aspp1 preserves hematopoietic stem cell pool integrity and ... · data shown are mean ± sd. *p...
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Article
Aspp1 Preserves Hematop
oietic Stem Cell PoolIntegrity and Prevents Malignant TransformationGraphical Abstract
Highlights
d Aspp1 is predominantly expressed in HSCs and restricts self-
renewal capacity
d Aspp1 induces apoptosis in damaged HSCs and inhibits
persistent DNA damage foci
d Aspp1 coordinates with p53 to limit HSC self-renewal and
DNA damage accumulation
d Double deletion of Aspp1 and p53 in HSCs leads to
hematological malignancy in vivo
Yamashita et al., 2015, Cell Stem Cell 17, 23–34July 2, 2015 ª2015 Elsevier Inc.http://dx.doi.org/10.1016/j.stem.2015.05.013
Authors
Masayuki Yamashita, Eriko Nitta,
Toshio Suda
[email protected] (E.N.),[email protected] (T.S.)
In Brief
Suda and colleagues show that the
hematopoietic stem cell (HSC) pool
integrity is coordinately controlled by p53
and its proapoptotic activator Aspp1.
Aspp1 has both p53-dependent and
-independent functions in regulating HSC
self-renewal and DNA damage tolerance,
and concomitant deletion of Aspp1 and
p53 in HSCs leads to hematological
malignancies.
Accession Numbers
GSE69032
Cell Stem Cell
Article
Aspp1 Preserves Hematopoietic Stem Cell PoolIntegrity and Prevents Malignant TransformationMasayuki Yamashita,1 Eriko Nitta,1,2,* and Toshio Suda1,3,*1Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University,
35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan2Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku,Chiba 260-8670, Japan3Cancer Science Institute, National University of Singapore, 14 Medical Drive MD6, Centre for Translational Medicine, 117599, Singapore
*Correspondence: [email protected] (E.N.), [email protected] (T.S.)
http://dx.doi.org/10.1016/j.stem.2015.05.013
SUMMARY
Quiescent hematopoietic stem cells (HSCs) areprone to mutagenesis, and accumulation of muta-tions can result in hematological malignancies. Themechanisms through which HSCs prevent suchdetrimental accumulation, however, are unclear.Here, we show that Aspp1 coordinates with p53 tomaintain the genomic integrity of the HSC pool.Aspp1 is preferentially expressed in HSCs and re-stricts HSC pool size by attenuating self-renewal un-der steady-state conditions. After genotoxic stress,Aspp1 promotes HSC cycling and induces p53-dependent apoptosis in cells with persistent DNAdamage foci. Beyond these p53-dependent func-tions, Aspp1 attenuates HSC self-renewal and accu-mulation of DNA damage in p53 null HSCs. Conse-quently, concomitant loss of Aspp1 and p53 leadsto the development of hematological malignancies,especially T cell leukemia and lymphoma. Together,these data highlight coordination between Aspp1and p53 in regulating HSC self-renewal and DNAdamage tolerance and suggest that HSCs possessspecific mechanisms that prevent accumulation ofmutations and malignant transformation.
INTRODUCTION
In adult mammals, a vast number of blood cells of all kinds are
derived from hematopoietic stem cells (HSCs), a rare population
in bone marrow (BM). HSCs are the only cells within the blood
system that have both self-renewal and multi-lineage potential,
giving rise to daughter stem cells and committed progenitor
cells, thus achieving lifelong hematopoiesis (Orkin and Zon,
2008). To avoid being lost by various stresses, HSCs are mostly
kept in a quiescent state (Arai et al., 2004; Wilson et al., 2008;
Yoshihara et al., 2007).
Growing evidence has suggested that HSCs have unique
characteristics in their DNA damage response owing to their
quiescence (de Laval et al., 2013; Mohrin et al., 2010). HSCs
tend to repair DNA double strand breaks (DSBs), the severest
form of DNA damage, by error-prone nonhomologous end
joining (NHEJ) rather than homologous recombination. Because
of the error-prone nature of NHEJ, surviving HSCs may retain
mutations (Mohrin et al., 2010). Once a mutation favorable for
cell survival is maintained, the mutation can be passed to
daughter stem cells and pooled in the HSC population and can
be transmitted to a variety of mature hematopoietic cells (Blan-
pain et al., 2011). Thus, HSCs can serve as a reservoir for muta-
tion accumulation, which may eventually lead to hematopoietic
failure or malignant transformation (Rossi et al., 2008). Recent
deep sequencing analyses suggest that clonal expansion of
HSCs with somatic mutation can precede the development of
leukemia and lymphoma, and these ‘‘pre-leukemic HSCs’’ can
survive after chemotherapy, contributing to relapse of the dis-
ease (Corces-Zimmerman et al., 2014; Quivoron et al., 2011;
Shlush et al., 2014).
Given that quiescent HSCs are vulnerable to mutagenesis due
to low-fidelity DNA repair, there should be some compensatory
mechanisms by which mutations are removed from the HSC
pool. One of the most plausible molecular pathways to guard
the HSC genome is through the well-known tumor suppressor
p53. Upon genotoxic stress, p53 can induce cell-cycle arrest,
senescence, or apoptosis to prevent mutation accumulation.
Greater p53 activation is observed in hematopoietic stem and
progenitor cells (HSPCs) than in myeloid progenitor cells (Mohrin
et al., 2010), and p53 also regulates HSC quiescence under
normal conditions (Liu et al., 2009).
The apoptosis-stimulating protein of p53 (Aspp) family pro-
teins Aspp1, Aspp2, and iAspp are co-factors of p53 that specif-
ically regulate its proapoptotic function (Bergamaschi et al.,
2003; Samuels-Lev et al., 2001). Aspp1 and Aspp2 enhance
the proapoptotic function of p53 by activating p53-mediated
transcription of proapoptotic genes, whereas iAspp plays the
opposite role. All three proteins contain ankyrin repeats, an
SH3 domain and a proline-rich region in their C termini, by which
they can associate with several proteins, including p53 (Trigiante
and Lu, 2006). Among the Aspp members, loss of ASPP1
expression due to aberrant methylation of its promoter has
been reported as an independent poor prognosis factor of hu-
man acute lymphoblastic leukemia (ALL) (Agirre et al., 2006).
Previous studies showed that Aspp1 was highly expressed in
murine hematopoietic cells as well as vascular endothelial cells
(Hirashima et al., 2004, 2008). In addition, a recent study identi-
fied Aspp1 as an apoptosis enhancer of human cord blood stem
Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc. 23
cells (Milyavsky et al., 2010). Together these findings suggest
that Aspp1 could be a potent regulator of hematopoiesis, espe-
cially of HSCs. However, the significance of Aspp1 in adult
hematopoiesis and the causal relationship between Aspp1
deficiency and hematological malignancy have not been
investigated.
Here we identify Aspp1 as a selective regulator of HSCs and
examine its function in protecting the integrity of the HSC pool
and the underlying mechanisms.
RESULTS
HSCs Are Predisposed to Activate p53-DependentProapoptotic Signaling after DNA DamageHSPCsactivate greater levels of p53 comparedwithmyeloidpro-
genitor cells after genotoxic stress (Mohrin et al., 2010). To further
assess the activity of p53 in HSCs, we treated mice with 2 Gy or
4 Gy ionizing radiation (IR), isolated myeloid progenitor-enriched
LKS� (lineage�Sca-1�c-Kit+), HSPC-enrichedLSK (lineage–Sca-
1+c-Kit+), and HSC-enriched long-term HSC (LT-HSC) (CD150+
CD41�CD48�LSK) populations, and evaluated p53 activation
and changes inmRNA levels of several p53 target genes (Figures
A
B
C D
E F G H
I J
Figure 1. Preferential Expression of Aspp1
in the HSC Population and Increased HSC
Pool Size in Aspp1–/– Mice
(A) Relative Aspp1 mRNA levels in purified BM
subpopulations, assessed by qRT-PCR (n = 4).
(B) Expression of LacZ in BM subpopulations of
Aspp1+/LacZ mice, measured by flow cytometry
after being stained with fluorescein di-b-D-gal-
actopyranoside (FDG).
(C) Quantification of FDG fluorescence shown in
(B). The fluorescence intensity was converted into
geometric mean, and the mean values were
normalized to Aspp1+/+ control (n = 3).
(D) Western blotting using lysates of lineage+,
LKS�, and LSK cells.
(E) BM cellularity of adult WT and Aspp1�/� mice
(n = 10).
(F–H) Absolute number of myeloid cells, B cells,
and T cells (F), common lymphoid progenitor
subpopulations (G), and myeloid progenitor sub-
populations (H) in the BM of adult WT and
Aspp1�/� mice (n = 10).
(I) Representative flow cytometry profiles of LSK
cells (left) and CD150+CD41�CD48�LSK cells (LT-
HSCs; middle) in adult WT and Aspp1�/� BM cells.
LT-HSCs are further classified based on CD150
expression levels (right).
(J) Absolute number of LSK and LT-HSC pop-
ulations in the BM of adult WT and Aspp1�/� mice
(n = 10; ��p < 0.01 [versus WT]).
Data shown are mean ± SD. *p < 0.05, **p < 0.01.
See also Figures S1 and S2.
S1A and S1C). LSK cells and LT-HSCs
consistently showed higher responsive-
ness to IR compared with LKS� cells, as
observed by strikingly enhanced phos-
phorylation of p53 and expression of p53
target genes after DNA damage (Figures
S1C and S1D). Of note, when treated with 4 Gy IR, LT-HSCs ex-
hibited stronger upregulation of proapoptotic genes Bax and
Noxa when compared with LKS� cells, whereas upregulation of
survival-contributing p21 and Mdm2 genes was comparable
between the two populations (Figure S1D). These observations
suggest a p53-dependent stem cell-specific machinery that
guides HSCs toward apoptosis rather than survival particularly
after severe DNA damage to protect the HSC pool against unre-
pairable DNA damage accumulation.
Aspp1 Is Highly Expressed in Adult HSCsThe driving force toward p53-dependent apoptosis in HSCs
encouraged us to investigate co-factors of p53, especially the
molecules that facilitate distinct responses between p53-depen-
dent apoptosis and survival. We therefore focused on Aspp1,
which is an apoptosis-specific co-activator of p53 (Trigiante
and Lu, 2006) that is preferentially expressed in the stem cell
population of human cord blood (Milyavsky et al., 2010). Aspp1
mRNA levels were evaluated by qRT-PCR in murine HSC, he-
matopoietic progenitor, and differentiated cell populations iso-
lated from BM of adult mice (Figures 1A, S1A, and S1B).
Aspp1 was highly expressed in LSK cells and LT-HSCs. To
24 Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc.
confirm Aspp1 expression in the HSC-enriched population, we
performed flow cytometric analysis using fluorescein di-b-D-gal-
actopyranoside (FDG) (Nolan et al., 1988) and Aspp1-LacZ
reporter (Aspp1+/LacZ) mice, in which part of the Aspp1 gene is
replaced with an IRES-LacZ cassette (Hirashima et al., 2008).
Consistent with the qRT-PCR data, FDG fluorescence was pref-
erentially detected in LSK cells and LT-HSCs (Figures 1B and
1C). Aspp1 protein was specifically detected in LSK cells, but
not in lineage+ or LKS� cells (Figure 1D). Among the Aspp family
of genes, LT-HSCs showed the highest enrichment of Aspp1
(Figure S1E). Evaluation of Aspp family expression by reanalysis
of RNA-seq and mass spectrometry data (Cabezas-Wallscheid
et al., 2014) also revealed the highest expression of Aspp1
among Aspp family members (Figure S1F).
Aspp1 Negatively Regulates the HSC Pool Size In VivoThe preferential expression of Aspp1 in HSCs led us to speculate
that Aspp1 might be responsible for significant regulation of
stem cell maintenance. To assess the physiological effect on he-
matopoiesis by disruption of the Aspp1 gene, we examined pe-
ripheral blood (PB) and BM status of 10- to 12-week-old mice
lacking Aspp1 because of homozygous replacement by the
LacZ gene (hereafter referred to as Aspp1�/� mice). Aspp1�/�
mice grew up healthy without distinctive abnormality from WT
mice in aC57BL/6 background, although the number of offspring
was significantly reduced for an unknown reason (Table S1).
However, complete blood count tests revealed a significant in-
crease in the number of white blood cells (WBCs) in Aspp1�/�
mice compared with WT mice (Table S2), accompanied with
increased numbers of myeloid cells (Gr1+Mac1+), B cells
(B220+CD19+), and T cells (CD3+) (Figure S2A). The mean
corpuscular hemoglobin (MCH) and platelet counts of Aspp1�/�
mice were also slightly elevated (Table S2). The number of BM
mononuclear cells (MNCs) was slightly increased in Aspp1�/�
mice (Figure 1E). Flow cytometric analysis revealed that the in-
crease in BM cellularity was derived from an increase in myeloid
cell population (Figure 1F). In addition, whereas the number
of lymphoid cells and common lymphoid progenitor cells
(CLPs; IL-7Ra+Flt3+lineage�Sca-1lowc-Kitlow) was comparable
between WT and Aspp1�/� mice (Figures 1F and 1G), the gran-
ulocyte-macrophage progenitor cell (GMP; CD34+FcgRhigh
lineage�Sca-1�c-Kit+) population was significantly increased in
Aspp1�/� mice BM (Figures 1H and S2B). Most significantly,
we observed an approximate 1.5- and 2-fold increase in the ab-
solute number of LSK cells and LT-HSCs in Aspp1�/� mice,
respectively (Figures 1I, 1J, and S2C). Of note, the increased
HSC pool size was accompanied with increased numbers of
CD150high LT-HSCs (Figure 1J), which are myeloid biased and
have a more robust self-renewal potential (Beerman et al.,
2010; Morita et al., 2010). Taken together, these data indicate
that Aspp1 negatively controls HSC pool size.
Sustained Self-Renewal Capacity of Aspp1–/– HSCs inSerial BM TransplantationThe increase in HSC population of Aspp1�/� mice can theoreti-
cally be caused by changes in cell-intrinsic factors (e.g., self-
renewal, differentiation, cell-cycle regulation, and apoptosis)
and/or cell-extrinsic factors (such as niche factors). To evaluate
long-term self-renewal capacity of bona fide HSCs, we per-
formed serial competitive BM transplantation using Aspp1-defi-
cient HSCs.Aspp1�/� LSK cells displayedmoderately enhanced
reconstitution capacity in the first round of transplantation, as
measured by the percentage of CD45.2+ cells monthly in PB
MNCs (Figure S3A) and 4 months after transplantation in BM
MNCs, LSK cells, and CD150+ LSK cells (Figure S3B). Further-
more, the reconstitution capacity of Aspp1�/� LSK cells was
significantly sustained, while WT LSK cells displayed diminished
reconstitution capacity in the second round of transplantation
(Figures S3C and S3D). Serial competitive transplantation further
clarified that the reconstitution capacity of Aspp1�/� LT-HSCs
was significantly sustained especially after tertiary transplanta-
tion (Figure 2A), suggesting sustained self-renewal capacity in
Aspp1-deficient HSCs. These results indicate that Aspp1 nega-
tively regulates the self-renewal capacity of HSCs in a cell-
intrinsic manner, especially under cumulative stress after serial
BM transplantation.
Aspp1 Negatively Regulates HSC Quiescence uponExposure to StressesNext we analyzed the mechanisms through which Aspp1 pro-
motes the decreased self-renewal capacity of HSCs in serial
BM transplantation. We examined the gene expression profile
of WT and Aspp1�/� LSK cells before and after BM transplanta-
tion using a multiplex qRT-PCR system. Consistent with the
sustained self-renewal capacity of Aspp1�/� LSK cells, gene
expression analysis revealed that multiple genes important for
the maintenance of HSCs were significantly upregulated in
Aspp1�/� LSK cells (Figure 2B). Among them, several quies-
cence-related genes including Tek, Ndn, and Cdkn1c were
significantly upregulated in Aspp1�/� LT-HSCs isolated after
transplantation (Figure S3E).
Quiescence is an important mechanism for the resistance to
stress stimuli and therefore for the maintenance of self-renewal
capacity of HSCs (Arai et al., 2004). Thus, we assessed the
cell-cycle status of Aspp1�/� HSCs. BrdU uptake was signifi-
cantly decreased in Aspp1�/� LT-HSCs in vivo (Figure 2C).
Furthermore, when we analyzed the quiescence of donor-
derived LSK cells in primary and secondary recipients using
the Ki67 proliferation marker and Hoechst33342 to stain DNA
content, we found that serial transplantation caused a progres-
sive decrease in Ki67-negative cells in control donor-derived
LSK cells, whereas Aspp1�/� LSK cells retained more Ki67-
negative cells (Figure 2D). These results indicate that Aspp1�/�
HSPCs were more quiescent and resistant to the loss of
quiescence after serial transplantation.
To further investigate the resistance to stress stimuli, we
administered a single dose of 5-FU, a chemotherapeutic agent
that selectively kills proliferating hematopoietic cells (Lerner
and Harrison, 1990; Van Zant, 1984), into Aspp1�/� mice and
control mice and monitored the recovery of PB and BM status.
Aspp1�/� mice displayed an enhanced recovery of WBCs, he-
moglobin, and platelets (Figure S3F). Consistent with this, we
observed rapid recovery in the number of BM CD150+
CD41�CD48�lineage�Sca-1+ cells prior to the recovery of PB
in Aspp1�/� mice (Figure S3G). Furthermore, Aspp1�/� LT-
HSCs remained quiescent upon 5-FU treatment, whereas the
majority of WT LT-HSCs entered the cell cycle (Figure S3H). In
contrast, no significant changes were observed in the apoptosis
Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc. 25
ratio in Aspp1�/� WBCs or LT-HSCs (Figure S3I). These findings
support the hypothesis that Aspp1�/� HSCs may remain quies-
cent upon exposure to stress stimuli and are more resistant to
myelotoxic agents.
Aspp1Promotes Apoptosis andControls Persistent DNADamage in HSCsAspp1 has been shown to induce apoptosis in response to DNA
damage in cancer cell lines and human cord blood stem cells
(Milyavsky et al., 2010; Samuels-Lev et al., 2001). Therefore,
we evaluated the apoptosis of donor-derived LSK cells induced
by primary and secondary transplantation using the apoptosis
marker Annexin V. The percentage of Annexin V-positive cells
in donor-derived LSK cells was comparable between both
groups of primary recipients, but a marked reduction was
observed in donor-derived LSK cells in secondary recipients
that received Aspp1�/� LSK cells as donors (Figures 3A and
3B). Similar to the findings under the transplantation stresses,
the percentages of early apoptotic cells were significantly
decreased in Aspp1�/� LT-HSCs and LSK cells upon 4 Gy IR
in vivo (Figures 3C, S4A, and S4B). LKS� cells, which contain
myeloid progenitor cells but no HSCs, showed no obvious
change in the apoptotic ratio between WT and Aspp1�/� cells
(Figure S4A). Importantly, there was no significant decrease of
apoptosis in Aspp1�/� LT-HSCs under steady state (Figure 3C).
Immunofluorescence revealed that p53 was normally stabilized
in Aspp1�/� LSK cells and LT-HSCs upon IR (Figure S4C).
Nevertheless, expression profiling of apoptosis-related genes
revealed that the expressions of multiple proapoptotic genes,
whether direct transcriptional targets of p53 or not, were signif-
icantly decreased in Aspp1�/� LSK cells upon IR (Figures 3D
and S4D). Likewise, Aspp1�/� LT-HSCs showed a marked
reduction in the expression levels of proapoptotic genes (Figures
3E and S4E), corresponding with the decreased apoptosis in
Aspp1�/� HSC populations. These results clearly demonstrate
that Aspp1 is an important intrinsic regulator of HSC apoptosis
especially under genotoxic conditions.
Apoptosis is an important mechanism for eliminating severely
damaged cells. To elucidate the relationship between Aspp1
deficiency and DNA damage, LSK cells from irradiated WT and
Aspp1�/� mice were cultured in serum-free medium and immu-
nostained for 53BP1, which forms subnuclear foci at the site of
DSBs and participates mainly in NHEJ-based DSB repair (Moh-
rin et al., 2010; Schultz et al., 2000). Immediately after LSK cells
were isolated from irradiated mice, comparable numbers of
53BP1 foci formed in both WT and Aspp1�/� LSK cells (Figures
3F and 3G). However, a larger portion (�12.2%) of the Aspp1�/�
LSK cells still survived with considerable (>10) 53BP1 foci even
at 36 hr post-irradiation, whereas the majority of the cells have
only a few foci at that time (Figure 3G). These findings support
our hypothesis that Aspp1may play a significant role in removing
severely damaged HSCs and that Aspp1�/� HSCs cannot be
eliminated when suffering from unrepairable DNA damage
because of defective apoptosis.
A
B
C D
Figure 2. Sustained Self-Renewal Capacity
of Aspp1–/– HSCs
(A) Serial competitive BM transplantation usingWT
and Aspp1�/� LT-HSCs. Two hundred LT-HSCs
isolated from adult WT and Aspp1�/� mice
(CD45.2) were transplanted into lethally irradiated
recipient mice (CD45.1) with 2 3 105 competitor
BM MNCs (CD45.1). Four months later, 2 3 106
BM MNCs of primary recipients were re-trans-
planted into lethally irradiated recipient mice.
Tertiary transplantation was performed in the
same way as secondary transplantation. The
percentage of donor-derived cells in PB was
measured monthly (n = 5–6/group).
(B) Expression levels of HSC-related genes in WT
andAspp1�/� LSK cells before (pre) and 16 hr after
(post) transplantation were measured using the
multiplex qRT-PCR array (50 LSK cells/sample,
n = 16–30).
(C) In vivo BrdU short labeling analysis ofAspp1�/�
mice. The percentages of BrdU+ LT-HSCs are
shown (n = 10).
(D) Flow cytometric analysis of cell cycle of donor-
derivedWT and Aspp1�/� LSK cells 4months after
primary (1st) or secondary (2nd) BM trans-
plantation. Representative flow cytometry profiles
(left) and the average percentages ± SD of G0, G1,
and S/G2/M fractions in donor-derived LSK cells
(right) are shown (n = 6).
The horizontal lines represent the mean in (A), (B),
and (C). Data shown are mean ± SD in (D). *p <
0.05, **p < 0.01. See also Figure S3.
26 Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc.
Aspp1 Coordinates with p53 to Regulate Self-Renewaland Persistent DNA Damage of HSCsAspp1 is a p53-binding protein that activates p53-dependent
apoptosis (Samuels-Lev et al., 2001). However, several recent
studies have demonstrated multiple p53-independent roles for
Aspp1 (Hirashima et al., 2008; Wang et al., 2013). To clarify the
relationship between Aspp1 and p53 in the regulation of HSCs
in vivo, we evaluated HSCs doubly deficient for Aspp1 and p53
(Aspp1�/�p53�/�). Because Aspp1�/�p53�/� offspring were
difficult to obtain from conventional mating (Table S3), we bred
Aspp1�/� mice with both conditional p53flox/flox mice (Marino
et al., 2000) andMx1-Cremice (Kuhn et al., 1995). Cre recombi-
nase activity was induced by the administration of polyinosinic-
polycytidylic acid (pIpC) to obtain Aspp1�/�p53�/�BM cells. BM
cellularity was slightly elevated in Aspp1�/�p53�/� mice (Fig-
ure 4A). Furthermore, flow cytometric analysis revealed that
the numbers of LSK cells and LT-HSCs were most significantly
A B C
D
E
F
G
Figure 3. Impaired Apoptosis and Persistent
DNA Damage Foci in Aspp1–/– HSCs
(A) Flow cytometric analysis of apoptosis of donor-
derivedWT and Aspp1�/� LSK cells 4 months after
primary (1st) or secondary (2nd) BM trans-
plantation. Representative flow cytometry profiles
are shown.
(B) The average percentages ± SD of Annexin V+
fraction in donor-derived LSK cells (n = 6).
(C) Apoptosis analysis of WT and Aspp1�/� LT-
HSC cells before and after exposure to in vivo
ionizing radiation (IR). WT and Aspp1�/� mice
treated with 4 Gy irradiation were analyzed at 16 hr
post-irradiation. The average percentages ± SD
of Annexin V+ DAPI� fraction in LT-HSCs are
shown (n = 6).
(D) Representative heat map of the gene expres-
sion levels collected bymultiplex qRT-PCR array in
LSK cells from WT and Aspp1�/� mice 1 hr after
4 Gy irradiation. LSK cells from nonirradiated mice
were used as controls.
(E) Expression levels of proapoptotic genes in
nonirradiated and irradiated LT-HSCs derived from
WT and Aspp1�/� mice were analyzed by qRT-
PCR (n = 4). Irradiated LT-HSCswere isolated 16 hr
after 4 Gy irradiation in vivo.
(F) Immunofluorescence of irradiated WT and
Aspp1�/� LSK cells using antibodies against
53BP1. Representative pictures of 53BP1 staining
of LSK cells collected immediately (0 h) or 36 hr
after irradiation are shown.
(G) Histograms displaying the number of 53BP1
foci per cell in LSK cells after 4 Gy irradiation.
A total of 90–100 nuclei were scored and the per-
centages of nuclei expressing more than 10 foci
were calculated for each time point.
Data shown are mean ± SD. *p < 0.05, **p < 0.01.
See also Figure S4.
increased in Aspp1�/�p53�/� mice
compared with other congenic controls
(Figures 4B and 4C). Notably, the number
of LT-HSCs, especially CD150high LT-
HSCs, was significantly increased in
Aspp1�/�p53�/� mice compared with
WT, as well as in p53�/� single mutant mice (Figures 4C
and S5A).
We next performed single-cell culture using Aspp1�/�p53�/�
HSCs ex vivo to evaluate proliferation and apoptosis (Ema
et al., 2006). Single-cell culture revealed that proliferation capac-
ity was not significantly changed in Aspp1�/�p53�/� LT-HSCs
(Figures S5C and S5D). Importantly, Aspp1�/�p53�/� LT-HSCs
did not show a significant decrease in the ratio of negative wells,
which are derived from negative sorting or apoptosis (Fig-
ure S5C), indicating that apoptosis may not be a main cause
for the increased number of LT-HSCs. To evaluate self-renewal
capacity of Aspp1�/�p53�/� HSCs, we performed serial
competitive transplantation using Aspp1�/�p53�/� LSK cells.
Despite comparable proportions of LT-HSCs in the LSK
population in Aspp1�/�, p53�/�, and Aspp1�/�p53�/� mice (Fig-
ure S5B), Aspp1�/�p53�/� LSK cells showed enhanced engraft-
ment potential compared with Aspp1�/� or p53�/� single mutant
Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc. 27
LSK cells, especially after secondary transplantation (Figure 4D).
These results were consistent with the results in LSK cells
with p53, suggesting that Aspp1 has a p53-independent role in
the regulation of HSC self-renewal. Furthermore, competitive
reconstitution analysis using LT-HSCs with limiting dilution
revealed that Aspp1�/�p53�/� LT-HSCs have enhanced HSC
activity compared with single mutant LT-HSCs, and the long-
term competitive repopulation unit in Aspp1�/�p53�/� BM was
3.4-fold higher compared with 1.6-fold higher in Aspp1�/� and
p53�/� BM (Figure 4E). Thus, these results indicate that Aspp1
negatively regulates the self-renewal of HSCs that lack p53
and acts coordinately with p53 as a limiter for HSC self-renewal
upon stress stimuli.
We then addressed the question of whether Aspp1 deficiency
facilitates the survival of damaged HSCs in a p53-deficient
background by analyzing gH2AX foci as a marker of DSB sites
(Rogakou et al., 1998). Consistent with the 53BP1 staining,
Aspp1 deficiency also caused a significant increase in the
number of LSK cells with numerous (>10) gH2AX foci at 36 hr
A B
C
D
E
Figure 4. Aspp1 and p53 Coordinately
Regulate HSC Pool Size and Self-Renewal
(A) BM cellularity of adult WT, Aspp1�/�, p53�/�,and Aspp1�/�p53�/� mice (n = 3).
(B) Representative flow cytometry profiles of LSK
cells and LT-HSCs in adult WT, Aspp1�/�, p53�/�,and Aspp1�/�p53�/�BM cells. Data shown are the
mean fractions of LSK cells and LT-HSCs in BM
MNCs.
(C) Absolute numbers of LSK and LT-HSC pop-
ulations in the BM of adult WT, Aspp1�/�, p53�/�,and Aspp1�/�p53�/� mice (n = 3; �p < 0.05, ��p <
0.01 [versus WT]; #p < 0.05 [versus p53�/�]).(D) Serial competitive BM transplantation using
Aspp1�/�, p53�/�, and Aspp1�/�p53�/� LSK cells.
The percentages of donor-derived cells in PB
measured monthly after primary and secondary
transplantation are shown (n = 4–6/group).
(E) Limiting dilution analysis using LT-HSCs. Three
different doses of LT-HSCs (WT and Aspp1�/�:4, 10, and 30; p53�/� and Aspp1�/�p53�/�: 2, 5,and 15; n = 9–10/group) were transplanted into
lethally irradiated recipient mice together with
2 3 105 competitor BM MNCs. Recipients were
considered reconstituted if the percentage of
donor-derived cells in PB showed no less than 1%
at 4months post-transplantation. The frequency of
non-reconstituted mice was plotted against the
dose of transplanted LT-HSCs (left). The frequency
of functional HSCs was calculated using ELDA
software at http://bioinf.wehi.edu.au/software/
elda (middle), and the numbers of functional
HSCs per mouse were calculated using the fre-
quency (right).
The horizontal lines represent the mean in (D). Data
shown are mean ± SD in (A), (C), and (E). *p < 0.05,
**p < 0.01. See also Figure S5.
post-IR (Figures 5A, S6A, and S6B).
Notably, loss of p53 also significantly
increased the number of damaged LSK
cells, and double deletion of Aspp1 and
p53 further enhanced the survival of
severely damaged LSK cells (Figures 5A, S6A, and S6B).
Furthermore, the tolerance for DNA damage in the mutant cells
was still observed in LSK cells and LT-HSCs isolated 4 weeks af-
ter in vivo IR (Figures 5B and 5C). Consistent with the results
of ex vivo analysis, the percentages of severely damaged cells
in LSK and LT-HSC populations were significantly increased
in Aspp1�/� and p53�/� mice and were further increased in
Aspp1�/�p53�/� mice (Figure 5D), indicating that Aspp1 still
contributes to the removal of severely damaged HSCs in vivo
in the absence of p53. Taken together, these results highlight
the coordinated regulation of DNA damage accumulation in the
HSC pool by Aspp1 and p53.
Aspp1 and p53 Double-Deficient HSCs DevelopHematological MalignancyThe coordinated regulation of self-renewal and persistent DNA
damage prompted us to investigate tumorigenicity. To focus on
the development of hematological disorders, we followed up
overall survival and tumor development after competitive BM
28 Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc.
transplantation. Aspp1-deficient LSK cells did not cause a
significant increase in susceptibility to malignant lymphoma or
leukemia development (Figure 6A). However, within 6 months
after transplantation, whereas 46% of the recipients receiving
p53�/� LSK cells died of thymic lymphoma, recipient mice
transplanted with Aspp1�/�p53�/� LSK cells showed 100%
lethality (Figure 6A). These mice succumbed to an acute illness,
presenting rapid weight loss, lethargy, and dyspnea. The ma-
jority of the diseased mice exhibited thymic enlargement
(Figure S7C) and some displayed splenomegaly (Figure S7D).
Histological analysis revealed massive growths of lympho-
blast-like cells in PB, BM, and thymus (Figure 6B). Flow
cytometric analysis confirmed that the tumor cells were all
donor-derived cells, and in most cases (88%), they expressed
both CD4 and CD8 or CD8 alone (Figure 6C), revealing that
the illness was akin to T cell lymphoblastic lymphoma/
leukemia. We also observed B cell lymphoblastic leukemia
(6%) and myeloproliferative neoplasia (6%) (Figure 6D and
Table S4), indicating that the hematological disorders caused
by Aspp1�/�p53�/� LSK cells were heterogeneous. These find-
ings clearly demonstrate that double deletion of Aspp1 and p53
in HSCs eventually results in leukemia and lymphoma, and
A
B
C
D
Figure 5. Coordinated Regulation of
Persistent DNA Damage Foci in HSCs by
Aspp1 and p53
(A) Immunofluorescence of irradiated WT,
Aspp1�/�, p53�/�, and Aspp1�/�p53�/� LSK cells
using antibodies against gH2AX. The percentages
of nuclei expressing more than 10 foci immediately
(0 h) or 36 hr after 4 Gy irradiation are shown (n = 3).
(B) Representative pictures of gH2AX immunoflu-
orescence of LSK cells and LT-HSCs isolated from
WT, Aspp1�/�, p53�/�, and Aspp1�/�p53�/� mice
4 weeks after 4 Gy irradiation.
(C) Histograms displaying the number of gH2AX
foci per cell in LSK cells and LT-HSCs of each
genotype 4 weeks after 4 Gy irradiation. Approxi-
mately 102–109 nuclei were scored and the
percentages of nuclei with more than 10 foci are
displayed.
(D) The percentages of nuclei expressing more
than 10 foci 4 weeks after 4 Gy irradiation (n = 3).
Data shown are mean ± SD. *p < 0.05, **p < 0.01.
See also Figure S6.
Aspp1 protects HSCs from malignant
transformation in conjunction with p53.
To delineate the mechanism by which
Aspp1 coordinately regulates HSCs with
p53, we conducted microarray analysis
using Aspp1�/�p53�/� LSK cells. Gene
ontology (GO) analysis revealed sig-
nificant enrichment of GO terms mainly
in downregulated genes in mutant
mice (Figure 7A). GO terms associated
with phosphorylation, inflammation, and
apoptosis were highly enriched in down-
regulated genes in Aspp1�/�p53�/� LSK
cells compared with p53�/� LSK cells
(Figure 7B). We speculated that the
downregulated genes with significant enrichment of GO terms
could offer functional signatures in each comparison, and we
extracted 38 gene sets as a common signature of Aspp1 defi-
ciency by analyzing two sets of the genes that could be downre-
gulated by Aspp1 deficiency in the presence or absence of p53
(Figure 7A and Table S5). Gene set enrichment analysis (GSEA)
revealed that 9 out of the 38 gene sets were downregulated in
all three mutant LSK cells compared with WT LSK cells and
also in Aspp1�/�p53�/� LSK cells compared with p53�/� LSK
cells (Table S5). These gene sets, which are likely candidate
targets of coordinated regulation by Aspp1 and p53, include
genes associatedwith HSCs and angioimmunoblastic T cell lym-
phoma signature (Figure 7C), suggesting that Aspp1 functions
with p53 to suppress transcriptional programs associated with
HSC activity and T cell malignancy. Furthermore, corresponding
with the GO analysis, our screening also identified significant
downregulation of several immune response signatures in
Aspp1�/�p53�/� LSK cells (Figure 7C), suggesting a possible
involvement of the immune response in the p53-independent
regulation by Aspp1. Collectively, these data highlight p53-
independent pathways through which Aspp1 regulates HSC
self-renewal and tumorigenicity in coordination with p53.
Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc. 29
DISCUSSION
Accumulation of DNA damage in HSCs is correlated with aging,
hematopoietic failure, and development of hematological malig-
nancies (Beerman et al., 2014; Blanpain et al., 2011; Garaycoe-
chea et al., 2012). Thus, the management of DNA damage in
HSCs is a matter of critical importance for lifelong hematopoie-
sis. Here, we revealed that Aspp1, which is preferentially ex-
pressed in the HSC population, regulates both the size and the
soundness of the HSC pool by restricting HSC self-renewal
and quiescence in steady state, along with activating p53-
dependent apoptosis of HSCs after cellular stress. Our results
also revealed a p53-independent function of Aspp1 in HSC
self-renewal and DNA damage tolerance. Our findings under-
score the importance of the tumor suppressor network at the
stem cell level for prevention of tumorigenesis, as HSCs deficient
for both Aspp1 and p53massively accumulate DNA damage and
subsequently develop hematological malignancies.
Our findings provide amolecular basis for the preferential acti-
vation of p53-mediated apoptosis in HSCs. Although HSCs are
deemed apoptosis resistant and survive after BM injury (Mohrin
et al., 2010), HSPCs react more strongly than myeloid progenitor
cells against irradiation as shown by increased p53 phosphory-
lation and upregulation of p53 target genes ex vivo (Mohrin
et al., 2010). In addition, our results further confirmed that
LT-HSCs show even stronger activation of p53 and preferential
induction of its proapoptotic targets after exposure to severe
DNA damage in vivo. These observations indicate that p53 activ-
ity is highly potentiated and thus DNA damage is rigorously
controlled through p53 in the HSC pool, once HSCs are exposed
to cellular stress. Our results demonstrate that HSCs deficient
for Aspp1 show impaired induction of apoptosis and proapopto-
tic genes after irradiation, suggesting that Aspp1 contributes to
the potent upregulation of p53-dependent proapoptotic genes
in the HSC population after DNA damage.
A B
C
D
Figure 6. Aspp1 Coordinates with p53 to
Prevent Malignant Transformation
(A) Kaplan-Meier survival curves of mice trans-
planted withWT (n = 16), Aspp1�/� (n = 17), p53�/�
(n = 20), and Aspp1�/�p53�/� (n = 32) LSK cells.
(B) Representative histology of PB, BM, and
thymus in mice transplanted with WT and
Aspp1�/�p53�/� LSK cells.
(C) Representative flow cytometry profiles for CD4
and CD8, or CD45.1 and CD45.2, in cells from
thymus of the recipient mice transplanted with WT
or Aspp1�/�p53�/� LSK cells.
(D) Frequency of each tumor type observed in
the recipients of Aspp1�/�p53�/� LSK cells (left,
n = 16) and frequency of each immunophenotype
of thymic tumor cells observed in the recipients of
Aspp1�/�p53�/� LSK cells (right, n = 11). *p < 0.05,
**p < 0.01. See also Figure S7.
Our study highlights a differential role of
Aspp1 in fate decision of HSCs under
basal and genotoxic conditions. We
showed that Aspp1 restricts the size of
the HSC pool both phenotypically and
functionally. This is likely attributed to limited self-renewal of
HSCs, as Aspp1 does not have a major impact on their prolifer-
ation capacity and apoptosis rate during ex vivo culture,
although we cannot exclude the possibility that some changes
observed might be still attributable to potentially reduced fre-
quency of apoptotic events in the HSC compartment that is
under the detection limit. Furthermore, Aspp1 facilitates exit
from quiescence, which itself causes DNA damage that in turn
induces differentiation and attrition of HSCs (Walter et al.,
2015; Wang et al., 2012). Thus, Aspp1 regulates pool size and
self-renewal of HSCs through a cell-cycle-induced DNA damage
response. Our data also suggest that Aspp1 induces apoptosis
in the HSPC population, particularly after serial transplantation,
indicating that Aspp1 potentially regulates cell death of HSCs
after replication stress, which is a potent driver of functional
decline in aging HSCs (Flach et al., 2014). Furthermore, Aspp1
contributes to the removal of severely damaged cells from the
HSC pool by the induction of apoptosis after severe DNA dam-
age. This is reminiscent of the recent study of iAspp in HSCs
and leukemia, which revealed that overexpression of iAspp
results in blockage of apoptosis and an increase in the propor-
tion of damaged HSPCs after irradiation (Jia et al., 2014). Thus,
the balance among Aspp family members may be critical to
the fine-tuned control of DNA damage accumulation in the
HSC pool.
Our study also uncovered a p53-independent role of Aspp1 in
the regulation of HSCs. In addition to effects similar to those of
p53 deficiency, Aspp1 deficiency further increased the number
of functional HSCs in vivo, enhanced self-renewal capacity of
HSCs, and facilitated survival of HSCs carrying considerable
DNA lesions in the absence of p53. Thus, Aspp1 regulates
HSCs in both p53-dependent and -independent fashions. Our
microarray analyses identified multiple potential molecular
pathways that could be responsible for the p53-independent
function of Aspp1. We extracted several pathways relating to
30 Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc.
phosphorylation, immune response, and hypoxia-induced
response by GO analysis and GSEA. Aspp family members
can associate with protein phosphatase 1 (Helps et al., 1995),
suggesting a possibility for Aspp1 to regulate the dephosphory-
lation of various proteins. TNF-a signaling, which has a negative
impact on HSC self-renewal (Pronk et al., 2011), activates
NF-kB, and its major component RelA/p65 can associate with
Aspp1 (Yang et al., 1999). In addition, our microarray indicates
a possible involvement of the hypoxia-induced response, which
is another key component of HSC regulation and regulates the
protein level of the Aspp1-paralogue Aspp2 (Kim et al., 2014).
Whether these pathways are involved in the p53-independent
regulation of HSCs by Aspp1 should be examined in future
studies.
Finally, our results offer direct evidence that Aspp1 coordi-
nates with p53 to prevent HSCs from developing hematological
malignancies. In humans, loss of ASPP1 expression due to
hypermethylation of its promoter region has been observed in
approximately 25% of ALL patients, mostly in T cell leukemia,
and has been proven by multivariate analysis to be an indepen-
dent poor prognosis factor in ALL patients (Agirre et al., 2006;
Roman-Gomez et al., 2005). In line with this, we have biologically
proven that Aspp1 functions as a tumor suppressor for hemato-
logical malignancies in the context of p53-deficiency-driven
tumorigenesis. Aspp1 deficiency alone did not increase the
incidence of hematological tumors, seemingly because of
the contribution of p53. The coordinated tumor-suppressive
function of Aspp1 can possibly be mediated by its ability to
activate the proapoptotic function of p73 (Bergamaschi et al.,
2004), which has been implicated in a variety of hematological
A B
C
Figure 7. p53-Independent Regulation of
HSCs by Aspp1
(A) Schematic procedure of microarray analysis.
Differentially expressed genes (more than 1.5-fold)
were subjected to gene ontology (GO) analysis.
Genes downregulated in Aspp1�/� compared with
WT LSK cells and Aspp1�/�p53�/� to p53�/� LSK
cells were then subjected to the ‘‘compute over-
lap’’ tool at http://www.broadinstitute.org/gsea/
index.jsp to screen gene sets highly relevant to
the downregulated genes. The top 100 gene sets
were obtained, and overlapped gene sets are listed
in Table S5. Red-colored numbers in left-hand
boxes show the number of upregulated genes and
blue-colored numbers show the number of down-
regulated genes. Asterisks indicate significant
enrichment of GO terms in the indicated genes
revealed by GO analysis.
(B) Top 10 GO terms significantly enriched in genes
downregulated in Aspp1�/�p53�/� compared with
p53�/� LSK cells.
(C) Coordinated changes in Aspp1�/�p53�/� LSK
cells compared with p53�/� LSK cells determined
by gene set enrichment analysis (GSEA) using the
overlapped gene sets.
malignancies (Alexandrova and Moll,
2012). The preference toward T cell
malignancies was underpinned by our
microarray analysis showing that Aspp1
coordinately downregulates the signature associated with
angioimmunoblastic T cell lymphoma, a common type of mature
T cell lymphoma with poor prognosis (Palomero et al., 2014;
Sakata-Yanagimoto et al., 2014; Yoo et al., 2014), in the HSPC
population. However, several of the tumors developed from
Aspp1�/�p53�/� LSK cells were of B cell or myeloid lineage.
Because Aspp1 and p53 deficiency causes non-homogeneous
tumors, it is possible that loss of both Aspp1 and p53 may
trigger eventual acquisition of driver mutations because of ge-
netic instability in HSCs during expansion rather than through
differentiation.
Together our findings reveal that Aspp1 and p53 coordinately
maintain integrity of the HSC pool and prevent hematological
malignancies. Our study highlights Aspp1 as well as p53 as a
potential target for stem cell-directed therapy of leukemia and
lymphoma. Our BM transplantation model using HSCs deficient
for both Aspp1 and p53 may provide beneficial findings for a
comprehensive understanding of how HSCs acquire mutations
and expand their clones before the onset of hematological
malignancies.
EXPERIMENTAL PROCEDURES
Mice
Aspp1�/� mice were generated as described (Hirashima et al., 2008) and
backcrossed to C57BL/6 mice for more than six generations. p53�/� mice
(Zhang et al., 2004), Mx1-Cre mice (Kuhn et al., 1995), and p53flox/flox mice
(Marino et al., 2000) were used to obtain p53-deficient LSK cells. Deletion of
floxed p53 alleles was induced as described (Yamashita et al., 2013). Litter-
mates or age- and gender-matched mice were used as controls unless other-
wise indicated. C57BL/6 mice congenic for the CD45 locus (C57BL/6-CD45.1)
Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc. 31
were purchased from Sankyo Labo Service (Tokyo, Japan). All procedures
were performed in accordance with the guidelines for animal and recombinant
DNA experiments of Keio University.
Flow Cytometry Analysis and Isolation of Hematopoietic Cells
Cell staining and enrichment for isolation of HSCs and other hematopoietic
cells were performed as described (Arai et al., 2004; Nitta et al., 2011). Cell-
cycle analysis using BrdU and anti-Ki67 antibody and apoptosis detection
using Annexin V were performed as described (Nitta et al., 2011). For detection
of b-gal activity, BM MNCs were stained with antibodies for HSC surface
markers and then loaded with FDG (Molecular Probes) by hypotonic shock
as described (Nolan et al., 1988).
BM Transplantation
C57BL/6-CD45.1 mice were used as recipients and also as a source of
competitor BM MNCs. Serial competitive reconstitution assays were per-
formed as described (Nitta et al., 2011). For cell-cycle and apoptosis analysis
after transplantation, 200 LT-HSCs were transplanted competitively into
lethally irradiated recipient mice and 2 3 106 BM MNCs from the primary
recipients were transplanted into lethally irradiated secondary recipients.
For gene expression analysis, 2 3 107 BM MNCs were injected into lethally
irradiated recipient mice and CD45.2+ LSK cells were isolated from BM of
the recipients at 16 hr post-transplantation.
Gene Expression Analysis
RNA extraction, cDNA synthesis, and qRT-PCR analysis were performed
as described (Yamashita et al., 2013). qRT-PCR arrays after BM transplan-
tation and after irradiation in vivo were performed with a BioMark 96.96
Dynamic Array and BioMark 48.48 Dynamic Array, respectively (Fluidigm)
using the TaqMan Gene Expression Assay (Applied Biosystems) as
described (Nakamura et al., 2010). For microarray analysis, isolated RNA
from approximately 3 3 105 LSK cells of each genotype was amplified,
labeled, and hybridized to an Agilent SurePrint G3 Mouse GE 8x60K
Microarray. Normalization of signals and initial GO analyses were performed
using Agilent GeneSpring version 12.6.1. GO analysis using DAVID, and
GSEAs were performed as described (Huang et al., 2009; Subramanian
et al., 2005).
Immunofluorescence Microscopy and Western Blotting
Immunofluorescence staining of hematopoietic cells was performed as
described (Yamashita et al., 2013). Briefly, cells were attached onto a MAS-
coated glass slide (Matsunami Glass), fixed with ice-cold methanol followed
by ice-cold acetone, and stainedwith anti-phospho-p53 (Ser15) (Cell Signaling
Technology; 1:400), anti-53BP1 (Novus Biologicals; 1:200), and anti-gH2AX
(Millipore; 1:500) antibodies. Nuclei were identified by being stained with
DAPI or TOTO-3 (Invitrogen). For western blotting, antibodies against mouse
Aspp1 were generated in rabbits against a peptide corresponding to amino
acid 87–106 (IBL). Cell lysates were separated by electrophoresis and trans-
ferred to polyvinylidene fluoride (PVDF) membranes (Millipore), which were
then incubated with antibodies against Aspp1 (1:10,000) and b-actin (Sigma-
Aldrich; 1:5,000) followed by horseradish-conjugated secondary antibodies.
Protein bands were visualized using ECL Prime (GE Healthcare) and
LAS4000 (Fuji Film).
Statistical Analysis
Unpaired two-tailed Student’s t test was used for comparison between two
groups. The survival rates of mice were compared using a log-rank nonpara-
metric test. Data are shown as the means ± SD. The value of p < 0.05 was
considered significant and p < 0.01 was considered highly significant.
ACCESSION NUMBERS
The microarray data reported in this manuscript have been deposited in
the GEO databank (http://www.ncbi.nlm.nih.gov/geo) under the accession
number GEO: GSE69032.
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes seven figures, five tables,
and Supplemental Experimental Procedures and can be found with this article
online at http://dx.doi.org/10.1016/j.stem.2015.05.013.
AUTHOR CONTRIBUTIONS
M.Y. and E.N. designed the study and performed the experiments. M.Y.
analyzed and interpreted the data and wrote the manuscript. E.N. and T.S.
assisted in manuscript preparation. T.S. directed the research and secured
funding.
ACKNOWLEDGMENTS
We thank Dr. Tohru Minamino (currently Niigata University, Niigata, Japan) for
providing the p53flox/flox mice; Dr. John E. Dick (University of Toronto, Toronto,
Canada), Dr. Michael Milyavsky (Tel Aviv University, Ramat Aviv, Israel), and
Dr. Masanori Hirashima (Kobe University, Kobe, Japan) for insights and
constructive suggestions; and all members of the Suda laboratory for com-
ments and discussion. We are grateful to the Collaborative Research
Resources, School of Medicine, Keio University for technical support and re-
agents. This work was supported by a Grant-in-Aid for Scientific Research
and a Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Cancer
Stem Cells’’ from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of Japan; the Global COE program ‘‘Education and
Research Center for Stem Cell Medicine’’ of Keio University; a Grant-in-Aid
for Japan Society for the Promotion of Science Fellows; and a grant-in-aid
from Takeda Science Foundation. M.Y. and E.N. are Research Fellows of
the Japan Society for the Promotion of Science.
Received: July 25, 2014
Revised: April 2, 2015
Accepted: May 26, 2015
Published: June 25, 2015
REFERENCES
Agirre, X., Roman-Gomez, J., Jimenez-Velasco, A., Garate, L., Montiel-Duarte,
C., Navarro, G., Vazquez, I., Zalacain, M., Calasanz, M.J., Heiniger, A., et al.
(2006). ASPP1, a common activator of TP53, is inactivated by aberrant
methylation of its promoter in acute lymphoblastic leukemia. Oncogene 25,
1862–1870.
Alexandrova, E.M., andMoll, U.M. (2012). Role of p53 family members p73 and
p63 in human hematological malignancies. Leuk. Lymphoma 53, 2116–2129.
Arai, F., Hirao, A., Ohmura, M., Sato, H., Matsuoka, S., Takubo, K., Ito, K., Koh,
G.Y., and Suda, T. (2004). Tie2/angiopoietin-1 signaling regulates hematopoi-
etic stem cell quiescence in the bone marrow niche. Cell 118, 149–161.
Beerman, I., Bhattacharya, D., Zandi, S., Sigvardsson, M., Weissman, I.L.,
Bryder, D., and Rossi, D.J. (2010). Functionally distinct hematopoietic stem
cells modulate hematopoietic lineage potential during aging by a mechanism
of clonal expansion. Proc. Natl. Acad. Sci. USA 107, 5465–5470.
Beerman, I., Seita, J., Inlay, M.A., Weissman, I.L., and Rossi, D.J. (2014).
Quiescent hematopoietic stem cells accumulate DNA damage during aging
that is repaired upon entry into cell cycle. Cell Stem Cell 15, 37–50.
Bergamaschi, D., Samuels, Y., O’Neil, N.J., Trigiante, G., Crook, T., Hsieh,
J.K., O’Connor, D.J., Zhong, S., Campargue, I., Tomlinson, M.L., et al.
(2003). iASPP oncoprotein is a key inhibitor of p53 conserved from worm to
human. Nat. Genet. 33, 162–167.
Bergamaschi, D., Samuels, Y., Jin, B., Duraisingham, S., Crook, T., and Lu, X.
(2004). ASPP1 and ASPP2: common activators of p53 family members. Mol.
Cell. Biol. 24, 1341–1350.
Blanpain, C., Mohrin, M., Sotiropoulou, P.A., and Passegue, E. (2011).
DNA-damage response in tissue-specific and cancer stem cells. Cell Stem
Cell 8, 16–29.
32 Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc.
Cabezas-Wallscheid, N., Klimmeck, D., Hansson, J., Lipka, D.B., Reyes, A.,
Wang, Q., Weichenhan, D., Lier, A., von Paleske, L., Renders, S., et al.
(2014). Identification of regulatory networks in HSCs and their immediate prog-
eny via integrated proteome, transcriptome, and DNA Methylome analysis.
Cell Stem Cell 15, 507–522.
Corces-Zimmerman, M.R., Hong, W.J., Weissman, I.L., Medeiros, B.C., and
Majeti, R. (2014). Preleukemic mutations in human acute myeloid leukemia
affect epigenetic regulators and persist in remission. Proc. Natl. Acad. Sci.
USA 111, 2548–2553.
de Laval, B., Pawlikowska, P., Petit-Cocault, L., Bilhou-Nabera, C., Aubin-
Houzelstein, G., Souyri, M., Pouzoulet, F., Gaudry, M., and Porteu, F. (2013).
Thrombopoietin-increased DNA-PK-dependent DNA repair limits hematopoi-
etic stem and progenitor cell mutagenesis in response to DNA damage. Cell
Stem Cell 12, 37–48.
Ema, H., Morita, Y., Yamazaki, S., Matsubara, A., Seita, J., Tadokoro, Y.,
Kondo, H., Takano, H., and Nakauchi, H. (2006). Adult mouse hematopoietic
stem cells: purification and single-cell assays. Nat. Protoc. 1, 2979–2987.
Flach, J., Bakker, S.T., Mohrin, M., Conroy, P.C., Pietras, E.M., Reynaud, D.,
Alvarez, S., Diolaiti, M.E., Ugarte, F., Forsberg, E.C., et al. (2014).
Replication stress is a potent driver of functional decline in ageing haemato-
poietic stem cells. Nature 512, 198–202.
Garaycoechea, J.I., Crossan, G.P., Langevin, F., Daly, M., Arends, M.J., and
Patel, K.J. (2012). Genotoxic consequences of endogenous aldehydes on
mouse haematopoietic stem cell function. Nature 489, 571–575.
Helps, N.R., Barker, H.M., Elledge, S.J., and Cohen, P.T. (1995). Protein phos-
phatase 1 interacts with p53BP2, a protein which binds to the tumour suppres-
sor p53. FEBS Lett. 377, 295–300.
Hirashima,M., Bernstein, A., Stanford,W.L., and Rossant, J. (2004). Gene-trap
expression screening to identify endothelial-specific genes. Blood 104,
711–718.
Hirashima, M., Sano, K., Morisada, T., Murakami, K., Rossant, J., and Suda, T.
(2008). Lymphatic vessel assembly is impaired in Aspp1-deficient mouse em-
bryos. Dev. Biol. 316, 149–159.
Huang, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integra-
tive analysis of large gene lists using DAVID bioinformatics resources. Nat.
Protoc. 4, 44–57.
Jia, Y., Peng, L., Rao, Q., Xing, H., Huai, L., Yu, P., Chen, Y., Wang, C., Wang,
M., Mi, Y., et al. (2014). Oncogene iASPP enhances self-renewal of hematopoi-
etic stem cells and facilitates their resistance to chemotherapy and irradiation.
FASEB J. 28, 2816–2827.
Kim, H., Claps, G., Moller, A., Bowtell, D., Lu, X., and Ronai, Z.A. (2014). Siah2
regulates tight junction integrity and cell polarity through control of ASPP2
stability. Oncogene 33, 2004–2010.
Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995). Inducible gene tar-
geting in mice. Science 269, 1427–1429.
Lerner, C., and Harrison, D.E. (1990). 5-Fluorouracil spares hemopoietic stem
cells responsible for long-term repopulation. Exp. Hematol. 18, 114–118.
Liu, Y., Elf, S.E., Miyata, Y., Sashida, G., Liu, Y., Huang, G., Di Giandomenico,
S., Lee, J.M., Deblasio, A., Menendez, S., et al. (2009). p53 regulates hemato-
poietic stem cell quiescence. Cell Stem Cell 4, 37–48.
Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J., and Berns, A. (2000).
Induction ofmedulloblastomas in p53-null mutantmice by somatic inactivation
of Rb in the external granular layer cells of the cerebellum. Genes Dev. 14,
994–1004.
Milyavsky, M., Gan, O.I., Trottier, M., Komosa, M., Tabach, O., Notta, F.,
Lechman, E., Hermans, K.G., Eppert, K., Konovalova, Z., et al. (2010). A
distinctive DNA damage response in human hematopoietic stem cells reveals
an apoptosis-independent role for p53 in self-renewal. Cell Stem Cell 7,
186–197.
Mohrin, M., Bourke, E., Alexander, D., Warr, M.R., Barry-Holson, K., Le Beau,
M.M., Morrison, C.G., and Passegue, E. (2010). Hematopoietic stem cell
quiescence promotes error-prone DNA repair and mutagenesis. Cell Stem
Cell 7, 174–185.
Morita, Y., Ema, H., and Nakauchi, H. (2010). Heterogeneity and hierarchy
within the most primitive hematopoietic stem cell compartment. J. Exp.
Med. 207, 1173–1182.
Nakamura, Y., Arai, F., Iwasaki, H., Hosokawa, K., Kobayashi, I., Gomei, Y.,
Matsumoto, Y., Yoshihara, H., and Suda, T. (2010). Isolation and characteriza-
tion of endosteal niche cell populations that regulate hematopoietic stem cells.
Blood 116, 1422–1432.
Nitta, E., Yamashita, M., Hosokawa, K., Xian, M., Takubo, K., Arai, F., Nakada,
S., and Suda, T. (2011). Telomerase reverse transcriptase protects ATM-defi-
cient hematopoietic stem cells from ROS-induced apoptosis through a telo-
mere-independent mechanism. Blood 117, 4169–4180.
Nolan, G.P., Fiering, S., Nicolas, J.F., and Herzenberg, L.A. (1988).
Fluorescence-activated cell analysis and sorting of viable mammalian cells
based on beta-D-galactosidase activity after transduction of Escherichia coli
lacZ. Proc. Natl. Acad. Sci. USA 85, 2603–2607.
Orkin, S.H., and Zon, L.I. (2008). Hematopoiesis: an evolving paradigm for
stem cell biology. Cell 132, 631–644.
Palomero, T., Couronne, L., Khiabanian, H., Kim, M.Y., Ambesi-Impiombato,
A., Perez-Garcia, A., Carpenter, Z., Abate, F., Allegretta, M., Haydu, J.E.,
et al. (2014). Recurrent mutations in epigenetic regulators, RHOA and FYN ki-
nase in peripheral T cell lymphomas. Nat. Genet. 46, 166–170.
Pronk, C.J., Veiby, O.P., Bryder, D., and Jacobsen, S.E. (2011). Tumor necro-
sis factor restricts hematopoietic stem cell activity in mice: involvement of two
distinct receptors. J. Exp. Med. 208, 1563–1570.
Quivoron, C., Couronne, L., Della Valle, V., Lopez, C.K., Plo, I., Wagner-Ballon,
O., Do Cruzeiro, M., Delhommeau, F., Arnulf, B., Stern, M.H., et al. (2011). TET2
inactivation results in pleiotropic hematopoietic abnormalities in mouse and is
a recurrent event during human lymphomagenesis. Cancer Cell 20, 25–38.
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., and Bonner, W.M. (1998).
DNA double-stranded breaks induce histone H2AX phosphorylation on serine
139. J. Biol. Chem. 273, 5858–5868.
Roman-Gomez, J., Jimenez-Velasco, A., Agirre, X., Prosper, F., Heiniger, A.,
and Torres, A. (2005). Lack of CpG island methylator phenotype defines a clin-
ical subtype of T-cell acute lymphoblastic leukemia associated with good
prognosis. J. Clin. Oncol. 23, 7043–7049.
Rossi, D.J., Jamieson, C.H., and Weissman, I.L. (2008). Stems cells and the
pathways to aging and cancer. Cell 132, 681–696.
Sakata-Yanagimoto, M., Enami, T., Yoshida, K., Shiraishi, Y., Ishii, R., Miyake,
Y., Muto, H., Tsuyama, N., Sato-Otsubo, A., Okuno, Y., et al. (2014). Somatic
RHOA mutation in angioimmunoblastic T cell lymphoma. Nat. Genet. 46,
171–175.
Samuels-Lev, Y., O’Connor, D.J., Bergamaschi, D., Trigiante, G., Hsieh, J.K.,
Zhong, S., Campargue, I., Naumovski, L., Crook, T., and Lu, X. (2001). ASPP
proteins specifically stimulate the apoptotic function of p53. Mol. Cell 8,
781–794.
Schultz, L.B., Chehab, N.H., Malikzay, A., and Halazonetis, T.D. (2000). p53
binding protein 1 (53BP1) is an early participant in the cellular response to
DNA double-strand breaks. J. Cell Biol. 151, 1381–1390.
Shlush, L.I., Zandi, S., Mitchell, A., Chen, W.C., Brandwein, J.M., Gupta, V.,
Kennedy, J.A., Schimmer, A.D., Schuh, A.C., Yee, K.W., et al.; HALT
Pan-Leukemia Gene Panel Consortium (2014). Identification of pre-leukaemic
haematopoietic stem cells in acute leukaemia. Nature 506, 328–333.
Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L.,
Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., and
Mesirov, J.P. (2005). Gene set enrichment analysis: a knowledge-based
approach for interpreting genome-wide expression profiles. Proc. Natl.
Acad. Sci. USA 102, 15545–15550.
Trigiante, G., and Lu, X. (2006). ASPP [corrected] and cancer. Nat. Rev. Cancer
6, 217–226.
Van Zant, G. (1984). Studies of hematopoietic stem cells spared by 5-fluoro-
uracil. J. Exp. Med. 159, 679–690.
Walter, D., Lier, A., Geiselhart, A., Thalheimer, F.B., Huntscha, S., Sobotta,
M.C., Moehrle, B., Brocks, D., Bayindir, I., Kaschutnig, P., et al. (2015). Exit
Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc. 33
from dormancy provokes DNA-damage-induced attrition in haematopoietic
stem cells. Nature 520, 549–552.
Wang, J., Sun, Q., Morita, Y., Jiang, H., Gross, A., Lechel, A., Hildner, K.,
Guachalla, L.M., Gompf, A., Hartmann, D., et al. (2012). A differentiation
checkpoint limits hematopoietic stem cell self-renewal in response to DNA
damage. Cell 148, 1001–1014.
Wang, Y., Godin-Heymann, N., Dan Wang, X., Bergamaschi, D., Llanos, S.,
and Lu, X. (2013). ASPP1 and ASPP2 bind active RAS, potentiate RAS sig-
nalling and enhance p53 activity in cancer cells. Cell Death Differ. 20,
525–534.
Wilson, A., Laurenti, E., Oser, G., van der Wath, R.C., Blanco-Bose, W.,
Jaworski, M., Offner, S., Dunant, C.F., Eshkind, L., Bockamp, E., et al.
(2008). Hematopoietic stem cells reversibly switch from dormancy to self-
renewal during homeostasis and repair. Cell 135, 1118–1129.
Yamashita, M., Nitta, E., Nagamatsu, G., Ikushima, Y.M., Hosokawa, K., Arai,
F., and Suda, T. (2013). Nucleostemin is indispensable for the maintenance
and genetic stability of hematopoietic stem cells. Biochem. Biophys. Res.
Commun. 441, 196–201.
Yang, J.P., Hori, M., Sanda, T., and Okamoto, T. (1999). Identification of a
novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor. J. Biol.
Chem. 274, 15662–15670.
Yoo, H.Y., Sung, M.K., Lee, S.H., Kim, S., Lee, H., Park, S., Kim, S.C., Lee, B.,
Rho, K., Lee, J.E., et al. (2014). A recurrent inactivating mutation in RHOA
GTPase in angioimmunoblastic T cell lymphoma. Nat. Genet. 46, 371–375.
Yoshihara, H., Arai, F., Hosokawa, K., Hagiwara, T., Takubo, K., Nakamura, Y.,
Gomei, Y., Iwasaki, H., Matsuoka, S., Miyamoto, K., et al. (2007).
Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence
and interaction with the osteoblastic niche. Cell Stem Cell 1, 685–697.
Zhang, D., Hirota, T., Marumoto, T., Shimizu, M., Kunitoku, N., Sasayama, T.,
Arima, Y., Feng, L., Suzuki, M., Takeya, M., and Saya, H. (2004). Cre-loxP-
controlled periodic Aurora-A overexpression induces mitotic abnormalities
and hyperplasia in mammary glands of mouse models. Oncogene 23, 8720–
8730.
34 Cell Stem Cell 17, 23–34, July 2, 2015 ª2015 Elsevier Inc.