Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma...

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Peter J. Valk, Jurgen Lohler, Robert K. Slany, Eric N. Olson and Carol StockingMaike Schwieger, Andrea Schuler, Martin Forster, Afra Engelmann, Michael A. Arnold, Ruud Delwel, MEF2CHoming and invasiveness of MLL/ENL leukemic cells is regulated by

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Homing and invasiveness of MLL/ENL leukemic cells is

regulated by MEF2C

Maike Schwieger,1,* Andrea Schüler,1,* Martin Forster,1 Afra Engelmann,1 Michael A. Arnold,2

Ruud Delwel,3 Peter J. Valk,3 Jürgen Löhler,1 Robert K. Slany,4 Eric N. Olson,2 and Carol

Stocking1

1Heinrich-Pette-Institute, 20251 Hamburg, Germany; 2Department of Molecular Biology, University of

Texas Southwestern Medical Center, Dallas, TX 75390, USA; 3Department of Hematology, Erasmus

University Medical Center, 3000 DR Rotterdam, The Netherlands; 4Department of Genetics, University

of Erlangen, 91058 Erlangen, Germany

Keywords: MADS transcription factors, chemokine receptor, mixed-lineage leukemia, retroviral

insertional mutagenesis

Running title: MEF2C imparts invasiveness to MLL leukemic cells

Category: Myeloid Neoplasia

* These authors contributed equally to this work

Correspondence should be addressed to: Carol Stocking, Ph.D., Heinrich-Pette-Institut, PO Box 201652,

D-20206 Hamburg, Germany. Tel: +49-40-480 51 273; Fax: +49-40-480 51 187; email:

[email protected]

Blood First Edition Paper, prepublished online July 7, 2009; DOI 10.1182/blood-2008-05-158196

Copyright © 2009 American Society of Hematology




2

Abstract

Acute myelogenous leukemia (AML) is driven by leukemic stem cells (LSC) generated by

mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation

program. Using retroviral mutagenesis, we identified genes that generate LSC in collaboration

with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a

myeloproliferation in vivo. Amongst the targeted genes, we identified Mef2c, encoding a MADS

transcription factor, and confirmed that over-expression induced a myelomonocytic leukemia in

cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have

been reported to be upregulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C

expression levels were confirmed in AML patient samples with MLL gene disruptions,

prompting an investigation of the causal interplay. Using a conditional mouse strain, we

demonstrated that Mef2c deficiency does not impair the establishment nor maintenance of LSC

generated in vitro by MLL/ENL fusion proteins – however, its loss led to compromised homing

and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding

matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to

increased homing and motility. Thus an early event in LSC generation may be responsible for the

aggressive nature of this leukemia subtype.




3

Introduction

Acute myelogenous leukemia (AML) is maintained by a small population of leukemic stem cells

(LSC), which share two main characteristics with hematopoietic stem cells (HSC): 1) self-renew

ability, thereby creating daughter cells with the capacity to replicate almost indefinitely; and 2)

differentiation capacity, by which progressively differentiating cells are generated – and which is

responsible for the cellular heterogeneity found in leukemia.1,2 Fusion proteins generated from

translocations involving the MLL gene on chromosome 11q23, which are associated with infant

mixed-lineage leukemia (MLL) and adult AML with poor prognosis, have been shown to

generate LSC from committed myeloid progenitors.3,4 MLL regulates gene transcription at the

chromatin level and MLL leukemias are invariably associated with aberrant expression of

selected HOX and TALE homeobox genes, which have been implicated in the establishment of

the LSC in a cooperative manner.5-7 More recently, the Mef2c transcription factor, encoded by a

member of the (MCM1-agamous-deficiens-serum response factor) MADS family of homeotic

genes, has also been implicated in the establishment of MLL-induced LSC.3 Nevertheless, the

role of these proteins or other downstream effectors in the conversion of a progenitor cell into an

LSC capable of inducing an aggressive clinical leukemia is not well defined.

We initiated this study to identify novel mechanisms in the generation of AML LSC by

using a genetic approach, in which mice deficient for the interferon regulatory factor-8 (Irf8)

were infected with murine leukemia virus (MuLV) to induce acute leukemia. Irf8-/- mice

spontaneously develop a chronic myeloproliferative syndrome,8 most likely due to the combined

effects of impaired apoptosis and increased sensitivity to proliferation stimuli in the myeloid

compartment.9-11 Infection with MuLV would be expected to accelerate the progression to an

acute phase by activating cooperating oncogenes (or inactivating tumor suppressors) through

random integration into the cellular DNA, resulting in the selective outgrowth of LSC that feed




4

an overt AML. Using this approach, we identified integrations in several genes that have been

implicated in the generation of LSC by MLL-fusion protein, such as HoxA9, Meis1, and Myb, but

also Mef2c. This result prompted us to not only confirm the cooperative action of Mef2c

overexpression and Irf8 deficiency, but also to explore the correlation between Mef2c and MLL.

Mef2c is one of four "myocyte enhancer factor 2" (MEF2) proteins, which make up one

of three distinct classes of MADS-box transcription factors. Mef2 proteins are central regulators

of diverse developmental programs,12 although a role in hematopoiesis was unappreciated until

recently. We have previously demonstrated that Mef2c is an important determinant of myeloid

cell fate,13 a result supported by the demonstration that Mef2c is regulated by the microRNA-223

during granulocytic differentiation.14 In addition, a critical role of Mef2c in mature B-cell

proliferation, survival, and homeostasis has recently been demonstrated.15-17

We show here that Mef2c induces myelomonocytic leukemia in cooperation with Irf8

deficiency, and also confirm that high Mef2c expression levels are found in patient samples with

disrupted MLL. However, taking advantage of mice in which Mef2c can be inducibly

inactivated, we show that Mef2c is dispensable for both establishment and maintenance of LSC

generated by MLL fusion proteins. Instead, we provide evidence that demonstrates the

importance of Mef2c for modulating the homing and invasive properties of MLL-AML.




5

Material and methods

Mouse strains

The generation of conditional (Mef2cfl) and non-conditional (Mef2c-) inactivating alleles of Mef2c have

been previously described.18,19 Mef2cfl/+ and Mef2c-/+ mice were backcrossed into the C57BL/6J

PtprccPepca (here called B6) mouse strain (for over 10 generations) and then crossed to generate Mef2cfl/-

and Mef2c+/+ B6 littermates, which were used for these studies. To induce Mef2c excision, mice were

crossed to MxCre B6 mice20 and newborns were injected with a double dose of 500 µg polyinosinic-

polycytidylic (pIpC) acid (Sigma-Aldrich Chemicals, Munich, Germany) dissolved in PBS. Alternatively,

bone marrow (BM) cells were infected with a retroviral vector coexpressing the CRE recombinase and

eGFP (pSF91-CRE-iGFP; R860).21 In transplantated mice, pIpC (300 µg) was injected three times at two

day intervals, as indicated. To validate Mef2c excision, DNA from blood or harvested cells was subjected

to PCR, as previously described.13 B6.SJL-PtprcaPep3b/BoyJ (here called B6-Ptprca) mice were used as

hosts for all BM transplantations. Irf8-/- B6 mice8 (also known as Icsbp-/-) and NOD.CB17-Prkdcscid/J

mice (here called NOD/scid) were originally obtained from Ivan Horak (Berlin, Germany) and Charles

River Laboratories (Sulzfeld, Germany), respectively, and were maintained in the Heinrich-Pette-Institut

animal facilities. All animal experiments were approved by the Commission for Animal Experiments at

the Heinrich-Pette-Institute together with Hamburg Office of Health. Survival curves of experimental

animals were calculated by the method of Kaplan-Meier and statistical analysis was performed using the

SPSS 15.0 software (SPSS, Chicago).

MuLV infections and isolation of integration sites

Newborn Irf8-/- B6 mice were infected i.p. with circa 5x105 infectious units of Moloney (Mo)-MuLV

harvested from SC1 fibroblasts as previously described.22 Retroviral integration sites were isolated from

genomic DNA of tumors using a modified protocol of the “ligase-mediated extension-primer tag

selection” PCR reaction.23




6

Retroviral gene transduction and BM transplantation

Retroviral vectors were used to transduce the murine Mef2c-α1 isoform (SF91-iGFP)24 or the MLL/ENL

fusion protein (MSCVneo25, MYs-GFP26or an MYs-Venus variant) into murine BM progenitors.

Retroviral pseudotypes were generated in 293T cells transiently transfected with the retroviral vector

plasmid plus plasmids expressing MoMuLV env and gagpol genes. For BM infections, virus particles

were fixed onto 35mm plates coated with Retronectin® (Cambrex, Verviers, Belgium) before the addition

of BM progenitors suspended in StemSpan SFEM medium (StemCells Technologies, Vancouver)

supplemented with 1% glutamine, 50ng/ml mSCF (Peprotech, Hamburg), 100ng/ml each hFlt3-L and

hIL-11 (StemCells Technologies), and 10ng/ml mIL-3 (Strathmann, Hamburg). Infections were repeated

after 24 hours. For Mef2c transductions, BM was isolated from mice that received i.p. injections of 5-

fluorouracil (150mg/kg) four days prior to isolation. For MLL/ENL transductions, BM was isolated from

untreated mice and progenitors (Linneg) were enriched using the Midi MAX Lineage Depletion Kit

(Miltenyi Biotec, Bergisch-Gladbach, Germany).

Transduced BM cells (1x106) supplemented with untreated whole BM cells (1x105) were

transplantated into lethally irradiated (9Gy) B6-Ptprca mice. For retransplantation experiments, 5x106

cells isolated from spleens of diseased mice were injected i.v. into either non-treated recipients. Mice

were monitored regularly for disease symptoms. FACS and histological analysis of hematopoietic cells

and organs were performed as previously described.24,27 The following antibody clones were used: mouse

anti-CD44 APC-conjugated (clone IM7), mouse anti-CD184 PE-conjugated (clone CXCR4), mouse anti-

CD11b APC-conjugated (clone M1/70), mouse anti-Gr1 PE-conjugated (clone RB6-8C5), and mouse

anti-F4/80 PE-conjugated (clone MCA497PEB). Unconjugated anti-CD16/32 (clone 93) was used to

block unspecific binding.

Establishment of cell M/E cell lines and colony forming assay

Two independent sets of MLL/ENL expressing cell lines (M/E cells) were generated. In the first set, BM

from Mef2cfl/- or Mef2c+/+ littermates were transformed with MSCVneo-MLL/ENL, as previously




7

described.24,28 These were subsequently infected with either with SF91-iGFP or SF91-CREiGFP. The

second set was generated from BM from Mef2cfl/-MxCRE and Mef2c+/+MxCRE littermates (treated with

pIpC as newborns) and infected with MYs-MLL/ENL-GFP. Transformed cells were isolated by serial

replating (7-day intervals) in Methocult M3434 (StemCell Technologies). Single colonies from the

quaternary cloning assay were picked at culture in RPMI 1640 medium, supplemented with 10% FCS, 1%

Glutamine, 10ng/ml each mIL3 (Strathmann), mGM-CSF, hIL-6 (Peprotech) and 100ng/ml mSCF

(generously supplied by Ursula Just, University Kiel). Several independent clones were analyzed, but no

difference in phenotype or proliferation was observed. For subsequent studies Mef2cΔ/-M/E-cells were

infected with MYs-iPac vectors, expressing a puromycin resistance marker alone, or with wild-type

Mef2c or a previously characterized HDAC-binding Mef2c mutant29 (Mef2c-VLL65-67ASR, here called

Mef2cASR), which retains the ability of Mef2c to induce differentiation (A.S. and C.S., unpublished

results). Both sets of cell lines were analyzed extensively in vitro (differentiation markers, proliferation,

and migration assays). The first set (M/E-Mef2cΔ/--CRE/GFP and M/E-Mef2c+/+-CRE/GFP cells) were

used to test tumorigenicity in vivo by i.p. injection of 106 cells into non-irradiated NOD/scid mice.

Gene expression analysis of AML samples and MLL/ENL-transformed cells

A total of 285 AML cases have been analyzed using Affymetrix HGU133A GeneChips (Affymetrix,

Santa Clara, CA).30 Relative expression values for IRF8 and MEF2C for each patient was used to

determine mean values for patient clusters. For gene expression profiling of M/E cells, RNA was isolated

from M/E-Mef2cΔ/- cells infected with MYs-iPAC or MYs-pMef2cASR (see above) after puromycin

selection at three different time points, pooled, and hybridized against the Agilent Whole Mouse Genome

Microarray 4x44K using the one-color service of Miltenyi. Transcript levels were verified by real-time

RT-PCR using the SYBRGreen Reaction Mix (Roche Mannheim) in a Roche Light-Cycler. cDNA levels

were normalized against Hprt transcript levels. Primers and amplification conditions are available upon

request. The microarray data is available in the GEO public database under accession number GSE1159.




8

In vivo and in vitro homing and proliferation assays

In vivo hematopoietic cell homing assay was performed as described.31 Briefly, freshly isolated BM cells

were labeled with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes,

Eugene, OR), washed, and then 107 cells were injected per recipient mouse irradiated (9Gy) 1h before

transplantation. Mice were killed 4 hours after the transplantation, and the percentage of CFSE+ cells in

the BM and spleen were determined by FACS. In order to obtain a statistical reproducibility, at least

7×106 events were analyzed for each sample. Transmigration-assays were performed using Transwells (5

µm pore size; Costar, Corning, NY) in 24-well tissue culture plates. Briefly, 1-2x105 M/E cells (see

above) were plated in upper chamber of each Transwell in medium without chemoattractant. The medium

in the lower chambers was supplemented with either 100ng/ml SDF1α or 1xPBS. Cell numbers were

determined after 3-5h. Cobblestone formation assays were performed by plating 106 M/E-Mef2c/GFP or

M/E-GFP cells onto a monolayer of stromal MS-5 cells in 6 well plates (Falcon) for 24h.

To determine the proliferation rate in vivo, Linneg BM progenitors isolated from pIpC treated

Mef2c+/+MxCre and Mef2c-/flMxCre littermates were infected with MYs-MLL/ENL-Venus. Infected BM

cells (1.2x106) were transplanted into lethally irradiated (9Gy) B6-Ptprca mice. Thirteen days after

transplantation, mice were injected i.p. with 2mg BrdU and analyzed two hours later. BM cells were

sorted for Venus positive cells and these cells were subsequently stained with the APC BrdU Flow Kit

(BD Biosciences, Heidelberg) following the manufacturer's instructions.

Results

Identification and confirmation of Mef2c oncogenic activity in a mouse model

To identify novel genes that cooperate with other genetic mutations to generate AML, we

performed retroviral insertional mutagenesis on Irf8-/- mice, which spontaneously develop a mild

chronic myeloproliferative disease due to Irf8 deficiency.8 Newborn Irf8+/+ and Irf8-/- B6 mice

were infected with Mo-MuLV and monitored for leukemia development. MuLV-induced




9

leukemia was accelerated in Irf8-/- as compared to Irf8+/+ B6 mice (Figure 1A). Furthermore,

although Mo-MuLV induced a T-cell leukemia (CD3+) in 100% of the control mice, myeloid

leukemia (CD11b+) was observed in 29% of Irf8-/- mice. These results confirm a cooperative

effect between Irf8 deficiency and MuLV insertional mutagenesis. Retroviral integration sites

mapping to the HoxA7, HoxA9, Meis1, and Myb gene loci were found in several independent

myeloid tumors, similar to other mouse models.32 In addition, integrations in the Mef2c gene

locus, mapping within 4kb upstream of the first coding exon, were found in circa 20% of the

tumors analyzed (Figure 1B). A 5-fold increase in Mef2c mRNA was observed in tumors

carrying proviral integrations in the Mef2c locus, as compared to tumors with the same

phenotype but without apparent disruption of the Mef2c gene (Figure 1C).

To determine whether constitutive and high expression levels of Mef2c were causative in

leukemia development, BM cells from Irf8-/- and Irf8+/+ were infected with retroviral vectors

expressing Mef2c and transplanted into syngenic hosts. With a median latency of 79 days post-

transplantation, all mice receiving Irf8-/- BM expressing Mef2c succumbed to AML (Figure 2A).

Moribund mice were characterized by cachexia and dyspnea, and suffered from

hepatosplenomegaly. Consistent with a diagnosis of acute leukemia, most mice were anemic and

peripheral blood (PB) and BM contained a high proportion of blasts and immature monocytic and

granulocytic forms (Figure 2B-D). Peripheral white blood counts were highly elevated in 90% of

the animals (mean value of 170x106 cells/ml; range 5x106 to 800x106 cells/ml) compared with

the GFP controls (range 3.5x106 to 9x106). The leukemia was highly invasive, and a striking

accumulation of crystal-storing macrophages was observed (Figure 2E-K). Flow cytometric

analyses of PB and BM confirmed that 80 to 95% of the organs were positive for both GFP (as a

surrogate marker for Mef2c), and the majority of the tumor cells were CD11bhiGr1lo and

expressed the monocytic marker F4/80 in the BM compartment – consistent with a high




10

incidence of immature monocytes and blasts (Figure 2L). Notably, no disease was observed over

a 9-month period in mice receiving Irf8+/+ BM cells that had been transduced with Mef2c vectors

with similar efficiencies (18 and 40% GFP+ cells, two independent experiments). These results

demonstrate a striking synergy between high Mef2c expression and Irf8 deficiency in the

induction of invasive acute myelomonocytic leukemia.

Relatively high levels of MEF2C transcripts in specific AML subtypes

To address the potential relevance of our findings to human disease, we analyzed MEF2C

transcript levels in a data set of leukemic BM and PB cells of 285 individuals with AML.30

Previous analysis of this data set identified 16 groups of AML with distinct gene expression

profiles. Surprisingly, we found a strong positive correlation (r = 0.40) between IRF8 and

MEF2C gene expression. These results contrast to our experimental system, in which Irf8

deficiency synergized with Mef2c over-expression, and thus suggests alternative mechanisms or

cooperating partners in human AML. A likely cooperating mechanism is the deregulation of the

MLL gene, as suggested by the high mean expression levels of MEF2C in patients carrying MLL

gene disruptions, which are found in both patient clusters #1 and #16 (Figure 3). MLL-associated

AML is predominately associated with a monocytic phenotype, however, high levels of MEF2C

expression was not found in clusters #5 or #9, which also contain high levels of monocytic and

myelomonocytic cells, refuting the theory that high MEF2C levels correlate strictly with the

differentiation phenotype. A relatively high mean expression level was also found in cluster #10

(with a predominantly M1 phenotype) but this was significantly less than that found in MLL-

associated AML. Interestingly, other common integration sites found in our screen (Meis1,

HoxA7, HoxA9, and Myb) have also been shown to be important players in MLL-associated

leukemogenesis.6,33 Furthermore, the high levels of MEF2C expression in MLL-leukemia




11

samples agrees with recent findings, in which MEF2C was identified as a direct target of the

MLL/AF9 fusion protein within the LSC compartment.3

Mef2c expression is not necessary for MLL/ENL transformation in vitro

To test the relevance of high MEF2C expression levels in MLL-deregulated AML, we used

littermates from a cross between mice carrying a conditional Mef2c null allele (Mef2cfl),18 which

is flanked by loxP sites for deletion by CRE recombinase, and mice carrying a constitutive Mef2c

null allele (Mef2c-; homozygous Mef2c null mice are embryonic lethal).19 In a first set of

experiments, BM cells from Mef2c+/+ or Mef2cfl/- littermates were infected with MLL/ENL

retroviral vectors to establish transformed cell lines (M/E cells). The MLL/ENL fusion protein,

which is the product of a common translocation involving the MLL gene, readily immortalizes

myelomonocytic progenitors in vitro, which are tumorigenic in vivo.28 The M/E cells were then

infected with a CRE/GFP expression vector to determine if Mef2c was necessary for the

maintenance of the transformed cell lines. Cells were sorted for GFP expression, and excision

was confirmed by PCR analysis. Notably, cells lacking functional Mef2c continued to

proliferate at the same rate as Mef2c+/+ transformed cells, also receiving the CRE vector, as

measured by incorporation of thymidine over a 48h period and cell counts over 6 days (data not

shown). In a second set of experiments, BM was isolated from Mef2cfl/--Mx1CRE mice, in which

the floxed allele was deleted by activating CRE expression by polyI:C, generating Mef2cΔ/-.

Transformed M/E cell lines could be readily established for both genotypes, as demonstrated in a

replating assay (Figure 4A). The morphology and the immunophenotype of the cells were similar

for all genotypes, although a slight but reproducible increase in Gr1 expression levels was

consistently observed in all cells lacking Mef2c (Figure 4B). Incubation in different myeloid




12

cytokines (singly or as cocktails) did not change either their morphology or surface antigen

expression. In all cases, the excised allele was confirmed by PCR analysis of cellular DNA (data

not shown). Thus Mef2c is not necessary for either the establishment or maintenance of the

immortalized state, nor for the block in terminal differentiation in MLL/ENL transformed

cultures.

Mef2c is not required for MLL/ENL-induced leukemia, but shortens the disease latency

and increases the dissemination of leukemic cells in vivo.

To determine if Mef2c expression was necessary for tumorigenesis in vivo, mice were injected

i.p. with cells from the established Mef2cΔ/- or Mef2c+/+ M/E cell lines (both carrying a CRE

retroviral vector) and monitored for disease induction. Mice of each cohort developed large

tumor masses within the peritoneal cavity with similar size and kinetics (Figure 4C and data not

shown). These results demonstrated that loss of functional Mef2c did not impair tumor/leukemia

formation in vivo, even if the M/E cells were initially transformed in the presence of Mef2c

However, post mortem inspection of hematopoietic organs within these mice revealed a striking

difference between mice receiving Mef2cΔ/- or Mef2c+/+-M/E cells. In contrast to mice receiving

M/E-Mef2c+/+ cells, in which infiltrating tumor cells were readily observed in the spleen, as

monitored by GFP positivity (median 89%; range 66% to 100%) and size (Figure 4D), mice

receiving M/E-Mef2cΔ/- cells had smaller spleens with lower GFP levels (median 29%, range 8%

to 85%; Figure 4D). A similar reduction in the spread to BM was also observed, as measured by

proportion of GFP+ cells (mean 13.5±8% for Mef2cΔ/- cells, n=6, compared to 82±16% for

Mef2c+/+ cells, n=7). Normal hematocrit levels (range 34% to 43%) and leukocyte counts (range

2-12x 06 cells / ml) were observed in both cohorts.




13

To verify that the impaired homing and dissemination was not due to a specific

characteristic of the immortalized cells, Mef2cΔ/-MxCRE or Mef2c+/+MxCRE BM cells (from

mice previously treated with pIpC) were freshly infected with MLL/ENL-GFP viral vectors with

similar efficiencies (29% and 25%, respectively) and transplanted i.v. into conditioned mice. All

mice receiving Mef2c+/+ or Mef2cΔ/- BM cells infected with MLL/ENL vectors succumbed to a

rapid disease, characterized by expansion of myeloid CD11b+ cells in the BM and spleen, but

with different latencies (median latency of 52 and 75 days, respectively for Mef2c+/+ or Mef2cΔ/-

BM recipients) (Figure 5A). Retransplantation of tumorigenic cells into secondary (non-

irradiated) recipients led to a rapid disease, regardless of the genotype, although a slight but

significant increase in disease latency was observed in mice receiving Mef2cΔ/- leukemic cells

(Figure 5B).

To better compare the disease with and without Mef2c expression, another MLL/ENL

transduction / BM transplantation was performed, in which the infection frequency was adjusted

to obtain 5% GFP BM cells. After the first signs of mortality (circa day 50), mice from the two

cohorts (mice receiving MLL/ENL transduced BM from either Mef2c+/+ or Mef2cΔ/- mice; n=8

per genotype) were analyzed. Inspection of sternal BM sections of all animals revealed a rather

homogenous picture due to the predominance of transformed myeloid cells and striking

hypercellularity, leading to almost complete suppression of the sinusoidal vascular system (data

not shown). Morphological and surface expression analysis confirmed that the BM was

comprised almost exclusively of CD11b+ myeloblasts and immature forms (Figure 5C). No

significant difference between Mef2c+/+ or Mef2cΔ/- BM recipients was observed, as quantified by

determining the proportion of GFP+ (i.e. MLL/ENL expressing) cells by FACS analysis (Figure

5D). Indeed, the very high proportion of transformed cells in this organ suggests that this is the




14

origin of the leukemic cell transformation. However, the spread of the transformed cells to the

periphery, and to other lymphoid or non-hematopoietic organs was strikingly different between

the two cohorts, manifesting in two distinct clinical diseases in the two genotypic cohorts. First

of all, whereas all mice showed splenomegaly, the median spleen weight of Mef2c+/+ recipients

was significantly higher than mice receiving Mef2cΔ/- (Figure 5E). Although, in both cohorts,

histological analysis of the splenic sections showed complete effacement of the normal

architecture due to myeloid hyperplasia of the red pulp (Figure 5F). FACS analysis demonstrated

a high incidence of MLL/ENL (GFP+) transformed myeloblasts (CD11b+) in the both cohorts,

although a significantly lower proportion was observed in Mef2cΔ/- BM recipients (Figure 5G).

Furthermore, whereas hepatomegaly was observed in 6 out of 7 mice receiving MLL/ENL

Mef2c+/+ BM, this was observed in only 2 out of 8 Mef2cΔ/- recipients. Histological inspection

showed that the normal liver lobular architecture was destroyed in all mice with enlarged livers;

the periportal and pervenous liver parenchyma being replaced by infiltrating leukemic

myeloblasts and immature forms (Figure 5H). In contrast, the non-enlarged livers of Mef2cΔ/-

BM recipients showed only limited myeloblasts infiltration (Figure 5H). Similarly,

lymphadenopathy was only observed in 2 out of 8 mice of the MLL/ENL Mef2cΔ/- cohort, but in

5 out of 7 Mef2c+/+. Finally, analysis of the blood also revealed a significant difference in the

spread of the transformed cells, as evidenced by the extremely high leukocyte counts in the

Mef2c+/+ but not Mef2cΔ/- cohort (Figure 5I). Blood smears and FACS analysis confirmed that

this was due to abnormal egression of MLL/ENL-infected Mef2c+/+ myeloblasts and abnormal

myeloid forms from the BM (Figure 5J, and data not shown). Taken together these results

support the hypothesis that Mef2c is important for either the engraftment / mobilization / or

proliferation of leukemic cells into permissive environments.




15

Mef2c expression correlates with increased homing and motility properties and with

increased expression levels of chemokines and receptors

The importance of Mef2c in proliferation, homing, engraftment and/or mobilization was

investigated in a number of different assays. Firstly, although in vitro proliferation assays of M/E

cells did not reveal a difference between Mef2c+/+ and Mef2cΔ/- cells, we sought to investigate the

proliferation of MLL/ENL transformed cells in the BM. Two weeks after transplantation of

freshly infected MLL/ENL-Venus Mef2c+/+ and Mef2cΔ/- cells BM progenitors, mice were

injected with BrdU and the levels of incorporation were assayed in Venus+ cells isolated from

the BM two hours later. As shown in Figure 6A, no significant difference in BrdU uptake and

thus proliferation between Mef2c+/+ and Mef2cΔ/- cells was observed.

In a second set of experiments, the homing potential of Mef2cΔ/- or Mef2c+/+ BM cells was

directly assayed by tracking transplanted cells labeled with a fluorescent dye (CFSE). Four hours

after transplantation into conditioned mice, significantly lower numbers of transplanted Mef2cΔ/-

as compared to Mef2c+/+ cells could be detected in both BM and spleen (Figure 6B), suggesting

impaired homing in the absence of Mef2c. Similarly, a strikingly reduced ability of Mef2c-

deficient MLL/ENL transformed cells to form cobblestone clusters underneath a stroma cell

layer in vitro was observed (Figure 6C), a characteristic ascribed to the interaction of the homing

factor SDF1α (CXCL12) secreted by stroma and its receptor CXCR4.34 To quantitate this

observation, and to ensure that the difference was due to Mef2c itself, a migration assay in the

presence of the SDF1α chemoattractant was performed on M/E Mef2c+/+, M/E Mef2cΔ/-, as well

as M/E Mef2cΔ/-+Pac (conferring resistance to puromycin) and M/E Mef2cΔ/-+Mef2c/Pac (in

which Mef2c was reintroduced into the Mef2c deficient cells). These experiments demonstrated




16

that loss of Mef2c lead to decreased migration capacity, whereas reintroduction of Mef2c could

rescue this effect (Figure 6D). As Mef2c protein activity is highly dependent on type IIa HDAC

binding (which is controlled by extracellular cues), we sought to determine if relief of HDAC

inhibition may reveal a stronger migration activity. To test this, a Mef2c mutant (Mef2cASR),

which retains transcription activity but can no longer be repressed by type IIa HDACs,29 was

tested in our assay. Expression of this HDAC-binding mutant in Mef2c-deficient cells resulted in

a high migration index relative to control cells in the absence of SDF1α (Figure 6E), possibly

reflecting an autocrine mechanism. However, this activity was inhibited by addition of SDF1α

(Figure 6E), suggesting reciprocal inhibition with other chemokines, as previously described.35

To determine if loss of effective homing to various hematopoietic organs was the key

mechanism by which loss of Mef2c function impaired MLL/ENL induced disease, a

transduction/ BM transplantation assay was performed on Mef2cfl/fl MxCRE BM cells not

exposed to pIpC. One week after transplantation of MLL/EENL-transduced BM cells, half of

the mice (n =7) were injected with pIpC to induce Mef2c excision. The disease latency of these

mice were comparable to that of mice expressing functional Mef2c (Figure 6F). PCR analysis

confirmed the efficient recombination of the floxed alleles in the developing tumors (Figure 6G).

No significant difference in spleen weights or GFP percentage was observed in these two mouse

cohorts (data not shown), although a higher incidence of hepatomegaly was observed in mice

with tumors expressing functional versus inactivated Mef2c (+pIpC) (0.8 versus 0.3). These

results demonstrate that defective homing capacity of MLL/ENL BM cells lacking functional

Mef2c is a key factor in the increased disease latency of MLL/ENL induced leukemia in these

cells.




17

Microarray gene expression profiling reveals upregulation of several chemokine ligands

and receptors, as well as matrix metalloproteinase genes in Mef2c+ cells

To determine the mechanism behind the defective homing capacity of Mef2c deficient cells, the

surface expression of CXCR4 and CD44, both known to be important in homing and engraftment

of hematopoietic cells, was determined (Figure 7A). CD44 was detected at high levels on all

MLL/ENL transformed cells, regardless of Mef2c status. In contrast, a small but consistent

subpopulation (up to 15%) of MLL/ENL-transformed Mef2c+/+ cells expressed high levels of

CXCR4, whereas CXCR4 could not be detected on transformed cells lacking Mef2c (Figure 7A).

Somewhat surprisingly, although CXCR4 was not detected on the surface of Mef2c-/- cells, high

levels of CXCR4 transcripts were detected (see below), suggesting a more complex regulation of

CXCR4 surface expression.

To gain insight into the possible mechanism by which Mef2c may impart increased

homing and invasion properties to the MLL/ENL transformed cells, microarray analysis was

used to compare gene expression profiles. For this assay, we used the Mef2cASR mutant, in which

the protein sites known to interact with Type IIa HDACs were mutated, to ensure that Mef2c

transcriptional activity was not repressed.29 A striking upregulation of transcripts encoding

chemokine ligands and receptors was observed in cells engineered to express Mef2cASR, which

was confirmed by RT-PCR in most cases (Figure 7B and C). Most prominent was the close to 3-

fold upregulation of CCR2, a shared receptor for several ligands of the CC family, including

CCL2. The closely related CCR5 receptor was also upregulated, but overall expression levels

were one-tenth of that of CCR2, as judged by signal intensity on microarrays. Indeed, CCR2 and

CXCR4 were the only two chemokine-receptor genes expressed at high levels in the MLL/ENL

transformed cells, giving signals that were more than 70-fold higher than background levels on

the arrays. In agreement with our analysis of M/E Mef2cΔ/- and Mef2c+/+cells, only a minor




18

difference in CXCR4 transcript levels were observed when Mef2c was expressed in the null

background (Figure 7A and B).

In addition to chemokine receptors, we observed increased expression of a number of CC

ligands (Figure 7B). In addition to CCL2, which belongs to the monocytic chemotactic protein

(MCP) family of CC-ligands, several members (CCL3, CCL4, and CCL6) of the macrophage

inflammatory protein (MIP) of CC ligands were highly expressed and upregulated in Mef2c+

cells. No significant signals for CXCL12 (SDF1α) transcript levels, or for other CXC ligands,

were observed in these cells.

Finally, analysis of the microarray data also revealed significant increases in transcripts

encoding several matrix metalloproteinases (Figure 7D), which have also been repeatedly

implicated in cell motility, tissue invasion, and metastasis.36 Highest expression levels were

observed for Mmp8 and Mmp9 (over 30-fold above background signals). Although only slightly

higher levels of Mmp9 was observed in Mef2c+ cells, an over 5-fold increase of Mmp8

transcripts were observed in these cells. In addition, up to 4-fold higher levels of Mmp12 and

Mmp25 was also observed.

Discussion

The study presented here provides new insight into the role of the MADS Mef2 transcription

factors in leukemia induction. Two observations were made in our study: 1) In the absence of the

tumor suppressor Irf8 (but not in its presence), deregulation of the Mef2c gene induces a rapid

and aggressive myelomonocytic leukemia with close to 100% penetrance, demonstrating the

Mef2c can act as a cooperating oncogene; and 2) High levels of Mef2c are found in the

myelomonocytic leukemia associated with MLL transformation, but Mef2c was neither




19

necessary for the establishment nor maintenance of the LSC that drives MLL leukemia induction

in an experimental model, but rather modulated the clinical manifestations of the disease by

imparting motility and invasive properties. It is of interest to note that many of the downstream

oncogenes of MLL fusion proteins characterized to date have been identified by retroviral

insertional mutagenesis in Irf8 deficient mice (either in the mouse strain we used or in BXH-2

mice.)37 We propose that this primarily reflects the relatively large number of genes that are

deregulated by MLL disruption and that alone cannot induce leukemia – however, either in

concert with other MLL targets or in cooperation with the Irf8-deficient "pre-leukemic"

phenotype are potent disease inducers. A key component of the Irf8 system is most likely related

to its decrease sensitivity to apoptotic stimuli, providing a cellular environment where secondary

events leading to acute leukemia can occur.9-11

By what mechanism does Mef2c act as an oncogene in leukemia induction?

The strong oncogenic potential of Mef2c was clearly manifested in the Irf8 deficient background.

Earlier work has also demonstrated cooperation between ectopic Mef2c expression and Sox4

activation in leukemia induction.39 Somewhat unexpectedly, our study showed that Mef2c

expression is not required for MLL/ENL transformation in vitro or in vivo, although disruption of

the MLL locus is associated with high levels of Mef2c expression. Even if transformation in

vitro initially occurred in the presence of Mef2c+ cells, subsequent excision of functional Mef2c

did not impact on its ability to induce tumors in vivo. The most likely explanation is that Mef2c

oncogenic function is redundant with other downstream targets of MLL fusion proteins, such as

HoxA9, Meis1, and Myb. Notably, many of the downstream targets of MLL fusion proteins

have complementary but also overlapping transforming properties.5,6,38 Alternatively, loss of

Mef2c may be compensated by upregulation of other Mef2 proteins in these cells. We did not




20

observe upregulation of Mef2d expression in these tumors (MS, S. Roscher, CS, unpublished

results), but we cannot rule out that expression of Mef2a or Mef2b is affected.

What is the mechanism behind the transforming capacity of Mef2c observed in the Ir8

deficient BM? Recent studies of both AML and ALL have shown that transcription factors

playing pivotal roles in differentiation are frequently deregulated in acute leukemia.40,41 We thus

hypothesize that upregulated Mef2c expression also contributes to the leukemogenic process by

deregulating the normal controls of myelomonocytic differentiation. This hypothesis is primarily

based on our recent findings showing that Mef2c is a critical modulator of monocytic

differentiation in response to external stimuli13 – in the absence of Mef2c, a reduction in

macrophage/monocytic progenitors is observed in the presence of various cytokines; whereas

ectopic Mef2c expression leads to increased levels of monopoiesis at the expense of

granulopoiesis. The ability of Mef2c to modulate monocytic differentiation is also observed in

the striking monocytic phenotype of the leukemia induced by enforced Mef2c expression in Irf8-

/- BM cells. In light of the fact that the Irf8-/- background is associated with increased

granulopoiesis and defective monopoiesis,8,42,43 this was clearly unexpected. It may be these

contradicting or incomplete signaling pathways, stipulating decisions between monocytic and

granulocytic differentiation, that lead to the observed block in myeloid differentiation in our

mouse model, and which is the hallmark of acute myelogenous leukemia. An important

downstream target of Mef2c for modulating monocytic differentiation is Jun,13 which has also

been identified as a critical target in the differentiation block observed in leukemic mice with

hypomorphic mutations affecting the PU.1 transcription factor.44 However, other genes that are

normally upregulated in activated macrophages were also shown to be upregulated in Mef2c+

transformed cells (see below), which may also be key to the aggressive nature of these tumors.




21

Finally, it is important to note that high levels of Mef2c transcripts are found in the

hematopoietic stem cell compartment,3,13 and thus Mef2c may impart specific features found in

HSC to the LSC. Competitive repopulation assays have revealed a small but consistent

disadvantage to Mef2cΔ/- hematopoietic stem cells, which is at least partly attributable to their

reduced homing capacity of these cells (AE, MS, and CS, unpublished results). Although studies

by Krivtsov et al. suggested the importance of Mef2c in imparting "self-renewal," as

demonstrated in a single plating assay of MLL/AF9 transformed BM progenitors, our studies do

not support this hypothesis. – although we cannot rule out that this discrepancy is due to the

different transforming fusion proteins or perhaps different target cells.

How may Mef2c regulate homing and invasiveness?

Although functional Mef2c was found to be not necessary for either initiating or maintaining

MLL/ENL induced transformation, its expression did impact on the clinical symptoms (e.g.

increased dissemination to other organs) and disease latency of the leukemia. The earlier study

of Krivtsov et al. also observed an increased latency of leukemia induction by MLL/AF9 in the

absence of Mef2c (using siRNA technology).3 Although they predicted that Mef2c up-regulation

is necessary for establishment of MLL-induced LSC, our cumulative results suggest that a more

likely interpretation is that Mef2c expression modulates the clinical disease by impinging on

homing, motility, and invasive properties of the leukemic cells. This hypothesis is supported by

the highly aggressive and metastatic nature of the AML induced by Mef2c expression in Irf8-

deficient cells, but also by various in vitro assays and animal experiments demonstrating

impaired homing and motility of cells deficient for Mef2c. Homing and engraftment of

hematopoietic stem cells to the bone marrow and extramedullary tissue – and presumably also




22

the homing and spread of LSCs – involves common mechanisms of adhesion, migration

signaling through cytokines and chemoattractants, and MMP activation.45-47 Whereas we

observed no difference in surface expression levels of CD44 on MLL/ENL transformed cells, we

consistently observed upregulated surface expression of CXCR4, the receptor for CXCL12

(SDF1α), in a subset of Mef2c+ cells, which was not observed in Mef2cΔ/- cells. However, this

regulation was not at the transcriptional level, as similar high levels of CXCR4 transcripts was

observed in Mef2c+/+ or Mef2cΔ/-, with or without enforced expression of Mef2c. Surface

expression levels of CXCR4 has been shown to be regulated by a number of different

mechanisms, including ubiquitination,48 cytokine stimulation,49,50 and by the formation of

heterodimers.48 Notably, both CCR2 and CCR5, which were down-regulated in the absence of

Mef2c, have been demonstrated to form dimers with CXCR4, leading to increased cell surface

expression and/or increased activity,51,52 suggesting a mechanism by which surface levels of

CXCR4 may be differentially regulated in cells expressing Mef2c.

We cannot rule out, however, the contribution of other Mef2c target genes. For instance,

the increased expression levels of several CC chemokines observed in our analysis may

contribute to the increased motility and invasiveness of the Mef2c+ tumors by either auto- or

paracrine mechanism by increasing chemotaxis, stimulating proliferation and/or survival, or

altering the stroma environment.53-55 Notably, increased levels of CCR2 and its ligands have

been observed in AML with a myelomonocytic or monocytic phenotype, and correlated with

increased extramedullary involvement.54 Furthermore, CCL3, 4, 6 and 9 are all highly expressed

in the MLL/ENL transformed cells, and also in activated macrophages, providing an additional

link between the leukemic phenotype and the importance of Mef2c during normal macrophage

differentiation. Similarly, our microarray analysis also revealed a number of MMPs that were




23

upregulated in Mef2c+ cells, which have often been linked to tumor metastasis and leukemic

survival rates.56,57 In analogy to the highly related MMP10 gene, which has been shown to be

directly activated by Mef2 transcription factors,58 a conserved Mef2c-binding site within the first

intron of MMP8 suggests that it may also be targeted by Mef2c.

Implications for deregulated Mef2 transcription factors and human leukemia

Taken together, our study has clearly demonstrated the oncogenic role of Mef2c in acute

myeloid leukemia, which is most likely attributable to its important role in modulating myeloid

differentiation, but also regulating migration and homing of hematopoietic progenitors to the

bone marrow and spleen. Although high expression levels of MEF2C in AML patients samples

was limited to a few patient clusters, it is well established that Mef2c is tightly regulated at a

post-transcriptional level by a number of different mechanisms.14,59 This includes regulation by

the microRNA-223, a newly recognized target of AML1-ETO, the fusion product of the

relatively common t(8;21) AML.60 Thus deregulation of Mef2 proteins may be critically

involved in a wide range of acute leukemias.

Acknowledgments

We acknowledge all members of the Stocking laboratory for help in this study, with special

thanks to Ulla Bergholz, Arne Düsedau, Gundula Pilnitz-Stolze, Ulla Müller, Susanne Roscher,

Sylvia Wegerich, and Marion Ziegler for in valuable assistance in many of the experiments. We

also acknowledge the expert assistance of Daniel Bayer in the statistical analysis. Financial

support from the Deutsche José Carreras Leukämie Stiftung and the Japanese Human Science

Foundation (in collaboration with T. Hara) is gratefully acknowledged. The Heinrich-Pette-




24

Institut is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für

Gesundheit und soziale Sicherung.

Authorship

Contribution: M.S., A.S., M.F., and A.E. designed and performed the majority of the

experiments, analyzed data, and helped write the manuscript; R.D. and P.J.V. performed analysis

of AML patient material; M.A.A. and E.N.O. provided the Mef2c conditional mouse strain; J.L.

provided pathology and histology expertise; R.S. designed and performed experiments and

analyzed data; C.S. designed, performed, and supervised the research and wrote the manuscript.

The authors have no conflicts of interest to declare.

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28

Figure Legends

Figure 1. Identification of Mef2c as a common integration site in MuLV-induced myeloid tumors of

Irf8-/- mice.

(A) Kaplan-Meier survival curves of Irf8-/- (n = 47) or Irf8+/+ (n = 28) B6 mice infected with Mo-MuLV.

The log-rank test for comparison of cumulative incidence curves confirmed a significant (p < 0.001)

increase in disease latency in the Irf8-deficient background. The mean survival was 125 ± 6 (± s.e.) and

184 ± 15 days, respectively.

(B) Cloning and analysis of sequences flanking retroviral integration sites revealed integrations upstream

of the first coding exon of Mef2c, in both 5' and 3' orientation to the gene, as indicated. Southern blot

analysis of HindIII digested genomic DNA isolated from myeloid tumors originating from MuLV-

infected Irf8-/- mice confirmed disruption of the Mef2c locus, as indicated by arrows.

(C) Tumor samples with Mef2c integrations have high levels of Mef2c transcripts. The mean levels of

Mef2c transcripts in different tumors (IC) relative to the level in heart tissue were determined by

quantitative RT-PCR in two independent experiments performed in duplicate. Sequence analysis of RT-

PCR fragments demonstrated coding sequences for both α1 and α1-β-γ isoforms in normal BM and

tumors, the former being the more prominent form.

Figure 2. Ectopic expression of Mef2c into Irf8-/- BM progenitors induces acute myelomonocytic

leukemia in recipient mice

(A) Kaplan-Meier survival curves of mice receiving Irf8-/- BM cells after infection with retroviral vectors

carrying Mef2c/GFP or GFP alone, or uninfected controls, are shown.

(B) Blood smear of moribund mouse receiving Mef2c-Irf-/- BM cells shows leukocytosis composed

predominately of immature blast-like monocytic and granulocytic cells. The heterochromatic erythrocytes

reflect a moderate anemia, confirmed by hematocrit values between 27 and 42 (normal values 42 to 48).

Pappenheim stain.




29

(C and D) Cytospin of BM cells from mice transplanted with either Mef2c/GFP (C) or GFP (D) Irf8-/-

BM. The accumulation of blast-like cells and immature monocytes is observed in BM with ectopic Mef2c

expression, in contrast to controls, which show the typical increase in granulopoiesis and immature

erythropoiesis characteristic of Irf8-/- mice. In addition to juvenile forms, mature banded granulocytes are

the prominent form. Pappenheim stain.

(E and F) Sternal BM of mouse transplanted with Mef2c-Irf8-/- BM showing considerable hypercellularity

associated with left-shifted mono- and granulocytosis and reduced numbers of erythroid cells. The

sinusoidal vascular system is compressed and obscured and an eruption of leukemic cells through the

nutrient foramina, which normally allows vascularization of the bone marrow cavity, is observed –

leading to the invasion of leukemic BM cells into the neighboring thoracical musculature (upper half of

microphotograph). Higher magnification clearly shows myeloid blast and immature forms of the invasive

cells and the numerous phagocytic cells with an eosinophilic storage phenomenon in their cytoplasm. H &

E stain.

(G – I) Splenic sections of mice receiving either Mef2c (G,I) or control GFP (H) Irf8-/- BM. The regular

splenic architecture observed in control mice (H) is completely effaced in Mef2c mice due to the myeloid

hyperplasia of the red pulp. As in the BM, an abundance of pseudo pseudo-Gaucher cells with

intracellular accumulation of para-crystalline substance at different stages of development can be

observed. Large storage cells showing hemophagocytosis are also present (I). Pockets of erythroid cells

are observed, which compensates for the loss of erythropoiesis in the BM. H & E stain.

(J and K) The highly invasive behavior of the leukemic cells is evidenced by infiltration into non-

hematopoietic tissue. (J) Similar to the spleen, the normal liver lobular architecture is completely

destroyed and periportal and perivenous liver parenchyma is replaced by infiltrating leukemic myeloblasts

and immature forms, as well as phagocytic histocytes. H & E stain. (K). Pulmonary infiltration with

hematopoietic cells was observed in all animals analyzed. Focal peribronchial and perivascular

accumulation of immature myeloid and phagocytic histocytes is seen, as well as the diffuse thickening of

the avelolar septa by leukemic infiltration. H & E stain.




30

(L) Flow cytometirc analysis of cells isolated from BM and PB from Mef2C and control GFP transduced

mice. Expression analysis of CD11b, Gr1, and F4/80 confirm the prominent monocytic component of the

AML induced by Mef2c. Only GFP-positive cells are shown. The results are representative of all mice

analyzed.

Figure 3. High levels of MEF2C gene expression found in clusters of AML patients with high

incidence of MLL. Shown are pair-wise correlations of gene expression profiles between AML patients

calculated by OmiViz software (version 3.6; Maynard, MA) and viewed by HeatMapper, as previously

described.30 The cells in the visualization are colored by Pearson correlation coefficient values, with

deeper colors indicating higher positive (red) or negative (blue) correlations. Histograms next to each

tumor represent expression levels for the IRF8 or MEF2C probe sets (bars are proportional to the level of

expression). Six of 16 clusters identified in the cohort of 285 AML patients on the basis of the

unsupervised clustering results and other parameters, such as clinical and molecular characteristics of the

AML patients, are indicated.30 These correspond to clusters with a high incidence of t(11q23) (percentage

is indicated), FAB classifications for myelomonocytic (M4) or monocytic (M5) AML, or aberrant high

EVI1 expression. The bar diagram shows the mean expression levels (±s.e.) of MEF2C for patient

samples with known MLL gene rearrangements (MLL) are compared with all other patiient samples

(other), or with that in the patients samples within the indicated clusters. Patient numbers are indicated.

P-values were determined by the Student`s t-test; **, p<0.0001; *, p =0.02 as compared to patients with

11q23 (MLL) disruptions.

Figure 4. Mef2c is not necessary for MLL/ENL-induced transformation or maintenance in culture,

but for homing and/or spread of the MLL/ENL leukemic cells in vivo.

(A) Establishment of transformed cell lines with MLL/ENL is not impaired after deletion of the floxed

Mef2c allele by CRE recombinase. Results show the mean colony number (±s.d.) of serially replated

cultures seeded with 2 x 104 cells in methylcellulose each week. The first plating was done immediately




31

after MLL/ENL transduction, so that variation in the colony numbers reflects also infection efficiencies.

Similarly results were obtained for two independent M/E cell lines.

(B) Inactivation of Mef2c in MLL/ENL transformed Mef2cfl/- cells did not alter their morphology

(Pappenheim stain) or gross immunophenotype, as assessed by FACS analysis.

(C) Survival curves of NOD/scid mice injected i.p. with 106 M/E cells with indicated genotype and

infected with a CRE vector. Mice were sacrificed when large tumors in abdominal cavity were clearly

visible.

(D) Spleen weights of animals receiving M/E cells with the indicated genetic background. Vertical line

indicates median value. P-value was calculated by the Student's t-test.

Figure 5. Mef2c is not required for in the induction of leukemia in vivo

(A) Kaplan-Meier survival curves of B6 mice receiving BM from mice with indicated genotype after

transduction with retroviral vectors coexpressing MLL/ENL and GFP. The log-rank test for comparison

of cumulative incidence curves confirmed a significant (p < 0.008) increase in disease latency in mice

receiving Mef2 deficient BM.

(B) Survival curves of B6 mice receiving 5x106 tumor cells (i.v.) from one of three independent mice (for

each genotype) from experiment shown in Panel A. The log-rank test for comparison of cumulative

incidence curves confirmed a significant (p < 0.001) increase in disease latency in mice retransplaned

with Mef2 deficient tumors.

(C) Cytospin of BM cells from mice transplanted with MLL/ENL infected Mef2cΔ/- BM (5% efficiency)

and sacrificed 50 days later. An accumulation of blast-like cells and immature myeloid forms is clearly

seen. Pappenheim stain.

(D) Flow cytometric analysis of cells isolated from BM of mice 50 days after receiving BM (with the

indicated genotype) infected with MLL/ENL vector (5% efficiency). High proportion of GFP+ cells was

observed in all mice. Each symbol in the dot diagram to the right represents an independent mouse.

Horizontal line indicates median value. P-value was calculated by the Student's t-test. Gating on the




32

GFP+ population of the BM (lower panels) demonstrates that the MLL/ENL/GFP+ cells are

CD11b+Gr1neg /lo.

(E) Dot diagram showing the spleen weights of mice analyzed 50 days after BM transplantation.

Horizontal line indicates median value. P-value was calculated by the Student's t-test.

(F) Splenic section of mice receiving Mef2cΔ/- BM infected with MLL/ENL vectors. The regular splenic

architecture is completely effaced in mice receiving MLL/ENL-infected BM due to the myeloid

hyperplasia of the red pulp. Pockets of erythroid cells are observed, which compensates for the loss of

erythropoiesis in the BM. H+E stain.

(G) Flow cytometric analysis of cells isolated from spleens of mice 50 days after receiving BM (with the

indicated genotype) infected with MLL/ENL vector (5% efficiency). A higher proportion of GFP+ cells

was observed in mice receiving Mef2c+/+ BM as compared to Mef2cΔ/- BM. Each symbol in the dot

diagram to the right represents an independent mouse. P-value was calculated by the Student's t-test.

Gating on the GFP population of the BM (lower panels) demonstrates that the MLL/ENL/GFP+ cells are

CD11b+Gr1neg /lo.

(H) The highly invasive behavior of the MLL/ENL Mef2c+/+ leukemic cells is evidenced by infiltration

into the liver. The normal liver lobular architecture is completely destroyed and periportal and perivenous

liver parenchyma is replaced by infiltrating leukemic myeloblasts and immature forms (upper panel). In

contrast, invading cells are limited to periportal regions in mice receiving MLL/ENL Mef2cΔ/- BM. H+E

staining.

(I) Leukocyte counts of mice 50 days after receiving BM (with the indicated genotype) infected with

MLL/ENL vector (5% efficiency).

(J) Blood smears of mice receiving MLL/ENL Mef2c+/+ (upper panel) or Mef2cΔ/- BM (lower panel). The

leukocytosis is predominantly composed of immature blast-like monocytic and granulocytic cells.

Pappenheim stain.




33

Figure 6. Mef2c deficient cells proliferate in vivo but show reduced homing and motility

(A) Percentage of BrdU incorporation after a two-hour pulse in MLL/ENL-YFP-infected Mef2cΔ/-

MxCRE or Mef2c+/+ MxCRE BM cells transplanted into irradiated mice. Each square represents an

independent mouse receiving BM with the indicated phenotype. Cells were sorted for YFP expression

(19 – 34% of total BM) before staining for BrdU.

(B) BM cells from Mef2cΔ/- MxCRE or Mef2c+/+ MxCRE mice (treated with polyIC) were labeled in vitro

with CFSE and then injected via the tail vein into lethally irradiated syngeneic recipient mice. Shown is

the relative mean proportion (±s.e.) of dye positive cells from BM or spleen isolated 4h after

transplantation from 4 mice from two independent experiments. An average of 68±9.5 and 78±32 CFSE+

Mef2c+/+ cells per 105 total spleen or bone marrow cells, respectively, were detected.

(C) Visualization of MS-5 stroma cell cultures 24h after 106 M/E Mef2c+/+ or Mef2cΔ/- cells were seeded

onto monolayer. Cells transcending the monolayer are clearly seen as phase-dark cells (indicated by

arrows). White translucent cells are above the stroma layer. Experiment was repeated three times with

identical results.

(D) Migration studies were compared between M/E cells in culture in which Mef2c was either deleted (by

infection with CRE vector) or in which Mef2c was reintroduced with a Mef2c vector. Migration index

was calculated 3-5h after cells from freshly infected and selected cultures were seeded into the upper well

of a Transwell with SDF1α in the bottom chamber. Shown is the mean (±s.e.) of two independent

experiments, each performed in duplicate. P-values were calculated by the Student's t-test for each pair.

(E) Migration index of M/E cells deficient for Mef2c that were either infected with an empty (pac) vector

or a vector expressing the HDAC-biding mutant Mef2cASR. Migration index was calculated 3-5h after

cells from freshly infected and selected cultures were seeded into the upper well of a Transwell with

either PBS or SDF1α in the bottom chamber, as indicated. Shown is the mean (±s.e.) of two independent

experiments, each performed in duplicate. P-values were calculated by the Student's t-test for each pair.




34

(F) Survival curves of mice receiving MLL/ENL transduced Mef2fl/fl BM, half of which received pIpC

injections at the indicated times.

(G) Verification of complete excision of the Mef2c floxed alleles after pIpC injection, PCR analysis was

performed on DNA isolated from BM cells of diseased mice (3 from each cohort). Primers were designed

to detect either the floxed (fl) or wild-type (wt) allele. Faint wt alleles can be detected in all animals, due

to residual host cells. Tail DNA from different genotypes was used as positive controls.

Figure 7. Mef2c expression in MLL/ENL Mef2cΔ/- transformed cells results in increased expression

of several genes implicated in homing and invasive growth.

(A) FACS analysis detected increase levels of CXCR4 expression on a subset of M/E cells of the

indicated genotype immediately after recloning in methylcellulose cultures, which enriches for clonogenic

or engrafting cells. Shown is one representative experiment of three.

(B-D) Microarray analysis was performed on RNA isolated from M/E Mef2cΔ/- cells infected with

Mef2cASR/pac or pac vectors and subjected to puromycin selection. The relative expression levels (±s.d.)

of the indicated gene for cultures expressing either Mef2cASR/pac (grey bars) or pac (black bars) vectors

are indicated. For some genes (marked with a Q), the results shown are that from quantitative RT-PCR,

which in all cases confirmed the results obtained from the microarray. Results are shown for only genes

that showed signals >4-fold above background. (B) Several chemokine receptors are upregulated in

Mef2c+ cultures. Asteriks denote genes with mean signal intensities greater than 50-fold over

background. (C) Several chemokines are highly expressed in MLL/ENL cultures and show upregulation

in Mef2c+ cultures. Asteriks denote genes with mean signal intensities greater than 100-fold above

background. (D) The expression of several MMP genes is upregulated in Mef2c+ cultures. Asteriks

denote genes with mean signal intensities greater that 20-fold above background.




Figure 1 – Schwieger et al.

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Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma...

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