lineage-negative Kit
1
Sca1
1
CD48
2
CD150
1
HSCs and approximately 30% of lineage-
negative Kit
1
Sca1
1
cells are quiescent in phase G
0
of the cell
cycle, as measured by Ki67 staining [16]. Small synthetic PHD
inhibitors mimic hypoxia throughout the entire BM, regardless
of perfusion and oxygen levels, by stabilizing HIF-1ain an oxygen-
independent manner. Consequently, PHD inhibitors pharmacolog-
ically stabilize HIF-1ain HSCs located in more perfused vascular
niches as well as hypoxic regions. Thus, following treatment with
PHD inhibitors, approximately 85% of long-term reconstituting
HSCs and 70% of lineage-negative Kit
1
Sca1
1
cells become quies-
cent [16].
PHD INHIBITORS AND HIFS
As described previously, the expression of HIF-asubunits is pre-
dominantly regulated by PHD-mediated hydroxylation. PHD inhib-
itors block the enzymatic activity or interfere with its substrate
binding site. There are well-known commercially available chemi-
cals that block PHD in vivo such as dimethyloxalylglycine and
N-oxalylglycine thatmimic the PHD cosubstratestructure of 2-oxo-
glutarate[43], thus blockingenzyme activity [6, 7](Fig. 2). More re-
cently, several companies have developed PHD inhibitors with
different chemical structures. Although the chemical structures
of most of these compounds are not described in the publically
available literature, some are. FibroGen (San Francisco, CA,
http://www.fibrogen.com), for instance, has developed a line of
compounds derived from isoquinoline bound to a dipeptide that,
similarly to oxalylglycine derivatives, mimic 2-oxoglutarate and
block PHD enzymatic activity with much higher efficacy [16] (Fig.
2). Amgen (Thousand Oaks, CA, http://www.amgen.com) has de-
veloped similar dipeptides bound to quinolone scaffolds, some
of which with half maximal inhibitory concentration values for
PHD2 as low as 35 nM and on the order of 5 nM for PHD1 and
PHD3 [44] (Fig. 2).
THERAPEUTIC INTERVENTION WITH PHD INHIBITORS
PHD inhibitors have been mostly trialed to correct anemia caused
by kidney diseases, with 22 clinical trials registered at the
ClinicalTrials.gov website (http://www.clinicaltrials.gov) at the
time of this review (Table 1). To date, no toxic effects have been
reported.
Anemia
Because EPO transcription and production are directly controlled
by HIF-2aprotein in the kidney, the proerythropoietic effect of
PHD inhibitors mediated by HIF-2aprotein stabilization and in-
creased EPO production is well documented in all mammalian
species tested. This elevated endogenous EPO production in-
creases red cell and hemoglobin blood concentration [45]. Conse-
quently, PHD inhibitors are being tested for treatment of anemia
in chronic renal disease, in which renal failure dramatically
reduces EPO production [46, 47]. Five orally available PHD inhib-
itors (i.e., AKB-6548, BAY85-3934, GSK1278863, FG-4592, and
FG-2216) are being or have been tested in clinical trials for this
specific application (as shown at ClinicalTrials.gov) (Table 1). FG-
2216 hasdemonstratedefficacy in increasing endogenous EPOcon-
centration in rhesus macaques [48] and in a phase II clinical trial on
hemodialysis patients with kidneys or who are anephric [47].
Forristal, Levesque 137
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In addition to boosting EPO production, PHD inhibitors such as
FG-4497 have also been found to improve acute kidney injury in
rats [49]. Pretreatment with the PHD inhibitor FG-4497 reduced
renal tissue injury and apoptosis following kidney ischemia, thus
suggesting a renoprotective role. The PHD inhibitor GSK1278863
is in a clinical trial to test efficacy to reduce ischemia injury during
surgery to repair aortic aneurysm (Table 1).
Because EPO has also been reported to have a number of
effects on nonhematopoietic cells and organs such as the brain,
glial cells, endothelial cells, skin, and the digestive tract [50], it
is possible that PHD inhibitors could have protective effects in
a number of other tissues [5153].
It must also be noted that the erythropoietic effect of PHD
inhibitors is bringing new challenges to sports medicine because
they are possibly being used as doping agents. Because the use of
performance enhancement with erythropoiesis-enhancing drugs
is prohibited, mass spectrometry methods are being developed to
detect PHD inhibitors in athletes [54].
Protection Against Irradiation
Quiescent cells in G
0
are more radiation resistant, with radiation
sensitivity peaking in phase G2/M of the cell cycle [55]. Pharmaco-
logical stabilization of HIF-1awith PHD inhibitors also increases
HSC resistance to severe irradiation (9.0 Gy), as measured by the
gold standard long-term competitive transplantation assay. Mice
treated with the PHD inhibitor dimethyloxalylglycine or FG-4497
prior to irradiationhad 100% of their HSCssurviving in the BM after
9.0-Gyirradiation,whereas in controlsaline-treatedmice, the num-
ber of long-termcompetitive repopulating HSCswas decreased 27-
fold by 9.0-Gy irradiation. Consequently, PHD inhibitor-treated
mice recovered significantly faster from radiation-induced neutro-
penia and thrombocytopenia and had less profound anemia than
irradiated control mice [16]. Importantly, increased HSC and HPC
quiescence in response to PHD inhibitors was not mediated by el-
evated endogenous EPO because administration of EPO has no ef-
fect on HSC and HPC cycling [16]. Consequently, treatment with
PHD inhibitors can protect HSCs from high, sublethal doses of irra-
diation; can accelerate hematological recovery; and can prevent
the rapid exhaustion of the hematopoietic system [16]. This sug-
gests that PHD inhibitors could be used preventatively in persons
at risk of being exposed to severe irradiation. This could be partic-
ularly useful in the management of catastrophic nuclear accidents
like Chernobyl or Fukushima. Additional studies are necessary to
determine whether PHD inhibitors administered shortly after irra-
diation would still be radioprotective.
HSC Mobilization
An important proportion of cancer patients fail to mobilize suffi-
cient numbers of HSCs in response to G-CSF precluding subsequent
autologous HSC transplantation. Plerixafor, a small inhibitor of the
chemokine receptor CXCR4, used for 4 days in combination with
G-CSF enables this minimal threshold to be reached in up to 60%
patients who previously failed to mobilize in response to G-CSF
alone; however, the remainder of the 40% of patients who failed
to mobilize in response to G-CSF alone still fail to mobilize ade-
quately with the combination of G-CSF plus plerixafor.
As mentioned previously, the endosteal region of the BM is
thought to be hypoxic because of binding of pimonidazole and
HIF-1aprotein expression [14, 15] in steady state; however, at
the peak of mobilization induced by G-CSF, the entire BM cavity
becomeshypoxic becauseof extensive myeloid progenitor prolifer-
ation, which leads to enhanced O
2
consumption without a detect-
able increase in blood supply [15, 38]. Coadministration of PHD
inhibitor FG-4497 with G-CSF further enhanced HIF-1aprotein sta-
bilization in mouse BM. Administration of FG-4497 in combination
with G-CSF and plerixafor increased 20-fold the number of long-
term repopulating HSCs mobilized into the peripheral blood [56].
This highlights the importance of HIF-1ain HSC mobilization and
provides a novel therapeutic strategy for increasing HSC mobiliza-
tion above levels obtained with combinations of G-CSF and plerix-
afor.Thus, PHD inhibitorscould be usefulagents in patients whostill
fail to mobilize in response to G-CSF and plerixafor.
CONCLUSION
BloodperfusionandhypoxiaviaHIF-1aare key regulators of HSCs in
their BM niches. Similarly, the hypoxia-sensing pathway via HIF-2ais
a key regulator of EPO production by the kidney and thus of eryth-
ropoiesis from HSCs. Consequently, the recent discovery of small
molecules that stabilize HIFs independently of tissue oxygenation
opens the possibility of pharmacological interventions in the hema-
topoietic system, particularly to treat anemia, to enhance HSC mo-
bilization for transplantation, and to increase HSC radioresistance.
HIFs also play a critical role in inflammation [57], expansion
of myeloid-derived suppressor cells [58], and differentiation
of antigen-presenting cells [59, 60]; therefore, PHD inhibitors
Table 1. Clinical trials using prolyl hydroxylase domain inhibitors registered on ClinicalTrials.gov
Company Drug Condition Status Phase
Akebia Therapeutics AKB-6548 Anemia caused by chronic
kidney disease
2 completed, 1 recruiting II
Bayer BAY85-3934 Anemia caused by chronic
kidney disease
3 completed I
FibroGen FG-2216 Renal anemia 1 completed II
FibroGen FG-4592 Anemia caused by chronic
kidney disease
7 completed, 2 active, 3
recruiting
II
GlaxoSmithKline GSK1278863 Ischemia reduction during
elective surgery of aortic
aneurysm
1 not yet recruiting II
GlaxoSmithKline GSK1278863 Anemia caused by chronic
kidney disease
2 completed II
138 HIF-Stabilizing Therapies in Hematology
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may be useful to treat immune diseases or to modulate immune
responses, particularly in the context of allogeneic transplantations.
ACKNOWLEDGMENTS
This work was supported by a project grant (604303) from the Na-
tional Health and Medical Research Council of Australia. J.-P.L.
is supported by a senior research fellowship (APP1044091) and
C.E.F is supported by a project grant (APP1046590) from the Na-
tional Health and Medical Research Council of Australia.
AUTHOR CONTRIBUTIONS
C.E.F.: conception and design, manuscript writing and editing;
J.-P.L.: conception and design, financial support, manuscript writ-
ing and editing, final approval of the manuscript.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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... Other HIF-PHIs such as dimethyloxalylglycine (Trichonas et al., 2013;Xie et al., 2014), L-mimosine (L-mim) (Trimmel et al., 2015;Janjic et al., 2019), MK8617 (Debenham et al., 2016;Li et al., 2019) and an FIH selective inhibitor N-oxalyl-D-phenylalanine (Meng et al., 2018) are still in preclinical research. Since the action and mechanism of HIF-PHIs in CKD anemia are well summarized in reviews published before (Forristal and Levesque, 2014;Gupta and Wish, 2017;Haase, 2017;Locatelli et al., 2017;Del Vecchio and Locatelli, 2018;Sanghani and Haase, 2019), this article focuses on milestones in the development of these clinical available HIF-PHIs and their potential in treating non-anemic diseases. ...
Article
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Hypoxia inducible factors (HIFs) and their regulatory hydroxylases the prolyl hydroxylase domain enzymes (PHDs) are the key mediators of the cellular response to hypoxia. HIFs are normally hydroxylated by PHDs and degraded, while under hypoxia, PHDs are suppressed, allowing HIF-α to accumulate and transactivate multiple target genes, including erythropoiesis, and genes participate in angiogenesis, iron metabolism, glycolysis, glucose transport, cell proliferation, survival, and so on. Aiming at stimulating HIFs, a group of small molecules antagonizing HIF-PHDs have been developed. Of these HIF-PHDs inhibitors (HIF-PHIs), roxadustat (FG-4592), daprodustat (GSK-1278863), vadadustat (AKB-6548), molidustat (BAY 85-3934) and enarodustat (JTZ-951) are approved for clinical usage or have progressed into clinical trials for chronic kidney disease (CKD) anemia treatment, based on their activation effect on erythropoiesis and iron metabolism. Since HIFs are involved in many physiological and pathological conditions, efforts have been made to extend the potential usage of HIF-PHIs beyond anemia. This paper reviewed the progress of preclinical and clinical research on clinically available HIF-PHIs in pathological conditions other than CKD anemia.
... Other key transcription regulators included HIF1A (and its cofactor ARNT), whose binding is facilitated by demethylation of the binding motif 123 ; HIF1A/ARNT are critical factors for HSC quiescence, through maintenance of the anaerobic glycolysisdependent metabolic activity in the bone marrow niche [124][125][126][127][128][129][130] . USF1/2 were also among the highlighted TFs, which have been shown to regulate chromatin architecture in erythroid differentiation and the betaglobin locus 131,132 . ...
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Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.
... 115 Several prolyl hydroxylase inhibitors have been tested in phase-II and phase-III trials as orally active drugs, mainly for the treatment of anemia induced by chronic kidney disease. 116,117 To date, no studies have evaluated the effects of this pharmacological class on anemia in patients with cancer owing to possible effects on tumor growth. Indeed, HIF stimulates angiogenesis via the transcription of pro-angiogenetic factors, such as vascular endothelialgrowth factor (VEGF). ...
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Anemia in cancer patients is a relevant condition complicating the course of the neoplastic disease. Overall, we distinguish the anemia which arises under chemotherapy as pure adverse event of the toxic effects of the drugs used, and the anemia induced by the tumour-associated inflammation, oxidative stress, and systemic metabolic changes, which can be worsened by the concomitant anticancer treatments. This more properly cancer-related anemia depends on several overlapping mechanism, including impaired erythropoiesis and functional iron deficiency, which make its treatment more difficult. Standard therapies approved and recommended for cancer anemia, as erythropoiesis-stimulating agents and intravenous iron administration, are limited to the treatment of chemotherapy-induced anemia, preferably in patients with advanced disease, in view of the still unclear effect of erythropoiesis-stimulating agents on tumour progression and survival. Outside the use of chemotherapy, there are no recommendations for the treatment of cancer-related anemia. For a more complete approach, it is fundamentally a careful evaluation of the type of anemia and iron homeostasis, markers of inflammation and changes in energy metabolism. In this way, anemia management in cancer patient would permit a tailored approach that could give major benefits. Experimental drugs targeting hepcidin and activin II receptor pathways are raising great expectations, and future clinical trials will confirm their role as remedies for cancer-related anemia. Recent evidence on the effect of integrated managements, including nutritional support, antioxidants and anti-inflammatory substances, for the treatment of cancer anemia are emerging. In this review article, we show standard, innovative, and experimental treatment used as remedy for anemia in cancer patients.
... The addition of FG-4497 significantly increased the mobilization of HSPCs induced by G-CSF [86,106]. In addition, FG-4497 exerts protective effects in ischemia-induced kidney injury and high-dose irradiation-induced BM failure [115]. ML141 is an inhibitor of cell division control protein 42 (Cdc42). ...
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Background Mobilization failure may occur when the conventional hematopoietic stem cells (HSCs) mobilization agent granulocyte colony-stimulating factor (G-CSF) is used alone, new regimens were developed to improve mobilization efficacy. Multiple studies have been performed to investigate the efficacy of these regimens via animal models, but the results are inconsistent. We aim to compare the efficacy of different HSC mobilization regimens and identify new promising regimens with a network meta-analysis of preclinical studies. Methods We searched Medline and Embase databases for the eligible animal studies that compared the efficacy of different HSC mobilization regimens. Primary outcome is the number of total colony-forming cells (CFCs) in per milliliter of peripheral blood (/ml PB), and the secondary outcome is the number of Lin ⁻ Sca1 ⁺ Kit ⁺ (LSK) cells/ml PB. Bayesian network meta-analyses were performed following the guidelines of the National Institute for Health and Care Excellence Decision Support Unit (NICE DSU) with WinBUGS version 1.4.3. G-CSF-based regimens were classified into the SD (standard dose, 200–250 μg/kg/day) group and the LD (low dose, 100–150 μg/kg/day) group based on doses, and were classified into the short-term (2–3 days) group and the long-term (4–5 days) group based on administration duration. Long-term SD G-CSF was chosen as the reference treatment. Results are presented as the mean differences (MD) with the associated 95% credibility interval (95% CrI) for each regimen. Results We included 95 eligible studies and reviewed the efficacy of 94 mobilization agents. Then 21 studies using the poor mobilizer mice model (C57BL/6 mice) to investigate the efficacy of different mobilization regimens were included for network meta-analysis. Network meta-analyses indicated that compared with long-term SD G-CSF alone, 14 regimens including long-term SD G-CSF + Me6, long-term SD G-CSF + AMD3100 + EP80031, long-term SD G-CSF + AMD3100 + FG-4497, long-term SD G-CSF + ML141, long-term SD G-CSF + desipramine, AMD3100 + meloxicam, long-term SD G-CSF + reboxetine, AMD3100 + VPC01091, long-term SD G-CSF + FG-4497, Me6, long-term SD G-CSF + EP80031, POL5551, long-term SD G-CSF + AMD3100, AMD1300 + EP80031 and long-term LD G-CSF + meloxicam significantly increased the collections of total CFCs. G-CSF + Me6 ranked first among these regimens in consideration of the number of harvested CFCs/ml PB (MD 2168.0, 95% CrI 2062.0−2272.0). In addition, 7 regimens including long-term SD G-CSF + AMD3100, AMD3100 + EP80031, long-term SD G-CSF + EP80031, short-term SD G-CSF + AMD3100 + IL-33, long-term SD G-CSF + ML141, short-term LD G-CSF + ARL67156, and long-term LD G-CSF + meloxicam significantly increased the collections of LSK cells compared with G-CSF alone. Long-term SD G-CSF + AMD3100 ranked first among these regimens in consideration of the number of harvested LSK cells/ml PB (MD 2577.0, 95% CrI 2422.0–2733.0). Conclusions Considering the number of CFC and LSK cells in PB as outcomes, G-CSF plus AMD3100, Me6, EP80031, ML141, FG-4497, IL-33, ARL67156, meloxicam, desipramine, and reboxetine are all promising mobilizing regimens for future investigation.
... hydroxylase inhibitors may have a potential to improve the effects of treatment in transplant recipients or oncology patients [67]. HIF also participates in the regulation of multiple processes including: apoptosis, angiogenesis, cell proliferation, pH regulation, cellular energy metabolism, and glucose transport [9]. ...
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The incidence of anemia of chronic disease (ACD) is underestimated, increases with age, and affects about 30% of the elderly. ACD treatment is currently based on the pharmacotherapy of diseases that caused anemia, erythropoiesis-stimulating agents, and parenteral administration of iron supplementation in case of iron deficiency. Increasing knowledge on the pathophysiology of ACD has resulted in the burst of research on the development of new drugs that are focused on three main areas. The first group of drugs includes substances that inhibit hepcidin transcription, namely direct and indirect bone morphogenetic protein 6 (BMP6) inhibitors and/or SMAD signaling pathway inhibitors, and drugs that regulate hepcidin transcription through STAT3 signaling pathway. The second group of drugs includes direct hepcidin inhibitors (e.g., aptamers, anticalin proteins, monoclonal antibodies) or substances that inhibit the binding of hepcidin to ferroportin. The third group of drugs improves erythropoiesis mainly by upregulation of erythropoietin and/or inhibition of proinflammatory cytokines. In the latter group, hypoxia-inducing factor stabilizers and IL-6 or TNFα antagonists are particularly important. This article discusses new drug groups and substances that are in different phases of development, including both preclinical and clinical studies, and focuses on the prospects of their use in ACD. © 2020 Polish Society of Hematology and Transfusion Medicine, Insitute of Hematology and Transfusion Medicine.
... Previous studies have demonstrated the ability of aconitine to depress I K(DR) amplitude and to fasten the inactivation rate of the current [39,46]. Therefore, because of their similar actions on the amplitude and gating of I K(DR) , to what extent changes in the amplitude and gating of I K(DR) by aconitine or roxadustat could produce any stimulatory effects on the function of hematopoietic cells in bone marrow [57] is worthy of being further investigated. ...
Article
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Roxadustat (FG-4592), an analog of 2-oxoglutarate, is an orally-administered, heterocyclic small molecule known to be an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase. However, none of the studies have thus far thoroughly investigated its possible perturbations on membrane ion currents in endocrine or heart cells. In our studies, the whole-cell current recordings of the patch-clamp technique showed that the presence of roxadustat effectively and differentially suppressed the peak and late components of IK(DR) amplitude in response to membrane depolarization in pituitary tumor (GH3) cells with an IC50 value of 5.71 and 1.32 μM, respectively. The current inactivation of IK(DR) elicited by 10-sec membrane depolarization became raised in the presence of roxadustatt. When cells were exposed to either CoCl2 or deferoxamine (DFO), the IK(DR) elicited by membrane depolarization was not modified; however, nonactin, a K+-selective ionophore, in continued presence of roxadustat, attenuated roxadustat-mediated inhibition of the amplitude. The steady-state inactivation of IK(DR) could be constructed in the presence of roxadustat. Recovery of IK(DR) block by roxadustat (3 and 10 μM) could be fitted by a single exponential with 382 and 523 msec, respectively. The roxadustat addition slightly suppressed erg-mediated K+ or hyperpolarization-activated cation currents. This drug also decreased the peak amplitude of voltage-gated Na+ current with a slowing in inactivation rate of the current. Likewise, in H9c2 heart-derived cells, the addition of roxadustat suppressed IK(DR) amplitude in combination with the shortening in inactivation time course of the current. In high glucose-treated H9c2 cells, roxadustat-mediated inhibition of IK(DR) remained unchanged. Collectively, despite its suppression of HIF prolyl hydroxylase, inhibitory actions of roxadustat on different types of ionic currents possibly in a non-genomic fashion might provide another yet unidentified mechanism through which cellular functions are seriously perturbed, if similar findings occur in vivo.
... 20 Since inhibition of PHDs results in HIFa stabilization and modulation of HIF-controlled gene products (including EPO), PHD inhibition represents a promising therapeutic approach for inducing the production of physiologic levels of endogenous EPO and alleviating anemia. 21 While the potential benefits of PHD inhibition for treatment of anemia in patients with CKD are under evaluation, some investigators have suggested that this mechanism of action could also potentially increase the risk of carcinogenesis and/or contribute to tumor progression due to upregulation of HIF/hypoxiaregulated gene products that have activities associated with cancer biology, such as vascular endothelial growth factor (VEGF). This view is based on findings in experimental xenograft models suggesting a role of HIF in tumor progression, and clinical data associating increased HIF with angiogenesis, metastasis, and poor prognosis. ...
Article
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Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat’s biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat’s 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration–time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.
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Cardiovascular diseases are the leading cause of death worldwide. During the development of cardiovascular diseases, hypoxia plays a crucial role. Hypoxia-inducible factors (HIFs) are the key transcription factors for adaptive hypoxic responses, which orchestrate the transcription of numerous genes involved in angiogenesis, erythropoiesis, glycolytic metabolism, inflammation, and so on. Recent studies have dissected the precise role of cell-specific HIFs in the pathogenesis of hypertension, atherosclerosis, aortic aneurysms, pulmonary arterial hypertension, and heart failure using tissue-specific HIF-knockout or -overexpressing animal models. More importantly, several compounds developed as HIF inhibitors or activators have been in clinical trials for the treatment of renal cancer or anemia; however, little is known on the therapeutic potential of these inhibitors for cardiovascular diseases. The purpose of this review is to summarize the recent advances on HIFs in the pathogenesis and pathophysiology of cardiovascular diseases and to provide evidence of potential clinical therapeutic targets.
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Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.
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Introduction: Anemia has and will continue to be a central theme in medicine particularly as clinicians are treating a burgeoning population of complex multi-organ system processes. As a result of multiple randomized controlled trials (RCTs), meta-analyses, and societal recommendations overly restrictive paradigms and under-administration of erythropoiesis stimulating agents (ESAs) have likely been followed by clinicians among all specialties. Areas covered: A review of anemia in the context of chronic kidney disease, hematologic malignancies, and cancer is presented with focus on the establishment of ESAs as integral in the treatment of anemia. Multiple RCTs and meta-analyses studying the use of ESAs are presented with focus upon their application to clinical practice. A 'compendium' is proffered describing the evolution, establishment, and implications of ESA administration initially among those with CKD with rapid subsequent application to the Hematology-Oncology population of patients. Literature search methodologies have included MEDLINE (1985-2020), PubMed (1996-2020), Cochrane Central Trials (1985-2020), EMBASE (2000-2020), and ClinicalTrials.gov (2000-2020). Expert opinion: Upon evaluation of risks and benefits of ESAs focused opinion and commentary is made supporting more liberal use of these agents and strongly suggesting that the current underlying treatment 'pendulum' has perhaps shifted too far to the 'under-treatment' side in many cases.
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Intravital microscopy of the calvarium is the only noninvasive method for high-resolution imaging of the bone marrow (BM) and hematopoietic stem cell (HSC) niches. However, it is unclear if the calvarium is representative of all BM compartments. Using the combination of whole body optical imaging, intravital microscopy, and "in vivo fluorescence trapping," a thorough comparison of HSCs and putative HSC niches in the calvaria, epiphyses, and diaphyses, at steady state or after HSC transplantation, can be made. We report substantial heterogeneity between different BM compartments in terms of bone-remodeling activity (BRA), blood volume fraction (BVF), and hypoxia. Although BVF is high in all BM compartments, including areas adjacent to the endosteum, we found that compartments displaying the highest BVF and BRA were preferentially seeded and engrafted upon HSC transplantation. Unexpectedly, the macroanatomical distribution of HSCs at steady state is homogeneous across these 3 areas and independent of these 2 parameters and suggests the existence of "reconstituting niches," which are distinct from "homeostatic niches." Both types of niches were observed in the calvarium, indicating that endochondral ossification, the process needed for the formation of HSC niches during embryogenesis, is dispensable for the formation of HSC niches during adulthood.
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Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.
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