Leukemia Research 65 (2018) 49–54

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Leukemia Research
journal homepage: www.elsevier.com/locate/leukres

Research paper

Leukemia cells impair normal hematopoiesis and induce functionally loss of T

hematopoietic stem cells through immune cells and inflammation
⁎
Ping Cui , Yuhua Zhang, Maoxiang Cui, Zhihong Li, Guang Ma, Rufeng Wang, Ning Wang,
Shujuan Huang, Jie Gao
Department of Pathology, Cangzhou Medical College, China

A R T I C L E I N F O A B S T R A C T

Keywords: Bone marrow (BM) failure is often seen in leukemia patients, indicating an abnormal hematopoietic process.
Leukemia However, hematopoiesis in leukemic milieus is largely unknown. In the present study, we utilized one of the
MLL-AF9 most frequent leukemogenic translocations MLL-AF9 to induce leukemia and investigated the hematopoiesis and
Hematopoiesis the activity of hematopoietic stem and progenitor cells (HSPCs) in a leukemic milieu. We found that the phe-
Hematopoietic stem and progenitor cells
notypes of the non-leukemic population in leukemic BM were drastically different than normal BM, including
Inflammation
blockage of differentiation and a drastically reduced Lin-/Sca+/c-kit+ (LSK) population that contains all HSPCs
in leukemic BM. Further, transplantation assays demonstrated that stem cell function of HSPCs from leukemic
BM was significantly compromised. Intriguingly, BM from a patient-derived xenograft leukemia model and from
immunocompromised mice transplanted with murine MLL-AF9 cells, showed comparable percentage of hema-
topoietic stem cells (HSCs) to normal controls, indicating that an immunocompetent microenvironment is cri-
tical for leukemia-induced loss of HSPCs. Mechanistically, we found that the non-leukemic cells from leukemic
BM possessed a more inflammatory profile than either leukemic cells or normal BM counterparts. Co-culturing or
co-transplantation with non-leukemic cells from leukemic BM impaired the stem cell function of normal HSPCs
in vitro and in vivo respectively, suggesting that the highly inflammatory non-leukemic population in leukemic
BM not only is functionally abnormal but displayed a ‘leukemia-like’ characteristic. Finally, we tested the effect
of the anti-inflammation drug diclofenac on leukemia mice. However, no phenotypic changes of HSPCs were
observed upon diclofenac treatment due to only mild repression of inflammatory genes by diclofenac, further
indicating that inflammation is a powerful negative regulator of HSPCs. Together, our results suggest that
leukemia impairs normal hematopoiesis and inflammation as well as immune cells play a critical role in leu-
kemia-induced BM failure.

1. Introduction leukemia samples indicate functional preservation of HSPCs in leu-

The function of hematopoietic stem and progenitor cells (HSPCs)
are essential for a steady state of hematopoiesis. Abnormalities in
HSPCs lead to bone marrow (BM) failure or malignant transformation.
Therefore, the activity of HSPCs is tightly regulated and closely mon-
itored. Biological disturbance to HSPCs such as dysregulation of cell
cycle, which could be triggered by inflammation, results in exhaustion
of HSPCs [1].
BM failure is often observed in leukemia patients and is one of the
leading causes for leukemia-related death. Thus, there seems to be a
causal relation between the malfunction of HSPCs and leukemia-in-
duced BM failure. However, the biological activity of HSPCs and he-
matopoietic process in leukemic milieus remains largely unknown.
Studies using immunocompromised mice transplanted with human
kemic BM [2]. However, since the activity and function of HSPCs are
highly regulated and maintained by immune cells and BM micro-
environment [3,4], utilization of immunocompromised mice whose
immune system and microenvironment are differing than im-
munocompetent mice, may not fully reflect the hematopoiesis in leu-
kemic milieus. Therefore, using immunocompetent models is required
for this line of investigation.
MLL translocations are found in leukemias across all ages especially
in childhood leukemias [5,6]. Patients with MLL translocations are
usually classified into an intermediate to poor risk groups [7,8]. In the
present study, we explored the hematopoiesis in a MLL-AF9 transloca-
tion induced leukemia model. MLL-AF9 is one of the most frequent MLL
rearrangements especially in childhood acute myeloid leukemia (AML)
[5]. We found severely impaired hematopoiesis in leukemic BM. We

⁎
Corresponding author at: Cangzhou Medical College, Cangzhou, Heibei, 061001, China.
E-mail address: ping.t.cui@gmail.com (P. Cui).

https://doi.org/10.1016/j.leukres.2018.01.002
Received 27 August 2017; Received in revised form 21 November 2017; Accepted 1 January 2018
Available online 02 January 2018
0145-2126/ © 2018 Elsevier Ltd. All rights reserved.
P. Cui et al. Leukemia Research 65 (2018) 49–54

further demonstrated stem cell function of non-leukemic compartment
was significantly compromised. Additionally, we found that the non-
leukemic compartment had a more inflammatory profile than leukemia
cells and might result in defected HSPCs. Interestingly, we observed
unaltered percentage of HSCs in immunocompromised mice trans-
planted with either human leukemia samples or murine MLL-AF9 leu-
kemia cells, suggesting that the immune microenvironment was in-
volved in leukemia-induced impairment of hematopoiesis.
2.6. Flow cytometry
All antibodies used for this study were purchased from BD bios-
ciences. Analyses were performed using FACSCelesta (BD biosciences)
and Fluorescence-activated cell sorting were done by FACSAria II sorter
(BD biosciences).
2.7. Competitive transplantation and LSK cells coculturing

A CD45 chimerism system was utilized to generate leukemia for

2. Material and methods
following competitive transplantation. Briefly, primary leukemia cells
(GFP+ cells on a CD45.1 background) were transplanted into uni-
2.1. Generation of leukemia models
rradiated recipient mice (CD45.2 background) to generate secondary
leukemias. GFP- (CD45.2+) cells from leukemic or normal BM
MLL-AF9 leukemic model was generated by transplantation of Sca
(CD45.2+) were isolated and mixed at 1:1 ratio with CD45.1+ cells
+/c-kit+/lin- (LSK) cells infected with GFP-MLL-AF9 retrovirus into
isolated from normal BM respectively. Cell mixtures were injected into
normal recipients (C57BL/6J). In this study, a secondary leukemic
lethally irradiated recipient mice (1 million cells/mouse). Another
transplantation was generated to study the hematopoiesis in a leukemic
group of recipients were transplanted with only CD45.1+ cells isolated
milieu. Briefly, LSK cells were isolated from normal BM by fluores-
from normal BM at a half cell dose (0.5 million cells/mouse).
cence-activated cell sorting (FACS) and cultured overnight in LSK
For LSK cells coculturing experiment, GFP- (CD45.2+) cells from
medium (10% FBS IMDM containing 10 ng/ml IL3, 10 ng/ml IL6,
leukemic or normal BM (CD45.2+) were isolated and mixed at 10:1
50 ng/ml SCF, 50 ng/ml Flt3-ligand). MLL-AF9 retrovirus was then in-
ratio with CD45.1+ LSK cells isolated from normal BM respectively.
troduced to LSK cells the following day. One day after infection, LSK
Cell mixtures were cultured in LSK medium (10% FBS IMDM containing
cells were injected into recipient mice retro-orbitally. Primary leukemia
10 ng/ml IL3, 10 ng/ml IL6, 50 ng/ml SCF, 50 ng/ml Flt3-ligand) for
cells from spleen and bone marrow (BM) were frozen for future usage.
10 days, and CD45.1 cells were isolated after coculturing and re-
To generate secondary leukemia, GFP+ cells from primary leukemia
suspended in methylcellulose complete media (50000 cells/plate). LSK
were isolated by FACS and transplanted into unirradiated recipient
cells cultured alone were served as control. Colony numbers were
mice (1 million cells/mouse).
counted at 10 days post methylcellulose culturing.
To generate MLL-AF9 leukemia in NOD scid gamma (NSG) mice,
GFP+ cells from primary leukemia were isolated and transplanted into
2.8. ELISA analysis
unirradiated naïve NSG mice (1 million cells/mouse).
AML patient sample derived xenograft model was created by
Equal number of normal BM cells, GFP- (non-leukemia) cells and
transplantation of AML patient samples to NOD scid gamma (NSG) mice
GFP+ (leukemia) cells were isolated and cultured in 10% FBS IMDM
(5 million cells/mouse). Recipients were sacrificed at 6 weeks post
for 24 h (1 million cells/ml). Culturing medium was collected and spin
transplantation.
down to get rid of cell debris. Supernatant was subjected to ELISA
All animal experiments were approved by Cangzhou medical
analysis per manufacturer’s instruction (R & D system).
College.
2.9. Statistical analysis
2.2. In vivo BrdU labeling (Cell cycle analyses)
Two-tailed student’s t-test with unpaired analysis was performed for
BrdU incorporation experiments were performed as described pre- relevance between biological factors. P < 0.05 was considered sig-
viously [9,10]. nificant.

2.3. In vivo drug treatment
Starting at day 6 after leukemic transplantation, leukemia mice
were treated with diclofenac (10 mg/kg/day, Sigma) intra-peritoneally.
Treatment was stopped one day before sacrificing the mice.
2.4. Colony forming assay
Equal number of lin-/GFP- cells were sorted from leukemic and
normal BM and resuspended in methylcellulose complete media (R&D
system) (2000 cells/plate). Numbers of colonies were counted at day 10
after culturing. For 2nd plating, cells were recovered from methylcel-
lulose media and equal number of cells (10000 cells/plate) were re-
suspended in methylcellulose complete media. Numbers of colonies
were counted at day 10 after culturing.
2.5. HSPCs transplantation
Equal number of lin-/GFP- cells were sorted from leukemic and
normal BM. Cells were then injected into lethally irradiated recipient
mice (1 million cells/mouse). Survival of recipients was monitored.
3. Results
3.1. Abnormal phenotypes of the non-leukemia population in the MLL-AF9
leukemia BM
To explore hematopoiesis in a leukemic milieu, we first examined
the phenotype of the non-leukemic compartment in a MLL-AF9 trans-
location induced leukemia model. We transplanted primary MLL-AF9
leukemic cells into unirradiated mice to generate secondary leukemias
and explored the hematopoiesis in these secondary leukemia mice. The
expression of MLL-AF9 and thus the leukemia cells were monitored by
green fluorescent protein (GFP) in this model [11]. We confirmed that
MLL-AF9 was exclusively expressed in the GFP+ population not in the
GFP- population (Supplemental Fig. 1).
We observed that the percentage of lineage- (lin-) cells in the non-
leukemic (GFP-) compartment was significantly increased (Fig. 1A and
B) when the leukemic burden was about 40% in BM (Supplemental
Fig. 2A), indicating blockage of the normal hematopoietic differentia-
tion. Cell cycle analyses showed that lin-/GFP- cells were more actively
cycling than their counterparts in normal BM (Fig. 1C and D), which
corroborated the expansion of lin-/GFP- cells in leukemic BM. Ad-
ditionally, we analyzed the composition of lin+ population and found
that the percentage of B cells was remarkably decreased while the

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P. Cui et al. Leukemia Research 65 (2018) 49–54

Fig. 1. Abnormal phenotypes of the non-leukemic compartment in leukemic BM. A and B. Increased percentage of lin- non-leukemia (GFP-) cells in leukemic BM. C and D. Increased BrdU
+ cells in the lin-/GFP- population from leukemic BM. E-G. Percentage of B cells (B220+ cells) (E), myeloid cells (Gr1+/Mac1+ cells) (F) and T cells (CD3e+ cells) (G) in leukemic and
normal BM. H and I. Depletion of LSK population in leukemic BM. n = 5–7. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Fig. 2. Stem cell function of HSPCs from leukemic BM is impaired. A. Colony forming ability of lin-/GFP- cells from leukemic BM was reduced. B. A schematic diagram showing
transplantation experiment. C. Survival rate of recipients transplanted with lin-/GFP- cells from normal or leukemic BM. D. A schematic diagram showing the competitive transplantation
experiment. E. Survival rate of recipients showed in D. n = 9. ***p < 0.0005. F. A schematic diagram showing the LSK coculturing experiment. G. Colony numbers generated from LSK
cells shown in F. n = 3. **p < 0.005.

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P. Cui et al.
Fig. 3. An immunocompetent milieu contributes to leukemia induced HSPCs loss. A. Flow plot showing the engraftment of human AML cells in NSG mice. B-E. Phenotypic comparisons of
non-leukemic compartment (mCD45+ cells) including lin- cells (B), LSK cells (C) and HSCs (D) between leukemic and normal BM. E. HSC cell cycle comparison between leukemic and
normal BM. F-H. NSG mice were transplanted with mouse MLL-AF9 leukemia cells. Percentage of lin- cells (F), LSK cells (G) and HSCs (H) was compared between leukemic and normal
BM. n = 7. n.s., not significant. **, p < 0.005; ***, p < 0.0005.
percentage of myeloid cells was increased (Fig. 1E and F). T cells were
slightly by significantly reduced in leukemic BM (Fig. 1G) indicating a
biased differentiation in leukemic BM [12]. We next examined LSK (Sca
+/c-kit+/lin-) population, which contains all the hematopoietic stem
and progenitors (HSPCs), in leukemic BM. Strikingly, LSK cells were
almost depleted in leukemic BM with reduced expression of both c-kit+
and Sca+ (Fig. 1H and I). Together, these data suggest that the normal
hematopoiesis in leukemic BM is disrupted.
3.2. Activity of HSCs is functionally compromised in leukemic BM
Based on the above phenotypic findings, we hypothesized that the
stem cell function of HSPCs in leukemia mice was impaired. To test this
hypothesis, we first performed colony forming assays. Equaxl numbers
of lin-/GFP- cells were isolated from leukemic and normal BM and their
abilities to form colonies were tested. We found the colony numbers
generated from leukemic BM were significantly less compared to
normal BM (Fig. 2A). This difference was more exacerbated when the
2nd plating was performed (Fig. 2A). To evaluate the stem cell function
of HSPCs in vivo, we transplanted lin-/GFP- cells from leukemic and
normal BM to lethally irradiated recipients (Fig. 2B). Surprisingly, all
recipients receiving lin-/GFP- cells from leukemic BM were dead in less
than 3 weeks, while recipients transplanted with normal BM stayed
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Leukemia Research 65 (2018) 49–54
alive for more than 17 weeks (Fig. 2C).
To confirm that the death of recipients was due to the impaired
function of HSPCs, we performed competitive transplantation assay
(Fig. 2D). Strikingly, recipient mice transplanted with cell mixture of
GFP- cells (CD45.2+ cells) from leukemic BM and normal BM
(CD45.1+ cells) died within 3 weeks (group A), similar to what we
observed in Fig. 2C, while recipients transplanted with either normal
BM cell mixture (CD45.1 and CD45.2 BM cell mixture, group B) or
normal BM cells (CD45.1, group C) at a half cell dose survived for more
than 12 weeks (Fig. 2E), suggesting that the non-leukemia cells from
leukemic BM were not only defective in HSPCs but impaired the
function of normal HSPCs. We further tested this hypothesis by ex-
amining the colony forming ability of normal LSK cells cocultured with
either non-leukemia cells (GFP- cells) from leukemic BM (group A) or
normal BM cells (group B) or cultured alone (group C) (Fig. 2F). As we
expected, the number of colonies generated from LSK cells cocultured
with non-leukemia cells from leukemic BM were significantly reduced
(Fig. 2G). Together, these data strongly support that the stem cell
function of HSPCs in leukemia mice is severely and permanently im-
paired.
P. Cui et al. Leukemia Research 65 (2018) 49–54

Fig. 4. Non-leukemic cells are the major inflammatory contributor. A. Expression of inflammatory genes in GFP- and GFP+ cells from leukemic BM and in normal BM (NBM). B.
Quantitative measurement of inflammatory cytokines in conditioned medium (CM) from normal BM (NBM) cells, GFP+ leukemia cells and GFP- non-leukemic cells from leukemic BM by
ELISA. C. BM leukemic burden in leukemic mice treated with saline and diclofenac. D. Body weight changes in leukemic mice treated with saline and diclofenac. E. Percentage of lin-/
GFP- cells in leukemic mice treated with saline and diclofenac. F. Flow plot showing that treatment with diclofenac did not prevent loss of LSK population in leukemia mice. G. Expression
of inflammatory genes in BM GFP- population from saline and diclofenac treated leukemia mice. n = 5. n.s., not significant; ND, not detectable. *, p < 0.05; **, p < 0.005; ***,
p < 0.0005.

3.3. An immunocompetent microenvironment is required for leukemia- that the percentage of LSK cells was decreased in MLL-AF9 NSG mice
induced loss of HSCs compared to normal NSG mice (Fig. 3G). However, compared to im-
munocompetent mice, the loss of LSK cells in MLL-AF9 NSG mice was
To explore whether human leukemia cells would have similar ef- less severe. Interestingly, we found that there was a trend of increased
fects on normal hematopoiesis, we examined the phenotype of BM non- percentage of HSCs (LSK/CD150+/CD48- cells) in MLL-AF9 NSG mice
leukemic compartment (mouse CD45+ cells) from NSG mice trans- (Fig. 3H), indicating that stem cell function of HSPCs in MLL-AF9 NSG
planted with two human AML samples harboring MLL rearrangements mice was preserved. Together, these data suggest that an im-
(AML1 and AML2) (Supplemental Table 1 and Fig. 3A). Intriguingly, we munocompetent milieu contributes to leukemia-induced loss of HSPCs.
did not find any significant differences in the percentage of lin- cells
(Fig. 3B) and LSK cells (Fig. 3C) between leukemic and normal BM. To
3.4. The non-leukemia population is highly inflammatory
further compare the HSPC population, we utilized CD150 and CD48 to
dissect this population to multipotent progenitors (MPPs) and hema-
Studies have shown that inflammation results in abnormal hema-
topoietic stem cells (HSCs) [13]. No significant differences in the per-
topoiesis [14]. And systemic inflammation is observed in leukemia
centage HSCs were observed between leukemic and normal BM
mice, which induces weight loss [10]. Intriguingly, we found that the
(Fig. 3D). Additionally, no differences in cell cycle were observed be-
non-leukemic (GFP-) population in MLL-AF9 leukemic BM possessed a
tween HSCs in leukemic and normal BM (Fig. 3E). Together, these data
more inflammatory profile than either leukemic cells (GFP+ cells) or
indicate leukemia mice transplanted with human leukemia cells have
normal BM cells (Fig. 4A and B). To test the involvement of in-
phenotypically normal HSPCs.
flammation in the leukemia-induced malfunction of HSPCs, leukemia
The above results suggest that either an immunocompetent milieu is
mice were treated with diclofenac, a nonsteroidal anti-inflammatory
critical for leukemia-induced impairment of hematopoiesis or human
drug. We found that although diclofenac treatment reduced weight loss
leukemia cells are not able to produce certain factors to affect murine
in leukemia mice, it did not alter BM leukemic burden (Fig. 4C and D).
hematopoiesis. To test whether an intact immune system is required for
Further, there were no phenotypic differences in the non-leukemic
leukemia-induced impairment of hematopoiesis, NSG mice were
(GPF-) compartment between diclofenac-treated and saline-treated
transplanted with mouse MLL-AF9 leukemia cells and phenotypes of
groups (Fig. 4E and F). Analysis of the non-leukemic population (GFP-
non-leukemic compartment (GFP- cells) were examined when leukemia
cells) showed that diclofenac only partially reduced some of the in-
burden was about 40% (Supplemental Fig. 3). We observed increased
flammatory genes expression (Fig. 4G), which may explain the lack of
percentage of lin- cells in MLL-AF9 NSG mice (Fig. 3F). We also noticed
efficiency of diclofenac on hematopoiesis in leukemia mice.

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4. Discussion
In the present study, we found that the stem cell function of HSPCs
was permanently lost in a MLL-AF9 translocation induced leukemia
model. Phenotypically, we found increased percentage of immature
(lin-) non-leukemic (GFP-) cells in leukemic BM. Additionally, a biased
differentiation was observed in leukemic BM. More importantly, the
LSK population that contains all HSPCs was almost depleted in leu-
kemic BM. Further, our functional readouts including colony forming
ability as well as the ability to reconstitute the hematopoietic system by
transplantation corroborated our phenotypic findings. Additionally, cell
cycle analyses revealed the abnormal cycling of lin-/GFP- cells in leu-
kemic BM, which may contribute to the functional loss of HSPCs.
Indeed, cell cycle of HSPCs is tightly regulated and disturbance to HSPC
cell cycle leads to exhaustion of stemness [15].
Interestingly, our result from competitive transplantation suggests
that the non-leukemic population from leukemic BM impaired the
function of normal HSPCs. Normal LSK cells cocultured with these non-
leukemic cells showed reduced ability to form colonies, further in-
dicating an inhibitory effect of the non-leukemic cells on normal HSPCs.
We speculate that the negative regulatory role of non-leukemic cells is
due to their highly inflammatory profile as inflammation leads to loss of
stemness of HSPC and mediated biased differentiation.
Intriguingly, we did not find similar effects of leukemia on hema-
topoiesis in the leukemia patient derived xenograft model.
Additionally, there were no differences in cell cycle between HSCs from
leukemic and normal BM, further indicating minimal effects of leu-
kemia on HSCs in this model. These results are consistent with studies
from other groups [2]. We speculated that an immunocompetent milieu
is required for leukemia-induced abnormalities in hematopoiesis. In-
deed, the role of immune cells in HSPCs maintenance has been in-
dicated [3,4]. Further, our phenotypic analysis of NSG mice trans-
planted with murine MLL-AF9 leukemia cells confirmed the importance
of immune cells in leukemia-induced loss of HSPCs.
Inflammation induces malfunction of HSPCs partially through cell
cycle regulation [12,15]. Recent studies show several inflammatory
cytokines are produced by leukemia cells and thus result in systemic
inflammation, which leads to body weight loss [10]. In the present
study, we found in the BM of MLL-AF9 induced leukemia, the non-
leukemic population was the major inflammatory contributor. When
leukemia mice were treated with an anti-inflammation drug diclofenac,
although reduced body weight loss was observed, loss of HSPCs was not
rescued. Analysis showed that diclofenac only mildly reduced some of
the inflammatory genes expression in the non-leukemic population,
further indicating inflammation is a powerful negative regulator of
hematopoiesis.
Collectively, our results show that hematopoiesis as well as the
function of HSPCs in a leukemic milieu is abnormal. Future studies
searching for the effective means to rescue function loss of HSPC in
leukemic BM will be a meaningful and interesting line of investigation.
Authors’ contribution
Ping Cui designed this study. Ping Cui performed all the experi-
ments with the help from other authors. Ping Cui wrote the manuscript.
Conflicts of interest
The authors have no conflict of interest to declare.
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Leukemia Research 65 (2018) 49–54
Acknowledgement
This study is supported by a startup grant given to Ping Cui by
Cangzhou Medical College.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.leukres.2018.01.002.
References
[1] L.G. Schuettpelz, D.C. Link, Regulation of hematopoietic stem cell activity by in-
flammation, Front. Immunol. 4 (2013) 204.
[2] F. Miraki-Moud, F. Anjos-Afonso, K.A. Hodby, E. Griessinger, G. Rosignoli,
D. Lillington, L. Jia, J.K. Davies, J. Cavenagh, M. Smith, H. Oakervee, S. Agrawal,
J.G. Gribben, D. Bonnet, D.C. Taussig, Acute myeloid leukemia does not deplete
normal hematopoietic stem cells but induces cytopenias by impeding their differ-
entiation, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 13576–13581.
[3] C. Riether, C.M. Schurch, A.F. Ochsenbein, Regulation of hematopoietic and leu-
kemic stem cells by the immune system, Cell Death Differ. 22 (2015) 187–198.
[4] C.C. Zhang, Hematopoietic stem cells: interplay with immunity, Am. J. Blood Res. 2
(2012) 219–227.
[5] A.V. Krivtsov, S.A. Armstrong, MLL translocations, histone modifications and leu-
kaemia stem-cell development, Nat. Rev. Cancer 7 (2007) 823–833.
[6] C. Meyer, J. Hofmann, T. Burmeister, D. Groger, T.S. Park, M. Emerenciano,
M. Pombo de Oliveira, A. Renneville, P. Villarese, E. Macintyre, H. Cave,
E. Clappier, K. Mass-Malo, J. Zuna, J. Trka, E. De Braekeleer, M. De Braekeleer,
S.H. Oh, G. Tsaur, L. Fechina, V.H. van der Velden, J.J. van Dongen, E. Delabesse,
R. Binato, M.L. Silva, A. Kustanovich, O. Aleinikova, M.H. Harris, T. Lund-Aho,
V. Juvonen, O. Heidenreich, J. Vormoor, W.W. Choi, M. Jarosova, A. Kolenova,
C. Bueno, P. Menendez, S. Wehner, C. Eckert, P. Talmant, S. Tondeur, E. Lippert,
E. Launay, C. Henry, P. Ballerini, H. Lapillone, M.B. Callanan, J.M. Cayuela,
C. Herbaux, G. Cazzaniga, P.M. Kakadiya, S. Bohlander, M. Ahlmann, J.R. Choi,
P. Gameiro, D.S. Lee, J. Krauter, P. Cornillet-Lefebvre, G. Te Kronnie, B.W. Schafer,
S. Kubetzko, C.N. Alonso, U. zur Stadt, R. Sutton, N.C. Venn, S. Izraeli,
L. Trakhtenbrot, H.O. Madsen, P. Archer, J. Hancock, N. Cerveira, M.R. Teixeira,
L. Lo Nigro, A. Moricke, M. Stanulla, M. Schrappe, L. Sedek, T. Szczepanski,
C.M. Zwaan, E.A. Coenen, M.M. van den Heuvel-Eibrink, S. Strehl, M. Dworzak,
R. Panzer-Grumayer, T. Dingermann, T. Klingebiel, R. Marschalek, The MLL re-
combinome of acute leukemias in 2013, Leukemia 27 (2013) 2165–2176.
[7] L. Munoz, J.F. Nomdedeu, N. Villamor, R. Guardia, D. Colomer, J.M. Ribera,
J.P. Torres, J.J. Berlanga, C. Fernandez, A. Llorente, M.P. Queipo de Llano,
J.M. Sanchez, S. Brunet, J. Sierra, C.G. Spanish, Acute myeloid leukemia with MLL
rearrangements: clinicobiological features, prognostic impact and value of flow
cytometry in the detection of residual leukemic cells, Leukemia 17 (2003) 76–82.
[8] B.V. Balgobind, C.M. Zwaan, R. Pieters, M.M. Van den Heuvel-Eibrink, The het-
erogeneity of pediatric MLL-rearranged acute myeloid leukemia, Leukemia 25
(2011) 1239–1248.
[9] S.J. Neering, T. Bushnell, S. Sozer, J. Ashton, R.M. Rossi, P.Y. Wang, D.R. Bell,
D. Heinrich, A. Bottaro, C.T. Jordan, Leukemia stem cells in a genetically defined
murine model of blast-crisis CML, Blood 110 (2007) 2578–2585.
[10] H. Ye, B. Adane, N. Khan, T. Sullivan, M. Minhajuddin, M. Gasparetto, B. Stevens,
S. Pei, M. Balys, J.M. Ashton, D.J. Klemm, C.M. Woolthuis, A.W. Stranahan,
C.Y. Park, C.T. Jordan, Leukemic stem cells evade chemotherapy by metabolic
adaptation to an adipose tissue niche, Cell Stem Cell 19 (2016) 23–37.
[11] A.V. Krivtsov, D. Twomey, Z. Feng, M.C. Stubbs, Y. Wang, J. Faber, J.E. Levine,
J. Wang, W.C. Hahn, D.G. Gilliland, T.R. Golub, S.A. Armstrong, Transformation
from committed progenitor to leukaemia stem cell initiated by MLL-AF9, Nature
442 (2006) 818–822.
[12] L.V. Kovtonyuk, K. Fritsch, X. Feng, M.G. Manz, H. Takizawa, Inflamm-aging of
hematopoiesis hematopoietic stem cells, and the bone marrow microenvironment,
Front. Immunol. 7 (2016) 502.
[13] H. Oguro, L. Ding, S.J. Morrison, SLAM family markers resolve functionally distinct
subpopulations of hematopoietic stem cells and multipotent progenitors, Cell Stem
Cell 13 (2013) 102–116.
[14] K.Y. King, M.A. Goodell, Inflammatory modulation of HSCs: viewing the HSC as a
foundation for the immune response, Nat. Rev. Immunol. 11 (2011) 685–692.
[15] A. Matsumoto, K.I. Nakayama, Role of key regulators of the cell cycle in main-
tenance of hematopoietic stem cells, Biochim. Biophys. Acta 1830 (2013)
2335–2344.