Aggressive acute myeloid leukemia in PU.1&p53 double ... Basova et al IF7.5.pdf · ORIGINAL ARTICLE...

ORIGINAL ARTICLE

Aggressive acute myeloid leukemia in PU.1/p53 double-mutantmiceP 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 ofacute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 isa well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to theiraggressiveness; 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 significantly lowerPU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1levels 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 toPU.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 p53knockdown. 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 characterizedby p53 mutation.

Oncogene advance online publication, 14 October 2013; doi:10.1038/onc.2013.414

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

INTRODUCTIONHaematopoietic cell determination is instructed by transcriptionfactors such as the Ets-domain transcription factor PU.1 (Sfpi1,Spi1). PU.1 levels are very important for development of variousblood lineages.1 PU.1 is expressed at threshold levels inhematopoietic stem and progenitor cells (HSPCs). Highestexpression is within monocytes, intermediate levels ingranulocytes and lower levels in lymphocytes.1 Erythroidprogenitors undergo progressive silencing of PU.1 level that if itis not completed results in acute myeloid leukemia (AML).2 PU.1expression is determined by interactions of its promoter withupstream regulatory element (URE) at � 14 kb relative totranscription start.3 Disruption of PU.1 gene in mouse is lethaldue to the block of the majority of blood cell development,whereas the deletion of URE (PU.1ure/ure) downregulates PU.1 to20% and promotes AML (marked by clonal accumulation ofmyeloid c-KitþMac-1lowGr-1low blast cells) preceded bypreleukaemic phase at 2–3 months (marked by two-fold increaseof c-Kitþ precursors, Gr-1þ granulocytes and splenomegaly).4

Indeed, the PU.1ure/ure AML displays clonal features characterizedby recurring chromosomal abnormalities and is retransplantableinto immunodeficient recipients.4

PU.1 is negatively regulated posttranscriptionally by micro-RNA(miR)-155.5 Constitutive expression of miR-155 in HSPCsdownregulates 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 bytemporal PU.1 occupation in HSPCs.7 This cross-regulationestablishes a regulatory loop providing a mechanism for preciseregulation of PU.1 expression. Notably, disruption of this finelytuned loop may contribute to AML pathology.8 Mir-155hg istranscriptionally activated by the Myeloblastoma protein (Myb)9

whose primary function is to stimulate progenitor cell proliferationand impede differentiation. Expression of Myb must bedownregulated during cell maturation.10

Tumor suppressor p53 has a central role in cellular response toDNA damage and other stresses by controlling the G1/S phasetransition. Activation of p53 leads to cell cycle arrest or cellsenescence and/or apoptosis. In turn, loss of p53 promotesproliferation, 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 predisposesmice to solid tumors, mostly lymphomas, by 6 months of age.13,14

While the loss of p53 does not result in myeloid dysplasias, its lossin combination with other defects, such as Kras mutations,promotes AML.15 p53 inactivation stimulates leukemia-initiatingcell survival15 together with E-box proteins (Myb and Myc) that areinvolved in leukaemogenesis.4,16,17 Approximately 10–15% AMLpatients bear p53-inactivating mutations and display shortersurvival and chemoresistance;15,18–20 however, the particular

1Department of Pathophysiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; 2Department of Experimental Biomodels, First Faculty ofMedicine, Charles University in Prague, Prague, Czech Republic; 3Department of Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic;4Department of Medicine—Haematology, 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 James’s 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: [email protected] authors contributed equally to this work.Received 12 February 2013; revised 23 August 2013; accepted 2 September 2013

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mechanism of how the loss of p53 leads to more aggressive AMLis not fully understood.

In this study, we crossed murine PU.1 knockdown model(PU.1ure/ure) and p53� /� mutant mice to address the question ofhow loss of p53 modulates AML that is caused by a defect of PU.1.The composite PU.1ure/urep53� /� mice developed highly aggres-sive AML that is coupled with further downregulation of PU.1.Mechanism between loss of p53 and downregulation of PU.1seems to be indirect and involves deregulation of E-box proteinsand miR-155. In addition, the described mechanism in PU.1ure/ure

p53� /� murine AML was also observable in human AML.

RESULTSPU.1/p53 double-mutant mice develop aggressive AMLPU.1ure/ure mice were mated with p53� /� strain to producePU.1ure/urep53� /� mice. At 3 months of age the significantdifferences between PU.1ure/ure mice and PU.1ure/urep53� /� mice

included weight loss, lethargy and a dorsal hump (Figures 1Aa andb). These symptoms indicated that PU.1ure/urep53� /� micesuffered from a more severe disease. Examination of theperitoneal cavity revealed that PU.1ure/urep53� /� mice containedprominent hepatosplenomegaly (enlarged liver and spleen); largerin comparison with the PU.1ure/ure (that also were hepatospleno-megalic) or control mice (Figures 1Ab–d).

Histological examinations of the spleens indicated that thePU.1ure/urep53� /� mice are infiltrated by cells of immaturemorphology strongly resembling human AML. Particularly,hyperemic spleens showed marked hyperplasia. AML-like myelo-blasts with heterogeneity of nucleocytoplasmic ratio observed inthe PU.1ure/ure and PU.1ure/urep53� /� mice replaced normalspleen architecture (Figures 1Ae–g). AML spleens contained tracesof dysplastic extramedullary haematopoiesis, from which mega-karyocytes were severely dysplastic in the PU.1ure/urep53� /� mice,whereas less dysplastic changes were observed in PU.1ure/ure

spleens (Figures 1Ae–g, Supplementary Figure 1).

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Figure 1. PU.1ure/urep53� /� mice develop aggressive AML. (A, a) Mice at 3 months. (A, b–c) 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 (N¼ 32), PU.1ure/ure (N¼ 65), andPU.1ure/urep53� /� mice (N¼ 35). (A, e–g) 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 weight±s.d. (WT(N¼ 38), PU.1ure/ure (N¼ 65) and PU.1ure/urep53� /� mice (N¼ 38) at 2 and 4 months. (C) Survival of WT (N¼ 20), p53� /� (N¼ 66), PU.1ure/ure

(N¼ 85) and PU.1ure/urep53� /� (N¼ 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 (N¼ 8), p53� /� (N¼ 6), PU.1ure/ure (N¼ 10) and PU.1ure/ure

p53� /� mice (N¼ 6) at 3 months. Mean±s.d., P-values (Kruskal–Wallis).

Aggressive AML in PU.1/p53 double-mutant miceP Basova et al

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Oncogene (2013) 1 – 11 & 2013 Macmillan Publishers Limited

As indicated in Figure 1c and Tables 1 and 2, the overall survivaldata confirmed that PU.1ure/urep53� /� mice die significantlyearlier than PU.1ure/ure or p53� /� mice (median B3 months).Other than 3 out of 38 cases the PU.1ure/urep53� /� micedeveloped no solid tumors.

Next, we determined peripheral blood (PB) counts of the diseasedmice (Figure 1d) and observed that the PU.1ure/urep53� /� micedisplayed markedly lower numbers of mature red blood cells(reflected by lower hemoglobin and hematocrit) as well as platelets.

Flow cytometric analyses4 of bone marrow (BM) demonstratedthat the PU.1ure/ure and PU.1ure/urep53� /� mice displayed similarexpression pattern of Mac1, c-Kit and Gr-1. Particularly, the AMLphenotype was characterized by accumulation of c-KitþMac-1lowGr-1þcells. The presence of c-Kit antigen on majority of Mac-1- and Gr-1-positive cells indicated abundance of myeloid cellswith immature phenotype. Most prominent difference was thatAML phenotype developed in PU.1ure/urep53� /� mice muchearlier (B3 months) compared with that of PU.1ure/ure (B6months, Figure 2). The preleukaemic phase, typical for PU.1ure/ure

mice, was not observed in the compound mice (Figure 2a andTable 1).

Histology examination (see Supplementary Figure 2) showedthat immature AML blast-like cells encompassed most of the BMof PU.1ure/ure and PU.1ure/urep53� /� mice, respectively. We againnoted frequent dysplastic features in the megakaryocytes ofPU.1ure/urep53� /� mice.

To test biological properties of AML derived from PU.1ure/ure andPU.1ure/urep53� /� mice we plated magnetically sorted BM-c-Kitþ

cells into semisolid media. While wild-type (WT) cells formedprogenitor CFU colonies, the AML cells of both genotypes formedmostly 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 ofPU.1ure/urep53� /� compared with PU.1ure/ure immature colonieswere significantly increased (Figure 2i). Preleukaemic progenitorsisolated at 3 month of age from PU.1ure/ure formed comparable(with WT) numbers of CFUs and negligible number of colonies ofimmature cells that increased upon replating (Figure 2i).

In summary, PU.1ure/urep53� /� mice develop a more aggressiveAML marked by (1) rapidly expanding and infiltrating blasts,severe anemia, thrombocytopenia associated with BM and splenicmegakaryocytic dysplasia, (2) in vitro aggressive behavior and (3)adverse symptoms and decreased overall survival.

Downregulation of PU.1 and deregulation of miR-155 in PU.1ure/ure

p53� /� AMLExpression levels of PU.1 are a crucial determinant of haemato-poietic differentiation and its downregulation causes AML inPU.1ure/ure mice.4 Thus, we first determined PU.1 expression intumor cells of all genotypes. PU.1 mRNA was expressed similarly inthe p53� /� to that of the WT in total c-Kitþ cells, whereas in thePU.1ure/ure mice it was B20%. This observation indicates that p53loss alone does not affect PU.1 expression. Notably, PU.1expression was further decreased (B10% compared to WT) inthe PU.1ure/urep53� /� mice (Figure 3a). In addition we measuredPU.1 transcripts in c-KitþSca-1þ lin� population of HSPCs and inc-KitþSca-1� lin� progenitors isolated by fluorescence-activatedcell sorting (FACS). Notably, HSPCs and progenitor populationswere four to fivefold under-represented in PU.1ure/urep53� /� mice(compared with PU.1ure/ure, not shown). PU.1 expression in thePU.1ure/urep53� /� HSPCs was two- to threefold lower comparedwith the same cells isolated from PU.1ure/ure mice. Similar datawere observed in c-KitþSca-1� lin� progenitors (Figure 3a).

We then quantified intracellular PU.1 protein.21 Correspondinglyto RNA level, the c-Kitþ BM progenitors of p53� /� miceexpressed comparable levels of PU.1 protein as WT (not shown).In the PU.1ure/ureprogenitors, the PU.1 protein was decreasedcompared with WT and p53� /�progenitors and further reducedin the PU.1ure/urep53� /�progenitors (Figure 3b). Thus PU.1 level,both mRNA and protein, is decreased in the c-Kitþ population inthe 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 acandidate factor that could account for the repression of PU.1 inour AML models. We quantified miR-155 expression and foundthat it is significantly upregulated in BM-derived c-Kitþ

progenitors in PU.1ure/ure mice (Figure 3c). Furthermore, inPU.1ure/urep53� /� mice the miR-155 level is further elevated(more than threefold, compared with WT) and is significantlyhigher than in PU.1ure/ure mice. The increased levels of miR-155were also demonstrated within the HSPCs and progenitorpopulations of the respective genotypes (Figure 3c).

Next, we determined whether the miR-155 upregulation in AMLis regulated at epigenetic level. We performed chromatinimmunoprecipitation at the Mir-155hg locus using antibodiesagainst H3K9Ac, H3K9Me and H3K4Me. We observed that thechromatin state in c-Kitþ blast cells of the PU.1ure/urep53� /� micesignificantly differs from PU.1ure/ure or WT mice. We detectedenhanced mark of active chromatin, histone H3K9Ac, at the distalregulatory region (near � 6 kb, Figure 3d; D-6), whereas thereciprocal state was noted at the proximal promoter (near� 0.6 kb, Figure 3d; P-0.6,). Additionally, region D-6 was alsoenriched (along with D-7 and D-1.5) by H3K4Me3 (SupplementaryFigure 3A). Active marks at the amplicons D-6 and P-0.6 werereflected by inverse state of the repressive mark, H3K9 trimethyla-tion (Figure 3d). Furthermore, previous studies have reported thatthe region near � 15 kb (D-15) is occupied by PU.1 in developingmyeloid progenitors to temporally activate and subsequentlyallow inhibition of Mir-155hg locus.7 The D-15 region was enrichedby H3K9Me3 in both PU.1ure/ure and PU.1ure/urep53� /� mice(Figure 3d), indicating that under low PU.1 levels it is unable toactivate Mir-155hg transcription. Collectively, histone modifica-tions analysis demonstrates an actively transcribed chromatin atthe Mir-155hg locus in PU.1ure/urep53� /�AML blasts.

Data in this section indicate that progenitors from thePU.1ure/urep53� /� mice have, in comparison to PU.1ure/ure, afurther downregulated expression of PU.1 and concurrentlyupregulated miR-155 that is associated with dysregulatedchromatin structure in AML of PU.1ure/ure and, to a greater extent,in PU.1ure/urep53� /� murine progenitors.

Table 1. Frequency of developing MPS and AML in PU.1ure/ure andPU.1ure/urep53� /� within 6 months

Age (months) Frequency

PU.1ure/ure PU.1ure/ure p53� /�

1–2 0% 100% AML2.5–3.5 100% preleukaemic 90% AML4–5.5 100% AML 100% AML

Table 2. Frequency of developing preleukaemia and AML and tumorformation in PU.1ure/ure, p53� /� and PU.1ure/urep53� /� mice

Frequency

PU.1ure/ure p53� /� PU.1ure/ure p53� /�

Preleukaemic 10/115 0/40 0/38AML 105/115 0/40 38/38Solid tumor 0/115 19/40 3/38


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& 2013 Macmillan Publishers Limited Oncogene (2013) 1 – 11

Myb and Myc oncogenes are deregulated in PU.1ure/urep53� /�

AMLPU.1 is a direct target of miR-155.6 As such, the observeddownregulation of PU.1 expression can be accounted for by theconcurrent increase of miR-155. However, what is regulating miR-155? The E-box transcription factors MYB and MYC are associatedwith transcriptional regulation of miR-155.9,22 In accordance with

these observations these two oncogenes are increased withinc-Kitþ progenitors of p53� /� mice (Figure 3e); however, theirlevels neither stimulated miR-155 expression (Figure 3c) norcaused AML. Furthermore, Myb, and to lower extent also Myc, areupregulated in PU.1ure/ure HPSCs. We note upregulation of Myb atsimilar levels in the individual mutants (p53� /� or PU.1ure/ure).Notably, Myb (as well as Myc) were further upregulated in

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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 areindicated on top. Double c-KitþMac-1þ myeloid cells represent immature AML phenotype. Quadrants display cell%. (b–h) Flow cytometry ofindicated surface markers (y axis) in PB, BM and spleen (WT (N¼ 20), p53� /� (N¼ 40), PU.1ure/ure (N¼ 115), PU.1ure/urep53� /� (N¼ 38)).Mean±s.e.m., P-values (Kruskal–Wallis). (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-Kitþ cells at day 5–7 before (ex vivo) andafter replating. Mean±s.e.m., P-values (t-test, unpaired for immature colonies).


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compound mutants (PU.1ure/urep53� /� ) compared with eitherPU.1ure/ure or to WT (Figure 3e). Upregulation of Myb in PU.1ure/ure

p53� /�AML blasts was relatively uniform as demonstrated byspleen immunohistochemistry (Supplementary Figure 3B).

In summary, Myb and also Myc are further upregulated in thecomposite PU.1ure/urep53� /� mutation and together with ele-vated miR-155 may add to aggressiveness of AML.

Functional tests reveal significance of p53/Myb/miR-155/PU.1pathway in murine AMLTo determine the individual contributions of Myb, miR-155 andPU.1 upon differentiation and leukemic growth, as well as testing

possible mutual regulation among these factors, we performedcomplementary experiments.

First, we used our animal model to manipulate the expression ofmiR-155, Myb or PU.1 in ex vivo isolated AML blasts. Down-regulation of miR-155 using anti-miR-155 resulted in a markedupregulation of PU.1 transcripts in progenitors of both PU.1ure/ure

and PU.1ure/urep53� /� mice (Figure 4a). PU.1 induction wasreflected by upregulation of several target mRNAs including Cbfb,Itgam (Mac1), Cebpa, Egr2, Cd14, Fos and Csf1r (SupplementaryFigure 4A). Next, we inhibited Myb by siRNA and again observedthe upregulation of PU.1 and its targets together with theconcurrent downregulation of miR-155 (Figure 4b, SupplementaryFigure 4B). These experiments establish that PU.1 expression is

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Figure 3. Low PU.1 level and transcriptional induction of Mir-155hg locus in AML. (a) PU.1 mRNA expression in BM c-Kitþ blasts (WT (N¼ 12),p53� /� (N¼ 28), PU.1ure/ure (N¼ 75), PU.1ure/urep53� /� mice (N¼ 24)). HSPCs and progenitors (WT (N¼ 2), PU.1ure/ure (N¼ 2) and PU.1ure/urep53� /�

(N¼ 2) mice) determined by qPCR. Fold change (y axis), controls set to 1, mean±s.e.m. (b) PU.1 flow cytometry analysis of Mac1þc-Kitþ cells ofindicated genotypes. Blocking peptide control experiment is indicated. (c) miR-155 expression (samples as in a). Mean±s.e.m., P-values (t-test,unpaired, two-tailed). (d) Chromatin immunoprecipitation using H3K9Ac and H3K9Me3 antibodies at the Mir-155hg locus (positions of ampliconsare relative to TSS, distal (D) at � 6kb and � 15kb and proximal (P) at � 0.6 kb, all data normalized to H3 content. Mean±s.e.m. (N¼ 2), P-values(t-test, unpaired). (e) Myb and Myc mRNA expression in BM c-Kitþ cells (samples as in a). Mean±s.e.m., P-values (Kruskal–Wallis).


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inhibited by both miR-155 and Myb. Furthermore, inhibition ofMyb also resulted in a decrease in miR-155 expression, indicatingthat Myb positively regulates miR-155 and is an upstreamregulator of miR-155.

To test whether ectopic expression of PU.1 can rescue themyeloid development of mutant PU.1ure/ure and PU.1ure/urep53� /�

progenitors, the c-Kitþ AML blasts were transfected with a PU.1-encoding plasmid (Figure 4c). Indeed levels of PU.1, as well as of

PU.1 target genes were upregulated (Supplementary Figure 4C).Interestingly, the levels of miR-155 and Myb were not affected byPU.1 ectopic expression (Figure 4c). These data indicate that whilePU.1 is a downstream effector of Myb and miR-155 the reciprocaldoes not hold.

Next, we determined whether Myb and miR-155 are involved inthe leukemic cell growth of the blast cells and whether restorationof PU.1 could block these effects. Leukemic growth of the c-Kitþ

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Figure 4. Functional characterization of Myb/miR-155/PU.1 axis in murine AML. mRNA expression in c-Kitþ cells transfected with: (a) anti-miR-155, (b) Myb siRNA, (c) PU.1- plasmid. qPCR data at 72 h, mean±s.e.m. (N¼ 3). (d) Immature cell- (empty bars) and CFU colonies derived fromc-Kitþ cells from AML or WTmice transfected with anti-miR-155, Myb siRNA or PU.1 plasmid. P-values are related to colonies of immature cells.Mix¼CFU�GEMM colonies, Ery¼ BFU� E, G/M¼CFU�M, CFU�G, CFU�GM, scored after 7–12 days of culture. Mean±s.e.m. of duplicatecultures. (e) Immature cell- and CFU colonies in replated semisolid cultures (day 7) following PU.1 overexpression in PU.1ure/urep53� /� c-Kitþ

blasts. Mean of blast±s.e.m., P-values (t-test, unpaired). (f ) mRNA expression in c-Kitþ cells transfected with p53 siRNA (at 120 h). (g) Immaturecell- (empty bars) and CFU colonies derived from c-Kitþ cells transfected with p53 siRNA. Mean±s.e.m., P-values (t-test, unpaired for immaturecolonies).


6


cells (used in Figures 4a–c) was assessed using semisolidmethylcellulose cultures. While WT progenitors formed only CFUscolonies, the non-treated PU.1ure/ure and PU.1ure/urep53� /� cellsformed large numbers of colonies of immature cells (Figures 4d(left) and 2i; note higher numbers of blast cell and less CFUcolonies in the PU.1ure/urep53� /� ). In contrast, the cells trans-fected with either Myb siRNA, anti-miR-155 or PU.1 plasmidformed significantly less colonies of immature cells with arestoration in the frequency (similar to WT) of all types of CFUcolonies (Figure 4d, right), consistent with expression data (Figures4a–c). Interestingly, the rescue was most effective in PU.1transfectants, confirming key importance of PU.l levels. Theeffect of PU.1 overexpression on PU.1 targets was furtherdocumented by FACS analysis of increased Mac1 and Gr-1positivity in colonies formed by PU.1ure/urep53� /�blasts(Supplementary Figure 4E). Furthermore, the replating of thePU.1-transfected PU.1ure/urep53� /�blasts revealed that increasedPU.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 bethe result of either enhanced genome instability followed byclonal selection in mice after the p53 deletion or a lack of directregulation of these factors by p53. To test these possibilities weused transient p53-siRNA knockdown. We observed, surprisingly,that the temporal inhibition of p53 level in PU.1ure/ure progenitorsto 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 ofPU.1 targets was achieved. Furthermore, p53 knockdown resultedin increased leukemic growth indicated by an increase in thenumber of colonies of immature cells and concurrent decrease indifferentiating CFU colonies (Figure 4g). These data indicate thatp53 is directly involved in the regulation of MYB/miR-155/PU.1mechanism and deregulation of this pathway is likely an earlyevent 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 p53knockdown in PU.1ure/ure c-Kitþ blasts. Decreased levels of mRNAcompared to pre-mRNA indicates indeed that PU.1 is regulated atpostrancriptional levels (as pre-mRNA localized in the nucleus isnot subjected to microRNA-mediated degradation that occurs inthe cytoplasm) (Supplementary Figure 4D).

This section confirms that (i) both Myb and miR-155 arerequired for the downregulation of PU.1 levels, (ii) suggests ahierarchical order of these factors (Myb/miR-155/PU.1) during AMLpathogenesis and (iii) the role of p53 loss as an upstream factor ofMyb expression in aggressive AML. The manipulations of thesefactors—either inhibition of the oncogenes (Myb and miR-155) orrescue of PU.1—lead to leukemia blast differentiation.

Dysregulation of p53/Myb/miR-155/PU.1 pathway is observable inhuman AMLTo validate and correlate our findings from mice to human clinicalAML, we determined expression of miR-155, PU.1 and E-boxproteins (MYC, MYB) in a cohort of 36 AML patient peripheralblood mononuclear cell (PBMCs) and 14 healthy controls(Supplementary Figure 5A).

Majority of patients (27 out 36, 75%) had decreased levels ofPU.1 (Po0.01), with a range of two- to eightfold less comparedwith controls (Figures 5a and b). First, based on the expressionlevels of PU.1, the AML patients were subdivided into two groupswith 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%

patients in PU.1-LOW group showed upregulation of miR-155(Po0.05), displaying a negative PU.1/miR-155 correlation (Spear-man, r¼ � 0.347, P¼ 0.038; Supplementary Figure 5A). Further-more, the MYB oncogene (unlike MYC) was overexpressed in 96%of PU.1-LOW patients and displayed a significant negativecorrelation with PU.1 (r¼ � 0.582, P¼ 0.0006). In addition, MYBand miR-155 were positively correlated (r¼ 0.223, P¼ 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 CD34þ cells of healthydonors are provided in Supplementary Figure 5C.

To further elaborate on the role of p53 inactivation in humanAML in relation to MYB/miR-155/PU.1 circuit, we considered thecytogenetic status of p53 in our AML patients. The deletion of17p13 locus carrying p53 gene was found in three AML patientsand strikingly all of them were among the PU.1-LOW patients withhighest MYB and miR-155 expression (indicated by arrows inFigure 5b). We note that detection of p53 deletion by FISH waslimited to a few patients’ samples and therefore the number ofp53 deletions in the cohort of patients could be higher.

The correlation between the levels of Myb, miR-155 andexpression of PU.1 in human AML may not necessarily dictatethat they are directly regulating the AML aggressiveness asobserved in the mice. Therefore, we used human AML cell lineNB4 and manipulated levels of MYB/miR-155/PU.1 axis and p53.Knockdown of MYB decreased miR-155 and reciprocally increasedPU.1; similarly as in murine AML (Figure 5c). Knockdown of miR-155 upregulated PU.1; however, likely due to restored PU.1, it ledto decreased MYB, indicating that PU.1 may function as arepressor of MYB (Figure 5d). The PU.1 overexpression in NB4cells resulted in the downregulation of MYB and its downstreamtarget miR-155 (Figure 5e). In addition, we treated NB4 cells withp53 siRNA that resulted in the derepression of MYB and miR-155coupled with the downregulation of PU.1 and its targets (Figure 5fand Supplementary Figure 5D-G), similarly as in murine AML.These data indicate that p53/MYB/miR-155/PU.1 regulatory axis isfunctional in human AML.

In summary, the pathological upregulation of miR-155 and MYBin accordance with the downregulation of PU.1 expressionsuggests that MYB/miR-155/PU.1 regulatory circuit is amongthose mechanisms that contribute to the downregulation ofPU.1 in a subset of human AML, especially in aggressive casescharacterized by p53 inactivation.

DISCUSSIONWe herein present evidence that PU.1ure/urep53� /� double-mutantmice express very low PU.1 levels and develop highly aggressiveAML (compared with PU.1ure/ure mice), thus supporting the overallnotion that regulation of PU.1 levels is involved in acuteleukaemogenesis. Extremely low levels of PU.1 can be found inHPSCs and progenitors; however, this pattern changes markedlyduring myeloid development,1 therefore low levels of PU.1 aresufficient to stimulate development up to the level of myeloid blastcells. Our current (Figures 4c and 5e) and previous data in otherAML models23 show that complementation with PU.1 rescuesmyeloid differentiation of leukemic blasts. Rescue models alsoindicate that PU.1 at low levels is not effectively stimulatingtranscription of PU.1 target genes required for myelopoiesis and atthe same time is not able to effectively repress oncogenictranscription factors: Myb and Myc, known activators of cellproliferation and inhibitors of myeloid maturation.24 Comparedwith PU.1ure/ure mice the double mutants develop aggressive AMLthat is marked by rapidly expanding and infiltrating blasts, severeanemia, thrombocytopenia associated with BM and splenicmegakaryocytic dysplasia, more aggressive leukemic in vitrogrowth and shorter overall survival (Figures 1 and 2). Somephenomena are however overlapping between the single- and


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double-mutant AML such as surface marker expression. Observedmolecular mechanisms herein associated with the AML aggressive-ness (such as expression and regulatory relationships of Myb/miR-155/PU.1 axis that lead to two different PU.1 levels) appear to bepresent in both single- and double-mutant AML, however in thedouble mutants it appears to be markedly more active (Figure 3).

AML in the PU.1ure/urep53� /� mice associates with upregula-tion of E-box proteins (Myb and also Myc) and miR-155.Specifically, in the compound mutant the upregulation of Myband Myc is significantly more robust compared with PU.1ure/ure orp53� /� mice. But why does an increased level of Myb notactivate miR-155 in p53� /� mice as it does in PU.1ure/ure and thecompound mutant? We suggest that the major factor that guides

miR-155 level is a lack of PU.1-mediated repression of Myb andmiR-155 in PU.1ure/ure and PU.1ure/urep53� /� mice (enhanced byloss of p53), which allows the activation of miR-155 that in turnfurther downregulates PU.1, resulting in AML. Indeed, experimentsusing siRNA-mediated inhibition of p53 in the PU.1ure/ure

progenitors support this hypothesis as resulting in an increasedMyb and miR-155 expression and further inhibition of PU.1program (Figure 4f and Supplementary Figure 4D).

The short-term p53 siRNA-mediated knockdown experimentprovide evidence that the effect of p53 on Myb expression (andMyb/miR-155/PU.1 axis) is likely a direct one and possibly an earlyevent in the hierarchy of the subsequent events initiated by p53deletion/mutation in mouse and not a stochastic cumulative

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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) RT–PCR for PU.1, miR-155, MYB and MYC of human AML PBMCs.Values are relative to average expression of healthy controls (CTRL, N¼ 5, for additional controls see Supplementary Figure 5C). Mean, P-values(Kruskal–Wallis, two-tailed). (c) RT–PCR 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).


8


consequence caused by genomic instability followed by clonalselection. Although p53 deficiency leads to multiple changes atthe HSPC level such as regulation of quiescence and self–renewal,25 it alone is not sufficient to downregulate PU.1,upregulate miR-155 or cause AML.

Work by others suggests that PU.1 can deregulate theexpression of E-box proteins.4,26 In accordance with theseobservations, our data using PU.1 overexpression in human NB4AML 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 influence Myb or miR-155(Figure 4c), documenting differences between murine andhuman AML.

We demonstrated that expression of MYB, miR-155 and PU.1levels correlates within a relatively large proportion of human AMLpatients (Figures 5a and b). At the same time we provide evidencethat MYB and miR-155 functionally cooperate to downregulatePU.1 in mouse model of AML. This indicates that MYB, miR-155and PU.1 axis is functional in human AML and represents themechanism likely responsible for downregulation of PU.1. Inter-estingly, the PU.1-LOW patients with p53 deletion expressed thehighest levels of miR-155 and MYB, indicating that furtherupregulation of MYB and miR-155 is associated with aggressivefeatures of p53� /�AML.

The higher number of AML patients with elevated MYB (B92%)than the patients with upregulated miR-155 (58%) indicates thatMYB has broader regulatory potential than miR-155 and possiblyactivates in AML set of other factors than solely miR-155. By insilico predictions (Targetscan.org) miR-155 has the potentialto postranscriptionally downregulate B440 conserved targetmRNAs. As such, in addition to PU.1, miR-155 likely downregulatesother genes that can contribute to AML. Similarly, in addition tomiR-155, other microRNAs that interfere with PU.1 transcriptionalprogram are likely to contribute to AML pathology includingoncogenic cluster miR-17–92 (Supplementary Discussion)27 ormiR-34 (30), both regulated by p53.

Consistent with our observations, the upregulation of miR-155as well as the downregulation of PU.1 have been previouslydescribed in human AML6 and myelodysplasia,28,29 respectively.Interestingly, the role of MYB within the MYB/miR-155/PU.1regulatory circuit is also functional in lymphoid cells derivedfrom CLL;9 however, histone marks analyses within the MIR-155HGindicate 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 representgeneral mechanism that operates in HSPCs and associated withthe development of several hematologic malignancies. Inhibitionof 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 fordifferentiation therapy of human myeloid leukemias.9,22

We propose a novel mechanism for the progressive pathologyof AML (Figure 6 and more detailed Supplementary Figure 6).Primary AML is initiated by a dysregulation of the myeloid genenetwork, such as a reduction in the expression levels of the PU.1transcription factor. Upon a ‘second hit’ mutation such as the lossof p53 additional dysregulation to the network occurs. Specifically,upon the loss of p53 the two oncogenes Myb and miR-155 areupregulated concomitantly with progressive PU.1 downregulation.The further downregulation of PU.1 advances the disease into amore 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 levelprevents full deregulation of Myb. miR-155 (regulated by PU.1 viahomeostatic feedback loop) at normal PU.1 level is notderegulated. As a result, AML does not occur. In contrast, in thePU.1ure/ure mice (Figure 6, Supplementary Figure 6 middle)downregulation of PU.1 to 20% leads to the upregulation ofE-box oncogenes (Myb and Myc) and together with abrogatedcontrol of miR-155hg by low PU.1 these two events lead to the

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Figure 6. Mechanism of aggressive murine AML. (Left) in p53� /� mice Myb are mildly upregulated yet fail to be highly expressed due to thepresence of normal PU.1 levels simultaneously repressing Myb. miR-155 (that is controlled by PU.1 by homeostatic feedback loop) at normalPU.1 level is not deregulated by Myb (dashed arrow). Upregulation of miR-155 is not observable. As a result, AML does not occur. (Middle) inPU.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 ofmiR-155. Upregulation of oncogenic miR-155 is known to inhibit PU.16 and both downregulation of PU.1 and upregulation of miR-155promote AML. (Right) in the compound PU.1ure/urep53� /� mice Myb is highly upregulated as a result of inefficient repression by the twotumor suppressors: PU.1 and p53 (dashed inhibition arrows). Highly upregulated Myb strongly activates miR-155 expression (bold arrow) thatin 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 regulatoryelement of PU.1 gene, -indicates activation, > indicates repression.


9


upregulation of miR-155. Upregulation of oncogenic miR-155 isknown to inhibit PU.1,6 and both downregulation of PU.1 andupregulation of miR-155 promote AML. Finally, in the compoundPU.1ure/ure p53� /� mice (Figure 6, Supplementary Figure 6 right)Myb is further upregulated as a result of inefficient repression byp53 and PU.1. High expression of Myb activates miR-155 that inturn progressively downregulates PU.1. Highly activated Myb/miR-155/PU.1 pathway results in accelerated pathogenesis of AMLmarked by several features of aggressiveness.

In summary, we herein established a mouse model to study therole and impact of p53 loss in AML pathogenesis. We identified acandidate mechanism that is responsible for PU.1 downregulationin murine AML and showed its possible involvement in thepathogenesis and aggressiveness of human AML.

MATERIALS AND METHODSHaematologyMAgnetiC c-Kitþ Separation (Miltenyi Biotec, Bergisch Gladbach, Germany)and FACS analysis on Canto II analyzer (BD Biosciences, San Jose, CA, USA)were performed follwing the instrument’s manual respectively. Flowcytometric PU.1 assay was performed as described elsewhere.21 Details ofthe antibodies used for MACS, IHC and FACS are in SupplementaryInformation. Venous PB films (May-Gru+nwald/Giemsa-Romanowsky onLeicaBME microscope) and counts (Advia60) were determined. Fixedparaffin-embedded tissues were sliced by microtome and haematoxylin/eosin-stained. Mouse handling and additional specific methods areextended in Supplementary materials and methods.

TransfectionMyb 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 CruzBiotechnology, sc-29436) together with pGFP were transfected into c-Kitþ

cells using DMRIE-c (Life Technologies) with 490% efficiency and platedfor CFU colonies (104cells/1 ml MethoCultGFM3434 (Stem CellTechnologies, 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), 0.2mgplasmid pEBB-PU.126 or p53 siRNA (60 nM, Santa Cruz Biotechnology, sc-29435) together with pGFP. FACS was used to separate transfected GFPþcells.

PCR assaysRNA 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, ThermoFisher Scientific, Wilmington, DE, USA) and reverse-transcribed. For PCR theABI 7900HT and TaqMan probe library (Roche, Rotkreuz, Switzerland) wasused. Chromatin immunoprecipitation assay was performed using themixture of mouse:yeast(carrier) cells (1:35) followed by formaldehyde-crosslink as published previously23,28 (antibodies and primers in Supplement).

StatisticsThe data sets were compared using one-way ANOVA, Kruskal–Wallis two-tailed tests, t-test (unpaired, two-tailed) and Spearman (confidenceintervals 95%: *p0.05, **p0.001, ***p0.0001. Mean and error bars (s.d.,s.e.m.) are shown.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSPrimary support: GACR P305/12/1033 Institutional: UNCE 204021, PRVOUK-P24/LF1/3,SVV-2013-266509, BIOCEV—Biotechnology and Biomedicine Centre of the Academyof Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the EuropeanRegional Development Fund. Other: PB: GAUK 251135 82210, GACR P301/12/P380;

MD: GAUK 251070 45410; VP: GACR 305 13-12449P; PL: Leukemia and LymphomaResearch 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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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)


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