A novel molecular mechanism involved in multiple myeloma development revealed by targeting MafB to haematopoietic progenitors

Abstract

Understanding the cellular origin of cancer can help to improve disease prevention and therapeutics. Human plasma cell neoplasias are thought to develop from either differentiated B cells or plasma cells. However, when the expression of Maf oncogenes (associated to human plasma cell neoplasias) is targeted to mouse B cells, the resulting animals fail to reproduce the human disease. Here, to explore early cellular changes that might take place in the development of plasma cell neoplasias, we engineered transgenic mice to express MafB in haematopoietic stem/progenitor cells (HS/PCs). Unexpectedly, we show that plasma cell neoplasias arise in the MafB-transgenic mice. Beyond their clinical resemblance to human disease, these neoplasias highly express genes that are known to be upregulated in human multiple myeloma. Moreover, gene expression profiling revealed that MafB-expressing HS/PCs were more similar to B cells and tumour plasma cells than to any other subset, including wild-type HS/PCs. Consistent with this, genome-scale DNA methylation profiling revealed that MafB imposes an epigenetic program in HS/PCs, and that this program is preserved in mature B cells of MafB-transgenic mice, demonstrating a novel molecular mechanism involved in tumour initiation. Our findings suggest that, mechanistically, the haematopoietic progenitor population can be the target for transformation in MafB-associated plasma cell neoplasias.

Full text

EMBO
open
A novel molecular mechanism involved in multiple
myeloma development revealed by targeting MafB
to haematopoietic progenitors
Carolina Vicente-Duen
˜as
1,2
, Isabel Romero-
Camarero
1,2
, Ine
´s Gonza
´lez-Herrero
1,2
,
Esther Alonso-Escudero
1,2
,
Fernando Abollo-Jime
´nez
1,2
,
Xiaoyu Jiang
3
, Norma C Gutierrez
2,4
,
Alberto Orfao
2,5
, Nieves Marı
´n
6
,
Luisa Marı
´a Villar
6
,M
aCarmen
Ferna
´ndez Criado
7
, Bele
´n Pintado
8
,
Teresa Flores
2,9
, Diego Alonso-Lo
´pez
10
,
Javier De Las Rivas
10,11
, Rafael Jime
´nez
2,12
,
Francisco Javier Garcı
´a Criado
2,13
,
Marı
´a Begon
˜a Garcı
´a Cenador
2,13
,
Izidore S Lossos
3,14
,Ce
´sar Cobaleda
15,
*
and Isidro Sa
´nchez-Garcı
´a
1,2,
*
1
Experimental Therapeutics and Translational Oncology Program,
Instituto de Biologı
´a Molecular y Celular del Ca
´ncer, CSIC/Universidad
de Salamanca, Salamanca, Spain,
2
Institute of Biomedical Research of
Salamanca (IBSAL), Salamanca, Spain,
3
Division of Hematology-
Oncology, Sylvester Comprehensive Cancer Center, University of Miami,
Miami, FL, USA,
4
Servicio de Hematologı
´a. Hospital Universitario de
Salamanca, Salamanca, Spain,
5
Servicio de Citometrı
´aand
Departamento de Medicina, Universidad de Salamanca, Salamanca,
Spain,
6
Servicio de Inmunologı
´a, Hospital Ramo
´n y Cajal, Madrid,
Spain,
7
Facultad de Medicina, Universidad Auto
´noma, Madrid, Spain,
8
Genetically Engineered Mouse Facility, CNB-CSIC, Madrid, Spain,
9
Departamento de Anatomı
´a Patolo
´gica, Universidad de Salamanca,
Salamanca, Spain,
10
Bioinformatics Unit, Cancer Research Center (CSIC-
USAL), Salamanca, Spain,
11
Bioinformatics and Functional Genomics
Research Group, Cancer Research Center (CSIC-USAL), Salamanca,
Spain,
12
Departamento de Fisiologı
´a y Farmacologı
´a, Universidad de
Salamanca, Edificio Departamental, Salamanca, Spain,
13
Departamento
de Cirugı
´a, Universidad de Salamanca, Salamanca, Spain,
14
Department
of Molecular and Cellular Pharmacology, University of Miami,
Miami, FL, USA and
15
Centro de Biologı
´a Molecular Severo Ochoa,
CSIC/Universidad Auto
´noma de Madrid, Madrid, Spain
Understanding the cellular origin of cancer can help to
improve disease prevention and therapeutics. Human
plasma cell neoplasias are thought to develop from either
differentiated B cells or plasma cells. However, when the
expression of Maf oncogenes (associated to human plasma
cell neoplasias) is targeted to mouse B cells, the resulting
animals fail to reproduce the human disease. Here, to
explore early cellular changes that might take place in
the development of plasma cell neoplasias, we engineered
transgenic mice to express MafB in haematopoietic stem/
progenitor cells (HS/PCs). Unexpectedly, we show that
plasma cell neoplasias arise in the MafB-transgenic mice.
Beyond their clinical resemblance to human disease, these
neoplasias highly express genes that are known to be
upregulated in human multiple myeloma. Moreover,
gene expression profiling revealed that MafB-expressing
HS/PCs were more similar to B cells and tumour plasma
cells than to any other subset, including wild-type HS/PCs.
Consistent with this, genome-scale DNA methylation pro-
filing revealed that MafB imposes an epigenetic program
in HS/PCs, and that this program is preserved in mature B
cells of MafB-transgenic mice, demonstrating a novel mo-
lecular mechanism involved in tumour initiation. Our
findings suggest that, mechanistically, the haematopoietic
progenitor population can be the target for transformation
in MafB-associated plasma cell neoplasias.
The EMBO Journal (2012) 31, 3704–3717. doi:10.1038/
emboj.2012.227; Published online 17 August 2012
Subject Categories: genome stability & dynamics; molecular
biology of disease
Keywords: cancer therapy; MafB; multiple myeloma mouse
model; oncogenes; reprogramming stem cells
Introduction
The identification of the cells of origin from which cancer
initially arises is of great importance, both for our under-
standing of the basic biology of tumours and for the transla-
tion of this knowledge to the prevention, treatment, and
precise prognosis of the human disease (Visvader, 2011).
Traditionally, the identity of the cancer cell-of-origin was
extrapolated from the histological characterization of
tumours, and assimilated to the most analogous physio-
logical cellular type. However, several transcriptome studies
have shown that the molecular characteristics of tumoral
cells do not correspond, in many cases, to what they seem
to be according to their appearance under the microscope
(Lim et al, 2009). For this reason, extrapolating the identity
of the cancer cell-of-origin without appropriate functional
lineage tracing analyses can lead us to the wrong
conclusions (Molyneux et al, 2010).
Multiple myeloma (MM) is a malignancy characterized by
the abnormal expansion of the terminally differentiated cells
of the B-cell lineage, the plasma cells (Jaffe et al, 2001), and
the nature of its cell-of-origin is still controversial. In fact, the
term tumour/cancer stem cell was first coined nearly 40 years
ago to highlight the observation that only a minority of MM
cells were capable of clonogenic growth (Hamburger and
Salmon, 1977). Two different functional approaches have
been used to directly investigate the identity of the cell of
origin of MM. One approach relies on the use of genetically
*Corresponding authors. C Cobaleda, Centro de Biologı
´a Molecular
Severo Ochoa, CSIC/Universidad Auto
´noma de Madrid, c/Nicola
´s
Cabrera, no 1, Campus de Cantoblanco, Madrid 28049, Spain.
Tel.: þ34 911964692; Fax: þ34 911964420;
E-mail: ccobaleda@cbm.uam.es or I Sa
´nchez-Garcı
´a, Experimental
Therapeutics and Translational Oncology Program, Instituto de Biologı
´a
Molecular y Celular del Ca
´ncer, CSIC/Universidad de Salamanca,
Campus M. de Unamuno s/n, Salamanca 37007, Spain.
Tel.: þ34 923238403; Fax: þ34 923294813; E-mail: isg@usal.es
Received: 17 May 2012; accepted: 20 July 2012; published online:
17 August 2012
The EMBO Journal (2012) 31, 3704–3717 |
&
2012 European Molecular Biology Organization |Some Rights Reserved 0261-4189/12
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engineered mouse models to induce the ectopic expression of
oncogenes associated to human plasma cell neoplasias in
different stages of B-cell development. Therefore, these
studies utilized B cell-specific promoters, like the IgH
promoter or the Emenhancer, to overexpress, in the B-cell
lineage, plasma cell neoplasia-associated oncogenes, like
c-Maf (Morito et al, 2011). However, what these mice
develop are B-cell lymphomas with some clinical features
that resembled those of MM (Morito et al, 2011). Thus, this
approach of targeting oncogenes associated to human plasma
cell neoplasias to mouse B cells has failed to fully recapitulate
the human MM disease. The other approach is to investigate
the tumorigenic potential of MM cells (either derived from
patients or from myeloma-prone mouse strains) by in-vivo
transplantation assays. Using this approach, it has been
demonstrated that memory B cells, rather than mature
plasma cells, are able to produce symptomatic disease
in immunodeficient mice (Matsui et al, 2004, 2008). These
in-vivo transplantation-based approaches are designed to
identify the tumour-propagating cells, but not the cell-of-
origin; hence, these studies cannot exclude that progenitor
cells (PCs) could also serve as the cells of origin for MM
in vivo. In summary, due to a lack of mouse models that
enable strict lineage targeting in the haematopoietic stem
(HS)/PC population, it has not been comprehensively
determined previously whether these cells can serve as
targets for transformation in MM. The present study aims to
reveal the role of PCs in MM initiation, using MafB as a model
of an oncogene associated to human plasma cell neoplasias.
MafB is a member of the Maf family proteins, which are
basic-leucine zipper transcription factors with important
functions both in early tissue specification and in terminal
differentiation (Eychene et al, 2008). The expression of Maf
proteins is tightly regulated in a spatio-temporal manner
during development (Eychene et al, 2008). MafB is an
inducer of monocytic differentiation that is expressed in
myeloid cells and precursors throughout haematopoietic
differentiation (Kelly et al, 2000). Expression of MafB in
erythroblasts inhibits erythroid differentiation (Sieweke
et al, 1996). However, MafB expression induces the
monocyte commitment of human CD34
þ
stem/progenitor
cells (Gemelli et al, 2006) and selectively restricts myeloid
commitment divisions at the haematopoietic stem cell (HSC)
in the mouse, hence contributing to the maintenance of a
balanced lineage potential in the HSCs (Sarrazin et al, 2009).
Maf proteins have been directly implicated in carcinogenesis,
both in cell culture systems and in human cancers (Eychene
et al, 2008). Among the different MAF proteins, MAFA and
c-MAF display the strongest oncogenic activity, whereas
MAFB is less effective in transforming cells (Nishizawa
et al, 2003; Pouponnot et al, 2006). Translocations affecting
either c-MAF (16q23) or MAFB (20q12) are present in 8–10%
of the cases of MM (Mitsiades et al, 2004; Tosi et al, 2006;
Hideshima et al, 2007). Even when it is not involved in
translocations, c-MAF overexpression has been found in
50% of MM bone marrow (BM) samples and several
human MM cell lines, suggesting an essential role for this
MAF family in the pathobiology of MM (Hurt et al, 2004).
Recently, it has been suggested that a threshold level of MAF
expression might be required for transformation, as only
mice carrying a high copy number of the MAF transgene in
the T-cell lymphoid compartment develop T-cell lymphoma
(Morito et al, 2006) or, in the B-cell lymphoid compartment,
B-cell lymphomas (Morito et al, 2011).
In this study, we have explored the early cellular changes
that might occur in plasma cell neoplasias by engineering
transgenic mice to express MafB in HS/PCs. Unexpectedly,
we show that plasma cell neoplasias arise in the MafB
transgenic mice. Besides their clinical resemblance to
human disease, these plasma cell neoplasias highly express
genes that are known to be upregulated in human MM.
Moreover, gene expression profiling revealed that MafB-ex-
pressing HS/PCs were more similar to B cells and tumour
plasma cells than to any other subset, including wild-type
HS/PCs. Consistent with this, genome-scale DNA methyla-
tion profiling revealed that MafB imposes an epigenetic
program in HS/PCs, and that this program is preserved in
mature B cells of Sca1-MafB mice, therefore showing that
MafB can act as a reprogramming factor to reset the genome
of stem/precursors cells to a terminally differentiated tumour
state. Overall, our findings suggest that a haematopoietic
progenitor population can be a target for transformation in
MafB-associated plasma cell neoplasias.
Results
Detection of chromosomal translocations by
fluorescence in-situ hybridization in BM CD34
þ
cells
of MM patients
Chromosomal translocations involving the immunoglobulin
heavy chain (IGH) gene are detected in 50–60% of MM
patients, using fluorescence in-situ hybridization (FISH).
These chromosomal rearrangements have been traditionally
used to identify tumour plasma cells. However, their presence
has not been investigated in primitive HS cells. Therefore, in
our aim of identifying the cell of origin for MM, we initially
searched for the presence of chromosomal translocations in
the haematopoietic/progenitor stem cells of MM patients. To
this aim, BM CD34
þ
cells (a marker for human haematopoie-
tic progenitor and stem cells) and CD38
þ
CD138
þ
cells (mar-
kers for plasma cells) were isolated from 15 MM patients.
FISH analysis of the CD38
þ
CD138
þ
cells showed the pre-
sence of IGH rearrangements in eight of the cases. However,
the CD34
þ
cells did not seem to show any of the transloca-
tions detected in CD38
þ
CD138
þ
cells (Supplementary
Figure 1; Supplementary Table I), although these aberrations
would be difficult to detect if the frequency of these putative
stem cells harbouring the translocation was low. These re-
sults, at face value, would seem to suggest that, either the MM
cell-of-origin where oncogene activation takes place as a
result of a chromosomal rearrangement is not a stem/pro-
genitor cell or that, being a stem/progenitor cell, the oncogene
might downregulate the stem cell markers (Mak et al,2012).
However, until now, all the experiments targeting the
expression of human plasma cell neoplasia-associated cMaf
oncogenes to the mouse B-cell compartment have failed to
reproduce the human disease in mice. From other types of
tumours, like for example acute leukaemias (Cobaleda and
Sanchez-Garcia, 2009), it is known that the functional impact
of the oncogenic translocations can manifest in the form of
cellular types whose markers of differentiation place them
either downstream or upstream of the point of origin of the
translocation. Therefore, it is potentially possible that, in
human patients, the occurrence of MM-associated oncogenic
MafB in MM pathogenesis
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alterations might happen in the HS/progenitor compartment,
and this cell-of-origin adopts/acquires afterwards a tumoral
plasma cell fate as a consequence of the oncogene’s activity.
This last possibility would reconcile the aforementioned
findings derived from both human and mouse models.
Therefore, in order to explore early cellular changes that
could/might occur in plasma cell neoplasias, we engineered
transgenic mice to express MafB in HS/PCs.
In-vivo ectopic expression of MafB in the HS/progenitor
compartment
Our initial strategy was therefore to determine whether
enforced expression of the MafB transcription factor was
sufficient to reprogram HS/PCs into tumour plasma cells.
Thus, in order to verify the feasibility of our experimental
approach, we initially examined the expression of the
endogenous MafB in normal HS/PCs (Sca1
þ
Lin

) cells
purified from the BM of control mice. MafB mRNA levels
were undetectable by RT–PCR analysis in the Sca1
þ
Lin

compartment of control mice (Supplementary Figure 2A).
Consequently, our strategy is an ideal in-vivo model to
study the consequences of ectopic MafB expression in
HS/PC Sca1
þ
cells.
We next generated and characterized transgenic mice en-
gineered to express the MafB cDNA under the control of the
Sca1 promoter, in order to determine the effect of ectopic
expression of MafB in HS/PCs (Figure 1). The Sca1-based
system ensures the expression of MafB into the HS/PCs
compartment (Miles et al, 1997; Perez-Caro et al, 2009;
Vicente-Duenas et al, 2012). Insertion of the MafB cDNA
under the control of the mouse Ly-6E.1 promoter (Miles et al,
1997) yielded the plasmid Sca1-MafB (Figure 1A), which was
used to drive Sca-1-directed expression of MafB in C57BL/
6CBA mice (Figure 1B). Two founders were obtained for
the Sca1-MafB transgene, and Southern blot comparison of
the endogenous and transgenic MafB hybridization signals
indicated transgene copy numbers ranging from B2to4
(Figure 1B). Both independent Sca1-MafB founder lines had
normal gestation and were viable and were used to examine
the phenotype further (Table I). Since c-kit is known from
previous studies to be downregulated in leukaemia stem cells
(Blair and Sutherland, 2000; Neering et al, 2007), our
functional definition of stem cell in this study does not
include c-kit as a surface marker.
In agreement with the known capacity of Sca1 regulatory
elements to drive and restrict transgenic expression to Sca1
þ
cells, Sca1-MafB transgene expression was detected in pur-
ified Sca1
þ
Lin

cells as determined by RT–PCR (Figure 1C;
Supplementary Figure 2B and C). On the contrary, Sca1-MafB
transgene expression was not detected in purified plasma
cells (B220
low
CD138
hi
FSC
hi
SSC
hi
) (Figure 1C; Supplementary
Figure 2B). Together, these results documented limited in-
creased levels of ectopic MafB expression only in Sca1
þ
cells,
enabling an in-vivo analysis of the functional impact of
enforced MafB expression in the stem/progenitor cell com-
partment. To this end, Sca1-MafB mice were assessed using
flow cytometric and histological analysis.
Reprogramming of HS/PC cells to terminally
differentiated plasma cells in the Sca1-MafB mice
Since MafB is an inducer of monocytic differentiation and
blocks other haematopoietic lineages (Sieweke et al, 1996;
Kelly et al, 2000), all the main haematopoietic compartments
were initially studied using flow cytometry in young Sca1-
MafB mice. At 8 weeks of age, no major abnormalities could
be detected neither in the myeloid nor in the T-lymphoid
compartments (as determined by Gr1, Mac1 or CD4, CD8
stainings) in the peripheral blood (PB), BM, spleen, and
thymus (Supplementary Figures 3–6). Also at 8 weeks,
B-lymphoid and plasma cell compartments in the PB were
comparable to those from wild-type control mice (as analysed
with B220, CD138, and IgM stainings). However, in the BM,
alterations of B-cell development could already be detected at
this time (8 weeks) in the form of changes in the proportions
of the different B220
þ
cell compartments. By 12 months of
age (Supplementary Figure 7), the IgM

B220
dull
B-cell pro-
genitor population had practically disappeared from the BM
of Sca1-MafB mice, and this was correlated with a decreased
percentage of B cells in the PB, BM, and spleen, indicating a
clear defect in B lymphopoiesis. The cells of human plasma
cell myeloma (the human haematopoietic cancer most fre-
quently associated with MafB overexpression) typically pre-
sent high levels of syndecan-1 (CD138) and low levels or
absence of expression of the pan-B cell antigen (CD19 in
humans or B220 in mice). In the BM and spleen of Sca1-MafB
mice, accumulations of B220
low
CD138
hi
FSC
hi
SSC
hi
plasma
cells could be clearly seen by 12 months of age
(Supplementary Figure 7B and C). In summary, the analysis
of Sca1-MafB mice by flow cytometry showed a normal
development of all the haematopoietic compartments that
was paralleled by the slow development of a plasma cell
accumulation in aging mice.
Reprogrammed plasma cells behave as tumour plasma
cells
Sca1-MafB mice exhibited an overall shortened lifespan when
compared with WT littermates (Figure 1D). The morphologic
analysis of the BM of Sca1-MafB showed the accumulation of
plasma cells (Figure 2A). In contrast to normal or reactive
plasma cells, which usually occur in small clusters of five or
six cells around marrow arterioles, plasma cell accumulations
in Sca1-MafB mice frequently occurred in larger loci, nodules
or sheets, in such a way that a significant BM volume was
comprised of plasma cells and plasmablasts. This strongly
supports the malignant nature of the plasma cells. As shown
in Figure 2A, reprogrammed plasma cells varied from mature
forms indistinguishable from normal plasma cells to imma-
ture, pleomorphic or anaplastic plasma cells. The mature
plasma cells were usually oval, with a round eccentric
nucleus with the typical ‘spoke wheel’ chromatic structure,
with abundant basophilic cytoplasm and a marked perinuc-
lear hof. In contrast, immature forms had dispersed nuclear
chromatin, a high nuclear/cytoplasmic ratio, and prominent
nucleoli (plasmablasts). Because nuclear immaturity and
pleomorphism rarely occur in reactive plasma cells, they
are reliable indicators of neoplastic plasmacytosis.
Upon necropsy of the sacrificed mice, macroscopic analysis
of the organs showed obvious splenomegaly and renal
pathology (Supplementary Figure 8). Histological examina-
tion of the Sca1-MafB kidneys revealed aggregates of eosino-
philic material (see below) in the lumen of renal tubules,
responsible for the tubular and glomerular damage
(Supplementary Figure 9A). The histological analysis
also revealed infiltration of either plasma cells or their
MafB in MM pathogenesis
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Figure 1 Transgenic expression of MafB in the mouse Sca1 lineage. (A) Schematic representation of the genomic structure of the mouse Sca1
locus and the Sca1-MafB transgenic vector used in this study. NotI sites used to excise the transgene fragments and EcoRI and HindIII sites used to
examine Southern blots are indicated. (B) Identification of the founder transgenic mice of the two lines used in this study by Southern analysis of
tail snip DNA after HindIII digestion. MafB cDNA was used for the detection of the transgene. Sca1-MafB and the endogenous MafB are indicated.
(C)MafB expression in stem and plasma cells of Sca1-MafB mice. RT–PCR analysis revealed MafB expression in stem (Sca1
þ
Lin

)cellsbutnot
in plasma (B220
low
CD138
hi
FSC
hi
SSC
hi
) cells purified from the BM of Sca1-MafB mice (line 71C). Actin was used to check cDNA integrity and
loading (bp, base pairs). The correct identity of the PCR product was analysed by hybridization using as a probe the MafB cDNA (not shown).
(D) Kaplan–Meier survival plots of Sca1-MafB mice (lines 71B or 71C). The total number of mice analysed in each group is indicated. Statistical
analysis of differences in survival was performed using the log-rank (Mantel-Cox) test, and the corresponding P-value is given.
Tabl e I Incidence and age of plasma cell neoplasia onset in Sca1-MafB mice
Tra nsg en ic
line
Mice
autopsied
a
Mice with
tumour
(%)
b
Age at tumour
onset (months)
Tumour type (%) Mice with plasma
cell accumulation
(%)
Mice with
amyloidosis
(%)
Osteolitic
lesions
(%) (n¼9)
Oligoclonal IgG
increase
(%) (n¼8)
71B 6 3 (5 0) 15.2±4.09 Plasma cell neoplasia (100) 3 (50) ND 1/1 (100) ND
71C 38 17 ( 44 .7) 15 .2±7.5 Plasma cell neoplasia (100) 17 (44.7) 18/38 (47.37) 4/8 (50) 7/8 (87)
a
Number of mice during or after the period of cancer.
b
Number of mice killed with cancer and percentage of tumour incidence; n, number of mice studied; ND, not determined.
MafB in MM pathogenesis
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plasmablast precursors in haematopoietic and non-haemato-
poietic organs, like BM, spleen, liver, kidney, and lungs
(Figure 2A). To prove the identity of the infiltrated cells
beyond their plasma cell morphology, immunohistochemical
analysis was performed using the plasma cell-specific markers
Mum1/IRF4 and CD138 (Figure 2B). The presence of IRF4
þ
or CD138
þ
cells was confirmed in spleen, kidney, intestine,
and lung. Also staining with an anti-IgG antibody revealed the
infiltration of plasma cells in all these organs (Figure 2B).
These infiltrations were associated with an increased number
of plasma cells in the BM of Sca1-MafB mice compared with
that of wild-type control mice (Figure 2A; Supplementary
Figure 7). Thus, the macroscopic and histological findings
define the malignant nature of the reprogrammed plasma cells
and further support the plasma cell accumulation that was
already detected by flow cytometry, especially considering
Figure 2 Reprogramming of Sca1 cells to terminally differentiated plasma cells. (A) Representative histological appearance of haematopoietic
(BM, spleen) and non-haematopoietic (liver, kidney, and lung) tissues of diseased Sca1-MafB mice versus WTcontrol mice after haematoxylin-
eosin staining. A total number of 44 Sca1-MafB mice were analysed, out of which 20 showed accumulations of plasma cells. Bottom panel
shows an enlargement of the BM of Sca1-MafB mice, where the infiltrating plasma cells and plasmablasts characteristic of multiple myeloma
can be clearly recognized. Examples of plasma cells and plasmablasts are indicated by black and yellow arrows, respectively.
(B) Representative immunohistochemical analysis of the tissues of diseased Sca1-MafB mice versus age-matched WT controls. To prove the
identity of the infiltrated cells beyond their plasma cell morphology, immunohistochemical analysis was performed in four different transgenic
mice using the plasma cell-specific markers Mum1/IRF4 or CD138 in different organs, as indicated. The presence of plasma cells was confirmed
in all the organs analysed. Furthermore, by staining with an anti-IgG antibody the infiltration of plasma cells could be detected in spleen,
kidney, intestine, and lung. The image magnification is 400 in all cases.
MafB in MM pathogenesis
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that it is well established that flow cytometry underestimates
the number of plasma cells due to their fragility and high
adhesivity (Orfao et al, 1994; Ng et al, 2006).
Lytic bone lesions and tumoral masses of plasma cells are
one of the most characteristic hallmarks of malignant plasma
cell behaviour. The most common sites of appearance of
these lesions are the BM areas where the haematopoiesis is
most active including, in order of frequency, the vertebrae,
ribs, skull, pelvis, femur, clavicle, and scapula (Jaffe et al,
2001). A detailed X-ray analysis of Sca1-MafB diseased
(between 9 and 16 months old) mice (n¼9) showed the
presence of clear osteolytic lesions in 55% of them (5/9)
(Figure 3A). These osteolytic lesions were produced as a
consequence of the accumulation of plasma cells around the
bone lytic area, as evidenced by histological analysis in the
Sca1-MafB mice (Figure 3B). Even more, in some instances
the extensive skeletal destruction by neoplastic plasma cells
resulted in pathological fractures (Figure 3).
The extensive BM replacement by neoplastic plasma cells,
together with the skeletal destruction and the renal damage,
resulted in anaemia in Sca1-MafB mice, as revealed
by the significantly lower values of red blood cell count,
haemoglobin, haematocrite, and mean corpuscular volume
(Supplementary Figure 10). Therefore, these results show
that Sca1-MafB mice exhibit reduced haematocrits, as it is
typically found in MM patients.
Tumour plasma cells in Sca1-MafB mice are oligoclonal
In order to evaluate the clonal expansion of reprogrammed
plasma cells, we evaluated the presence of an M-component
(abnormal monoclonal immunoglobulin accumulation) in
the serum of Sca1-MafB mice, as a result of the aberrant
overproduction of immunoglobulin by the clonal neoplastic
plasma cells. Western blottings of isoelectric focusing gels
were performed (Supplementary Figure 11). Anti-IgG showed
oligoclonal patterns in Sca1-MafB mice versus controls, as
revealed by the comparison with a human hydridoma mono-
clonal control (Supplementary Figure 11A–C). The presence
of this oligoclonal pattern becomes more evident in mice with
a more advanced disease. The finding that the oligoclonality
is only observed in the late stages of the disease is in
agreement with the fact that Sca1 targets MafB expression
to a significant percentage of the cells in the stem/progenitor
compartment, implying that more than one cell-of-origin of
the same type can contribute to the final reprogrammed
plasma cells. This is further supported by the results from
the analysis of V(D)J rearrangements in plasma cells from
young Sca1-MafB mice (Supplementary Figure 12), which
shows that there is not any predominant clone in this
population. This indicates that malignant plasma cells arise
from a population of equipotent and identically predisposed
progenitors.
In humans, primary amyloidosis can be caused by a
plasma cell neoplasm that secretes elevated titres of immuno-
globulins to the serum, which afterwards deposit in
various tissues. These amyloid deposits form a b-pleated
sheet structure that binds Congo Red dye with a characteristic
birefringence and induces changes in the fluorescence of
the dye thioflavin T, and that is the direct responsible for
the kidney failure frequently seen in the later stages of the
disease (Jaffe et al, 2001). In this regard, the determination
of the levels of the immunoglobulins in the serum of the
Sca1-MafB mice showed that there is not a diffuse
hypergammaglobulinemia although some of the transgenic
mice showed elevated titres of specific immunoglobulins
isotypes (Supplementary Figure 11D), as it is typically
found in MM patients. In Sca1-MafB mice, paraprotein
accumulation could be clearly detected in the spleen,
liver, kidney, or intestine (Supplementary Figure 9A–C).
The staining of the organs from diseased Sca1-MafB mice
using thioflavin T or Congo Red proved the amyloid nature
of the deposits, and provides further confirmation of the
myelomatous disease affecting the animals (and responsible
for the macroscopically visible organ alterations in the case of
the kidneys).
Tumour plasma cells in Sca1-MafB mice display gene
signatures analogous to those of human malignant
plasma cells
The above results surprisingly show that it is possible to
reprogram HS/PCs to malignant plasma cells by expressing
the MafB oncogene in a restricted manner, exclusively in
the Sca1
þ
HS/PCs. The MafB oncogene, according to the
mechanistic experimental design, is not expressed in more
differentiated, Sca1

, cell types, or even in the tumoral
plasma cells themselves. This indicates that, somehow, pro-
gramming of the malignant phenotype has already taken
place at the stem cell level. This is, in fact, very similar to
what it happens in the differentiation of normal haemato-
poietic lineages, where molecules like IL7-R or EpoR are only
necessary at early developmental stages in order to program a
given differentiation fate, but are not required afterwards,
once the program has been established. In order to identify
the molecular features of the pathological programming
imposed by MafB in the stem cells in Sca1-MafB mice, we
proceeded initially to compare the gene expression profiles of
plasma cells (B220
low
CD138
hi
FSC
hi
SSC
hi
) of Sca1-MafB mice
versus those from WT mice (Figure 4) using cDNA micro-
array analysis. Supervised analysis showed that, although the
global gene expression pattern is quite similar between both
populations, differences of ±2 expression levels could be
found (with a false discovery rate (FDR) ¼6.65%) in a small
percentage of genes, representing 0.8% of all the ones
analysed in the array (Figure 4A). Further transcriptional
analysis revealed that overexpression of genes known to be
associated with human malignant plasma cells, including
Mum1/IRF4,Vegfa,IL6,Muc1,Fgf3,CD44, and Bcl2 (Casey
et al, 1986; Williams, 1991; Matsumura and Tarin, 1992; Iida
et al, 1997; Silva et al, 2001; van de Donk et al, 2005; Wei
et al, 2006), were also expressed in tumour plasma cells of
Sca1-MafB mice (Figure 4B). These results prove that Sca1-
MafB tumour plasma cells share a genetic profile with human
malignant plasma cells.
Maf-B target genes are not expressed in tumour plasma
cells from Sca1-MafB mice
In these Sca1-MafB mice, oncogenic reprogramming takes
place within the stem cell/progenitor population and, accord-
ing to the experimental design, the MafB oncogene is not
expressed in the tumour plasma cells themselves. However, it
could be that MafB target genes continue to be expressed in
the absence of MafB. These genes could be targets of a ‘hit
and run’ mode of action in which MafB turns genes on in
stem cells but is not required for maintaining their expression
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at later stages of development. Remarkably, not only MafB
expression is not detected in the plasma cells of Sca1-MafB
mice, but what is more, neither MafB physiological target
genes (Ets-1,Hoxa3,Hoxb3,Gcg,Pax6,Maf,Fos ) (Kataoka
et al, 1994; Sieweke et al, 1996; Kelly et al, 2000; Blanchi
et al, 2003; Artner et al, 2007; Nishimura et al, 2008),
nor the pathological downstream ones (Ang,Blvra,Ccr1,
Itgb7,Notch2,Cx3cr1,Ccnd2,Nuak1/Ark5,Ntrk2,Arid5a,
Smarca1,Tlr4,Spp1,G6mb,Sfrp2,Tnfaip8,Dkk1) (Zhan
et al, 2002, 2003, 2006; van Stralen et al, 2009), were detected
either (Figure 4; Supplementary Table II). It might appear
then counter-intuitive and surprising that tumour plasma
cells develop efficiently in these mice, since in actual
human cancers all cancerous cells carry the oncogenic genetic
Figure 3 Bone lesions in Sca1-MafB mice. (A) Detection of osteolytic lesions and pathological fractures (indicated by orange arrows) by X-ray
analysis of Sca1-MafB mice. A total number of nine mice (between 9 and 16 months old) were analysed, out of which five presented bone
lesions. (B) Demonstration of specific plasma cell accumulation around the bone lytic areas in the Sca1-MafB mice after haematoxylin-eosin
staining. Histologic appearance of ribs of both control (a, b) and Sca1-MafB mice (c, d). Plasma cells were not detected in the BM of ribs of
control mice (e). However, plasma cells were present within the BM of ribs of Sca1-MafB mice (f). These plasma cells were infiltrating soft
tissues around the ribs with osteolytic lesions (g). The image magnification is indicated.
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lesions, and not only the cancer stem cells. Nevertheless,
tumour plasma cells arise in these mice, indicating that
continuous expression of MafB is not critical for the genera-
tion of tumour plasma cells and suggesting a ‘hands off’ role
for MafB in regulating tumour formation.
Neoplastic plasma cell commitment program is driven
by the oncogene MafB
The results shown above prove that stem cell-restricted
expression of MafB is enough to reprogram stem cells to-
wards a neoplastic plasma cell fate. This is similar to what
happens in other cases of normal fate programming or
experimentally induced reprogramming, where the repro-
gramming factor(s) does not need to be present anymore
once the initial fate-inducing change has taken place (e.g.,
induced pluripotency, see Discussion). Therefore, since the
effects of MafB in the transgenic mice are restricted to the
HS/PC compartment, we next sought for the differences in
gene expression patterns between BM Sca1
þ
Lin

progeni-
tors from Sca1-MafB mice when compared with those from
WT mice. The Sca1
þ
Lin

population was isolated from 16
independent Sca1-MafB mice and was compared by gene
expression profiling to normal Sca1
þ
Lin

cells from control
mice. This provided an expression signature for the MafB-
expressing HS/PCs, where a total of 4.3% of the genes (444
genes) analysed were upregulated or downregulated in
Sca1-MafB Sca1
þ
Lin

cells (FDR p8.9%; Supplementary
Figure 13; Supplementary Table III), revealing a common
expression signature of MafB-expressing HS/PCs. The analy-
sis identified a set of genes that are reproducibly differentially
regulated in MafB-expressing HS/PCs versus controls, indi-
cating that MafB expression does induce changes in the stem
cell transcriptome that can affect cellular differentiation at
later stages. Of particular interest was the upregulation of
factors responsible for the terminal differentiation of B cells
into plasma cells, like Blimp1,Xbp1,Mum1, and Syndecan1/
CD138 in MafB-expressing HS/PCs, results that were con-
firmed by real-time PCR analysis (Supplementary Figures 13
and 14). In order to rule out that these findings could be due
to an enrichment of some specific subpopulation (e.g., com-
mon lymphoid progenitors, CLPs), we analysed and quanti-
fied both LSK and CLP compartments in transgenic and wild-
type mice by flow cytometry (Supplementary Figure 4B and
C). The results showed that the LSK population is increased
in the transgenic mice. Using stainings including the Slam
antibodies, we could identify that this change was due to an
increase in the short-term HSC pool (Supplementary
Figure 4B). On the contrary, the CLP population is decreased
in transgenic mice (Supplementary Figure 4C). These
AB
–2.4 0 +2.4
–2.4 0 +2.4
Figure 4 Molecular identity of plasma cells in Sca1-MafB mice. To
molecularly characterize tumour plasma cells of Sca1-MafB mice,
we compared the gene expression profiles of BM plasma cells
(B220
low
CD138
hi
FSC
hi
SSC
hi
) purified from diseased Sca1-MafB
mice versus those from the BM of control mice. Each gene (identi-
fied at right) is represented by a single row of coloured boxes; each
mouse is represented by one single column. Data are displayed by a
colour code. Red indicates overexpression in tumour plasma cells
versus WT plasma cells, green indicates lower expression in the
tumour versus WT plasma cells. (A) Tumour plasma cells are
different from normal plasma cells. BM plasma cells were isolated
from two different control mice and from eight different Sca1-MafB
mice and independently extracted and amplified RNA from each
aliquot for gene expression profiling. Genes for which signal
intensities were up/down (threshold±2) regulated in Sca1-MafB
plasma cells are shown. The ratios refer to the expression levels in
Sca1-MafB plasma cells versus control plasma cells. (B) Gene
expression pattern in plasma cells of Sca1-MafB mice (n¼8) of
markers overexpressed in human malignant plasma cells. The
expression levels of Mum1/IRF4,Vegfa,IL6,Muc1,Fgf3,CD44,
and Bcl2 in plasma cells of Sca1-MafB mice resemble those for
plasma cells of human MM patients.
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findings would therefore reinforce the interpretation of the
results from the expression studies, and point towards the
existence of a plasma cell-biased stem cell transformation
signature that is dependent on the underlying MafB initiating
mutation. These observations provide evidence indicating
that different oncogenic proteins, when expressed in stem/
progenitor cells can have selective impacts that depend on
their intrinsic molecular properties. These results in turn
provide a rationale for the strikingly consistent associations
between different oncogenes and their corresponding cancer
phenotypes (Sanchez-Garcia, 1997).
The existence of a MafB-induced stem cell signature sug-
gests that deregulation of MafB can promote cancer through a
specific oncogenic mechanism. Of particular interest was the
fact that, as mentioned, neither the physiological MafB target
genes (Kataoka et al, 1994; Sieweke et al, 1996; Kelly et al,
2000; Blanchi et al, 2003; Artner et al, 2007; Nishimura et al,
2008) nor the pathological downstream MafB targets (Zhan
et al, 2002, 2003, 2006; van Stralen et al, 2009) were involved
in the MafB-induced reprogramming of stem cells in Sca1-
MafB mice (Supplementary Figure 13A). Therefore, the onco-
genic activity of the MafB protein at the HS/PC level should
most likely be due to alternative, ‘non-canonical’ functions
arising in this specific cellular context (stem cells).
B cells in Sca1-MafB mice retain an epigenetic memory
of their MafB-expressing HSCs of origin
Our results suggest that MafB imposes a gene regulatory state
in stem cells that persists during haematopoiesis and med-
iates a latent tumour phenotype that only becomes overtly
cancerous in the mature B-cell compartment, despite absence
of MafB expression, suggesting a possible aberrant epigenetic
programming. To confirm this hypothesis, and to determine
the molecular mechanisms mediating MafB-induced repro-
gramming of stem cells into tumour plasma cells, we per-
formed a complete, unbiased, and quantitative assessment of
cytosine methylation from purified populations of primary
cells. Using high-throughput reduced representation bisul-
phite sequencing (RRBS; Meissner et al, 2008; Bock et al,
2010; Gu et al, 2011), we generated DNA methylation maps
for pooled FACS-isolated, Sca1
þ
Lin

fractions from Sca1-
MafB mice and control mice, pooled B220
þ
cells from Sca1-
MafB mice and control mice, and pooled Gr1
þ
Mac1
þ
cells
from control mice (Figure 5). The heatmaps of the methyla-
tion values for the CpG islands and/or promoters allowed to
cluster and distinguish the different cell types studied, in-
dicating that the identified sites of increased/decreased
methylation heterogeneity are indeed cell type-specific
DNA-methylation regions (DMRs; Figure 5A and B). Several
differentially hypermethylated and hypomethylated regions
associated with promoters and with CpG islands were found
in the comparisons and are shown in Figure 5C: control WT
HS/PCs versus those from Sca1-MafB mice (MC1 compari-
son), mature B cells of Sca1-MafB mice versus WT control B
cells (MC2 comparison), mature B cells of Sca1-MafB mice
versus control WT HS/PCs (MC3 comparison), and WT
control B cells versus WT control HS/PCs (MC4 comparison).
The data from these comparisons are summarized in Table II
and the full data are available in Supplementary Tables IV–XI.
Remarkably, the majority of the methylation changes found
in the MC1 comparison were also found in MC3 and MC4
experiments. However, in the MC2 comparison only 50% of
this methylation pattern is maintained (Supplementary
Tables XII–XVII; Supplementary Figure 15). Moreover, when
plotting these identically methylated regions in a proportional
Venn diagram, several overlapping regions appear
(Figure 5D) showing 84 conserved methylation changes in
regions associated with CpG islands and 52 conserved methy-
lation changes in regions associated with promoters through
all the comparisons. These observations support the notion
that MafB imposes a specific epigenetic state in stem cells
that persists in the mature B-cell compartment of Sca1-MafB
mice, allowing us to identify an epigenetic DNA methylation
memory of reprogrammed stem cells in the mature B cells of
Sca1-MafB mice. Moreover, these results further indicate
that differentiated tumour cells were derived from the
Sca1-driven, MafB-expressing stem cells.
Discussion
MafB-induced reprogramming of the cellular identity of
a normal stem/progenitor cell into a tumour plasma cell
Reprogramming is the rewiring of the transcriptional and
epigenetic status of a cell to that of another cell type. It can
be driven by different molecules, in physiological develop-
ment, in tumoral development, or experimentally in the
laboratory. Interestingly enough, the factors most commonly
used in reprogramming to pluripotency have all been shown
to play an oncogenic role in different contexts (Rowland and
Bernards, 2005; Okita et al, 2007; Tanaka et al, 2007; Chen
et al, 2008; Abollo-Jimenez et al, 2010; Castellanos et al,2010;
Sanchez-Garcia, 2010), thus further linking reprogramming to
tumorigenesis. However, an essential question that remains
to be answered in order to prove the reprogramming capacity
of human oncogenes is whether normal progenitors can be
reprogrammed to give rise to terminally differentiated tumour
cells by the specifically associated oncogenes.
Our data provide proof-of-principle evidence of the fact
that, at least in the context of MM development, HS/PCs can
be directly reprogrammed into tumour plasma-like cells
in vivo by an oncogene that in human patients is associated
with plasma cell myeloma. This MafB-mediated reprogram-
ming is, however, permissive in that it allows the normal
differentiation of all haematopoietic cell types, and only
reveals its malignant nature in the plasma cell compartment.
In the oncogenic reprogramming model here presented, the
reprogrammed Sca1
þ
population can nevertheless complete
a multistage differentiation pathway involving an initial
commitment to the B-cell lineage and a subsequent differ-
entiation to plasma cells. A similar scenario has been de-
scribed in human CLL, where the propensity to generate
malignant B cells has already been acquired at the HSC
stage (Kikushige et al, 2011) and therefore, patient-derived
HSCs showed an abnormal expression of lymphoid-related
genes, presumably reflecting their cell-intrinsic pathologic
priming into the lymphoid lineage. Notably, these HSCs
did not show the typical chromosomal abnormalities of CLL
(Kikushige et al, 2011), like we have shown for MM
(Supplementary Figure 1).
But how does MafB instruct stem cells to give rise to a
malignant plasma cell?
In spite of their well-proven involvement in some subsets of
human MM, MAF proteins do not appear, on base of their
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physiological role in development, to be implicated in the
major cellular functions associated to tumoral plasma cells.
Indeed, MAF proteins are mostly inducers of terminal
differentiation (Eychene et al, 2008), and MafB selectively
restricts the sensitivity of stem cells to myeloid-inducing
cytokines, limiting the development of myeloid lineages and
contributing to the maintenance of a balanced lineage
potential in the HSCs (Sarrazin et al, 2009). These activities
cannot easily explain their oncogenic properties. Also,
none of their physiological target genes has been found to
be deregulated in Maf-transformed cells or to be involved
in oncogenic processes (Eychene et al, 2008). In our
experimental system, MafB targets are also not deregulated
in MafB-reprogrammed stem cells. Therefore, the oncogenic
activity of the MafB protein seems to be due to other, not yet
described functions.
In order to identify the genes that are associated with
MafB-induced reprogramming of stem cells, we performed a
supervised analysis of the transcriptional profiles of HS/PCs
Figure 5 Genome-scale DNA methylation maps of stem and mature B cells of Sca1-MafB mice. (A,B) Heatmaps with hierarchical clustering.
Samples were grouped based on differential methylation profiles for promoters (A) and CpG island loci (B). Yellow represents high levels of
DNA methylation, and red represents low levels of DNA methylation. Methylation classification definitions: hypermethylated: 0–33% more
methylated than reference; hypomethylated: 0–33% less methylated than reference; inconclusive: uneven or insufficient coverage for one or
both samples; insignificant: statistically insignificant difference in methylation (P-value40.05); strongly hypermethylated: 33–100% more
methylated than reference; strongly hypomethylated: 33–100% less methylated than reference. (C) Comparisons to detect methylation changes
in HP/PCs of Sca1-MafB mice versus wild-type HS/PCs (MC1), mature B cells of Sca1-MafB mice versus wild-type mature B cells (MC2),
mature B cells of Sca1-MafB mice versus control HS/PCs (MC3) and wild-type mature B cells versus wild-type HS/PCs (MC4). (D) Proportional
Venn diagrams of conserved methylation changes in CpG islands and promoters associated regions. Each circle corresponds to the conserved
changes between MC1 and MC2 (group a), MC1 and MC3 (group b) and MC1 and MC4 (group b). It also indicates the number of specific
regions in each overlapping and non-overlapping zones of the diagrams.
Tabl e II Number of differentially hypermethylated and
hypomethylated regions associated to promoters or CpG islands in
each comparison
MC1 MC2 MC3 MC4
Regions associated with promoters
Hypomethylated 858 2312 1927 1139
Hypermethylated 1390 1351 1674 1562
Regions associated with CpG islands
Hypomethylated 1158 2958 23 95 1363
Hypermethylated 1743 1484 1937 1948
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purified from Sca1-MafB mice and control mice. The data
identified a set of genes that are reproducibly differentially
regulated in MafB-targeted stem cells versus control stem cells,
showing that Sca1-MafB-derived HSCs presented an abnormal
expression of lymphoid-related genes, presumably reflecting
their cell-intrinsic priming into the lymphoid lineage. Overall,
these results show that (1) enforced MafB expression restricted
to stem cells is all that is required to generate tumoral plasma
cells in mice, therefore suggesting for the first time a role for
stem/progenitor cells in the pathogenesis of plasma cell neo-
plasias; (2) HS/PCs transformed by MafB share a transforma-
tion-specific gene expression signature.
Among the genes present in the cancer stem cell signature
there are some, like Bmi1 or b-catenin, that are known to play
relevant roles in the biology of both normal and cancer stem
cells (Supplementary Figure 13B; Taipale and Beachy, 2001;
Vonlanthen et al, 2001; Ema and Nakauchi, 2003; Lessard and
Sauvageau, 2003) or to embryonic stem cells (Supplementary
Figure 13C). Also, Xbp1, besides being essential for plasma
cell development, has recently been found to significantly
increase HSC activity in a functional screening based
on retroviral transduction of CD150
þ
CD48

Lin

mouse
BM cells (Deneault et al, 2009). These findings provide a
rationale of how reprogramming by MafB can induce changes
from normal to pathological stem cells capable of giving rise
to tumoral plasma cells.
In order to gain even more insight into the mechanism by
which MafB-induced reprogramming of stem cells into
tumour plasma cells, we generated in-vivo genome-scale
maps of DNA methylation in both stem cells and mature B
cells. We have found that a substantial number of CpG
islands and promoters are specifically hypermethylated or
hypomethylated in the stem cells of Sca1-MafB mice, setting a
pattern inherited throughout B-cell development, similar to
the DNA methylation changes taking place during somatic
erythroid cell differentiation (Shearstone et al, 2011). Thus,
these findings indicate that MafB oncogenic effect is driven in
part by epigenetic mechanisms. In support of these findings,
recent studies have shown that incorporation of targeted
epigenetic agents to the standard chemotherapy is a
promising approach to the treatment of relapsed paediatric
ALL (Bhatla et al, 2012).
Until now, differentiated tumour cells have been obtained
in mice mainly by targeting oncogene expression to mature
cells. The results presented in this study demonstrate a novel
molecular mechanism involved in tumour initiation, by
showing that HS/PCs can be epigenetically reprogrammed
to terminally differentiated tumour cells by defined onco-
genes associated to human neoplasias (MafB, in this case,
Figure 6). Our results also provide a proof-of-principle ex-
perimental model to understand how such a phenomenon
could be possible also in human patients. We believe that our
findings have critical implications for the understanding of
the etiopathogenesis of MM and for the development of novel
therapies for this disease.
Materials and methods
MM patient sample preparation and detection of
chromosomal translocations by FISH
PC (CD38
þ
CDE138
þ
) and CD34
þ
populations were sorted using
a FACSAria flow cytometer (BDB) with CD38-PerCP-Cy5.5
(Pharmingen), CD138-FITC (Pharmingen) and CD34-APC (BDB)
monoclonal antibodies, for further FISH analysis. Reanalysis of
the sorted cells showed purity 495%. Interphase FISH studies for
the detection of IGH rearrangements were carried out by means of
LSI IGH dual-colour, break-apart rearrangement probe (Vysis,
Downers Grove, IL, USA). Those MM samples with IGH transloca-
tions were explored for t(11;14)(q13;q32), t(4;14)(p16;q32), and
t(14;16)(q32;q23) with the corresponding dual-colour, dual-fusion
translocation probes from Abbott Molecular/Vysis, as previously
described (Lopez-Corral et al, 2011).
Generation of Sca1-MafB transgenic mice
The Sca1-MafB vector was generated as follows: The 1.3-kb EcoRI-
EcoRI fragment, containing the mouse MafB cDNA, was inserted
into the ClaI site of the pLy6 vector (Miles et al, 1997) to generate
the Sca1-MafB vector. The transgene fragment (Figure 1A) was
excised from its vector by restriction digestion with NotI, purified
for injection (2 ng ml
1
) and injected into CBA C57BL/6J ferti-
lized eggs. Transgenic mice were identified by Southern analysis of
tail snip DNA after EcoRI or HindIII digestion. MafB cDNA was used
for detection of the transgene. A total of 44 transgenic animals and
20 control animals were used to define the phenotype. Two inde-
pendent transgenic lines were generated (Figure 1B) and analysed
and similar phenotypic features were seen in both.
EpiQuest library construction
EpiQuest libraries were prepared from 200 to 500ng mouse genomic
DNA obtained from primary cells (Sca1
þ
Lin

cells from BM, and
B220
þ
cells from PB) purified from Sca1-MafB (n¼3) mice and/or
wild-type (n¼10) mice. The DNAwas digested with 60 units of Ta q I
and 30 units of MspI (NEB) sequentially. Size-selected Taq I-MspI
fragments (40–120 bp and 120–350bp) were filled-in and 30-term-
inal-A extended, extracted with Zymo Research DNA Clean and
Concentrator(tm) kit. Ligation to pre-annealed adaptors containing
50-methyl-cytosine instead of cytosine (Illumina) was performed
using the Illumina DNA preparation kit and protocol. Purified,
adaptor-ligated fragments were bisulphite treated using the EZ
DNA Methylation-Direct(tm) Kit (Zymo Research). Preparative-
scale PCR was performed and DNA Clean and Concentrator-purified
PCR products were subjected to a final size selection on a 4%
NuSieve 3:1 agarose gel. SYBR-green-stained gel slices containing
adaptor-ligated fragments of 130–210 or 210–460 bp in size were
excised. Library material was recovered from the gel
(Zymoclean(tm) Gel DNA Recovery Kit) and sequenced on an
Illumina GAIIx genome analyzer.
Sequence alignments and data analysis
Sequence reads from bisulphite-treated EpiQuest libraries were
identified using standard Illumina base-calling software and then
analysed using a Zymo Research proprietary computational
pipeline.
Residual cytosines (Cs) in each read were first converted to
thymines (Ts), with each such conversion noted for subsequent
analysis. A reference sequence database was constructed from the
36-bp ends of each computationally predicted MspI-Ta q I fragment
in the 40–220-bp size range. All Cs in each fragment end were then
converted to Ts (only the C-poor strands are sequenced in the RRBS
process). The converted reads were aligned to the converted
reference by finding all 12-bp perfect matches and then extending
to both ends of the treated read, not allowing gaps (reverse
complement alignments were not considered). The number of
mismatches in the induced alignment was then counted between
the unconverted read and reference, ignoring cases in which a T in
the unconverted read is matched to a C in the unconverted
reference. For a given read, the best alignment was kept if the
second-best alignment had two more mismatches, otherwise the
read was discarded as non-unique. The methylation level of each
sampled cytosine was estimated as the number of reads reporting a
C, divided by the total number of reads reporting a C or T. A
bioinformatics pipeline was used to score epigenetic alterations
according to strength and significance, and links them to potentially
affected genes. To that end, we collected a comprehensive set of
regions of interest, which includes promoters, CpG islands, and
repetitive elements. For each of these regions, the number of
methylated and unmethylated CpG observations is determined,
and a P-value is assigned using Fisher’s exact test. Once all P-values
are calculated, multiple-testing correction is performed separately
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for each region type using the q-value method, which controls the
FDR to be below a user-specified threshold (typically 10%). The
software pipeline is implemented in Python (alignment processing
module) and R (statistical analysis module).
Analysis of methylation levels
Once regions are classified, the information from all samples is
processed in a different pipeline, also implemented in R. After
filtering the inconclusive and insignificant values from each com-
parison, the common regions of control HS/PCs and those from
Sca1-MafB mice (MC1) and (A) tumour B cells of Sca1-MafB mice
versus B cells (MC2), (B) tumour B cells of Sca1-MafB mice versus
control HS/PCs (MC3) and (C) B cells versus control HS/PCs (MC4)
are extracted and combined (Supplementary Tables XII–XVII;
Supplementary Figure 15) as shown in Figure 5C. This allows
comparing the methylation levels for those common regions in
each pair of comparisons.
With these combined data, the ratio of regions with conserved
and changed methylation levels is calculated. Classes of conserved
methylation levels and patterns of changed methylation levels are
also determined. Overlapping and specific regions with conserved
methylation levels in the three pairs of comparisons are represented
in a Venn diagram. This analysis was carried out separately for
promoters and for CpG islands.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank all members of lab 13 at IBMCC for their helpful comments
and constructive discussions on this project and Professor Jesu
´sSan
Miguel for the samples and the critical reading of the manuscript. We
are indebted to patients who gave samples. We are grateful to the
Animal Care Facility staff members for their valuable help. We are
grateful to Dr E Dzierzak for the Sca1 promoter and Dr Michael
Sieweke for the mouse Maf-B cDNA. Research in ISG group was
partially supported by FEDER and by MICINN (SAF2009-08803 to
ISG), by Junta de Castilla y Leo
´n (REF. CSI007A11-2 and Proyecto
Biomedicina 2009-2010 to ISG), by MEC OncoBIO Consolider-Ingenio
2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), by
Sandra Ibarra Foundation, by Group of Excellence Grant (GR15) from
Junta de Castilla y Leon, and the ARIMMORA project (FP7-ENV-2011,
European Union Seventh Framework Program) and by proyecto en
red de investigacio
´n en celulas madre tumorales en cancer de mama
supported by Obra Social Kutxa y Conserjerı
´a de Sanidad de la Junta
de Castilla y Leon. All Spanish funding is co-sponsored by the
European Union FEDER program. CVD research is supported by
Junta de Castilla y Leo
´n (proyecto de investigacio
´n en biomedicina
SAN/39/2010). ISG is an API lab of the EuroSyStem project. Research
at CC’s lab was partially supported by FEDER, Fondo de
Investigaciones Sanitarias (PI080164), CSIC P.I.E., Junta de Castilla
yLeo
´n (SA060A09 and proyecto Biomedicina 2009-2010) and from an
institutional grant from the Fundacio
´n Ramo
´n Areces. IR-C and FA-J
were supported by an FPU fellowship from the Spanish Ministerio de
Ciencia e Innovacion. AO research is supported by a grant from the
Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo,
Madrid, Spain (IISCIII-RTICC RD06/0020/0035-FEDER).
Figure 6 A model by which ectopic expression of MafB reprogrammes HS/PCs into tumour plasma cells. (A) Normal lymphoid development
in human and mice. Blue circles represent normal gene regulatory events (activating or repressing) happening during B-lymphocyte
development. Green circles represent normal gene regulation events happening during terminal differentiation to plasma cells, initially
triggered by antigen recognition. (B) Current working model for the development of tumour plasma cells in humans. The existence of dormant
alterations previous to the terminal differentiation is unknown. Currently, MafB effects (closed circles) are thought to occur in a plasma cell or
in the final steps of the terminal differentiation from a B cell. The nature of the cancer cell-of-origin is therefore unknown. (C) Mechanism of
tumour plasma cell development in Sca1-MafB transgenic mice. Open red circles represent latent epigenetic regulatory events caused by Sca1-
driven expression of MafB. These epigenetic marks do not interfere with normal B-cell development, but become active (either activating or
repressing) in the process of terminal differentiation, thus leading to the appearance of tumour plasma cell. According to this model, tumour
plasma cell is the result of a cell reprogramming process (see text for details).
MafB in MM pathogenesis
C Vicente-Duen
˜as et al
3715&2012 European Molecular Biology Organization The EMBO Journal VOL 31 |NO 18 |2012

Author contributions: CV-D, CC, and ISG designed the study. CV-
D, IG-H, EA-E, FA-J, RJ, AO, FJG-C, and MBG-C performed experi-
ments. NG performed FISH analysis. NM and LMCG performed the
isoelectric focusing. MCFC performed the X-ray studies. BP gener-
ated the transgenic mice. TF analysed mouse and human histo-
pathology data. CV-D, EA-E, and TF performed IHC analyses in
mouse samples. DA-L and JD analysed methylation data. XJ and IL
determined Ig concentrations in serum and analysed data. CV-D,
CC, and ISG coordinated and supervised the project. CV-D, CC, and
ISG prepared figures and wrote the manuscript. All authors read
and agreed the paper content.
Conflict of interest
The authors declare that they have no conflict of interest.
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MafB in MM pathogenesis
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3717&2012 European Molecular Biology Organization The EMBO Journal VOL 31 |NO 18 |2012