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Blood First Edition Paper, prepublished online April 22, 2015; DOI 10.1182/blood-2014-11-610907
Overexpression of the Shortest Isoform of Histone Demethylase LSD1 Primes
Hematopoietic Stem/Progenitor Cells for Malignant Transformation
Taeko Wada1, Daisuke Koyama1, Jiro Kikuchi1, Hiroaki Honda2, and Yusuke Furukawa1*
1
Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University,
Shimotsuke, Tochigi 329-0498; 2Department of Disease Model, Research Institute for Radiation
Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan.
Running head: The fundamental role of LSD1 in leukemogenesis
*Correspondence: Yusuke Furukawa, M.D., Division of Stem Cell Regulation, Center for
Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498,
Japan.
E-mail: [email protected]; Tel.: +81-285-58-7400; Fax: +81-285-44-7501
Microarray data have been deposited in the MIAME-compliant GEO database under accession
number GSE66074.
Key points
LSD1 is barely expressed in normal hematopoietic stem cells, but is overexpressed in
leukemias especially those of a T-cell origin.
LSD1 overexpression forms pre-leukemic stem cells with an increased self-renewal potential
in a transgenic mice model.
1
Copyright © 2015 American Society of Hematology
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Abstract
Recent investigations indicate that epigenetic regulators act at the initial step of myeloid
leukemogenesis by forming pre-leukemic hematopoietic stem cells (HSCs), which possess the
increased self-renewal potential but retain multi-differentiation ability, and synergize with genetic
abnormalities in later stages to develop full-blown acute myeloid leukemias.
unknown whether this theory is applicable to other malignancies.
However, it is still
In this study, we demonstrate
that LSD1 overexpression is a founder abnormality for the development of T-cell lymphoblastic
leukemia/lymphoma (T-LBL) using LSD1 transgenic mice.
LSD1 expression is tightly
regulated via alternative splicing and transcriptional repression in HSCs, and is altered in most
leukemias especially T-LBL.
Overexpression of the shortest isoform of LSD1, which is
specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than
other isoforms, increases self-renewal potential via up-regulation of the HoxA family but retains
multi-differentiation ability with a skewed differentiation to T-cell lineages at transcriptome
levels in HSCs.
Transgenic mice overexpressing LSD1 in HSCs did not show obvious
abnormalities but developed T-LBL at very high frequency after γ-irradiation.
LSD1
overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into
novel strategies for early diagnosis and effective treatment of the disease.
Keywords: LSD1; T-cell lymphoblastic leukemia/lymphoma; pre-leukemic stem cell; transgenic
mouse; two-hit theory
2
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Introduction
Epigenetic modifications have been implicated in various biological processes of considerable
importance, such as cell proliferation and differentiation.1
Recent systematic investigations
revealed that epigenetic deregulation plays fundamental roles in oncogenesis, especially the
development of hematologic malignancies.2,
3
More importantly, epigenetic abnormalities
generally act as initiating mutations, which transform normal stem cells into cancer stem cells at
the initial step of oncogenesis.
For example, recurrent mutations of DNA methyltransferase 3A
(DNMT3A) or isocitrate dehydrogenase (IDH) 1/2 give rise to pre-leukemic hematopoietic stem
cells (HSCs), which possess a higher self-renewal potential than normal counterparts but retain
multi-differentiation capacity.4-6
Additional genetic abnormalities, such as NPM1c mutations
and FLT3-ITD, confer further growth advantages and malignant phenotypes to pre-leukemic
HSCs, leading to the development of full-blown acute myeloid leukemias (AML).
Similar
initiator functions have been demonstrated in other epigenetic regulators, such as the
methylcytosine hydroxylase TET2, histone methyltransferase EZH2 and Polycomb-related
protein ASXL2, in myeloid leukemogenesis.7-10
However, it is still unknown whether this
theory is applicable to other malignancies.
Lysine-specific demethylase 1 (LSD1 also named KDM1A/AOF2/BHC110) is an
FAD-dependent histone demethylase that specifically removes the mono-and di-methyl groups
from lysine-4 and lysine-9 residues of histone H3.11 Hence, LSD1 bifunctionally modulates the
enhancer/promoter functions of target genes in a context-dependent manner.12
LSD1 mediates a
repressive function via H3K4 demethylation as a component of the CoREST and NuRD
complexes13, whereas it activates hormone-regulated genes via H3K9 demethylation when
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associated with androgen or estrogen receptors.14 Moreover, LSD1 has four isoforms with
different histone demethylating activities and affinities to CoREST, offering an additional layer of
functional regulation.15
Recent genetic approaches revealed that LSD1 is indispensable for broad tissue homeostasis
and proper development.16
In the hematopoietic system, Sprüssel et al.17 demonstrated that
shRNA-mediated knockdown of LSD1 impairs maturation of blood cells in association with the
expansion of undifferentiated hematopoietic stem/progenitor cells (HSPCs) and committed
progenitors in mice. On the other hand, LSD1 was shown to be essential for the self-renewal
and proper differentiation of HSCs in conditional LSD1 knockout mice.18 Furthermore, it is of
note that LSD1 expression is elevated in neuroblastoma and bladder cancer19, 20 and is correlated
with a poor prognosis in hormone-dependent prostate and breast cancers.21
From these observations, we hypothesized that the expression levels and function of LSD1
are tightly regulated in HSPCs and their deregulation underlies the development of hematologic
malignancies.
This idea is compatible with the initiating role of epigenetic deregulation in
leukemogenesis, but has not yet been experimentally proven.
In the present study, we
investigated whether the abnormalities of LSD1 cause the malignant transformation of HSPCs by
generating transgenic mice that overexpress an LSD1 isoform in HSPCs under the control of the
Sca-1 promoter.
Methods
Cells
Human bone marrow mononuclear cells and CD34+ progenitors were purchased from Takara Bio
(Shiga, Japan). CD34−/lineage markers (Lin)− and CD34−/Lin+ cells were separated with CD34
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micro beads and lineage cell depletion kits (Miltenyi Biotec, Gladbach, Germany).
CD34+/CD38+ and CD34+/CD38− cells were purified with an anti-human CD38 antibody in the
Easy Sep system (StemCell Technologies, Vancouver, Canada).
handled as described previously.22
Leukemic cell lines were
Primary leukemic cells were isolated at the time of
diagnostic procedure from patients after otaining informed consent in accordance with the
Declaration of Helsinki and the protocol approved by the Institutional Review Board of Jichi
Medical University.
We used specimens containing >90% blasts on morphological and
phenotypic examinations. Fractionation of murine HSCs and HSPCs was performed according
to the protocol of Oguro et al.23
Colony formation and replating assays
Purified Lin–/Sca-1+/c-Kit+ (LSK) and Lin−/Sca-1−/c-Kit+ cells (5 × 105 cells) were plated in
triplicate in methylcellulose M3231 medium (StemCell Technologies) supplemented with stem
cell-oriented cytokines.
The colony number was scored after 7 days, followed by second and
third platings.
Polymerase chain reaction
We performed RT-PCR and real-time quantitative RT-PCR as described previously.15, 24
rearrangement was analyzed by genomic PCR according to Shen et al.25
TCRß
Primer sequences are
listed in Supplemental Table 1.
Plasmids and transfection
Full-length cDNAs for LSD1 transcript variants 1 (BC040194, E2a+/E8a+) and 2 (BC048134,
E2a−/E8a−) were purchased from Open Biosystems (Huntsville, AL).
5
We constructed cDNA for
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LSD1 isoform E2a+/E8a− from variants 1 and 2 as described by Zibetti et al.15
expression
vectors
were
constructed
by
inserting
full-length
cDNAs
Lentiviral
into
a
CSII-CMV-MCS-IRES DsRed vector as reported.26
Immunoblotting
We carried out immunoblotting using specific antibodies against LSD1 (Diagenode, Liege,
Belgium and Cell Signaling Technology, Beverly, MA), G9a, Suv39H1 (Cell Signaling
Technology), PHF8/JHDM1F (Active Motif, Carlsbad, CA), JARID1B (Sigma-Aldrich, St. Louis,
MO), di-methylated H3K4 (Active Motif), di-methylated H3K9 (Abcam, Cambridge, MA),
histone H3 and GAPDH (Cell Signaling Technology).
In vitro histone demethylase assay
In vitro translated LSD127 (0-20 μg) was incubated with 8 μg of calf thymus histones (Roche) or
0.3 μg of purified HeLa mononucleosomes (EpiCypher, Research Triangle Park, NC) with or
without 3.9 µM recombinant CoREST (BPS BioScience, San Diego, CA), followed by
immunoblotting using methyl-specific antibodies.
Mice
To generate the LSD1 transgenic mice C57BL/6J-Tg(Ly6a-KDM1A*v2), human LSD1 cDNA
encoding the shortest isoform (E2a−/E8a−) and an SV40 early polyadenylation (pA) signal were
inserted into the Ly-6A cassette (pLy-6A14) that contains the mouse Sca-1 gene and its
regulatory regions.28 The DNA fragment containing the mouse Sca-1 promoter and human
LSD1 cDNA with a pA signal was excised with NotI and microinjected into the pronuclei of
C57BL/6 mice as described.29
The copy number of the transgene was determined by blotting
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mouse tail DNA with human LSD1 cDNA as a probe and comparing the band intensity with copy
controls of the injection fragment. Pathological examination of mice was conducted by outside
experts.
All animal studies were approved by the Institutional Animal Ethics Committee and
performed in accordance with the Guide for the Care and Use of Laboratory Animals formulated
by the National Academy of Sciences.
Stem cell transplantation in syngeneic mice
Bone marrow mononuclear cells were isolated from LSD1 transgenic and control mice at 8-12
weeks of age, and injected via the tail vein into lethally irradiated (9.5 Gy) C57BL/6 (CD45.1)
recipient mice (8-12 weeks of age) with 2 × 105 backup cells.30
Engraftment was confirmed by
measuring the percentage of CD45.2+ cells in the peripheral blood.
Subsequent transplantations
were similarly performed using 2 × 106 bone marrow cells with a 12-week interval.
Cell cycle analysis in vivo
Bone
marrow
cells
were
harvested
40
minutes
after
intraperitoneal
injection
of
bromodeoxyuridine (1.5 mg/150 μl) and stained with the APC BrdU Flow Kit (BD Pharmingen,
San Diego, CA) to determine cell cycle distribution on flow cytometry.
Global analysis of mRNA expression
RNA samples (3 μg) were labeled using T4 RNA ligase in duplicate, and hybridized with the
Gene Chip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA). The hybridized arrays
were scanned with a GeneChip Scanner 3000, and the scanned images were processed using
GeneChip Operating Software (Affymetrix).
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Results
LSD1 is barely expressed in normal hematopoietic stem/progenitor cells, but is
overexpressed in leukemic cells
In an initial effort to clarify the role of LSD1 in leukemogenesis, we compared the expression of
LSD1 between normal human hematopoietic cells and leukemic cell lines at protein and mRNA
levels.
LSD1 protein was under the detection limit in normal human hematopoietic cells
including CD34+ HSPCs, CD34− bone marrow progenitors, lineage marker-positive bone marrow
cells and mature blood cells (Figure 1A, upper panel).
LSD1 expression was also very low in
CD34+ HSPCs but was slightly up-regulated in CD34−/Lin– progenitors at the mRNA level
(Figure 1A, lower panel).
In contrast, LSD1 was strongly expressed in most leukemic cell lines,
especially those derived from T-lymphoblastic leukemia/lymphoma (T-LBL) and those harboring
leukemic fusion proteins such as K562 (BCR-ABL+), THP-1 (MLL-AF9+), MV4-11
(MLL-AF4+) and PL-21 (PMA-RARα+) (Figure 1A, upper panel).
LSD1 overexpression was
confirmed at the mRNA level in leukemic cell lines (Figure 1A, lower panel).
In addition, we
analyzed LSD1 mRNA expression in primary samples using published datasets from the
Oncomine microarray database and verified LSD1 overexpression in primary leukemic cells
relative to normal bone marrow cells. Consistent with the results of the cell line studies, LSD1
expression was the highest in primary T-LBL cells, followed by BCR-ABL carrying chronic
myelogenous leukemia and AML in primary samples (Figure 1B).
LSD1 isofroms are differentially expressed in normal HSCs and leukemic cells
Mammals have four LSD1 isoforms generated by either the single or double inclusion of two
alternative exons 2a and 8a, which are located in the SWIRM and amine oxidase domains,
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respectively (Figure 1C).
Although the neuron-specific expression of exon 8a-retaining splice
variants has been reported previously15, no information is available regarding the expression
pattern and biological functions of LSD1 isoforms in the hematopoietic system.
To address this
issue, we attempted to detect LSD1 isoforms discriminately using isoform-specific RT-PCR
(Supplemental Table 1).
Hematopoietic cells expressed the isoform (–/–), which carries no
additional exons, and the isoform (+/–), which has the insertion of exon 2a (Figure 1D).
Neuron-specific isoforms (+/+) and (–/+) were undetectable in either normal or malignant
hematopoietic cells (Supplemental Figure 1A).
Among normal bone marrow cells, the isoform
(–/–) was barely detected in CD34+/CD38− HSCs and was up-regulated in CD34+/CD38+
multipotent progenitors and CD34−/Lin– committed progenitors by semi-quantitative RT-PCR
(Figure 1D/E) and real-time quantitative RT-PCR (Figure 1F).
In contrast, leukemic cells,
including primary samples from 3 patients with T-LBL, apparently overexpressed both (–/–) and
(+/–) forms (Figure 1D/E/F).
Finally, we investigated whether the same expression pattern of
LSD1 isoforms is retained in murine hematopoietic stem/progenitor cells. The isoform (–/–)
was faintly expressed in LSK and Lin–/Sca-1–/c-Kit+ fractions, as observed in human
CD34+/CD38– HSCs, whereas the expression of the isoform (+/–) was relatively strong (Figure
1G).
Neuron-specific isoforms (+/+) and (–/+) were also undetectable in murine hematopoietic
cells.
The shortest isoform of LSD1 displays robust histone H3K9 demethylating activity in vivo
To gain insights into functional differences between the LSD1 isoforms, we compared histone
demethylating activity of the two isoforms in vitro and in vivo.
The in vitro histone
demethylation assay revealed that the isoform (–/–) readily demethylated both H3K4 and H3K9
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in naked histones even in the absence of CoREST, whereas the isoform (+/–) had little if any
H3K9 demethylating activity and weak H3K4 demethylating activity (Figure 2A, left half).
The
same assay using nucleosomes yielded slightly different results: H3K4 demethylation was
detected only with the isoform (–/–) in the presence of CoREST and very weak H3K9
demethylation was observed with the isoform (+/–) in the absence of CoREST (Figure 2A, right
half).
As the experiments with nucleosomes might reflect the in vivo situation more accurately,
we attempted to confirm these results by analyzing the methylation status of endogenous histones
in HEK293 cells after the overexpression of each isoform.
The isoform (–/–) demethylated
H3K9 robustly and H3K4 moderately, whereas the isoform (+/–) displayed very weak activity for
both residues in HEK293 cells (Figure 2B, left panel and Supplemental Figure 1B).
The
discrepancy between the results of in vitro demethylation and in vivo transfection assays might be
due to the effects of LSD1 on other histone modification enzymes.
To investigate this
possibility, we examined the expression of H3K9 methyltransferases G9a and Suv39H1, H3K9
demethylase PHF8/JHDM1F and H3K4 demethylase JARID1B in LSD1-overexpressing
HEK293 cells.
As shown in Figure 2B (right panel), LSD1 readily increased and decreased the
expression levels of PHF8/JHDM1F and JARID1B, respectively.
Combined with the
expression data, we propose the model that low LSD1 activity is required to maintain HSCs in a
quiescent state and an increase in LSD1 activity, especially that against H3K9, via the
up-regulation of the isoform (–/–) shifts quiescent HSCs to an active/cycling state (Figure 2C).
The shortest isoform of LSD1 positively regulates the self-renewal of hematopoietic stem
cells
To verify the proposed model, we performed colony assays using mouse bone marrow cells
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transduced with each LSD1 isoform by the aid of the lentiviral expression system. When LSD1
isoform (–/–) was overexpressed, LSK cells formed 5-fold more colonies than mock-transduced
control (Figure 3A).
The isoform (+/–) also significantly increased the colony numbers of LSK
cells, but the effect was less prominent than that of the isoform (–/–). This isoform significantly
inhibited the clonogenic growth of c-Kit-positive differentiated progenitors, suggesting that the
expression level of the isoform (+/–) should be maintained at appropriate levels in progenitor
cells. These results prompted us to examine the expression of genes that govern the expansion
of HSCs in LSD1-overexpressing LSK cells using real-time RT-PCR.
LSD1 induced the
up-regulation of HoxA7, HoxA9, Meis1, Cxcr4, Gfi1b and c-Myb in LSK cells, which was
obviously stronger with the isoform (–/–) than the isoform (+/–) (Figure 3B).
Via the induction
of these genes, LSD1 especially the isoform (–/–) may increase the self-renewal capacity of
HSCs.
To confirm the ability of LSD1 isoform (–/–) to enhance the self-renewal of HSCs, we
conducted long-term reconstitution and replating assays. c-Kit-positive bone marrow cells were
prepared from donor mice, transduced with either LSD1 isoform (–/–) or (+/–), and injected into
lethally-irradiated recipient mice.
As illustrated in Figure 3C, we isolated bone marrow
mononuclear cells from recipient mice 10 months after transplantation and performed colony
assays.
The replating ability of bone marrow cells from LSD1 (–/–)-transduced mice was
significantly stronger than that of bone marrow cells from mock- and LSD1 (+/–)-transduced
mice.
Notably, LSD1 (–/–)-transduced cells were able to form colonies after third replating
(Figure 3D).
These results imply that the overexpression of the shortest isoform of LSD1 could
cause leukemic conversion of HSCs. However, recipient mice did not develop leukemias or any
hematopoietic deregulation by 16 months after transplantation, suggesting that LSD1
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overexpression alone is not sufficient for malignant transformation.
In support of this view,
LSD1-transduced cells were not able to survive beyond 3 passages in colony replating assays
(data not shown).
The shortest isoform of LSD1 enhances the stemness and self-renewal of hematopoietic
stem/progenitor cells in vivo
The results so far suggest that the shortest isoform of LSD1 is repressed in normal HSCs and its
overexpression predisposes to the development of acute leukemias via transactivation of genes
involved in self-renewal and leukemogenesis such as HoxA9.
To obtain compelling evidence
supporting this notion, we generated a transgenic mouse overexpressing LSD1 isoform (–/–) in
HSPCs under the control of the Sca-1 promoter (Figure 4A, upper panel). We established two
transgenic lines with different copy numbers and expression of integrated LSD1.
As shown in
Figure 4A (lower panel), the expression level of human LSD1 mRNA was 5-fold higher in Line 3
than in Line 6, possibly reflecting the copy numbers of the transgene: one copy in Line 6 and ~30
copies in Line 3 (Figure 4A, right panel; see Supplemental Table 2 for possible integration sites).
To investigate the effects of LSD1 overexpression on the self-renewal capacity of HSPCs in vivo,
we measured the size of LSK and Lin–/Sca-1+ fractions of these mice using flow cytometry.
The proportions of cells in both fractions were significantly increased in the high-expression line
(Figure 4B).
As LSK fractions contain HSCs and multipotent progenitors, we further
fractionated LSK cells using CD150 and CD48, and found that CD150+/CD48–/LSK cells, which
are highly enriched for long-term HSCs,23 were exclusively increased in the low-expression line
(Figure 4C). Consistent with these results, Lin–/Sca-1+ cells in the low-expression line showed
an increase in the G0/G1 phase and a decrease in sub-G0/G1 and S phases in vivo.
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high-expression line showed a significant decrease in the G0/G1 phase in both Lin–/Sca-1+ and
Lin– bone marrow cells and an increase in the S phase in the latter (Figure 4D).
To investigate whether LSD1 overexpression expands functionally active HSCs in vivo, we
conducted stem cell transplantation assays.
In Line 6 of LSD1 transgenic mice, long-term HSCs
were obviously expanded compared with Line 3 and control mice 24 weeks after transplantation
(Figure 4E).
Serial transplantation assays revealed that HSCs from LSD1 transgenic mice but
not control mice retained a high repopulating capacity at the third transplantation. Notably, the
repopulating activity was significantly stronger in Line 6-derived cells than in Line 3-derived
cells (Figure 4F).
Taken together, these results suggest that the shortest isoform of LSD1
regulates stem cell quiescence and activity depending on expression levels (Figure 4G).
HSCs
in the low-expression line show a quiescent phenotype with long-term repopulating ability,
whereas those in the high-expression line have higher self-renewal potential and commitment to
differentiation.
This is in agreement with the expression pattern and functions of LSD1
isoforms in HSPCs.
The shortest isoform of LSD1 modifies the gene expression program in favor of increasing
the self-renewal and T-cell priming of hematopoietic stem/progenitor cells
To more clearly understand the molecular basis responsible for the changes in HSC functions of
LSD1 transgenic mice, we performed microarray-based gene expression profiling using
Lin–/Sca-1+ bone marrow cells. The overall pattern of gene expression indicated that LSD1
up-regulated the genes with relatively low levels of expression (blue dots) and down-regulated
the genes with higher levels of expression (red dots) in both lines with a negligible difference
(Figure 5A and Supplemental Table 3). Gene ontology analyses revealed the unique pattern of
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gene regulation affecting various biological processes by LSD1 (Figure 5B).
Of these, we
focused on the homeobox group, lineage markers and cell signaling-related genes to estimate
stem cell functions.
In the homeobox gene cluster, HoxA2-10 were selectively up-regulated in
LSD1-overexpressing Lin–/Sca-1+ cells, whereas others, including HoxB4, were unaffected
relative to control cells (Figure 5C), confirming the results of RT-PCR in vitro (Figure 3B).
Among lineage markers, there was a considerable increase in the expression of genes encoding
stem cell and T-cell markers, such as c-Kit ligand, c-Mpl, DNMT3A, GATA3, IL-2-inducible T
cell kinase (ITK), TCF7 and Rag2. In contrast, B-cell related genes, including Bcl-6, Btg1,
Ly75 and Pbx1, were generally suppressed.
These results at least partly explain LSD1-mediated
increase in the self-renewal capacity of HSCs and further suggest that LSD1 overexpression may
skew the differentiation program of HSPCs into T-cell lineages.
LSD1 transgenic mice show the focal hyperplasia of T-lymphocytes, but do not develop
lymphoid malignancies for up to 16 months of observation
The shortest isoform of LSD1 is overexpressed in T-LBL, and its overexpression renders an
increase in the self-renewal potential and T-cell priming of HSPCs.
From these findings, it is
tempting to speculate that overexpression of the shortest isoform of LSD1 triggers
T-leukemogenesis.
The two lines of LSD1 transgenic mice serve as the ideal system to test this
hypothesis. Toward this end, we first analyzed the spleen and thymus of 12 week-old LSD1
transgenic mice and found that thymus but not spleen was significantly enlarged in Line 6 mice
compared with age-matched controls (Figure 6A/B).
Histopathological examination revealed no
proliferation or invasion of malignant cells in the thymus, spleen, kidney, liver or bone marrow of
LSD1 transgenic mice; however, the thymus of LSD1 transgenic mice, especially Line 6, showed
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the focal hyperplasia of T-lymphocytes. The pattern of TCRß rearrangements of thymocytes
was the same between LSD1 transgenic and normal B6J mice, suggesting that the proliferating
T-lymphocytes of LSD1 transgenic mice are non-malignant (Figure 6A, right panel).
A periodic
examination of peripheral blood showed a tendency of leukocytosis in 7-10 month-old LSD1
transgenic mice, but there was no statistically significant difference (Supplemental Figure 2).
In
addition, no increase in specific lineage cells was noted in the peripheral blood (data not shown),
bone marrow or thymus (Figure 6C).
Overall, LSD1 transgenic mice had the focal hyperplasia
of normal T-lymphocytes in the thymus, but did not develop hematological abnormalities,
including lymphoid malignancies, for up to 16 months of observation (Figure 7A, broken line).
LSD1 overexpression in quiescent HSCs accelerates γ-irradiation-induced development of
T-cell lymphoblastic leukemia/lymphoma
Recent investigations indicate that some epigenetic regulators act at the initial step of myeloid
leukemogenesis to form pre-leukemic HSCs.4-6
We assumed that HSCs of LSD1 transgenic
mice were also pre-leukemic because they exhibited an increased self-renewal potential but
retained the ability of normal differentiation.
It is therefore likely that LSD1 transgenic mice
develop hematological malignancies, especially T-cell leukemias, when they are exposed to
additional genotoxic insults.
To substantiate this hypothesis, we irradiated 4 week-old LSD1
transgenic and control mice with a dose of 1.7 Gy per week 4 times, followed by regular blood
cell counting and close inspections.
As anticipated, Line 6 mice, which overexpress LSD1 in
quiescent HSCs, developed T-LBL earlier and with higher frequency than control and Line 3
mice, which express LSD1 in activated HSCs and more differentiated progenitors, after
γ-irradiation (Figure 7A).
The mean overall survival periods were 26, 31 and 35 weeks for
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irradiated Line 6, Line 3 and control mice, respectively (p<0.01 between Line 6 and either Line 3
or control).
Malignant cells were detected in the thymus, spleen, bone marrow, liver and kidney
at autopsy (Figure 7B/C).
From a pathological standpoint, malignant cells seemed to be
originated in the thymus or bone marrow, and then invaded into other organs (Figure 7C).
Immunophenotypic analyses revealed a striking increase in the number of CD3+ T-lymphocytes
especially CD4/CD8 double-positive and CD8 single-positive cells in the bone marrow (Figure
7D). Moreover, preliminary studies detected Notch1 mutations, a hallmark of T-LBL, in thymic
tumors of LSD1 transgenic mice (Supplemental Table 4). These findings are fully consistent
with the pathological diagnosis of T-LBL.
Taken together, the shortest isoform of LSD1 drives
quiescent HSCs to pre-leukemic HSCs with an increased self-renewal potential and T-cell
priming, which are subject to a second hit such as γ-irradiation to develop T-LBL (Figure 7E).
Discussion
Recent studies indicate that certain epigenetic abnormalities, such as recurrent mutations of
DNMT3A and IDH1/2, generate pre-leukemic HSCs, which progress to full-blown leukemias by
additional oncogenic stimuli.4-6
In the present study, we demonstrate that another epigenetic
modifier LSD1 acts in a similar manner to generate pre-leukemic stem cells for the development
of T-cell malignancies.
To the best of our knowledge, this is the first demonstration of a
founder abnormality specific for T-cell leukemogenesis; however, it is possible that LSD1
overexpression predisposes to other types of malignancies depending on second hits, given the
fact that T-LBL is the most common tumor induced by γ–irradiation in B6 mice.31, 32
To test
this possibility, we are currently cross-breeding LSD1 transgenic mice with other cancer-prone
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mice such as p53-deficient mice, Lck-NotchIC9 transgenic mice33 and BCR-ABL transgenic
mice.34
We found that LSD1 increases self-renewal potential by up-regulating the expression of
HoxA family members such as A9, A7, A10 and A5 (Supplemental Figure 3 and Discussion).
This is fully compatible with the involvement of the HoxA family in stem cell expansion and
leukemogenesis.35-38
Using RNAi-based functional screening, Cellot et al.39 identified the
H3K4 demethylase JARID1B as a negative regulator of HSC functions among the Jumonji
domain-containing family of histone demethylases.
The shRNA-mediated knockdown of
JARID1B leads to the in vitro expansion of HSCs, which is accompanied by the up-regulation of
HoxA7, HoxA9 and HoxA10, with preserved long-term reconstitution potential.
They
speculated that PHF8/JHDM1F, an H3K9 demethylase of the Jumonji family, regulates HSC
functions in collaboration with JARID1B, but did not provide direct evidence. Our present
finding suggests that LSD isoform (–/–) is implicated in this process as another H3K9
demethylase and a modulator of the abundance of JARID1B and PHF8/JHDM1F in favor of
HoxA transactivation, thereby providing a clearer picture of stemness regulation within a
complex network of histone modifications.40
The mechanisms by which LSD1 (–/–) acts primarily as H3K9 demethylase in vivo is a
subject of future investigations. The discrepancy between the results of biochemical studies in
vitro and in vivo strongly suggests the presence of co-factors regulating the substrate specification
by LSD1 isoforms.
We speculate that some cells, including HSCs, possess a unique factor that
facilitates the tethering of LSD1 (–/–) to H3K9 as CoREST does so to H3K4.
It is also possible
that inhibitory molecules bind to the exon 2a sequence, thereby reducing the activity of the
isoform (+/–). This hypothesis is partly substantiated by a recent report of Laurent et al.41, in
17
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which the full length isoform of LSD1 fails to show demethylating activity against both H3K4
and H3K9 in vitro, but it effectively demethylates H3K9 but not H3K4 in neuronal cells.
They
identified the SVIL protein as an auxiliary molecule for LSD1 (+/+) to mediate specific H3K9
demethylation and subsequent transactivation of target genes in vivo.
We also demonstrated that
LSD1 (+/+) acts predominantly as H3K9 demethylase in HEK293 cells, which express the SVIL
protein (Supplemental Figure 1B).
The oncogenic LSD1 complexes could be novel therapeutic targets. As overexpressed
LSD1 impedes the repair of DNA double-strand breaks and may cause genomic instability
(Supplemental Figure 4), it is anticipated that LSD1 inhibition retards the progression of
pre-leukemic HSCs to full-blown leukemias.
Indeed, recent studies showed that small
molecular inhibitors for LSD1 induce differentiation and cell death in acute leukemias with
MLL-AF9 and PMA-RARα fusion proteins42-44, which overexpress LSD1 more prominently than
other AMLs.
Our present finding is not only confirmatory of these observations, but also
provides an insight into novel strategies for the early detection at pre-leukemic stages, prevention
of disease progression, and eradication of drug-tolerant persisters using LSD1 inhibitors in
hematologic malignancies.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Area
“Cell fate control”, Scientific Research (B) and (C), and grants from Asteras Foundation for
Research on Metabolic Disorders, Japan Leukemia Research Fund and Takeda Science
Foundation (to T.W. and Y.F.).
T.W. received a postdoctoral fellowship and the Young Scientist
18
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Award from Jichi Medical University.
We thank Dr. Elanine Dzierzak (Erasmus University
Rotterdam in Holland) for providing us with the Ly6A/Sca-1 transgenic cassette.
Authorship
Contribution: T.W. designed and performed experiments, analyzed data and drafted the
manuscript.
D.K. J.K. and H.H. provided materials and critically reviewed the manuscript.
T.W. and H.H. generated trnasgenic mice.
Y.F. designed and supervised research, and compiled
the manuscript.
Conflict of interest exposure: The authors declare no conflict of interest.
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Figure legends
Figure 1. LSD1 is barely expressed in normal hematopoietic stem/progenitor cells, but is
overexpressed in leukemic cells
(A) Upper panel: We isolated whole cell lysates from normal human bone marrow cells (CD34+
HSPCs, CD34−/Lin− progenitors and Lin+ cells), mature blood cells (granulocytes, monocytes,
uncultured T-lymphocytes and 24 h-stimulated T-lymphocytes), T-LBL cell lines (TALL-1,
MOLT-4, CEM, KOPT-K1 and Jurkat), myeloid leukemia cell lines (K562, HL-60, KG-1, THP-1,
MV4-11 and PL-21), normal human fibroblasts and the cervical carcinoma cell line HeLa for
immunoblot analyses on the expression of LSD1 and GAPDH (internal control).
Lower panel:
We isolated total cellular RNA from the indicated cells and subjected them to semi-quantitative
22
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RT-PCR analysis for the expression of LSD1 and GAPDH.
The PCR products of suboptimal
amplification cycles, 20-35 cycles, were electrophoresed (5 μl/lane).
(B) LSD1 mRNA
expression in primary samples according to the Oncomine database (http://www.oncomine.org).
Left panel: 0; normal bone marrow mononuclear cells (n=6), 1; AML (n=23), 2; B-cell acute
lymphoblastic leukemia (n=87), 3; T-LBL (n=11). Right panel: 0; various normal somatic cells
(n=146), 1; T-LBL (n=2), 2; AML (n=2), 3; Burkitt lymphoma (n=4), 4; colorectal cancer (n=2).
5; chronic myelogenous leukemia (n=2).
Asterisks denote p<0.05 determined by a one-way
ANOVA with the Bonferroni post hoc test.
(C) The structure of four LSD1 isoforms generated
by either the single or double inclusion of two alternative exons 2a (Ex2a) and 8a (Ex8a).
Asterisks denote the FAD-binding domain. We designed PCR primers 1/2F and 3R (arrows) to
detect LSD1 isoforms (–/–) and (+/–) discriminately based on differences in sizes (60 bp as
shown in the box).
(D) We performed semi-quantitative RT-PCR for the expression of LSD1
isoforms in normal human hematopoietic progenitors, leukemic cell lines, and primary T-LBL
cells from 3 patients (cases #2, #5 and #8).
The cDNAs were amplified with primers covering
the entire exons (1F and 19R; see Supplemental Table 1 for nucleotide sequences), followed by
nested PCR with primers 1/2F and 3R as reported by Zibetti et al.15
(E) The signal intensities of
each band at 35 cycles in (D) were quantified by the Image J software (National Institute of
Health, USA), normalized to those of corresponding GAPDH, and shown as relative values
setting the expression level of LSD1 (–/–) in CD34+/CD38– cells to 1.0. (F) We quantified the
mRNA expression of total LSD1, mostly (–/–) and (+/–), with 1/2F and 3R primers and the
isoform (+/–) with 1/2F and 2aR (within exon 2a) primers using the Power SYBR Green PCR
Master Mix (Life Technologies, Carlsbad, CA). The expression level was normalized to that of
GAPDH and quantified by the 2-ΔΔCt method.
The means ± S.D. (bars) of three independent
23
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experiments are shown. (G) We isolated total cellular RNA from murine Lin–/Sca-1+/c-Kit+ and
Lin−/Sca-1−/c-Kit+ cells, and subjected them to nested RT-PCR for the expression of murine
LSD1 isoforms with primer pairs of 2F and 3R (upper panel) or 2F and 8/9R (middle panel) (see
Supplemental Table 1 for nucleotide sequences).
Figure 2. Differential functions of LSD1 isoforms
(A) Calf thymus histones or HeLa mononucleosomes were incubated with purified LSD1 (0, 2
and 20 μg) with or without recombinant CoREST, followed by immunoblotting using specific
antibodies or silver staining (LSD1 and histones) (upper panel).
Signal intensities were
quantified and shown as relative values of corresponding untreated controls (lower panel).
(B)
Whole cell lysates were prepared from HEK293 cells transfected with expression vectors for
LSD1 isoform (–/–) or (+/–), and subjected to immunoblotting with the indicated antibodies.
Signal intensities were quantified and shown as relative values of corresponding mock
transfectants. (C) LSD1 expression and activity in hematopoietic stem and progenitor cells (see
text for the detail).
Figure 3. The shortest isoform of LSD1 positively regulates the self-renewal of
hematopoietic stem cells
(A) We transduced LSK and Lin−/Sca-1−/c-Kit+ cells from normal mouse bone marrow with an
empty plasmid (Mock) or expression vectors for LSD1 isoforms (–/–) and (+/–) for 48 h, and
cultured them in methylcellulose medium for 7 days to evaluate the clonogenic growth.
Signal
intensity of DsRed is shown in the left panel as a surrogate marker of LSD1 expression in starting
materials.
(B) Total cellular RNA was isolated from LSK cells transduced with LSD1 isoforms
(–/–) or (+/–), and subjected to real-time quantitative RT-PCR for the expression of the indicated
24
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genes.
Data were quantified by the 2-∆∆Ct method using simultaneously amplified GAPDH as a
reference and shown as fold increases against mock-transfected controls. (C) c-Kit-positive
bone marrow mononuclear cells were transduced with an empty vector (Control), LSD1 isoform
(–/–) or (+/–), and transplanted into lethally-irradiated recipient mice. We isolated bone marrow
mononuclear (BM) cells from recipient mice 10 months after transplantation and performed
colony-replating assays in the presence of murine stem cell factor (mSCF), FLT3 ligand (FL) and
murine interleukin-3 (IL-3) at 50, 50 and 10 ng/ml, respectively.
(D) The means ± S.D. (bars)
of colony numbers in each passage (left panel) and representative photographs of colonies (right
panel) are shown.
Asterisks denote p<0.01 (*) and p<0.05 (**) determined by a one-way
ANOVA with the Bonferroni post hoc test (n=3).
Figure 4. The shortest isoform of LSD1 enhances the stemness and self-renewal of
hematopoietic stem/progenitor cells in vivo
(A) Upper panel: Schematic view of the injected fragment for the generation of LSD1 transgenic
mice.
Lower panel: We quantified the expression of human LSD1 and murine GAPDH (internal
control) transcripts in two lines of LSD1 transgenic and B6J (Control) mice using specific
primers (Supplemental Table 1). Right panel: Genomic DNA (5 µg) was digested with BamHI
and hybridized with LSD1 cDNA as a probe to estimate copy numbers of transgene.
Lane 1:
DNA from wild-type mice + 2 copies of the injection fragment, lane 2: DNA from wild-type mice
+ 20 copies of the injection fragment, lane 3: DNA from wild-type mice, lane 4: DNA from Line
6 mice, lane 5: DNA from Line 3 mice.
(B) We determined the percentage of LSK and
Lin−/Sca-1+ cells in the bone marrow of two lines of LSD1 transgenic and B6J (Control) mice
using flow cytometry.
Representative data are shown in the right panel (P8 and P6 correspond
25
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to LSK and Lin−/Sca-1+ fractions, respectively).
(C) We determined the percentage of
CD150+/CD48− cells in LSK fractions of two lines of LSD1 transgenic and B6J mice using flow
cytometry.
Representative data are shown in the right panel (the boxed area corresponds to
CD150+/CD48−/LSK fractions).
(D) We determined the cell cycle profile of Lin−/Sca-1+ and
Lin− bone marrow mononuclear cells in two lines of LSD1 transgenic and B6J (Control) mice in
vivo.
(E) Bone marrow cells from two lines of LSD1 transgenic and B6J (Control) mice
(CD45.2) were trasplanted into lethally-irradiated C57BL/6 (CD45.1) mice.
We determined the
percentage of CD45.2+ cells in the peripheral blood 24 weeks after transplantation.
(F) Bone
marrow cells were collected from control- and LSD1-recipient mice 12 weeks after primary
transplantation, and transplanted into lethally-irradiated C57BL/6 mice.
was similarly performed 12 weeks after secondary transplantation.
three independent experiments are shown.
Tertiary transplantation
The means ± S.D. (bars) of
Asterisks denote p<0.05 (*) and p<0.01 (**)
determined by one-way ANOVA with the Bonferroni post hoc test.
(G) Cell cycle status of
HSPCs in LSD1 transgenic mice (see text for the detail).
Figure 5. The shortest isoform of LSD1 modifies the gene expression program in favor of
increasing the self-renewal and T-cell priming of hematopoietic stem/progenitor cells
(A) We performed microarray-based gene expression profiling in Lin−/Sca-1+ bone marrow cells
from LSD1 transgenic (Line 6 and 3) and control mice. A pair-wise comparison of 39000 genes
is shown with signal intensities in both axes.
(B) Gene ontology enrichment analysis for genes
whose expression was altered >2-fold in Line 6 (left panel) and Line 3 (right panel) of LSD1
transgenic mice compared with control mice (p<0.05 with an FDR threshold of 0.05). (C)
Examples of genes whose expression was altered in Line 3 (filled column) and Line 6 (shaded
26
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column) of LSD1 transgenic mice compared with control mice in three categories (Hox family,
hematopoietic cell lineage markers, and cell signaling-related).
Microarray data have been
deposited in the MIAME-compliant GEO database under accession number GSE66074.
Figure 6. LSD1 transgenic mice show the focal hyperplasia of lymphocytes but do not
develop lymphoid malignancies for up to 12 months of observation
(A) Left panel: Representative photographs of the spleen and thymus from two lines of LSD1
transgenic and age-matched (10-18 weeks) control mice and their histological sections. Right
panel: Genomic DNA was extracted from thymocytes of LSD1 transgenic and B6J control mice,
and subjected to PCR analysis for TCRß rearrangement. M: molecular size marker.
actual weights of thymus and spleen from 12-week old mice.
(B) The
The means ± S.D. (bars) of five
independent samples are shown (*p<0.05). (C) Bone marrow mononuclear cells (left panel) and
thymic cells (right panel) were subjected to flow cytometric analysis for the expression of the
indicated lineage markers.
The means ± S.D. (bars) of three independent experiments
performed between 10 and 12 months of age.
P-values were determined by a one-way ANOVA
with the Bonferroni post hoc test.
Figure 7. LSD1 overexpression in quiescent HSCs accelerates γ-irradiation-induced
development of T-LBL
(A) Kaplan-Meier survival curves of Line 6 (n=10) and Line 3 (n=8) of LSD1 transgenic and B6J
control mice (n=8) with (solid lines) or without (broken lines) γ–irradiation.
determined by the log-rank test.
P-values were
(B) Representative photographs of the spleen, thymus and
kidney dissected from LSD1 transgenic (Lines 6 and 3) and control mice.
photographs of histopathological sections of the indicated organs.
27
(C) Representative
The diagnosis of T-LBL was
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made by outside experts of animal pathology.
(D) Surface marker expression of bone marrow
cells from LSD1 transgenic and control mice.
Asterisks denote p<0.01 determined by a
one-way ANOVA with the Bonferroni post hoc test (n=3).
(E) Graphical abstract: LSD1
overexpression generates pre-leukemic HSCs with T-lineage priming, which are transformed to
T-cell malignancy by 2nd hits such as γ–irradiation.
28
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Figure 1
E
AML
35
Relative expression of LSD1
HL60
KG-1
K562
THP-1
MV4-11
PL21
Fibroblasts
HeLa
T-LBL
TALL-1
MOLT-4
CEM
KOPT
Jurkat
K562
CD34+
CD34−Lin−
CD34−Lin+
Granulocytes
Monocytes
T cells (T=0)
T cells (T=48)
K562
A
Total LSD1
GAPDH
CD34+
HL60
CD34−
20 25 30 35
20 25 30 35
K562
20 25 30 35 20 25 30 35
Total LSD1
GAPDH
(−/−)
30
(+/−)
25
20
15
10
5
0
B
F
AML
B-ALL
Normal
T-LBL
175
C
2a−/8a− (−/−)
175
2a−/8a+ (−/+)
2a+/8a−
417
247
SWIRM
T-LBL
522
AOD Tower
247
421
AML
BL
CRC
CML
852
★ AOD
526
856
★
(+/−)
2a+/8a+ (+/+)
175
267
437
542
★
872
175
267
437
546
★
876
3R
1/2F
12
(−/−) and (+/−)
10
Relative expression levels
Normal
Relative expression levels
12
8
6
4
2
0
Ex2a
G
Lin−Sca-1+
25
D
CD34+
CD38−
CD34+
CD38+
CD34−
K562
(+/−)/(−/−)
(+/−)/(−/−)
GAPDH
GAPDH
CEM
25 30 35
25 30 35
TALL-1
MV4-11
293
25 30 35 25 30 35 25 30 35 25 30 35
25 30 35 25 30 35 25 30 35
Molt4
HL60
(+/−)/(−/−)
(+/+)/(−/+)
h. GAPDH
#2
Jurkat
#5
#8
25 30 35 25 30 35 25 30 35
25 30 35 25 30 35
(+/−)/(−/−)
(+/−)/(−/−)
GAPDH
GAPDH
8
6
4
2
0
Ex8a
gca agcaggagga cttcaagacg acagttctgg
agggtatgga gacggccaag catcagg
GQAGGLQDDSSGGYGDGQAS(172-192)
10
30
35
Lin−Sca1−
25
30
h. Brain
35
35
(+/−)
Figure 2
A
Histone
−CoREST
−/−
Histone
+CoREST
+/−
−/−
+/−
Nucleosome
−CoREST
Nucleosome
+CoREST
−/−
−/−
+/−
+/−
H3K4me2
H3K9me2
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LSD1
Histone
Histone/H3K4me2
1.2
Relative signal intensity
1
0.8
0.6
0.4
−/− +CoREST
+/− +CoREST
−/− −CoREST
+/− −CoREST
0.2
0
2
LSD1 (µg)
20
Histone/H3K9me2
1
0.8
0.6
0.4
−/− +CoREST
+/− +CoREST
0.2
−/− −CoREST
+/− −CoREST
0
1.2
2
LSD1 (µg)
20
Nuc./H3K9me2
1
0.6
0.4
0.2
0
+/− +CoREST
0.2
−/− −CoREST
+/− −CoREST
0
2
LSD1 (µg)
20
0
+/−
−/−
0
−/− +CoREST
2
LSD1 (µg)
Mock
1.2
−/−
Relative signal intensity
LSD1
GAPDH
H3K4me2
H3K9me2
1
+/−
PHF8/
JHDM1F
0.6
0.4
JARID1B
0.2
LSD1
0
Histone H3
H3K4me2
H3K9me2
C
Quiescent
HSC
Low LSD1 activity
LSD1 (−/−) ↓
LSD1 (+/−) ↑
G9a
Suv39H1
0.8
Active
HSC
Progenitors
High LSD1 activity
LSD1 (−/−) ↑
LSD1 (+/−) ↓
20
GAPDH
+/−
−/− +CoREST
+/− +CoREST
−/− −CoREST
+/− −CoREST
−/−
0.4
Mock
0.8
0.8
0.6
B
1
Mock
Relative signal intensity
1.2
0
Nuc./H3K4me2
0
Relative signal intensity
Relative signal intensity
1.2
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
A
Figure 3
Mock
92.2%
120
LSK cells
120
**
(−/−)
94.2%
100
80
60
**
40
(+/−)
Colony number
Colony number
100
20
86.8%
**
80
60
40
20
0
B
lin−, Sca-1−, c-kit+ cells
Mock
−/−
0
+/−
LSD1 over-expressing mLSK cells
4.5
Mock
*
−/−
+/−
4.0
3.5
Fold increase
−/−
*
3.0
2.5
*
*
2.0
*
*
1.5
1.0
0.5
0.0
C
HoxA7 HoxA9 HoxB4 Meis1
BMT mice (10M)
Control, −/−, +/−
Cxcr4
1st
Bmi1
Gfi1b
Myb
2nd
3rd
mSCF
FL
mIL-3
BM cells
3rd replating
Control
D
30
Control
−/−
25
+/−
Colony number
*
(−/−)
20
15
*
10
5
0
(+/−)
1st
2nd
3rd
100µm
+/−
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Figure 4
D
A
1 2 3 4 5
NotI
NotI
0
18215
9K
35
40
30
35
Line3
40
30
35
40
h.LSD1
mGAPDH
4K
1.0
0.8
0.6
2K
60
50
40
30
20
0
0.2
0.0
B6J
Line6
E
Line3
*
sub-Go/G1
Go/G1
100
Cont.
7
*
*
Line 6
c-kit
6
5
**
Line 3
c-kit
G
Lin-Sca-1+
Lin−Sca-1+
sub-Go/G1
Go/G1
**
30
70
60
50
40
30
20
10
Control
Line6
0
Line3
1st
Quiescent
Control
Differentiation
CD48
**
10
8
High LSD1
(Line3)
6
Go/G1
Quiescent
Go/G1
Self-renewal Differentiation
Quiescent
Self-renewal
Differentiation
CD48
Line6
Low LSD1
(Line6)
4
2
0
Line3
B6J
Line6
Line3
CD48
CD150+CD48-LSK (%)
12
CD150
**
80
Self-renewal
Cont.
G2M
*
100
Sca-1
C
S
90
40
0
LSK
F
Control
Line6
Line3
50
3
0
30
0
G2M
60
10
1
S
70
4
*
40
10
20
2
50
Donor-derived cells (%)
8
% of lin- mBMCs
*
Control
Line 6
Line3
9
60
80
c-kit
10
*
20
*
90
B
Cont.
Line 6
Line3
70
10
0.4
80
70
Donor-derived cell (%)
30
Line6
Signal intensity (h.LSD1/mGAPDH)
control
6K
90
Cont.
Line 6
Line3
80
23K
1.2
*
% of Lin− mBMCs
Ly6A-h.LSD1
(the shortest isoform)
*
90
% of Lin− Sca-1+ mBMCs
h. LSD1 cDNA-pA
Quiescent
HSC
Active
HSC
Line6
Line3
Progenitors
2nd
3rd
cont.
Line6
Line3
Figure 5
Line 3
Line 6
Line 3
A
Control
B
Control
Line 6
Cont. vs Line6
Cont. vs Line3
biological_process
1223
signal transduction
450
anatomical structure development
416
biosynthetic process
396
cell differentiation
372
cellular nitrogen compound metabolic…
349
transport
299
response to stress
268
cellular protein modification process
262
immune system process
210
cell death From185
www.bloodjournal.org
cell proliferation
184
small molecule metabolic process
160
cell motility
147
homeostatic process
145
neurological system process
138
anatomical structure formation…
134
cell adhesion
125
reproduction
122
lipid metabolic process
119
catabolic process
119
embryo development
117
cell morphogenesis
115
cell cycle
111
C
by
biological_process
signal transduction
571
biosynthetic process
502
anatomical structure development
481
cellular nitrogen compound metabolic…
439
cell differentiation
424
transport
377
response to stress
348
cellular protein modification process
315
guest on June
15,
2017.
For
personal
use
immune system process
262
small molecule metabolic process
227
cell proliferation
220
cell death
219
homeostatic process
186
lipid metabolic process
167
cell motility
166
cell adhesion
162
neurological system process
160
catabolic process
149
vesicle-mediated transport
147
1599
only.
Lineage marker
Hox gene
D13
D12
D11
D10
D9
D8
D4
D3
D1
C13
C10
C9
C8
C6
C5
C4
B13
B9
B7
B6
B5
B4
B3
B2
B1
A13
A11
A10
A9
A7
A5
A4
A3
A2
A1
Line3
Tcrg-V4
Tcf7l2
Tgtp1 /// Tgtp2
Timd4
Line6
Tal1
Rag2
Trav6-3
Line3
Line6
Tet2
Pbx1
Pilrb1
Pilra
Mpl
Ly75
Lyve1
Kitl
Itk
Ikzf3
Ikzf2
HES1
GATA3
Dnmt3a
Cyld
Blk
Blnk
Btg1
Bcl6
0.0
0.5
1.0
1.5
Expression (Log2 Ratio)
2.5 -3.0
2.0
-2.0
-1.0
0.0
1.0
Expression (Log2 Ratio)
Cell signaling
Line3
Line6
Rhobtb3
Rhobtb1
Rhoa
Rhpn2
Arhgef26
Rab6b
Rab27b
Rab17
Rabl2
Arhgef25
Arhgef10
Arhgap32
Rab23
Arhgap24
Arhgap12
Gpr84
Gpr56
Gpr22
Gpr150
Gpr125
-3.0
-1.0
Gpr15
1.0
Expression (Log2 Ratio)
3.0
5.0
2.0
3.0
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
A
Spleen
Spleen
Thymus
Figure 6
Thymus
【Control】
M
100µm
100µm
100µm
100µm
B6J Line6 Line3
germline
D2-J2.1
D2-J2.2
D2-J2.3
D2-J2.4
D2-J2.5
【Line6】
D2-J2.6
【Line3】
100µm
100µm
Thymus
B
80
Spleen
80
*
70
70
50
40
30
100
90
50
40
30
20
20
10
10
0
C
60
Weight (mg)
Weight (mg)
60
Control
Line6
0
Line3
B6J
Line6
Line3
90
P>0.05
80
Line6
Line3
B6J
Line6
Line3
P>0.05
70
70
Lin marker+ cells (%)
Lin marker+ cells (%)
80
60
50
40
30
60
50
40
30
20
20
10
10
0
Control
0
c-kit
TER119 CD41
CD11b
CD3e
B220
DN
DP
CD4+CD8-
CD4-CD8+
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
**
**
0.80
100
0.60
90
80
0.40
Control-IR
Line6-IR
Line3-IR
Control, Line6, Line3
0.20
0.00
0
5
10
15
20
P<0.01
25
30
35
40
Lin marker+ cells (%)
Cumulative survival rate
Figure 7
D
1.00
P>0.05
45
50
Time (weeks)
B
Spleen
Thymus
Kidney
100
B6J
6l-IR
3l-IR
60
50
40
30
**
**
**
*
0
**
70
60
50
*
40
30
20
10
10
【Control-IR】
c-kit
CD41
0
CD3e
DP
CD4+CD8−
E
【Line6-IR】
Quiescent
HSC
MPP / CLP
T-cell (Polyclonal)
1st hit
LSD1
【Line3-IR】
Quiescent
HSC
C
Spleen
Thymus
Femur
Liver
Differentiation
T-cell (Polyclonal)
HSCs expansion
T-cell priming
Pre-leukemic HSC
Kidney
Quiescent
HSC
【Control-IR】
100µm
100µm
100µm
100µm
100µm
100µm
100µm
100µm
100µm
100µm
100µm
100µm
【Line6-IR】
【Line3-IR】
*
80
70
20
**
90
Lin marker+ cells (%)
A
1st hit
2nd hit
LSD1
Transformation
HSCs expansion
T-cell priming
Pre-leukemic HSC
T-cell malignancy
(Monoclonal)
CD4−CD8+
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Prepublished online April 22, 2015;
doi:10.1182/blood-2014-11-610907
Overexpression of the shortest isoform of histone demethylase LSD1
primes hematopoietic stem/progenitor cells for malignant transformation
Taeko Wada, Daisuke Koyama, Jiro Kikuchi, Hiroaki Honda and Yusuke Furukawa
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