Oncogene (2014) 33, 47354745

& 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14

www.nature.com/onc

ORIGINAL ARTICLE
Aggressive acute myeloid leukemia in PU.1/p53 double-mutant
mice
P Basova1,2, V Pospisil1, F Savvulidi1,7, P Burda1,7, K Vargova1, L Stanek1,3, M Dluhosova1, E Kuzmova1, A Jonasova1,4, U Steidl5,
P Laslo6 and T Stopka1,4

PU.1 downregulation within hematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of
acute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is
a well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to their
aggressiveness; however, its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure
p53  /  ) results in more aggressive AML with shortened overall survival. PU.1ure/urep53  /  progenitors express signicantly lower
PU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1
levels in PU.1ure/urep53  /  mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML.
Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukemic cell growth similarly to
PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53
knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML,
suggesting that MYB/miR-155/PU.1 mechanism may be involved in the pathogenesis of AML and its aggressiveness characterized
by p53 mutation.

Oncogene (2014) 33, 47354745; doi:10.1038/onc.2013.414; published online 14 October 2013

Keywords: PU.1; leukemia; differentiation; microRNA; p53; AML

INTRODUCTION
Haematopoietic cell determination is instructed by transcription
factors such as the Ets-domain transcription factor PU.1 (Sfpi1,
Spi1). PU.1 levels are very important for development of various
blood lineages.1 PU.1 is expressed at threshold levels in
hematopoietic stem and progenitor cells (HSPCs). Highest
expression is within monocytes, intermediate levels in
granulocytes and lower levels in lymphocytes.1 Erythroid
progenitors undergo progressive silencing of PU.1 level that if it
is not completed results in acute myeloid leukemia (AML).2 PU.1
expression is determined by interactions of its promoter with
upstream regulatory element (URE) at  14 kb relative to
transcription start.3 Disruption of PU.1 gene in mouse is lethal
due to the block of the majority of blood cell development,
whereas the deletion of URE (PU.1ure/ure) downregulates PU.1 to
20% and promotes AML (marked by clonal accumulation of
myeloid c-Kit Mac-1lowGr-1low blast cells) preceded by
preleukaemic phase at 23 months (marked by two-fold increase
of c-Kit precursors, Gr-1 granulocytes and splenomegaly).4
Indeed, the PU.1ure/ure AML displays clonal features characterized
by recurring chromosomal abnormalities and is retransplantable
into immunodecient recipients.4
PU.1 is negatively regulated posttranscriptionally by micro-
RNA(miR)-155.5 Constitutive expression of miR-155 in HSPCs
downregulates PU.1 and causes a phenotype resembling AML.6
While PU.1 transcripts are directly targeted by miR-155, the miR-
155 Host Gene (Mir-155hg) is itself transcriptionally activated by
temporal PU.1 occupation in HSPCs.7 This cross-regulation
establishes a regulatory loop providing a mechanism for precise
regulation of PU.1 expression. Notably, disruption of this nely
tuned loop may contribute to AML pathology.8 Mir-155hg is
transcriptionally activated by the Myeloblastoma protein (Myb)9
whose primary function is to stimulate progenitor cell proliferation
and impede differentiation. Expression of Myb must be
downregulated during cell maturation.10
Tumor suppressor p53 has a central role in cellular response to
DNA damage and other stresses by controlling the G1/S phase
transition. Activation of p53 leads to cell cycle arrest or cell
senescence and/or apoptosis. In turn, loss of p53 promotes
proliferation, survival, genomic instability and tumor progres-
sion.11 Fifty percent of all human cancers contain p53 mutations.12
p53 is dispensable for normal development while it predisposes
mice to solid tumors, mostly lymphomas, by 6 months of age.13,14
While the loss of p53 does not result in myeloid dysplasias, its loss
in combination with other defects, such as Kras mutations,
promotes AML.15 p53 inactivation stimulates leukemia-initiating
cell survival15 together with E-box proteins (Myb and Myc) that are
involved in leukaemogenesis.4,16,17 Approximately 1015% AML
patients bear p53-inactivating mutations and display shorter
survival and chemoresistance;15,1820 however, the particular

1
Department of Pathophysiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; 2Department of Experimental Biomodels, First Faculty of
Medicine, Charles University in Prague, Prague, Czech Republic; 3Department of Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic;
4
Department of MedicineHaematology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; 5Albert Einstein College of Medicine, Yeshiva University,
Bronx, NY, USA and 6Section of Experimental Haematology, Leeds Institute of Cancer and Pathology, St Jamess University Hospital, University of Leeds, Leeds, UK.
Correspondence: Dr T Stopka, Department of Pathophysiology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, Prague 2 12853, Czech Republic.
E-mail: tstopka@lf1.cuni.cz
7
These authors contributed equally to this work.
Received 12 February 2013; revised 23 August 2013; accepted 2 September 2013; published online 14 October 2013
 Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4736
mechanism of how the loss of p53 leads to more aggressive AML included weight loss, lethargy and a dorsal hump (Figures 1Aa and
is not fully understood. b). These symptoms indicated that PU.1ure/urep53  /  mice
In this study, we crossed murine PU.1 knockdown model suffered from a more severe disease. Examination of the
(PU.1ure/ure) and p53  /  mutant mice to address the question of peritoneal cavity revealed that PU.1ure/urep53  /  mice contained
how loss of p53 modulates AML that is caused by a defect of PU.1. prominent hepatosplenomegaly (enlarged liver and spleen); larger
The composite PU.1ure/urep53  /  mice developed highly aggres- in comparison with the PU.1ure/ure (that also were hepatospleno-
sive AML that is coupled with further downregulation of PU.1. megalic) or control mice (Figures 1Abd).
Mechanism between loss of p53 and downregulation of PU.1 Histological examinations of the spleens indicated that the
seems to be indirect and involves deregulation of E-box proteins PU.1ure/urep53  /  mice are inltrated by cells of immature
and miR-155. In addition, the described mechanism in PU.1ure/ure morphology strongly resembling human AML. Particularly,
p53  /  murine AML was also observable in human AML. hyperemic spleens showed marked hyperplasia. AML-like myelo-
blasts with heterogeneity of nucleocytoplasmic ratio observed in
the PU.1ure/ure and PU.1ure/urep53  /  mice replaced normal
RESULTS spleen architecture (Figures 1Aeg). AML spleens contained traces
PU.1/p53 double-mutant mice develop aggressive AML of dysplastic extramedullary haematopoiesis, from which mega-
PU.1ure/ure mice were mated with p53  /  strain to produce karyocytes were severely dysplastic in the PU.1ure/urep53  /  mice,
PU.1ure/urep53  /  mice. At 3 months of age the signicant whereas less dysplastic changes were observed in PU.1ure/ure
differences between PU.1ure/ure mice and PU.1ure/urep53  /  mice spleens (Figures 1Aeg, Supplementary Figure 1).

* ***
30 *** **
e e e re WT
ur ur ur

Body weight (g), +/- SD

u
e/ e/ e/ e/ PU.1ure/ure
ur ur - ur ur
.1 3 -/-
.1 .1 -
3
/
T .1 PU.1ure/ure p53-/-
PU PU p5 PU.1ure/ure PU.1ure/ure p53-/- W PU PU p5 20
0 *** 4
*** *
1
2 3
3 10
4

cm
cm

5 2
6 0
7 1 0-2 3-4
8 Age (Months)
9
0
WT PU.1ure/ure PU.1ure/urep53-/- 100

RP WT
75 PU.1ure/ure

*** ***
% Survival

PU.1ure/ure p53-/-
50 p53-/-

25
WP

0
0 1 2 3 4 5 6 7 8 9 10
Age (Months)

*** ** *
50 ** 20 50 12 1000 **
* *
WBC, x10^3/mm^3, +/- SD

PLT, x10^3/mm^3, +/- SD

BBC, x10^6/mm^3, +/- SD

40 40 800
15 9
HGB, g/dl, +/- SD

HTC, %, +/- SD

30 30 600
10 6
20 20 400

5 3
10 10 200

0 0 0 0 0
T /- /ure re T /- /ure re T /- /ure re T /- /ure re T /- /ure re
W 53- ure re/u W 53- ure re/u W 53- ure re/u W 53- ure re/u W 53- ure re/u
p .1 .1u p .1 .1u p .1 .1u p .1 .1u p .1 .1u
PU PU -/- PU PU -/- PU PU -/- PU PU -/- PU PU -/-
3 3 3 3 3
p5 p5 p5 p5 p5

Figure 1. PU.1ure/urep53  /  mice develop aggressive AML. (A, a) Mice at 3 months. (A, bc) Peritoneal cavity at 5 (left) and 3 (right) months.
Liver and spleen (arrows). (A, d) Ex vivo explanted spleens (P-values indicate weight differences) of WT (N 32), PU.1ure/ure (N 65), and
PU.1ure/urep53  /  mice (N 35). (A, eg) Spleen histology (hematoxylin/eosin,  1000). WT spleens display normal red and white pulp
(RP,WP). Myeloblasts (circles) replacing RP and WP, dysplastic megakaryocytes (arrows), lymphocytes (rhomboid) (B) Average weights.d. (WT
(N 38), PU.1ure/ure (N 65) and PU.1ure/urep53  /  mice (N 38) at 2 and 4 months. (C) Survival of WT (N 20), p53  /  (N 66), PU.1ure/ure
(N 85) and PU.1ure/urep53  /  (N 39) mice at 10 months. (D) PB counts with indicated units on y axis. WBC, white blood cells; HGB,
hemoglobin ; HCT, hematocrit RBC, red blood cells; PLT, platelets). Genotypes: WT (N 8), p53  /  (N 6), PU.1ure/ure (N 10) and PU.1ure/ure
p53  /  mice (N 6) at 3 months. Means.d., P-values (KruskalWallis).

Oncogene (2014) 4735 4745 & 2014 Macmillan Publishers Limited


ure/ure
Table 1. Frequency of developing MPS and AML in PU.1 and
PU.1ure/urep53  /  within 6 months
Age (months) Frequency
PU.1ure/ure PU.1ure/ure p53  / 
12 0% 100% AML
2.53.5 100% preleukaemic 90% AML
45.5 100% AML 100% AML
Table 2. Frequency of developing preleukaemia and AML and tumor
formation in PU.1ure/ure, p53  /  and PU.1ure/urep53  /  mice
Frequency
PU.1ure/ure p53  /  PU.1ure/ure p53  / 
Preleukaemic 10/115 0/40 0/38
AML 105/115 0/40 38/38
Solid tumor 0/115 19/40 3/38
As indicated in Figure 1c and Tables 1 and 2, the overall survival
data conrmed that PU.1ure/urep53  /  mice die signicantly
earlier than PU.1ure/ure or p53  /  mice (median B3 months).
Other than 3 out of 38 cases the PU.1ure/urep53  /  mice
developed no solid tumors.
Next, we determined peripheral blood (PB) counts of the diseased
mice (Figure 1d) and observed that the PU.1ure/urep53  /  mice
displayed markedly lower numbers of mature red blood cells
(reected by lower hemoglobin and hematocrit) as well as platelets.
Flow cytometric analyses4 of bone marrow (BM) demonstrated
that the PU.1ure/ure and PU.1ure/urep53  /  mice displayed similar
expression pattern of Mac1, c-Kit and Gr-1. Particularly, the AML
phenotype was characterized by accumulation of c-Kit Mac-
1lowGr-1 cells. The presence of c-Kit antigen on majority of Mac-
1- and Gr-1-positive cells indicated abundance of myeloid cells
with immature phenotype. Most prominent difference was that
AML phenotype developed in PU.1ure/urep53  /  mice much
earlier (B3 months) compared with that of PU.1ure/ure (B6
months, Figure 2). The preleukaemic phase, typical for PU.1ure/ure
mice, was not observed in the compound mice (Figure 2a and
Table 1).
Histology examination (see Supplementary Figure 2) showed
that immature AML blast-like cells encompassed most of the BM
of PU.1ure/ure and PU.1ure/urep53  /  mice, respectively. We again
noted frequent dysplastic features in the megakaryocytes of
PU.1ure/urep53  /  mice.
To test biological properties of AML derived from PU.1ure/ure and
PU.1ure/urep53  /  mice we plated magnetically sorted BM-c-Kit
cells into semisolid media. While wild-type (WT) cells formed
progenitor CFU colonies, the AML cells of both genotypes formed
mostly colonies of immature phenotype (see Figure 2i, right).
Interestingly, PU.1ure/urep53  /  immature colonies were remark-
ably larger suggesting their increased proliferation. Accordingly,
following the replating of the semisolid cultures the number of
PU.1ure/urep53  /  compared with PU.1ure/ure immature colonies
were signicantly increased (Figure 2i). Preleukaemic progenitors
isolated at 3 month of age from PU.1ure/ure formed comparable
(with WT) numbers of CFUs and negligible number of colonies of
immature cells that increased upon replating (Figure 2i).
In summary, PU.1ure/urep53  /  mice develop a more aggressive
AML marked by (1) rapidly expanding and inltrating blasts,
severe anemia, thrombocytopenia associated with BM and splenic
megakaryocytic dysplasia, (2) in vitro aggressive behavior and (3)
adverse symptoms and decreased overall survival.
& 2014 Macmillan Publishers Limited
Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4737
Downregulation of PU.1 and deregulation of miR-155 in PU.1ure/ure
p53  /  AML
Expression levels of PU.1 are a crucial determinant of haemato-
poietic differentiation and its downregulation causes AML in
PU.1ure/ure mice.4 Thus, we rst determined PU.1 expression in
tumor cells of all genotypes. PU.1 mRNA was expressed similarly in
the p53  /  to that of the WT in total c-Kit cells, whereas in the
PU.1ure/ure mice it was B20%. This observation indicates that p53
loss alone does not affect PU.1 expression. Notably, PU.1
expression was further decreased (B10% compared to WT) in
the PU.1ure/urep53  /  mice (Figure 3a). In addition we measured
PU.1 transcripts in c-Kit Sca-1 lin  population of HSPCs and in
c-Kit Sca-1  lin  progenitors isolated by uorescence-activated
cell sorting (FACS). Notably, HSPCs and progenitor populations
were four to vefold under-represented in PU.1ure/urep53  /  mice
(compared with PU.1ure/ure, not shown). PU.1 expression in the
PU.1ure/urep53  /  HSPCs was two- to threefold lower compared
with the same cells isolated from PU.1ure/ure mice. Similar data
were observed in c-Kit Sca-1  lin  progenitors (Figure 3a).
We then quantied intracellular PU.1 protein.21 Correspondingly
to RNA level, the c-Kit BM progenitors of p53  /  mice
expressed comparable levels of PU.1 protein as WT (not shown).
In the PU.1ure/ureprogenitors, the PU.1 protein was decreased
compared with WT and p53  /  progenitors and further reduced
in the PU.1ure/urep53  /  progenitors (Figure 3b). Thus PU.1 level,
both mRNA and protein, is decreased in the c-Kit population in
the more aggressive AML developed in PU.1ure/urep53  / 
compared with that in PU.1ure/ure mice.
miR-155 represents a key regulator that post-transcriptionally
downregulates PU.1 and its overexpression causes a myeloproli-
ferative disorder in mouse.6 We hypothesized that miR-155 is a
candidate factor that could account for the repression of PU.1 in
our AML models. We quantied miR-155 expression and found
that it is signicantly upregulated in BM-derived c-Kit
progenitors in PU.1ure/ure mice (Figure 3c). Furthermore, in
PU.1ure/urep53  /  mice the miR-155 level is further elevated
(more than threefold, compared with WT) and is signicantly
higher than in PU.1ure/ure mice. The increased levels of miR-155
were also demonstrated within the HSPCs and progenitor
populations of the respective genotypes (Figure 3c).
Next, we determined whether the miR-155 upregulation in AML
is regulated at epigenetic level. We performed chromatin
immunoprecipitation at the Mir-155hg locus using antibodies
against H3K9Ac, H3K9Me and H3K4Me. We observed that the
chromatin state in c-Kit blast cells of the PU.1ure/urep53  /  mice
signicantly differs from PU.1ure/ure or WT mice. We detected
enhanced mark of active chromatin, histone H3K9Ac, at the distal
regulatory region (near  6 kb, Figure 3d; D-6), whereas the
reciprocal state was noted at the proximal promoter (near
 0.6 kb, Figure 3d; P-0.6,). Additionally, region D-6 was also
enriched (along with D-7 and D-1.5) by H3K4Me3 (Supplementary
Figure 3A). Active marks at the amplicons D-6 and P-0.6 were
reected by inverse state of the repressive mark, H3K9 trimethyla-
tion (Figure 3d). Furthermore, previous studies have reported that
the region near  15 kb (D-15) is occupied by PU.1 in developing
myeloid progenitors to temporally activate and subsequently
allow inhibition of Mir-155hg locus.7 The D-15 region was enriched
by H3K9Me3 in both PU.1ure/ure and PU.1ure/urep53  /  mice
(Figure 3d), indicating that under low PU.1 levels it is unable to
activate Mir-155hg transcription. Collectively, histone modica-
tions analysis demonstrates an actively transcribed chromatin at
the Mir-155hg locus in PU.1ure/urep53  /  AML blasts.
Data in this section indicate that progenitors from the
PU.1ure/urep53  /  mice have, in comparison to PU.1ure/ure, a
further downregulated expression of PU.1 and concurrently
upregulated miR-155 that is associated with dysregulated
chromatin structure in AML of PU.1ure/ure and, to a greater extent,
in PU.1ure/urep53  /  murine progenitors.
Oncogene (2014) 4735 4745
 Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4738
WT PU.1ure/ure PU.1ure/ure PU.1ure/urep53-/-
(3 months) 3 months, preleukaemic) (6 months, AML) (3 months, AML)

37.3 7.9 29 26.6 9.7 73 9.2 75.7 100

***
***

% Mac-1+ c-Kit+ (BM)

75

Mac-1 5.7 5.5 6.7 9.0 50

c-Kit
0.5 60.7 0.3 55 0.2 82 0.3 72.5 25

0
wt 3-/- e/ure e/ure
p5 1u 1u
r r
9.8 27 10.8 5.8 . . /-
PU PU 3-
Mac-1 p5
Gr-1

100 100 100 40 *** 40 *** 40 ***

*** *** *** *** *** ***
*** *** *** ** *
*
% Mac-1+ Gr-1+ (Spleen)

75 75 75 30 30 30
% Mac-1+ Gr-1+ (BM)

% Mac-1+ Gr-1+ (PB)

% Ter119+ (BM)
% B220+ (BM)

% B220+ (PB)
50 50 50 20 20 20

25 25 25 10 10 10

0 0 0 0 0 0
wt -/- re
3 re/u re/u
re wt -/-
3 re/u re/u
re re wt -/- re
3 re/u re/u
re wt -/-
/u
3 re re
re
/u
re wt -/-
/u
3 re re
re
/u
re wt -/- re
3 re/u re/u
re
p5 1u 1u p5 1u 1u p5 1u 1u p5 1u 1u p5 1u 1u p5 1u 1u
. . /- . . /- . . /- . . /- . . /- . . /-
P PU 3-
U P PU 3-
U P PU 3-
U PU PU 3- PU PU 3- P PU 3-
U
p5 p5 p5 p5 p5 p5

*
* WT
*** ***
1600
CFU colonies
Colonies of immature cells
1400
cells

100x
c-Kit+

1200
PU.1ure/ure
1x105

1000
400
Colonies per

300 100x

200 PU.1ure/urep53-/-

100

0
ex vivo replating ex vivo replating ex vivo replating ex vivo replating
100x
Wt PU.1ure/ure PU.1ure/ure PU.1ure/ure
Preleukaemic P53-/-

Figure 2. Analysis of PU.1ure/urep53  /  AML haematopoiesis. (a) BM-FACS analysis for Mac-1 (y axis), Gr-1 and c-Kit (both x axis). Genotypes are
indicated on top. Double c-Kit Mac-1 myeloid cells represent immature AML phenotype. Quadrants display cell%. (bh) Flow cytometry of
indicated surface markers (y axis) in PB, BM and spleen (WT (N 20), p53  /  (N 40), PU.1ure/ure (N 115), PU.1ure/urep53  /  (N 38)).
Means.e.m., P-values (KruskalWallis). (i) Numbers and photographs of semisolid CFU and immature cell colonies (of indicated genotypes,
isolated from the PU.1ure/ure mice at 6 months and PU.1ure/urep53  /  at 3 months) derived from c-Kit cells at day 57 before (ex vivo) and
after replating. Means.e.m., P-values (t-test, unpaired for immature colonies).

Myb and Myc oncogenes are deregulated in PU.1ure/urep53  /  these observations these two oncogenes are increased within
AML c-Kit progenitors of p53  /  mice (Figure 3e); however, their
PU.1 is a direct target of miR-155.6 As such, the observed levels neither stimulated miR-155 expression (Figure 3c) nor
downregulation of PU.1 expression can be accounted for by the caused AML. Furthermore, Myb, and to lower extent also Myc, are
concurrent increase of miR-155. However, what is regulating miR- upregulated in PU.1ure/ure HPSCs. We note upregulation of Myb at
155? The E-box transcription factors MYB and MYC are associated similar levels in the individual mutants (p53  /  or PU.1ure/ure).
with transcriptional regulation of miR-155.9,22 In accordance with Notably, Myb (as well as Myc) were further upregulated in

Oncogene (2014) 4735 4745 & 2014 Macmillan Publishers Limited

 Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4739
PU.1
***
*** * *
** * ** * **
blocking peptide

Relative to 18S rRNA, +/- SEM

WT
p53-/- p53-/-
1000
1.0 PU.1ure/ure PU.1ure/ure
PU.1ure/urep53-/- PU.1ure/urep53-/-
750

Count
500
0.5
250

0
102 103
0.0
PU.1 expression
+) rs
sts s
PC -Kit ito t+ )
Bla it+ ) HS -1+ c en - -Ki
(c-
K
c a P rog a-1 c
nS
- - c
(Li nS
(Li

MiR-155
H3K9Ac H3K9Me3
*** *
** * **
** *
*** ** *
*
*

Relative occupancy to H3, +/- SEM

5 0.8
WT
Relative to snon02, +/- SEM

1.6 p53-/-
4 PU.1ure/ure
0.6 PU.1ure/urep53-/-
1.2
3
0.4
0.8
2

0.2 0.4
1

0 0 0
D-6 P-0.6 D-15 D-6 P-0.6
sts s
PC -Kit
+)
ors it+ )
Bla it+ ) nit
- K HS -1+ c r o ge 1- c-K
(c - c
a P ca-
nS nS
-
(Li (Li

Myb Myc
*** **
*
15 ** ** 15 ** ***
Relative to 18S, +/- SEM

10 10

5 5

0 0
T -/- re re T -/- ure ure
W 53 ure/u re/u W 53 ure/ ure/
p u - p .1 .1
.1 .1 -/ /-
PU PU p53 PU PU 3-
p5

Figure 3. Low PU.1 level and transcriptional induction of Mir-155hg locus in AML. (a) PU.1 mRNA expression in BM c-Kit blasts (WT (N 12),
p53  /  (N 28), PU.1ure/ure (N 75), PU.1ure/urep53  /  mice (N 24)). HSPCs and progenitors (WT (N 2), PU.1ure/ure (N 2) and PU.1ure/urep53  / 
(N 2) mice) determined by qPCR. Fold change (y axis), controls set to 1, means.e.m. (b) PU.1 flow cytometry analysis of Mac1 c-Kit cells of
indicated genotypes. Blocking peptide control experiment is indicated. (c) miR-155 expression (samples as in a). Means.e.m., P-values (t-test,
unpaired, two-tailed). (d) Chromatin immunoprecipitation using H3K9Ac and H3K9Me3 antibodies at the Mir-155hg locus (positions of amplicons
are relative to TSS, distal (D) at  6 kb and  15 kb and proximal (P) at  0.6 kb, all data normalized to H3 content. Means.e.m. (N 2), P-values
(t-test, unpaired). (e) Myb and Myc mRNA expression in BM c-Kit cells (samples as in a). Means.e.m., P-values (KruskalWallis).

compound mutants (PU.1ure/urep53  /  ) compared with either possible mutual regulation among these factors, we performed
PU.1ure/ure or to WT (Figure 3e). Upregulation of Myb in PU.1ure/ure complementary experiments.
p53  /  AML blasts was relatively uniform as demonstrated by First, we used our animal model to manipulate the expression of
spleen immunohistochemistry (Supplementary Figure 3B). miR-155, Myb or PU.1 in ex vivo isolated AML blasts. Down-
In summary, Myb and also Myc are further upregulated in the regulation of miR-155 using anti-miR-155 resulted in a marked
composite PU.1ure/urep53  /  mutation and together with ele- upregulation of PU.1 transcripts in progenitors of both PU.1ure/ure
vated miR-155 may add to aggressiveness of AML. and PU.1ure/urep53  /  mice (Figure 4a). PU.1 induction was
reected by upregulation of several target mRNAs including Cbfb,
Itgam (Mac1), Cebpa, Egr2, Cd14, Fos and Csf1r (Supplementary
Functional tests reveal signicance of p53/Myb/miR-155/PU.1 Figure 4A). Next, we inhibited Myb by siRNA and again observed
pathway in murine AML the upregulation of PU.1 and its targets together with the
To determine the individual contributions of Myb, miR-155 and concurrent downregulation of miR-155 (Figure 4b, Supplementary
PU.1 upon differentiation and leukemic growth, as well as testing Figure 4B). These experiments establish that PU.1 expression is

& 2014 Macmillan Publishers Limited Oncogene (2014) 4735 4745

 Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4740
anti-miR-155 siRNA Myb pEBB-PU.1

3 ** 3 ** * 20
* * * * ** ***
* *

Relative abundance, +/- SEM

Relative abundance, +/- SEM

Relative abundance, +/- SEM

15
2 2 6

5
1 1 1.5
1.0

0 0 0
m ctrl
55
yb

ne .1

iR l
55
yb

.1

rl
m yb
55
ne U.1

rl
m yb
55

.1

PU l
.1
m yb

ne 55

rl
.1
m yb
55
r

r
ct

ct

ct

ct

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PU.1ure/ure PU.1ure/ure PU.1ure/ure PU.1ure/ure PU.1ure/ure PU.1ure/ure

p53-/- p53-/- p53-/-

***
*** *** 1000 *
200 *** ***

Colonies per 1x105 c-Kit+ cells

***
Colonies per 1x105 c-Kit+ cells

* 800 Colonies of immature cells

Immature

CFU colonies
150
600

100
400

50 G/M
200
Ery
0 Mix
0
wt re re re re re re re re 1
U.

ne
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1 .1 /- U.
1 .1 /- U.
1 .1 /-
P PU 3- P PU 3- P PU 3- P PU 3- pEB
p5 p5 p5 p5
Replating
None anti-miR-155 siRNA Myb pEBB-PU.1
PU.1ure/ure p53-/-

siRNA p53
3 *** 200 **
*
Colonies per 1x105 c-Kit+ cells

**
Relative abundance, +/- SEM

**
** 150
Immature

100

1
50
G/M
Ery
Mix
0 0
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ur ur
rl

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ct

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siRNA p53

Figure 4. Functional characterization of Myb/miR-155/PU.1 axis in murine AML. mRNA expression in c-Kit cells transfected with: (a) anti-miR-
155, (b) Myb siRNA, (c) PU.1- plasmid. qPCR data at 72 h, means.e.m. (N 3). (d) Immature cell- (empty bars) and CFU colonies derived from
c-Kit cells from AML or WT mice transfected with anti-miR-155, Myb siRNA or PU.1 plasmid. P-values are related to colonies of immature cells.
Mix CFU  GEMM colonies, Ery BFU  E, G/M CFU  M, CFU  G, CFU  GM, scored after 712 days of culture. Means.e.m. of duplicate
cultures. (e) Immature cell- and CFU colonies in replated semisolid cultures (day 7) following PU.1 overexpression in PU.1ure/urep53  /  c-Kit
blasts. Mean of blasts.e.m., P-values (t-test, unpaired). (f ) mRNA expression in c-Kit cells transfected with p53 siRNA (at 120 h). (g) Immature
cell- (empty bars) and CFU colonies derived from c-Kit cells transfected with p53 siRNA. Means.e.m., P-values (t-test, unpaired for immature
colonies).

inhibited by both miR-155 and Myb. Furthermore, inhibition of PU.1 target genes were upregulated (Supplementary Figure 4C).
Myb also resulted in a decrease in miR-155 expression, indicating Interestingly, the levels of miR-155 and Myb were not affected by
that Myb positively regulates miR-155 and is an upstream PU.1 ectopic expression (Figure 4c). These data indicate that while
regulator of miR-155. PU.1 is a downstream effector of Myb and miR-155 the reciprocal
To test whether ectopic expression of PU.1 can rescue the does not hold.
myeloid development of mutant PU.1ure/ure and PU.1ure/urep53  /  Next, we determined whether Myb and miR-155 are involved in
progenitors, the c-Kit AML blasts were transfected with a PU.1- the leukemic cell growth of the blast cells and whether restoration
encoding plasmid (Figure 4c). Indeed levels of PU.1, as well as of of PU.1 could block these effects. Leukemic growth of the c-Kit

Oncogene (2014) 4735 4745 & 2014 Macmillan Publishers Limited


cells (used in Figures 4ac) was assessed using semisolid
methylcellulose cultures. While WT progenitors formed only CFUs
colonies, the non-treated PU.1ure/ure and PU.1ure/urep53  /  cells
formed large numbers of colonies of immature cells (Figures 4d
(left) and 2i; note higher numbers of blast cell and less CFU
colonies in the PU.1ure/urep53  /  ). In contrast, the cells trans-
fected with either Myb siRNA, anti-miR-155 or PU.1 plasmid
formed signicantly less colonies of immature cells with a
restoration in the frequency (similar to WT) of all types of CFU
colonies (Figure 4d, right), consistent with expression data (Figures
4ac). Interestingly, the rescue was most effective in PU.1
transfectants, conrming key importance of PU.l levels. The
effect of PU.1 overexpression on PU.1 targets was further
documented by FACS analysis of increased Mac1 and Gr-1
positivity in colonies formed by PU.1ure/urep53  /  blasts
(Supplementary Figure 4E). Furthermore, the replating of the
PU.1-transfected PU.1ure/urep53  /  blasts revealed that increased
PU.1 expression limited the leukemogenic potential of AML blasts
(Figure 4e).
The upregulation of both Myb and miR-155 with the down-
regulation of PU.1 observed in PU.1ure/urep53  /  mice can be
the result of either enhanced genome instability followed by
clonal selection in mice after the p53 deletion or a lack of direct
regulation of these factors by p53. To test these possibilities we
used transient p53-siRNA knockdown. We observed, surprisingly,
that the temporal inhibition of p53 level in PU.1ure/ure progenitors
to 40% affected expression of all members of the Myb/miR-155/
PU.1 axis (including PU.1 targets) as well as p53 target gene p21
(Figure 4f and Supplementary Figure 4D). Particularly, upregula-
tion of MYB (2-fold), upregulation of miR-155 (2-fold), down-
regulation of PU.1 (threefold) and simultaneous downregulation of
PU.1 targets was achieved. Furthermore, p53 knockdown resulted
in increased leukemic growth indicated by an increase in the
number of colonies of immature cells and concurrent decrease in
differentiating CFU colonies (Figure 4g). These data indicate that
p53 is directly involved in the regulation of MYB/miR-155/PU.1
mechanism and deregulation of this pathway is likely an early
event after p53 mutation in PU.1ure/ure mice.
To validate that p53 regulates PU.1 at posttranscriptional level,
we compared pre-mRNA and mRNA levels of PU.1 upon p53
knockdown in PU.1ure/ure c-Kit blasts. Decreased levels of mRNA
compared to pre-mRNA indicates indeed that PU.1 is regulated at
postrancriptional levels (as pre-mRNA localized in the nucleus is
not subjected to microRNA-mediated degradation that occurs in
the cytoplasm) (Supplementary Figure 4D).
This section conrms that (i) both Myb and miR-155 are
required for the downregulation of PU.1 levels, (ii) suggests a
hierarchical order of these factors (Myb/miR-155/PU.1) during AML
pathogenesis and (iii) the role of p53 loss as an upstream factor of
Myb expression in aggressive AML. The manipulations of these
factorseither inhibition of the oncogenes (Myb and miR-155) or
rescue of PU.1lead to leukemia blast differentiation.
Dysregulation of p53/Myb/miR-155/PU.1 pathway is observable in
human AML
To validate and correlate our ndings from mice to human clinical
AML, we determined expression of miR-155, PU.1 and E-box
proteins (MYC, MYB) in a cohort of 36 AML patient peripheral
blood mononuclear cell (PBMCs) and 14 healthy controls
(Supplementary Figure 5A).
Majority of patients (27 out 36, 75%) had decreased levels of
PU.1 (Po0.01), with a range of two- to eightfold less compared
with controls (Figures 5a and b). First, based on the expression
levels of PU.1, the AML patients were subdivided into two groups
with either normal (NORM, FClog2o  0.5) or decreased (LOW,
FClog24  0.5) PU.1 levels. Next, we compared expression of PU.1,
miR-155, MYB and MYC within in each group. Interestingly, 70%
& 2014 Macmillan Publishers Limited
Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4741
patients in PU.1-LOW group showed upregulation of miR-155
(Po0.05), displaying a negative PU.1/miR-155 correlation (Spear-
man, r  0.347, P 0.038; Supplementary Figure 5A). Further-
more, the MYB oncogene (unlike MYC) was overexpressed in 96%
of PU.1-LOW patients and displayed a signicant negative
correlation with PU.1 (r  0.582, P 0.0006). In addition, MYB
and miR-155 were positively correlated (r 0.223, P 0.069),
indicating that both miR-155 and MYB cooperate in the down-
regulation of PU.1 in clinical AML. Additional controls including B-
and T-depleted PBMCs and PB-derived CD34 cells of healthy
donors are provided in Supplementary Figure 5C.
To further elaborate on the role of p53 inactivation in human
AML in relation to MYB/miR-155/PU.1 circuit, we considered the
cytogenetic status of p53 in our AML patients. The deletion of
17p13 locus carrying p53 gene was found in three AML patients
and strikingly all of them were among the PU.1-LOW patients with
highest MYB and miR-155 expression (indicated by arrows in
Figure 5b). We note that detection of p53 deletion by FISH was
limited to a few patients samples and therefore the number of
p53 deletions in the cohort of patients could be higher.
The correlation between the levels of Myb, miR-155 and
expression of PU.1 in human AML may not necessarily dictate
that they are directly regulating the AML aggressiveness as
observed in the mice. Therefore, we used human AML cell line
NB4 and manipulated levels of MYB/miR-155/PU.1 axis and p53.
Knockdown of MYB decreased miR-155 and reciprocally increased
PU.1; similarly as in murine AML (Figure 5c). Knockdown of miR-
155 upregulated PU.1; however, likely due to restored PU.1, it led
to decreased MYB, indicating that PU.1 may function as a
repressor of MYB (Figure 5d). The PU.1 overexpression in NB4
cells resulted in the downregulation of MYB and its downstream
target miR-155 (Figure 5e). In addition, we treated NB4 cells with
p53 siRNA that resulted in the derepression of MYB and miR-155
coupled with the downregulation of PU.1 and its targets (Figure 5f
and Supplementary Figure 5D-G), similarly as in murine AML.
These data indicate that p53/MYB/miR-155/PU.1 regulatory axis is
functional in human AML.
In summary, the pathological upregulation of miR-155 and MYB
in accordance with the downregulation of PU.1 expression
suggests that MYB/miR-155/PU.1 regulatory circuit is among
those mechanisms that contribute to the downregulation of
PU.1 in a subset of human AML, especially in aggressive cases
characterized by p53 inactivation.
DISCUSSION
We herein present evidence that PU.1ure/urep53  /  double-mutant
mice express very low PU.1 levels and develop highly aggressive
AML (compared with PU.1ure/ure mice), thus supporting the overall
notion that regulation of PU.1 levels is involved in acute
leukaemogenesis. Extremely low levels of PU.1 can be found in
HPSCs and progenitors; however, this pattern changes markedly
during myeloid development,1 therefore low levels of PU.1 are
sufcient to stimulate development up to the level of myeloid blast
cells. Our current (Figures 4c and 5e) and previous data in other
AML models23 show that complementation with PU.1 rescues
myeloid differentiation of leukemic blasts. Rescue models also
indicate that PU.1 at low levels is not effectively stimulating
transcription of PU.1 target genes required for myelopoiesis and at
the same time is not able to effectively repress oncogenic
transcription factors: Myb and Myc, known activators of cell
proliferation and inhibitors of myeloid maturation.24 Compared
with PU.1ure/ure mice the double mutants develop aggressive AML
that is marked by rapidly expanding and inltrating blasts, severe
anemia, thrombocytopenia associated with BM and splenic
megakaryocytic dysplasia, more aggressive leukemic in vitro
growth and shorter overall survival (Figures 1 and 2). Some
phenomena are however overlapping between the single- and
Oncogene (2014) 4735 4745
 Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4742

PU.1LOW MYBHIGH
(N=27) (N=32)
8
0 5
18
1 1

1
MiR-155HIGH
(N=21)
AML patients N=36

PU.1 miR-155 MYB MYC

** * **
*** *
4 4 6 4
3
Relative abundance (log2)

3
3
2 4 2
2
1 1
0 1 2 0
-1 -1
0
-2 0 -2
-1
-3 -3
-4 -2 -2 -4
CTRL PU.1NORM PU.1LOW CTRL PU.1NORM PU.1LOW CTRL PU.1NORM PU.1LOW CTRL PU.1NORM PU.1LOW

siRNA MYB anti-miR-155 pEBB-PU.1 siRNA p53

* * *** *
** * **
12 ** 5 5 10 **
** ** * **
9 *
10
Relative abundance, +/- SEM

Relative abundance, +/- SEM

Relative abundance, +/- SEM
Relative abundance, +/- SEM

8
4 4
7
8
6
6 3 3 5
2 2 2 2

1 1 1 1

0 0 0 0
rl
YB

55

.1

rl

5
YB

.1

rl

.1
YB

55

rl

1
YB

1
ct

ct

ct

ct

p5

p2

.
PU

PU

PU

PU
-1

-1

-1

-1
M

M
g.

g.

g.

g.
iR

iR

iR

iR
ne

ne

ne

ne
m

Figure 5. Analysis of PU.1, miR-155 and E-box proteins in AML patients. (a) Venn diagram of mRNA expression (from b) of PU.1-LOW
(FClog2o  0.5), MYB HIGH (FClog241) and miR-155 HIGH (FClog241). (b) RTPCR for PU.1, miR-155, MYB and MYC of human AML PBMCs.
Values are relative to average expression of healthy controls (CTRL, N 5, for additional controls see Supplementary Figure 5C). Mean, P-values
(KruskalWallis, two-tailed). (c) RTPCR of indicated RNAs in NB4 cells treated with anti-miR-155 for 96 h, (d) MYB siRNA (96 h), (e) PU.1 plasmid
(48 h) or (f ) p53 siRNA (48 h).

double-mutant AML such as surface marker expression. Observed miR-155 level is a lack of PU.1-mediated repression of Myb and
molecular mechanisms herein associated with the AML aggressive- miR-155 in PU.1ure/ure and PU.1ure/urep53  /  mice (enhanced by
ness (such as expression and regulatory relationships of Myb/miR- loss of p53), which allows the activation of miR-155 that in turn
155/PU.1 axis that lead to two different PU.1 levels) appear to be further downregulates PU.1, resulting in AML. Indeed, experiments
present in both single- and double-mutant AML, however in the using siRNA-mediated inhibition of p53 in the PU.1ure/ure
double mutants it appears to be markedly more active (Figure 3). progenitors support this hypothesis as resulting in an increased
AML in the PU.1ure/urep53  /  mice associates with upregula- Myb and miR-155 expression and further inhibition of PU.1
tion of E-box proteins (Myb and also Myc) and miR-155. program (Figure 4f and Supplementary Figure 4D).
Specically, in the compound mutant the upregulation of Myb The short-term p53 siRNA-mediated knockdown experiment
and Myc is signicantly more robust compared with PU.1ure/ure or provide evidence that the effect of p53 on Myb expression (and
p53  /  mice. But why does an increased level of Myb not Myb/miR-155/PU.1 axis) is likely a direct one and possibly an early
activate miR-155 in p53  /  mice as it does in PU.1ure/ure and the event in the hierarchy of the subsequent events initiated by p53
compound mutant? We suggest that the major factor that guides deletion/mutation in mouse and not a stochastic cumulative

Oncogene (2014) 4735 4745 & 2014 Macmillan Publishers Limited


consequence caused by genomic instability followed by clonal
selection. Although p53 deciency leads to multiple changes at
the HSPC level such as regulation of quiescence and self
renewal,25 it alone is not sufcient to downregulate PU.1,
upregulate miR-155 or cause AML.
Work by others suggests that PU.1 can deregulate the
expression of E-box proteins.4,26 In accordance with these
observations, our data using PU.1 overexpression in human NB4
AML cell line indicated that PU.1 is a repressor of MYB (Figure 5e).
Interestingly, in murine PU.1ure/ure or PU.1ure/urep53  /  progeni-
tors restored expression of PU.1 did not inuence Myb or miR-155
(Figure 4c), documenting differences between murine and
human AML.
We demonstrated that expression of MYB, miR-155 and PU.1
levels correlates within a relatively large proportion of human AML
patients (Figures 5a and b). At the same time we provide evidence
that MYB and miR-155 functionally cooperate to downregulate
PU.1 in mouse model of AML. This indicates that MYB, miR-155
and PU.1 axis is functional in human AML and represents the
mechanism likely responsible for downregulation of PU.1. Inter-
estingly, the PU.1-LOW patients with p53 deletion expressed the
highest levels of miR-155 and MYB, indicating that further
upregulation of MYB and miR-155 is associated with aggressive
features of p53  /  AML.
The higher number of AML patients with elevated MYB (B92%)
than the patients with upregulated miR-155 (58%) indicates that
MYB has broader regulatory potential than miR-155 and possibly
activates in AML set of other factors than solely miR-155. By in
silico predictions (Targetscan.org) miR-155 has the potential
to postranscriptionally downregulate B440 conserved target
mRNAs. As such, in addition to PU.1, miR-155 likely downregulates
other genes that can contribute to AML. Similarly, in addition to
miR-155, other microRNAs that interfere with PU.1 transcriptional
program are likely to contribute to AML pathology including
oncogenic cluster miR-1792 (Supplementary Discussion)27 or
miR-34 (30), both regulated by p53.
Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4743
Consistent with our observations, the upregulation of miR-155
as well as the downregulation of PU.1 have been previously
described in human AML6 and myelodysplasia,28,29 respectively.
Interestingly, the role of MYB within the MYB/miR-155/PU.1
regulatory circuit is also functional in lymphoid cells derived
from CLL;9 however, histone marks analyses within the MIR-155HG
indicate differences between CLL and AML tumor cells (Figure 3d,
Supplementary Figure 3A). Despite these differences, the onco-
genic circuit involving MYB, miR-155 and PU.1 may represent
general mechanism that operates in HSPCs and associated with
the development of several hematologic malignancies. Inhibition
of miR-155 and/or restoration of PU.1 levels (Figures 5d and e)
could be a valuable strategy to restore progenitor cell differentia-
tion. Additionally, upstream factors regulating MIR-155HG tran-
scription, such as MYB or MYC, are also candidate targets for
differentiation therapy of human myeloid leukemias.9,22
We propose a novel mechanism for the progressive pathology
of AML (Figure 6 and more detailed Supplementary Figure 6).
Primary AML is initiated by a dysregulation of the myeloid gene
network, such as a reduction in the expression levels of the PU.1
transcription factor. Upon a second hit mutation such as the loss
of p53 additional dysregulation to the network occurs. Specically,
upon the loss of p53 the two oncogenes Myb and miR-155 are
upregulated concomitantly with progressive PU.1 downregulation.
The further downregulation of PU.1 advances the disease into a
more aggressive form.
The possible scenarios are as followed: in p53  /  mice, Myb
(and also Myc) are only mildly upregulated (see Figure 6,
Supplementary Figure 6 left). It is possible that normal PU.1 level
prevents full deregulation of Myb. miR-155 (regulated by PU.1 via
homeostatic feedback loop) at normal PU.1 level is not
deregulated. As a result, AML does not occur. In contrast, in the
PU.1ure/ure mice (Figure 6, Supplementary Figure 6 middle)
downregulation of PU.1 to 20% leads to the upregulation of
E-box oncogenes (Myb and Myc) and together with abrogated
control of miR-155hg by low PU.1 these two events lead to the

PU.1ure/urep53-/-
p53-/- PU.1ure/ure (AML)
(aggressive AML)

p53 Myb p53 Myb p53 Myb

PU.1 miR-155 PU.1 miR-155 PU.1 miR-155

URE URE URE

Figure 6. Mechanism of aggressive murine AML. (Left) in p53  /  mice Myb are mildly upregulated yet fail to be highly expressed due to the
presence of normal PU.1 levels simultaneously repressing Myb. miR-155 (that is controlled by PU.1 by homeostatic feedback loop) at normal
PU.1 level is not deregulated by Myb (dashed arrow). Upregulation of miR-155 is not observable. As a result, AML does not occur. (Middle) in
PU.1ure/ure mice deletion of URE inhibits PU.1 to 20%. Downregulation of PU.1 leads to unsatisfactorily inhibited Myb (dashed inhibition arrow)
leading to upregulation of Myb and abrogated control of miR-155hg by low PU.1 (dashed arrow); these two events lead to upregulation of
miR-155. Upregulation of oncogenic miR-155 is known to inhibit PU.16 and both downregulation of PU.1 and upregulation of miR-155
promote AML. (Right) in the compound PU.1ure/urep53  /  mice Myb is highly upregulated as a result of inefficient repression by the two
tumor suppressors: PU.1 and p53 (dashed inhibition arrows). Highly upregulated Myb strongly activates miR-155 expression (bold arrow) that
in turn progressively downregulates PU.1, leading to abrogated control of miR-155hg by low PU.1 (dashed arrow). Highly activated Myb/miR-
155/PU.1 pathway results in accelerated pathogenesis of AML marked by several features of aggressiveness. URE, upstream regulatory
element of PU.1 gene, -indicates activation, > indicates repression.

& 2014 Macmillan Publishers Limited Oncogene (2014) 4735 4745

 Aggressive AML in PU.1/p53 double-mutant mice
P Basova et al
4744
upregulation of miR-155. Upregulation of oncogenic miR-155 is
known to inhibit PU.1,6 and both downregulation of PU.1 and
upregulation of miR-155 promote AML. Finally, in the compound
PU.1ure/ure p53  /  mice (Figure 6, Supplementary Figure 6 right)
Myb is further upregulated as a result of inefcient repression by
p53 and PU.1. High expression of Myb activates miR-155 that in
turn progressively downregulates PU.1. Highly activated Myb/miR-
155/PU.1 pathway results in accelerated pathogenesis of AML
marked by several features of aggressiveness.
In summary, we herein established a mouse model to study the
role and impact of p53 loss in AML pathogenesis. We identied a
candidate mechanism that is responsible for PU.1 downregulation
in murine AML and showed its possible involvement in the
pathogenesis and aggressiveness of human AML.
MATERIALS AND METHODS
Haematology
MAgnetiC c-Kit Separation (Miltenyi Biotec, Bergisch Gladbach, Germany)
and FACS analysis on Canto II analyzer (BD Biosciences, San Jose, CA, USA)
were performed follwing the instruments manual respectively. Flow
cytometric PU.1 assay was performed as described elsewhere.21 Details of
the antibodies used for MACS, IHC and FACS are in Supplementary
Information. Venous PB lms (May-Gru+nwald/Giemsa-Romanowsky on
LeicaBME microscope) and counts (Advia60) were determined. Fixed
parafn-embedded tissues were sliced by microtome and haematoxylin/
eosin-stained. Mouse handling and additional specic methods are
extended in Supplementary materials and methods.
Transfection
Myb siRNA (30 nM, Santa Cruz Biotechnology, Dallas, TX, USA, sc-29856),
mmu-miR-155 (100 nM, Life Technologies, Grand Island, NY, USA, AM13058/
AM17110), 0.2mg plasmid pEBB-PU.126 or p53 siRNA (30 nM, Santa Cruz
Biotechnology, sc-29436) together with pGFP were transfected into c-Kit
cells using DMRIE-c (Life Technologies) with 490% efciency and plated
for CFU colonies (104cells/1 ml MethoCultGFM3434 (Stem Cell
Technologies, CA, USA)/well). For additional details see Supplement. NB4,
a human AML cell line, was electroporated using Amaxa (Lonza Group,
Basel, Switzerland) with Myb siRNA (60 nM, Santa Cruz Biotechnology, sc-
29855), mmu-miR-155 (100 nM, Life Technologies, AM17000), 0.2 mg
plasmid pEBB-PU.126 or p53 siRNA (60 nM, Santa Cruz Biotechnology, sc-
29435) together with pGFP. FACS was used to separate transfected GFP
cells.
PCR assays
RNA from Ficoll-isolated PBMCs (36 informed AMLs and 14 control subjects;
Supplementary Figure 5B) was isolated with RiboZol (Amresco, Solon, OH,
USA), quality-checked (Nano-Drop ND1000 Spectrophotometer, Thermo
Fisher Scientic, Wilmington, DE, USA) and reverse-transcribed. For PCR the
ABI 7900HT and TaqMan probe library (Roche, Rotkreuz, Switzerland) was
used. Chromatin immunoprecipitation assay was performed using the
mixture of mouse:yeast(carrier) cells (1:35) followed by formaldehyde-
crosslink as published previously23,28 (antibodies and primers in Supplement).
Statistics
The data sets were compared using one-way ANOVA, KruskalWallis two-
tailed tests, t-test (unpaired, two-tailed) and Spearman (condence
intervals 95%: *p0.05, **p0.001, ***p0.0001. Mean and error bars (s.d.,
s.e.m.) are shown.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGEMENTS
Primary support: GACR P305/12/1033 Institutional: UNCE 204021, PRVOUK-P24/LF1/3,
SVV-2013-266509, BIOCEVBiotechnology and Biomedicine Centre of the Academy
of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European
Regional Development Fund. Other: PB: GAUK 251135 82210, GACR P301/12/P380;
Oncogene (2014) 4735 4745
MD: GAUK 251070 45410; VP: GACR 305 13-12449P; PL: Leukemia and Lymphoma
Research grant.
Authorship: Mouse-AML (PB, EK, MD, US), human-AML (VP, AJ, KV), knock-
down (PB), FACS (FS, PB), histology (LS), chromatin immunoprecipitation (PB),
experimental design (TS, VP), writing (TS, VP, PB, PL).
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