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Published Ahead of Print on February 8, 2016, as doi:10.3324/haematol.2015.140749.
Copyright 2016 Ferrata Storti Foundation.
The LSD1 inhibitor RN-1 recapitulates the fetal pattern of
hemoglobin synthesis in baboons (P. anubis)
by Angela Rivers, Kestis Vaitkus, Vinzon Ibanez, Maria Armila Ruiz,
Ramasamy Jagadeeswaran, Yogen Saunthararajah, Shuaiying Cui, James D. Engel,
Joseph DeSimone, and Donald Lavelle
Haematologica 2016 [Epub ahead of print]
Citation: Rivers A, Vaitkus K, Ibanez V, Ruiz MA, Jagadeeswaran R, Saunthararajah Y, Cui S, Engel JD,
DeSimone J, and Lavelle D. The LSD1 inhibitor RN-1 recapitulates the fetal pattern of hemoglobin synthesis
in baboons (P. anubis). Haematologica. 2016; 101:xxx
doi:10.3324/haematol.2015.140749
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Title: The LSD1 inhibitor RN-1 recapitulates the fetal pattern of hemoglobin synthesis in
baboons (P. anubis)
Running Head: The LSD-1 Inhibitor RN-1 Increases HbF in Baboons
Angela Rivers1,2, Kestis Vaitkus2,3, Vinzon Ibanez2,3, Maria Armila Ruiz2,3 Ramasamy
Jagadeeswaran1,2, Yogen Saunthararajah4, Shuaiying Cui5, James D. Engel5, Joseph
DeSimone2,3, and Donald Lavelle2,3
1
Department of Pediatrics, University of Illinois at Chicago, Chicago, IL
2
Jesse Brown VA Medical Center, Chicago, IL
3
Department of Medicine, University of Illinois at Chicago, Chicago, IL
4
Department of Hematology and Oncology, Taussig Cancer Institute, Cleveland Clinic,
Cleveland, Ohio
5
Department of Cell and Developmental Biology, University of Michigan Medical School,
Ann Arbor, Michigan
Main Text Word Count = 3851
Number of Figures = 8
Abstract Word Count = 241
Supplemental PDF File = 1
Acknowledgements: This work was supported by NIH U01 HL117658 and NIH R01
HL114561.
Corresponding Author: Donald Lavelle, 820 S. Damen Ave., Jesse Brown VA Medical
Center, Chicago, Illinois 60612; Ph: 312-569-6623; FAX: 312-569-8114; email:
[email protected]
1
ABSTRACT
Increased fetal hemoglobin levels lessen the severity of symptoms and increase the
lifespan of patients with sickle cell disease.
Hydroxyurea, the only drug currently
approved for the treatment of sickle cell disease, is not effective in a large proportion of
patients and therefore new pharmacological agents that increase fetal hemoglobin
levels have long been sought. Recent studies identifying LSD-1 as a repressor of γglobin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1
increased γ-globin expression in the sickle cell mouse model. Because the
arrangement and developmental stage-specific expression pattern of the β-like globin
genes is highly conserved between man and baboon, the baboon model remains the
best predictor of activity of fetal hemoglobin-inducing agents in man. In this report, we
demonstrate that RN-1 increases γ-globin synthesis, fetal hemoglobin, and F cells to
high levels in both anemic and non-anemic baboons with activity comparable to
decitabine, the most potent fetal hemoglobin inducing agent known. RN-1 not only
restores high levels of fetal hemoglobin but causes the individual 5’ Iγ- and 3’ Vγ-globin
chains to be synthesized in the ratio characteristic of fetal development. Increased
fetal hemoglobin was associated with increased levels of acetylated Histone H3,
H3K4Me2, H3K4Me3, and RNA polymerase II at the γ-globin gene, and diminished γglobin promoter DNA methylation. RN-1 is likely to induce clinically relevant levels of
fetal hemoglobin in patients with sickle cell disease, although careful titration of the dose
may be required to minimize myelotoxicity.
2
INTRODUCTION
The term “haemoglobin switching” describes the sequential, highly regulated
pattern of expression of the α- and β-like globin genes during development.1 In humans,
the ε-globin gene is expressed during the first eight weeks of gestation, followed by high
level expression of the duplicated γ-globin genes during the fetal period.
The γ-globin
genes are expressed at very low levels (<1%) in the adult stage when expression of the
β-globin gene predominates.
A secondary level of developmental regulation
characterizes expression of the γ-globin genes. The duplicated γ-globin genes can be
distinguished by an amino acid difference at aa136 where the 5’ Gγ-globin gene
contains glycine while the 3’ Aγ-globin gene contains alanine (Figure 1).
During the
fetal period the Gγ- and Aγ genes are expressed in a ratio of 7:3, but in adult life this
ratio is 2:3.
The baboon (P. anubis) is an important animal model for studies of fetal
hemoglobin regulation because the structure and developmental regulation of the βglobin gene complex is nearly identical to man.2,3 In the baboon the duplicated γ-globin
genes are designated Iγ and Vγ because their amino acid sequence differs by the
presence of an Ile or Val at aa75.4 The ratio of Iγ/Vγ-globin expression is >9:5 in early
gestational age (50-60dpc) fetuses, 3:2 at birth and 1:2 in the adult.4,5 Therefore the
developmental pattern of expression of the baboon Iγ- and Vγ-globin genes is very
similar to the human Gγ- and Aγ-globin genes (Figure 1).
In sickle cell disease (SCD) the substitution of glutamic acid for valine at the
sixth amino acid of the β-globin protein leads to the formation of abnormal hemoglobin S
3
(HbS).6
Following deoxygenation in red blood cells (RBCs), HbS forms polymers
causing the RBCs to become deformed and adherent leading to painful vaso-occlusive
events and organ injury. In vitro studies have shown that both fetal hemoglobin (HbF)
(α2γ2) tetramers and (α2β Sγ) tetramers inhibit HbS polymerization.7 Because increased
levels of HbF lessen the severity of symptoms and increase the life expectancy of
patients with SCD, therapeutic approaches to increase HbF levels would be highly
desirable.8,9
While many drugs increase HbF in cultured erythroid cells, only a few
have been shown to increase HbF in vivo. Studies in simian primate animal models
that exhibit conservation of developmental globin gene regulation10, such as baboons,
are considered one of the best tests of the activity of HbF-inducing drugs for therapies
in patients designed to alleviate the symptoms of SCD and β-thalassemia.11
Early studies in anemic baboons showed that treatment with the DNA
methyltransferase inhibitor 5-azacytidine increased HbF to levels predicted to be
therapeutic in patients.12 Later studies confirmed that 5-azacytidine and its closely
related analog decitabine did indeed increase HbF to therapeutic levels in patients with
SCD and β-thalassemia.13-18 Based on experiments conducted in baboons, a phase 1
trial is currently in progress to test the safety and HbF-inducing activity of oral
administration of decitabine in combination with the cytidine deaminase inhibitor
tetrahydrouridine in SCD patients.19 Hydroxurea (HU), the first drug approved for the
treatment of SCD, was initially shown to increase HbF levels in monkeys 20 and
subsequent clinical trials also confirmed that HU increased HbF levels in patients with
4
SCD. 21 Because a large percentage of patients do not respond to HU, developing new
drugs that increase HbF levels remains a priority.
The TR2/TR4 heterodimer and Bcl11A have been identified as γ-globin gene
repressors that act by recruiting co-repressor complexes that establish repressive
epigenetic modifications at specific sites to inactivate gene expression.22 Recognition of
the role of Direct Repeat (DR) elements in the ε- and γ-globin gene promoters in
repression of these respective genes led to the identification of the TR2 and TR4
nonsteroidal nuclear receptor proteins that specifically bind these elements and
subsequently recruit a multi-protein co-repressor complex that includes DNA
methyltransferase 1 (DNMT1), lysine specific demethylase 1 (LSD1), and histone
deacetylases (HDACs) to repress gene expression.23-25
Both DNMT1 and LSD1 have
also been identified as components of co-repressor complexes also recruited by
Bcl11a.26,27
LSD1, the first histone demethylase identified, demethylates lysine 4
(H3K4) and lysine 9 (H3K9) residues of histone H3 and represses gene expression
during hematopoietic differentiation28-30. Tranylcypromine (TCP), an LSD-1 inhibitor,
induced HbF production in cultured human hematopoietic erythroid progenitors and
transgenic mice containing the entire human β-globin gene complex in the context of a
yeast artificial chromosome (β-YAC mice).31 Two recent studies have shown that RN1, a more potent and specific LSD-1 inhibitor than TCP, induced γ-globin expression in
human and baboon erythroid progenitors and a sickle cell mouse model.32,33 In this
study we have tested the effect of RN-1 in baboons and confirm that it is a highly potent
in vivo inducer of HbF synthesis.
5
METHODS
Baboons
Baboons were housed at the University of Illinois at Chicago Biologic Resources
Laboratory (UIC BRL) under conditions that meet the Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC) standards. Bone marrow aspirations
were performed from the hips of animals under ketamine/xylazine anesthesia (10
mg/kg; 1 mg/kg). Prior to bone marrow sampling, Buprenex (0.01 mg/kg IM) was given
and later in the afternoon a second dose of Buprenex (0.01 mg/kg IM) was administered
to alleviate potential pain and suffering.
The effect of RN-1 was tested in both anemic and non-anemic baboons.
Anemia was induced by repeated phlebotomies during a two-week period prior to
administration of drug to attain a hematocrit (HCT) of 20 and animals were maintained
at this HCT during the course of the experiment by periodic phlebotomies. Baboons
were subjected to ketamine/midazolam (10 mg/kg; 3-5 mg/kg) anesthesia to allow
removal of sufficient volumes of blood (15% of body weight) to induce and maintain
anemia. RN-1 was dissolved in phosphate-buffered saline and passed through a 0.45
micron filter prior to administration by subcutaneous injection.
All procedures were
approved by the Animal Care Committee of the University of Illinois at Chicago
6
F-cells and F-reticulocytes
Levels of F-cells and F-reticulocytes were analyzed by flow cytometry using a
Cytomics FC500 (Beckman Coulter) after staining with thiazole orange and PEconjugated anti-HbF (BD Bioscience).
HbF and globin chain synthesis
HbF levels in peripheral blood were determined by alkali denaturation.34
Measurement of globin chain synthesis in peripheral blood reticulocytes was performed
by biosynthetic radiolabeling of globin chains in the presence of [3H] leucine.35
For
non-anemic animals, a fraction enriched in reticulocytes was obtained by centrifugation
of 7 ml whole blood on Percoll step gradients as previously described.36 Globin chain
separation was achieved by high-performance liquid chromatography (HPLC) as
previously described (Supplemental Methods).37
RNA analysis
Real time PCR assays were performed as previously described38 to determine
levels of globin transcripts (Supplemental Methods).
Bisulfite Sequence Analysis
For bisulfite sequence analysis, DNA was purified from washed cell pellets
using QIAmp Blood DNA Minikits. Bisulfite modification was performed using Epitect
Bisulfite kits (Qiagen #59104) according to the instructions of the manufacturer.
7
Amplification of bisulfite modified DNA was performed as previously described
(Supplemental Methods).35
Chromatin Immunoprecipitation Analysis
Chromatin immunoprecipitation analysis was performed as previously
described39 with modifications (Supplemental Methods).
Statistics
Student's t-test was used for all the statistical analyses. Error bars represent
standard deviation.
RESULTS
Effect of RN-1 in anemic baboons
A series of dose-response experiments were conducted in anemic baboons to
investigate the effect of RN-1 on HbF levels, F cells, and F reticulocytes.
Anemic
baboons were treated with five different doses (0.125, 0.20, 0.25, 0.50, and 2.5
mg/kg/d) of RN-1 administered subcutaneously for 4-5d (Exp 1-5, Supplemental Table
2). The highest dose of RN-1 (2.5 mg/kg) increased HbF from 5.8% on the first day of
drug treatment to 27.3% 7d after the last injection (Exp 1, Supplemental Table 2; Figure
2). Peak γ-globin chain synthesis (0.78 γ/γ+β) was observed 3d following the last day of
drug administration demonstrating a near-complete reversion to fetal-stage synthesis
(Figure 3). At this time the ratio of chain synthesis of the 5’ Iγ gene and the 3’ Vγ gene
(Iγ/Vγ ratio) was >2, characteristic of the fetal stage.
F-reticulocytes increased from
34.6 to 92.3% while F-cells increased from 28.5 to 59.2% (Supplemental Table 2;
8
Figure 4). Neutropenia was observed with an absolute neutrophil count (ANC) nadir of
290/μL 10d after the last RN-1 dose (Supplemental Table 2; Figure 5). A second animal
(PA8549) was treated with a reduced dose of RN-1 (0.5 mg/kg/d).
The HbF level
increased from 2.4% on the first day of drug treatment to 29.4% 6d after the last
injection (Exp 2, Supplemental Table 2; Figure 2). Peak γ-globin chain synthesis (0.68
γ/γ+β) was attained 3 days after the last RN-1 treatment (Figure 3). The Iγ/Vγ synthesis
ratio at this time was >2, characteristic of fetal-stage synthesis. F cells increased from
19.1 to 60.8% (Supplemental Table 2; Figure 4) and F retics from 33.9 to 97.5%
(Supplemental Table 2; Figure 4).
Neutropenia was observed with an ANC nadir of
570/μL 10d after the last RN-1 dose (Supplemental Table 2; Figure 5). A third animal
(PA8000) was treated with a reduced RN-1 dose (0.25 mg/kg/d). The HbF increased
from 4.1% on the first day of treatment to 20.6% 6 days after the last injection (Exp 3,
Supplemental Table 2; Figure 2). Peak γ-globin chain synthesis (γ/γ+β) was 0.49 and
the Iγ/Vγ chain ratio was 1.18 (Figure 3). F-cells increased from 20.3% to 47.0% and Freticulocytes from 45.7% to 80.7% (Supplemental Table 2; Figure 4). The decrease in
ANC (nadir=870/μL) was less severe than at the higher doses (Supplemental Table 2;
Figure 5).
No changes in total hemoglobin production (α/γ+β=1.06+0.02) were
observed in these three animals treated with the highest doses of RN-1. In a fourth
experiment PA8548 was re-treated with a reduced RN-1 dose (0.2 mg/kg/d).
HbF
increased from 3.7% on the first day of treatment to 20.5% 6 days post-treatment (Exp
4, Supplemental Table 2; Figure 2). F-cells increased from 36.9% to 54.8% and Freticulocytes from 36.9% to 89.2% (Supplemental Table 2; Figure 4). Peak γ-globin
9
chain synthesis (γ/γ+β) was 0.52 (Supplemental Table 2).
Neutropenia (ANC
nadir=800/μL) was observed (Supplemental Table 2; Figure 5).
Another animal
(PA8001) was treated with a reduced RN-1 dose (0.125 mg/kg/d) for 5d. HbF increased
from 1.7% pre-treatment to 4.8% post-treatment, F cells increased from 13.5 to 21.8%
and F-reticulocytes from 22.9 to 31.7% (Supplemental Table 2; Figure 4). Neutropenia
was not observed in this animal (Supplemental Table 2; Figure 4).
These experiments defined a linear dose relationship between HbF, F cells, and
F retics and RN-1 dose within the 0.125 to 0.5 mg/kg range (Figure 4). Dose-related
decreases in ANC were also observed (Figure 5 and Supplemental Figure 1). Dosedependent decreases in platelets 8-9 days following the initial RN-1 treatment were
followed by thrombocytosis peaking 15-20d after the initial dose (Figure 5 and
Supplemental Figure 1). In addition, dose-dependent increases in monocytes were
observed that peaked 15-17d following the initial drug dose (Figure 5 and Supplemental
Figure 1). Increases in MCV were observed at the higher RN-1 doses coinciding with
the day of maximal HbF levels (Supplemental Figure 2A) and MCHC levels were
decreased at all doses (Supplemental Figure 2B). Significant increases in HbF were
observed at lower doses of RN-1 that were not associated with large increases in MCV
(Supplemental Figure 2C).
In an effort to achieve significant increases in HbF in the absence of neutropenia,
PA8549 was re-treated with a dose of 0.125 mg/kg for 10 days (Exp 6, Supplemental
Table 2). The HbF increased from 3.6% on the first day of treatment to 16% 5 days
after the last injection. F-cells increased from 17.6% to 42.7% and F-reticulocytes from
10
21% to 70%. Peak γ-globin chain synthesis (γ/γ+β) was 0.22.
Decreases in ANC were
still observed however, with an ANC nadir of 870/μL. Two other experiments were
performed to test the effects of a 2d per week dose schedule. When PA8000 was
treated with 0.25 mg/kg (Exp 7, Supplemental Table 2), HbF increased from 4.7 to
10.4%, F cells from 18.2 to 28.1%, and F reticulocytes from 29.2 to 52.2% associated
with an ANC nadir of 2360/μL, indicating that significant increases in HbF could be
attained with this schedule and dose in the absence of neutrophil toxicity. When the
dose was increased to 0.5 mg/kg at the 2d per week dose schedule in another animal
(PA8549) HbF increased from 6.2 to 22.1%, F cells from 22.2 to 53.6%, and F
reticulocytes from 24.1 to 86.4%, however an ANC nadir of 570/μL was observed (Exp
8, Supplemental Table 2).
The effect of RN-1 on globin mRNAs was determined in pre-and post-treatment
peripheral blood samples in three animals treated with different doses (Supplemental
Table 3). RN-1 treatment increased the level of γ-globin mRNA nearly fivefold (p<0.05)
in post-treatment samples (0.39+0.12 γ/ε+γ+β) compared to pre-treatment samples
(0.08+0.01 γ/ε+γ+β) while levels of ε-globin mRNA remained low.
Effect of RN-1 on histone modifications
To investigate the mechanism of action of RN-1, chromatin immunoprecipitation
(ChIP) assays were performed to determine the effect of the drug on histone acetylation
and methylation associated with the ε-, γ-, and β-globin genes. For these experiments,
anemic baboons (n=4) were treated for 2d with RN-1 (0.5 or 0.25 mg/kg/d).
11
Bone
marrow (BM) erythroid cells were purified by immunomagnetic column selection of bone
marrow samples obtained pre-treatment from bled baboons and post-treatment 48-72
hours after the last dose of drug.
Levels of pol II, histone H3K4Me2, H3K4Me3,
H3K9Ac, and H3K9Me2 associated with the ε-, γ-, and β-globin promoter and IVS II
regions were measured by real time PCR in DNA samples obtained from formaldehyde
fixed, immunoprecipitated chromatin of purified BM erythroid cells (Figure 6A).
Increased levels of pol II associated with the γ-globin promoter (4.7 fold; p<0.02) and γglobin IVS II region (3.2 fold; p<0.05) were observed. Levels of H3K9Ac were also
increased at the γ-globin promoter (6.2 fold; p<0.01) and γ-globin IVS II regions (4.2
fold; p<0.02).
Histone H3K4Me2 levels were increased at the γ-globin IVS II (2.1 fold;
p<0.05) but not at the γ-globin promoter while H3K4Me3 levels were increased at both
the γ-globin promoter (4.7 fold; p<0.02) and at the γ-globin IVS II region (4.6 fold;
p<0.02). No significant differences in the levels of these modifications at the ε- and βglobin promoter and IVS regions were observed between pre- and post-treatment
samples. Levels of Histone H3K9Me2 were increased at the β-globin promoter (2.8
fold; p<0.02) and IVS II region (5.3 fold; p<0.01) in post-treatment samples.
Effect of RN-1 on DNA methylation
Bisulfite sequence analysis of the baboon 5’ γ-globin promoter region in DNA
isolated from erythroid precursors purified from BM aspirates obtained pre-and posttreatment from 7 baboons treated with varying doses of RN1 (0.125 to 0.5 mg/kg/d) was
performed to investigate whether increased γ-globin expression in RN-1 treated
baboons was associated with loss of DNA methylation. The level of DNA methylation of
12
five CpG residues within this region located at -54, -51, +5, +16 and +48 with respect to
the transcription start site in purified erythroid precursors of post-treatment BM samples
(0.80+0.05% dmC) was reduced compared to pre-treatment samples (0.73+0.09%
dmC; p=0.053). CpG residues at -51 and +16 are polymorphic in the baboon with G to
A transitions eliminating the CpG site. Further analysis of only the 3 non-polymorphic
CpG sites within the 5’ γ-globin promoter region at -54, +5, and -48 confirmed a small
but significant (p<0.05) decrease in the level of DNA methylation in post-treatment
(0.79+0.05% dmC) compared to pre-treatment samples (0.71+0.09% dmC; p<0.05;
Figure 6B). Measurement of globin chain synthesis in peripheral blood reticulocytes in
6 of these baboons showed increased γ-globin synthesis in post-treatment (0.032+0.15
γ/γ+β) compared to pre-treatment samples (0.09+0.04 γ/γ+β; p<0.01; Figure 6B).
Effect of RN-1 in Non-anemic Baboons
To investigate whether the effect of RN-1 on γ-globin expression required
erythroid expansion, additional experiments were conducted in normal, non-anemic
baboons. In the first experiment treatment of PA8549 with RN-1 (0.5 mg/kg/d; 5d)
increased HbF synthesis in peripheral blood reticulocytes from 0.03 γ/γ+β pre-treatment
to 0.71 post-treatment (Exp 9, Supplemental Table 4; Figure 7). Neutropenia was not
observed.
In the second experiment a reduced dose of RN-1 (0.25 mg/kg/d; 5d)
administered to PA8549 increased γ-globin synthesis in peripheral blood reticulocytes
from 0.015 γ/γ+β pre-treatment to 0.26 post-treatment (Exp 10, Supplemental Table 4;
Figure 7) while again neutropenia was not observed. In the third experiment this same
RN-1 dose (0.25 mg/kg/d; 5d/wk) given for 2 weeks to PA8698 increased γ-globin
13
synthesis to 0.24 γ/γ+β (Exp 11, Supplemental Table 4; Figure 7). F cells increased > 4
fold from 2.0% pre-treatment to 9.2% post-treatment. A transient decrease in ANC to
<1500/μL (1210) was observed.
To investigate the effect of a longer term treatment
schedule, PA8696 was treated with RN-1 (0.2-0.25 mg/kg/d) for a period of 10 weeks
(Exp 12, Supplemental Table 4) while two other animals (PA8695 and P8698, Exp 13
and 14, Supplemental Table 4) were treated with 0.25mg/kg/d RN-1 for 6 weeks.
Increased F cells were observed in all animals and ranged from 5 to 8 fold
(Supplemental Table 4: Figure 8).
HbF levels increased 3-4 fold in all animals
(Supplemental Table 4, Figure 8). Within the F cell populations of the three baboons
the HbF levels rose >3 fold (Supplemental Figure 3).
Importantly, these increases in
HbF levels and F cells were associated with minimal neutropenia that resolved quickly
(Supplemental Table 4; Supplemental Figure 4).
In all three non-anemic animals
treated for either 10 or 6 weeks increases in monocytes and decreases in platelets were
also observed (Supplemental Figure 4).
Treatment of these non-anemic animals was
not associated with changes in MCV or MCHC (Supplemental Figure 5).
Discussion
Treatment of baboons with the LSD-1 inhibitor RN-1 substantially increased HbF,
F cells and γ-globin synthesis in peripheral blood reticulocytes. The results confirm and
extend our previous studies in the sickle cell mouse model showing that RN-1 increased
F cells, F reticulocytes, and γ-globin mRNA to levels similar to decitabine, the most
potent HbF-inducer known32,33. Both these drugs elicit only small increases of γ-globin
expression in the SCD mouse model compared to the magnitude observed in baboons
14
however, emphasizing the importance of using nonhuman primates such as baboons to
test the effect of HbF-inducing drugs.
Comparison of the level of γ-globin synthesis in RN-1-treated baboons with
previous results from our laboratory of baboons treated with DNMT inhibitors 3-5,12,38,40-42
shows that the HbF-inducing activity of RN-1 in the baboon model is comparable to
DNMT inhibitors.
In addition, RN-1 treatment of baboons resulted in the complete
reversion of the pattern of synthesis of the individual 5’ Iγ- and 3’ Vγ-globin genes (Iγ/Vγ
ratio) to that characteristic of fetal development in anemic baboons, an effect only
partially achieved by DNMT1 inhibitors.4 The preferential effect of RN-1 on expression
of the 5’ Iγ-globin gene was even greater in the non-anemic animals. This effect has not
been previously observed and appears to be a property unique to RN-1. The induction
of HbF in the absence of changes in total β-like globin production (α/β ratio) and MCV in
the treated animals suggests that increased γ-globin expression results from a direct
effect of the drug on the repressive chromatin environment of the γ-globin gene,
consistent with previous mechanistic studies demonstrating a critical role for LSD-1 in
mediating γ-globin repression31, although effects elicited through alteration of erythroid
differentiation kinetics cannot be excluded.
Further experiments will be required to
determine the mechanism of action of RN-1 in vivo. ChIP experiments showing RN-1
increased levels of H3K4Me2, H3K4Me3 and H3K9ac associated with the γ-globin but
not the ε-globin gene suggest a specific effect of the drug on γ-globin expression and
are consistent with previous studies in AML cell lines showing that LSD1 inhibition did
not induce genome-wide changes in H3K4Me2 but rather acted only at a specific subset
15
of genes.43 The lack of effect of RN-1 on ε-globin expression in the baboon contrasts
with increases in Εy and βh1 observed in the SCD mouse model. It is possible that this
difference is dose-related and that the ε-globin gene is less sensitive to de-repression
by RN-1 because the ε-globin promoter contains two DR elements that bind DRED with
higher affinity than the single DR1 element in the γ-globin promoter.22-24
LSD1 demethylates other proteins including DNMT1.
Demethylation of DNMT1
by LSD1 increases DNMT1 stability while targeted deletion of LSD1 in results in loss of
DNMT1 and DNA demethylation in ES cells.44 In this regard, we observed small effects
on γ-globin promoter DNA methylation in BM erythroid cells from RN-1-treated baboons
that were significantly less than changes previously observed in decitabine-treated
baboons.
DNA methylation changes are therefore not likely to be the major
mechanism responsible for increased HbF levels in RN-1 treated baboons, although
these small changes in DNA methylation could have contributed, in part, to the
magnitude of the HbF response.
Because RN-1 is not covalently incorporated into DNA, as is decitabine and 5azacytidine, it is potentially a less genotoxic and safer drug. The clinical usefulness of
RN-1 will likely depend on the design of a dose and schedule that limit its undesirable
effects on other hematopoietic lineages.
The anemic baboon has been traditionally
used and was initially developed to model the anemia and reticulocytosis observed in
SCD.12 In this model, anemia produced by repeated phlebotomies results in a nearcomplete replacement of the erythrocyte population within a two week period, removal
of all formed elements, and alterations in levels of multiple cytokines in addition to
16
erythropoietin.45 To complement studies in anemic baboons, experiments were also
performed in non-anemic animals where baseline hematopoiesis was not perturbed.
The neutropenia observed in phlebotomized baboons was lessened in non-anemic
animals where ANC>1500 were generally maintained. The greater decreases in ANC in
anemic animals may due to depletion of neutrophils by repeated phlebotomies or the
effect of these phlebotomies on growth factor levels.
Decreased platelets and
increased monocyte counts were observed in both the anemic and non-anemic animals
and likely are due to effects of the drug on hematopoietic differentiation. In an LSD1
conditional knockdown mouse model expansion of granulocytic, erythroid, and
megakaryocytic progenitors was observed while granulopoiesis, erythropoiesis, and
platelet production was inhibited suggesting that the similar effects observed in RN-1treated baboons are due to inhibition of LSD1 activity.30 RN-1 has been reported to
affect long term memory in mice and therefore this may be an additional possible
adverse effect of the drug.46
Our results show that the LSD1 inhibitor RN-1 induces high levels of HbF in
baboons and are consistent with previous studies showing that LSD1 plays a critical
role in γ-globin silencing as a component of the DRED complex25, 31. A possible role for
perturbation of erythroid differentiation in the mechanism of HbF reactivation cannot be
negated, however, and the in vivo mechanism of action will be investigated in future
studies. The level of the HbF response varies between different baboon species,47 and
therefore the HbF response in man and baboons may differ somewhat due species
differences and/or physiological differences between the experimental animal model
17
and patients with hemoglobinopathies. However, results in P. anubis are generally
predictive of HbF-inducing effects in man and have been successfully translated in
clinical studies in SCD and β-thalassemia patients.13-18 Therefore we suggest that RN-1
and/or other LSD1 inhibitors are excellent candidates for clinical evaluation as
therapeutic agents in β-thalassemia and sickle cell disease.
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25
Figure Legends
Figure 1: Amino acid substitutions in the baboon and human duplicated γ-globin
genes and the difference in their expression at birth and in adults.
Figure 2. Effect of RN-1 on Hemoglobin F in anemic baboons. Three individual
anemic baboons were treated with varying doses of RN-1 as indicated by subcutaneous
injection (arrows correspond to treatment days). HbF levels were determined by alkali
denaturation on the indicated days.
Figure 3.Effect of RN-1 on globin chain synthesis in anemic baboons. Globin
chain synthesis in reticulocytes of three baboons pre- and post-treatment with varying
doses of RN-1 was measured by biosynthetic radiolabeling with [3H] leucine followed by
HPLC separation of globin chains.
Figure 4. Effect of RN-1 on HbF, F cells, and F retics. Five baboons were treated
with varying doses of RN-1 (0.125-2.5 mg/kg/d for five days (Days 1-5). Left Panels:
Changes in HbF, F cells, and reticulocytes in treated baboons. Right Panels:
Relationship between the change in HbF, F cells, and F reticulocytes and RN-1 dose
(Maximal value post-treatment- pre-treatment value).
Figure 5. Effect of RN-1 on hematological parameters. Five baboons were treated
with varying doses of RN-1 (0.125-2.5 mg/kg/d for five days (Days 1-5). Changes in
neutrophils (ANC), monocytes, platelets (plt), and reticulocytes are shown.
26
Figure 6. Effect of RN-1 on histone modifications and γ-globin promoter DNA
methylation. A) ChIP analysis of levels of pol II, Histone H3K4Me2, H3K4Me3,
H3K9Ac, and H3K9Me2 associated with the ε-, γ-, and β-globin promoters and IVS
regions in purified BM erythroid cells from RN-1 treated baboon pre-and post-treatment.
Anemic baboons were treated with 0.5mg/kg/d RN-1 for two days and BM aspirates
were obtained 48 hours following the last RN-1 treatment. Asterisks (*) designate
significant differences (p<0.05) between pre and post-treatment samples. (B) Left
Panel: Levels of cytosine methylation at three non-polymorphic CPG residues in the 5’
γ-globin promoter region in purified BM erythroid cells from RN-1 treated baboons preand post-treatment. Paired pre- and post-treatment samples from individual baboons
are shown. The horizontal bar shows the mean % dmC value of all samples. Anemic
baboons were treated with either 0.5mg/kg/d (filled triangles, filled squares, filled circles,
filled diamonds, lined squares) or 0.25 mg/kg/d (open triangles, open circles). Right
Panel: Changes in γ-globin synthesis in peripheral blood reticulocytes of baboons preand post-treatment. Paired pre- and post-treatment samples from each individual are
shown. The horizontal bar shows the mean level of γ-globin synthesis (γ/γ+β) of all
samples.
Figure 7. Effect of RN-1 in on globin chain synthesis in non-anemic baboons.
HPLC analysis of globin chain synthesis in peripheral blood reticulocytes of normal,
non-anemic baboons treated with varying doses of RN-1 pre-treatment and posttreatment. Top Panel: Effect of RN-1 on globin chain synthesis in peripheral blood
reticulocytes of PA8549. This baboon was treated in two separate experiments for five
27
days with RN-1( 0.5 mg/kg/d or 0.25 mg/kg/d). Pre-treatment and post-treatment values
at each dose are shown. Bottom Panel: Effect of RN-1 on globin chain synthesis of
peripheral blood reticulocytes of PA8698. The baboon was treated with RN-1 (0.25
mg/kg/d; 10d). Analysis of chain synthesis pre-treatment and following 5 days and 10
days of treatment are shown.
Figure 8. Effect of RN-1 on HbF and F cells in non-anemic baboons treated with
RN-1. (A) Effect of RN-1 on HbF (left panel) and F cells (right panel) in three nonanemic baboons. PA8696 was treated for 10 weeks (0.2-0.25 mg/kg/d; 5d/wk; sc).
PA8698 and PA8695 were treated for 6 weeks (0.25 mg/kg/d; 5d/week/ sc). (B) Flow
cytometric analysis of F cells and F retics on d1 and d70 following RN-1 treatment in a
non-anemic baboon (PA8696).
28
Supplemental Information
I. Supplemental Methods
Supplemental Table 1. Primer and Probe Sequences
II. Supplemental Data
Supplemental Table 2. Experiments in Anemic Animals.
Supplemental Table 3. Effect on RN-1 on Globin mRNA
Supplemental Table 4. Experiments in Non-Anemic Animals.
Supplemental Figure 1. Changes in ANC, monocytes, and platelets at varying
doses of RN-1 in anemic baboons.
Supplemental Figure 2. (A) Changes in MCV of anemic baboons treated with
varying doses of RN-1. (B) Changes in MCHC in baboons treated with varying
doses of RN-1. Animals were treated on days 1-5. (C) Relationship between HbF
levels and changes in MCV in anemic baboons treated with varying doses of RN-1
Supplemental Figure 3. Difference in median fluorescence (PE-anti-HbF) in F cells
of three non-anemic baboons pre-treatment and following treatment with RN-1 for
either 10 weeks (n=1) or 6 weeks (n=2; mean + SD).
Supplemental Figure 4. Changes in ANC, platelets, monocytes, absolute
reticulocyte count, and WBC during treatment of a normal, non-anemic baboon with
RN-1. Animals were treated 5d/wk starting on d1.
Supplemental Figure 5. MCV and MCHC levels of three non-anemic baboons
treated with RN-1 for 10 weeks (PA 8696) or 6 weeks (PA8695, PA8698) on varying
days.
Supplemental Methods
Globin Chain Synthesis
Peripheral blood (20 µL) from anemic animals was washed three times in PBS
and incubated overnight at 37°C in 0.2 mL leucine-free α-minimum essential medium
(MEM; Invitrogen, Carlsbad, CA, USA) containing 20% dialyzed fetal bovine serum,
transferrin, ferric ammonium citrate, and 50 µCi/mL L-[4,5-3H] leucine (Perkin Elmer).
For non-anemic animals, a fraction enriched in reticulocytes was obtained by
centrifugation of 7 ml whole blood on Percoll step gradients as previously described.36
The reticulocyte rich fraction was centrifuged (500 g; 10 min) and washed three times
with PBS.
The reticulocyte-rich pellet radiolabeled with [3H] leucine in leucine-free
media as described above. Following radiolabeling, cells were washed three times in
PBS (500 g; 10 min).
Pellets were suspended in H 2 O and cells lysed by multiple
freeze-thaw cycles in dry ice-methanol baths.
Lysates were filtered using 0.4 mm
cellulose acetate syringe filter (Nalgene) and globin chain separation was achieved by
high-performance liquid chromatography (HPLC) using a LithoCART 250-4 column
(VWR) and a Spectra System HPLC (Thermo-Finnegan) with acetonitrile-methanol
gradients.37 Quantitation of radioactivity in collected fractions was determined by liquid
scintillation counting using a Packard Tricarb 1600TR liquid scintillation analyzer.
Results are expressed as γ/γ+β chain ratios.
RNA analysis
RNA was isolated using the RNeasy Minikit (Qiagen #74104) and treated with
RNase-free DNase I (Ambion #AM1906) according to the manufacturer’s instructions.
Purified RNA was used for the synthesis of cDNA using RevertAid First Strand CDNA
Synthesis Kits (Thermo Scientific #K1622). Real time PCR analysis was conducted
using custom designed primer-probe combinations were used for analysis of globin
transcripts (Supplemental Table 1).
Absolute numbers of globin transcripts were
determined by extrapolation from standard curves prepared from the cloned
amplicons.38
Bisulfite Sequence Analysis
For bisulfite sequence analysis, DNA was purified from washed cell pellets
using QIAmp Blood DNA Minikits. Bisulfite modification was performed using Epitect
Bisulfite kits (Qiagen #59104) according to the instructions of the manufacturer.
Amplification of bisulfite modified DNA was performed as previously described. 35with
using the BG2 5’- AATAACCTTATCCTCCTCTATAAAATAACC and BG5 5’GGTTGGTTAGTTTTGTTTTGATTAATAG primers.35 Amplicons from three separate
PCR reactions were pooled to reduce PCR bias and the amplicons cloned using Topo
TA cloning kits (Life Technologies #K4575-01SC). DNA was isolated from individual
clones and sequence analysis performed at the University of Illinois DNA Sequence
Laboratory.
Chromatin Immunoprecipitation Analysis
Chromatin immunoprecipitation analysis was performed as previously
described39 with modifications. Fixation was conducted by incubation of cells in
Iscove’s media containing 1% fetal bovine serum and 1% formaldehyde (methanol-free,
Thermo Scientific #28906) for 10 minutes at room temperature. The fixation reaction
was stopped by the addition of 0.125 M glycine followed by a second incubation at room
temperature for 5 minutes. Cells were lysed in 200 µL cell lysis buffer. Chromatin
shearing was performed using a Diagenode Bioruptor 300 sonicator (20 cycles; Power =
High). Sheared chromatin (400-600bp) was diluted 1:5 in IP dilution buffer and precleared overnight with normal rabbit serum. An equivalent of 10 µg chromatin lysate
was used for each IP condition. Anti-Histone H3K4Me2 (07-030), anti-H3K4Me3 (17614), anti-H3K9Me2 (17-648) and anti-H3K9ac (17-658) were obtained from Millipore.
Anti-pol II (sc-899x) was obtained from Santa Cruz Biotech. Immunoprecipitation was
performed overnight at 4oC followed by incubation with RNase A followed by a second
incubation with Proteinase K. DNA from the chromatin immunoprecipitates was
purified using ChIP DNA and Concentrators (Zymo Research). Quantitative real-time
PCR of triplicate samples was performed using primers specific for the necdin, ε-, γ-,
and β-globin promoters and ε-, γ-, and β-globin IVSII regions (Supplemental Table 1)
with SBYR Green reagent using a 7500 Real Time PCR instrument (Applied
Biosystems).5
Standard curves constructed for each primer set using dilutions of input
DNA were used to determine the relative amount of specific sequences recovered in
samples following immunoprecipitation. Results were expressed as the fraction of
input.
Supplemental Table 1. Primer and Probe Sequences
RT-PCR primer-probes
Gene
Forward Primer
Reverse Primer
Probe
ε-globin
AACTTCAAGCTCCTGGGTAACG GGGTGAACTCCCTGCCAAA
ATGGTGATTATTCTGGCTACT
γ-globin
CGGCAAGAAGGTGCTCACTT
GCCCTTGAGATCATCCAGGTT
CTTGGGAGATGCCG
β-globin
GCTGGTGGTCTACCCTTGGA
AGGAGAGGACAGATCCCCAAA CCAGAGGTTCTTTGATTC
ChIP Primers
Gene Location
Forward Primer
Reverse Primer
Necdin
GAGCTCCTGGACGCAGAGG
GCAAAGTTAGGGTCGCTCAGA
ε-globin promoter
TCAGCC TTGACCAATGACTTTTAA
GTTCTGGCCCCCTGTTCTC
ε-globin IVS II
CAGAAATCATGGGTCGAGTTTG
TCAGGTTAGTCTTGTTATGCTCACT
γ-globin promoter
AGGGATGAAGAATAAAAGGAAGCA
CAGAAGCGAGTGTGTGGAACTG
γ-globin IVS II
GAGGGCAAAATGTCAGGCTTT
CCCAGCTTCCACCCAGAAT
β-globin promoter
GGGCTGAGGGTTTGAAGTCC
CCTTGGCTCTTCTGGCACTG
β-globin IVS II
TCCCCTTCTTTTCTATGATTAACTTCA TTGTCTCTTCCCCATTCTAAACTGT
Supplemental Table 2. Experiments in Anemic Animals.
Exp Animal
Dose
Schedule
mg/kg/d
ANC
Plt
Mono
Abs Retics
F cells
F retics
(/µL)
(X 103/µl)
(/µL)
(X 109/L)
(%)
(%)
HbF
synthesis
HbF
(%)
(γ/γ+β)
Pre
Nadir
Pre
Nadir
Peak
Pre
Peak
Pre
Nadir
Pre
Peak
Pre
Peak
Pre
Peak
Pre
Peak
1
8548
2.5
4d
3020
290
750
130
1350
84
483
323
209
28.5
59.2
34.6
92.3
ND
0.78
5.8
27.3
2
8549
0.5
5d
3160
570
772
372
1445
95
380
526
209
19.1
60.8
33.9
97.5
0.06
0.68
2.4
29.4
3
8000
0.25
5d
3620
870
353
122
362
322
532
467
249
20.3
47
45.7
80.7
0.15
0.49
4.1
20.6
4
8548
0.2
5d
3870
800
772
514
1092
110
204
280
51
36.9
54.8
36.9
89.2
0.04
0.52
3.7
20.5
5
8001
0.125
5d
11120
3242
476
400
596
243
483
253
244
13.5
21.8
22.9
31.7
ND
0.06
1.7
4.8
6
8549
0.125
10d
3150
870
804
663
1108
120
195
390
242
17.6
42.7
21
70
0.04
0.22
3.6
16
7
8000
0.25
2d/wk
2580
2360
372
217
391
262
591
265
138
18.2
28.1
29.2
52.2
0.11
0.46
4.7
10.4
3830
570
896
1165
1165
98
198
360
200
22.2
53.6
24.1
86.4
0.13
0.51
6.2
22.1
4 wks
8
8548
0.5
2d/wk
4 wks
Supplemental Table 3. Effect on RN-1 on Globin mRNA
Exp
Dose
γ-globin mRNA
ε-globin mRNA
(mg/kg/d)
(γ/ε+γ+β)
(γ/ε+γ+β)
Animal
2
8549
0.5
0.07
0.52
0.007
0.016
4
8548
0.2
0.07
0.35
0.005
0.011
6
8549
0.125
0.09
0.30
0.002
0.003
Mean
0.08+0.01 0.39+0.12 0.004+0.002 0.001+0.008
Supplemental Table 4. Experiments in Non-Anemic Animals.
Exp
Animal
Dose
Schedule
mg/kg/d
ANC
Plt
Mono
Abs Retics
(/µL)
(X 10 /µL)
3
(/µL)
(X 10 /L
F cells
F retics
(%)
(%)
9
HbF
synthesis
HbF
(%)
(γ/γ+β)
Pre
Nadir
Pre
Nadir
Peak
Pre
Peak
Pre
Peak
Pre
Peak
Pre
Peak
Pre
Peak
Pre
Peak
9
8549
0.5
5d
7130
1910
394
169
1244
175
1269
39.7
105
ND
ND
ND
ND
0.03
0.71
1.2
2.1
10
8549
0.25
5d
3350
1910
377
219
839
236
720
53.6
135
ND
ND
ND
ND
0.02
0.26
1.6
2.6
11
8698
0.25
5d/wk
4190
1210
249
68
1340
151
719
30.5
105
2.0
9.2
ND
ND
0.01
0.24
0.6
2.3
3720
1440
328
64
288
106
378
35.8
112
3.5
29.0
2.4
74.4
ND
0.16
1.5
4.8
2110
1610
351
198
302
100
293
29.3
48.7
3.1
17.4
4.8
76.6
ND
ND
1.2
4.1
2690
1250
224
90
187
161
460
25.4
43.6
2.0
17.3
15.8
75.6
ND
ND
1.0
4.3
2 wks
12
8696
0.2-0.25
5d/wk
10wks
13
8695
0.25
5d/wk
6wks
14
8698
0.25
5d/wk
6wks
Supplemental Figure 1
D Plt at nadir
( x 109 /L)
0
-100
R² = 0.746
-200
-300
-400
0
0.1
0.2
0.3
0.4
RN-1 (mg/kg/d)
0.5
0.6
Supplemental Figure 2
A.
MCV
105
fL
100
95
2.5 mg/kg/d
90
0.5 mg/kg/d
85
0.25 mg/kg/d
80
0.2 mg/kg/d
75
0.125 mg/kg/d
0
5
10
15
20
DAY
B.
pg/cell
MCHC
35
30
25
20
15
10
5
0
2.5 mg/kg/d
0.5 mg/kg/d
0.25 mg/kg/d
0.2 mg/kg/d
0.125 mg/kg/d
0
5
10
15
20
DAY
C.
35
R² = 0.6753
30
HbF (%)
25
20
15
10
5
0
0
2
4
6
8
MCV (% change)
10
12
Supplemental Figure 3
p<0.02
Supplemental Figure 4
Plt
7000
6000
5000
4000
3000
2000
1000
0
400
300
PA8698
PA8695
( X 109/L)
(/mL)
ANC
PA8698
200
PA8695
100
PA8696
PA8698
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
DAY
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
DAY
Monocytes
120
500
100
400
(/mL)
80
PA8698
60
40
PA8695
20
PA8696
300
PA8698
200
PA8695
100
PA8696
0
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
DAY
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
DAY
WBC
10
8
( X 109/L)
( x 109/L)
Retics
6
PA8698
4
PA8695
2
PA8696
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
DAY
Supplemental Figure 5
PA8696
MCV (fL)
MCHC (g/dL)
100
80
60
MCV
40
MCHC
20
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
DAY
PA8695
MCV (fL)
MCHC (g/dL)
100
80
60
MCV
40
MCHC
20
0
0
5
10
15
20 25
DAY
30
35
40
PA8698
MCV (fL)
MCHC (g/dL)
100
80
60
MCV
40
MCHC
20
0
0
5
10
15
20 25
DAY
30
35
40