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doi:10.1182/blood-2012-06-438937
Prepublished online January 15, 2013;
2013 121: 2175-2185
Campo and Virginia Amador
Anna Enjuanes, Pedro Jares, Neus Villamor, Dolors Colomer, José Ignacio Martín-Subero, Elias
Alba Navarro, Guillem Clot, Alexandra Moros, Helena Suárez-Cisneros, Sílvia Beà, Luis Hernández,
Maria Carmela Vegliante, Jara Palomero, Patricia Pérez-Galán, Gaël Roué, Giancarlo Castellano,
differentiation in aggressive mantle cell lymphoma
SOX11 regulates PAX5 expression and blocks terminal B-cell
http://bloodjournal.hematologylibrary.org/content/121/12/2175.full.html
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Copyright 2011 by The American Society of Hematology; all rights reserved.
Washington DC 20036.
by the American Society of Hematology, 2021 L St, NW, Suite 900,
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly
personal use only. For at CRAI UNIVERSITAT DE BARCELONA on April 12, 2013. bloodjournal.hematologylibrary.orgFrom
Plenary Paper
SOX11 regulates PAX5 expression and blocks terminal B-cell
differentiation in aggressive mantle cell lymphoma
Maria Carmela Vegliante,
1
Jara Palomero,
1
Patricia P´erez-Gal´an,
1
Ga¨el Rou´e,
1
Giancarlo Castellano,
1
Alba Navarro,
1
Guillem Clot,
1
Alexandra Moros,
1
Helena Su´arez-Cisneros,
1
S´
ılvia Be`a,
1
Luis Hern´andez,
1
Anna Enjuanes,
1
Pedro Jares,
1
Neus Villamor,
1
Dolors Colomer,
1
Jos´e Ignacio Mart´
ın-Subero,
2
Elias Campo,
1,2
and Virginia Amador
1
1
Hematopathology Unit, Pathology Department, Hospital Cl´
ınic, Institut d’Investigacions Biom `ediques August Pi i Sunyer; and
2
Department of Anatomic
Pathology, Pharmacology and Microbiology, University of Barcelona, Barcelona, Spain
Key Points
• SOX11 silencing promotes
the shift from a mature B cell
into the initial plasmacytic
differentiation phenotype
in MCL.
• SOX11 promotes tumor
growth of MCL cells in vivo,
highlighting its implication
in the aggressive behavior
of conventional MCL.
Mantle cell lymphoma (MCL) is one of the most aggressive lymphoid neoplasms whose
pathogenesis is not fully understood. The neural transcription factor SOX11 is
overexpressed in most MCL but is not detected in other mature B-cell lymphomas or
normal lymphoid cells. The specific expression of SOX11 in MCL suggests that it may
be an important element in the development of this tumor, but its potential function is
not known. Here, we show that SOX11 promotes tumor growth in a MCL-xenotransplant
mouse model. Using chromatin immunoprecipitation microarray analysis combined
with gene expression profiling upon SOX11 knockdown, we identify target genes and
transcriptional programs regulated by SOX11 including the block of mature B-cell
differentiation, modulation of cell cycle, apoptosis, and stem cell development. PAX5
emerges as one of the major SOX11 direct targets. SOX11 silencing downregulates
PAX5, induces BLIMP1 expression, and promotes the shift from a mature B cell into the
initial plasmacytic differentiation phenotype in both primary tumor cells and an in vitro
model. Our results suggest that SOX11 contributes to tumor development by altering
the terminal B-cell differentiation program of MCL and provide perspectives that may have clinical implications in the diagnosis
and design of new therapeutic strategies. (Blood. 2013;121(12):2175-2185)
Introduction
Mantle cell lymphoma (MCL) is one of the most aggressive
lymphoid neoplasms.
1
This biological behavior has been attrib-
uted to the molecular mechanisms involved in its pathogenesis
combining the deregulation of cell proliferation, disruption of the
DNA-damage–response pathways, and the activation of cell-
survival mechanisms.
2
Recent studies have identified a subgroup
of patients with MCL that have an indolent clinical behavior and
long survival even without chemotherapy. These tumors have very
stable karyotypes and frequently carry hypermutated IGHV, indi-
cating an origin in cells that have experienced the follicular
germinal center microenvironment.
3-7
A gene expression profiling
(GEP) study has shown that these indolent MCL differ from
conventional MCL in a particular gene signature that lacks the
expression of some transcription factors of the high-mobility
group (HMG). One of the best discriminatory genes between these
2 subtypes of tumors is SOX11 (SRY [sex-determining region-Y]-
box11),
4,6
a neural transcription factor whose function in normal
and neoplastic B-cell development is unknown.
SOX11 belongs to the SOX gene family encoding for
transcription factors that play a critical role in the regulation
of cell fate and differentiation in major developmental pro-
cesses. In mice, Sox11 is important in organ development and
neurogenesis.
8,9
SOX11 upregulation has been detected in
various types of human solid tumors.
10,11
SOX4, the SOX
family member with the highest homology to SOX11, is a prom-
inent transcription factor in lymphocytes of both B- and T-cell
lineage
12
and is crucial for B-cell lymphopoiesis.
13
In contrast,
SOX11 has no known lymphopoietic function and it is not
expressed in lymphoid progenitors or in mature normal B-cells.
However, it is expressed in virtually all aggressive MCL and at
lower levels in a subgroup of Burkitt and acute lymphoblastic
lymphomas, but not in other lymphoid neoplasms.
14-16
These
findings suggest that SOX11 plays a relevant role in the
pathogenesis of these tumors. However, the function of SOX11
and its potential target genes in lymphoid cells remain unknown.
To identify direct target genes, transcriptional programs, and
oncogenic pathways regulated by SOX11 in malignant lymphoid
cells, we have used genome-wide promoter analysis and GEP after
SOX11 silencing in MCL cell lines, followed by the validation in
an in vivo murine model and in primary MCL tumors.
Submitted June 24, 2012; accepted December 10, 2012. Prepublished online
as Blood First Edition paper, January 15, 2013; DOI 10.1182/blood-2012-06-
438937.
M.C.V. and J.P. contributed equally to this study.
All microarray data are available in the Gene Expression Omnibus (accession
numbers GSE34763 and GSE3502 for the gene expression array and the
ChIP-chip array, respectively).
The online version of this article contains a data supplement.
There is an Inside Blood commentary on this article in this issue.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
©2013 by The American Society of Hematology
BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12 2175
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Materials and methods
Cell lines and patient samples
A total of 6 MCL cell lines, 5 SOX11 positive (Z138, GRANTA519, REC1,
HBL2, and JEKO1) and 1 SOX11 negative (JVM2),
17
were used for
chromatin immunoprecipitation (ChIP) microarray (ChIP-chip) and/or ChIP
quantitative polymerase chain reaction (qPCR) experiments, gene expression
analysis after SOX11 silencing, and western blot (WB) experiments. Human
embryonic kidney 293 (HEK293) cells were used for luciferase assays.
Microarray GEP data previously generated in 38 primary MCL (16
SOX11-positive and 22 SOX11-negative) were used for gene set
enrichment analysis (GSEA) (GSE36000).
7
Immunophenotypic surface
markers were investigated in 6 of these primary MCL (3 SOX11 positive
and 3 SOX11 negative) by flow cytometry.
Details on cell culture and primary tumor’s information are provided in
“supplemental Materials and methods.”
ChIP
Z138, GRANTA519, and JVM2 cell lines were used for ChIP-chip and/or
ChIP-qPCR analysis. Cells were crosslinked with 1% of formaldehyde for 10
minutes and chromatin sonicated using Branson Sonifer 250 followed by
Biorupter sonicator from Diagenode. SOX11 antibody (sc-17347; Santa Cruz
Biotechnology, Santa Cruz, CA) was used to immunoprecipitate protein-
DNA complexes. Immunocomplexes were amplified in 2 steps using
a GenomePlex Whole Genome Amplification WGA Kit (Sigma, St Louis,
MO), following the manufacturer’s instructions, and used for ChIP-chip
analysis and/or ChIP-qPCR. We performed ChIP assays in triplicate from
Z138-SOX11-positive cells and in a single experiment from JVM2 cell line
as negative control (supplemental Materials and methods).
ChIP-qPCR
Primers for the qPCR analysis of the SOX11 ChIP-enriched genomic
DNA regions were designed using Primer3 (http://frodo.wi.mit.edu/)
(supplemental Table 1). Amplified SOX11-ChIP DNA and 1:100 diluted
input samples were analyzed in triplicate by qPCR using Fast SYBR
Green Master Mix in a StepOnePlus PCR detection system (Applied
Biosystems, Foster City, CA).
Reporter plasmid constructs and luciferase assay
The SOX11-binding site from the regulatory region of PAX5 gene (ChIP-
peak region from 2435 bp to 21884 bp) was amplified by PCR using
GRANTA519 genomic DNA and primers are listed in supplemental Table 1.
Subsequently, the PCR fragment was cloned in front of a minimal promoter
luciferase reporter vector pGL4.23[luc2/minP] (Promega, Leiden, The
Netherlands). Constructs were sequenced using internal primers prior to use.
Lipofectamine 2000 system (Invitrogen, Life Technologies, Carlsbad,
CA) was used for transient cotransfection experiments in HEK293 cells
following the manufacturer’s instructions. Cotransfected HEK293 cells
were harvested and luciferase activity was measured 24 hours after
transfection with Dual-Glo Luciferase Assay System (Promega) and a
Modulus microplate luminometer (Turner BioSystems, Sunnyvale, CA).
All assays were performed in triplicate on separated days.
Gene expression profiling
Total RNA of Z138 cells stably transduced with short hairpins targeting
SOX11 and control short hairpin was extracted with the TRIzol reagent
following the manufacturer’s recommendations (Invitrogen). RNA integrity
was examined with the Agilent 2100 Bioanalyser (Agilent Technologies,
Palo Alto, CA) and hybridized to HG-U133plus2.0 GeneChips (Affyme-
trix) according to Affymetrix standard protocols. The analysis of the GEP is
detailed in “supplemental Materials and methods.”
GSEA
We performed GSEA
18
on 2 preranked lists of genes derived from in vitro
SOX11 silencing microarray data and from our series of 38 purified primary
MCL data. To generate these preranked lists, we analyzed the GEP of our
in vitro cell line model as well as the GEP of 38 primary MCL according
to their SOX11 expression levels. Details on GSEA are provided in
“supplemental Materials and methods.”
SOX11 silencing by lentiviral infection
SOX11 shRNA lentiviral particles (shRNASOX11.1, clone ID NM_
003108.3-982s1c1; shRNASOX11.2, clone ID NM_003108.3-1235s1c1;
and shRNASOX11.3, clone ID NM_003108.3-454s1c1) were purchased
from Sigma-Aldrich. Control shRNA lentiviral particle (Clone ID sc-
108080) was purchased from Santa Cruz Biotechnology.
SOX11 knockdown stable MCL cell lines were generated by
lentiviral transduction of PLKO1-puro–shSOX11 and PLKO1-puro–
scramble constructs in 3 MCL cell lines (Z138, GRANTA519, and
JEKO1). Details on SOX11 silencing are provided in “supplemental
Materials and methods.”
WB analysis
Protein extract preparation and WB experiments were performed as
previously described
19
using antibodies against hemagglutinin (Roche
Applied Science, Indianapolis, IN; 12CA5), SOX11 (Atlas Antibodies,
Stockholm, Sweden [HPA000536]; Santa Cruz Biotechnology [H-290
and C-20]), PAX5 (Santa Cruz Biotechnology; C-20), BLIMP1 (CNIO,
Madrid, Spain; Ros 195G/G5), IRF4 (Santa Cruz Biotechnology; M-17),
GAPDH (Santa Cruz Biotechnology; A-3), and a-tubulin (Oncogene,
Uniondale, NY; CP06-100UG). Membranes were developed with
chemiluminescence substrate Pierce ELC WB substrate (Thermo Fisher
Scientific, South Logan, UT) and visualized on a LAS4000 device
(Fujifilm, Tokyo, Japan). Protein quantification was done with Image
Gauge software (Fujifilm).
qRT-PCR
qRT-PCR was performed as described before
19
using a designed primer
set and TaqMan MGB probe for SOX11 using Primer Express Software
Version 2.0 (Applied Biosystems) (primers used are shown in supple-
mental Table 1). Inventoried TaqMan Gene Expression Assays were used
for HSPD1 (Hs01941522_u1), SUV39H2 (Hs00226595_m1), SEPT2
(Hs00189358_m1), MSI2 (Hs-00292670_m1), PAX5 (Hs00277134_m1),
VPREB3 (Hs00429452_m1), SPIB (Hs0062150_m1), EBF1 (Hs00365513_m1),
CD19 (Hs00174333_m1), IKZF3 (Hs00232635_m1), LEF1 (Hs01547250_m1),
and LCK (Hs00178427_m1). Relative quantification of gene expression
was analyzed with the 2
2DDCt
method using GUS (Hs00939627_m1) as
the endogenous control.
Flow cytometry studies
For immunophenotype analysis, 500 000 cells of interest were collected in
1.5-mL tubes, washed once in phosphate-buffered saline 0.5% bovine serum
albumin (BSA), and stained with 5 mL of antibody against CD20 (2H7),
CD24 (ML5), immunoglobulin (Ig) M (G20-127), IgD (IA6-2), CD5
(UCHT2), CD19 (HIB19), CD38 (HIT2) (all from BD Pharmingen, San
Diego, CA), and CD138 (B-A38; Beckman Coulter, Carlsbad, CA) for 30
minutes at 4°C in the dark. Cells were then washed 3 times in phosphate-
buffered saline 0.5% BSA and acquired and analyzed using the Attune
Acoustic Focusing Cytometer and Software (Applied Biosystems). For the
analysis, 10 000 events were collected in the lymphocyte gate. Details on
proliferation assay, cell-cycle analysis, and apoptosis assay are provided in
“supplemental Materials and methods.”
Xenograft mouse model and tumor phenotyping
CB17 severe combined immunodeficient (CB17-SCID) mice (Charles River
Laboratory, Wilmington, MA) were housed in the animal care facility under
2176 VEGLIANTE et al BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12
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a 12/12-hour light/dark cycle at 22°C, and they received a standard diet and
acidified water ad libitum. With the use of a protocol approved by the
animal testing Ethical Committee of the University of Barcelona, mice
were subcutaneously inoculated into their lower dorsum with 1 310
7
Z138shSOX11.1 (n 58), shSOX11.3 (n 56), shControl (n 57),
JEKOshSOX11.3 (n 55), JEKOshControl (n 55), GRANTA519sh-
SOX11.3 (n 55), and GRANTA519shControl (n 55) in Matrigel
basement membrane matrix (Becton Dickinson, San Jose, CA). The shortest
and longest diameters of the tumor were measured with external calipers every
3 days, and tumor volume (in mm
3
) was calculated with the use of the fol-
lowing standard formula: (the shortest diameter)
2
3(the longest diameter) 3
0.5. Animals were sacrificed according to institutional guidelines, and tumor
xenografts were paraffin embedded on silane-coated slides in a fully
automated immunostainer (Bond Max; Vision Biosystems, Mount Waverley,
Australia).
Immunohistochemical staining
Immunohistochemical studies were performed as previously described
16,20
using antibodies against human Ki67, kappa, lambda, and IgM (ready to use;
Dako, Glostrup, Denmark), anti-phosphohistone H3 (Epitomics, Burlin-
game, CA; polyclonal), anti–caspase-3 cleaved (Cell Signaling Technology,
Danvers, MA; 5A1E), SOX11 (Atlas Antibodies; HPA000536), cyclin D1
(Thermo Fisher Scientific; EPR2241IHC), PAX5 (Santa Cruz Biotechnol-
ogy; sc-1974), and BLIMP1 (CNIO; Ros 195G/G5).
Necrotic areas were quantified using the digitalized images of the
activated caspase-3 immunohistochemical staining acquired with an
Olympus BX51 microscope at original magnification 34 and analyzed
with an Olympus Cell B Basic Imaging Software. Necrotic areas were
delineated by cleaved caspase-3–positive staining and quantified as the
sum of all necrotic areas (mm
2
) divided by the total area of the core
analyzed (mm
2
).
Statistical analysis
Data are represented as mean 6standard deviation (SD) of 3
independent experiments. Statistical tests were performed using SPSS
v16.0 software. Comparison between 2 groups of samples was
evaluated by independent-sample ttest, and results were considered
statistically significant when P,.05.
Results
Genome-wide promoter analysis of SOX11 in MCL
We first investigated the direct target genes of SOX11 using ChIP
and DNA microarrays (ChIP-chip). We used a MCL cell line with
high SOX11 expression (Z138) and one negative for SOX11
(JVM2) (Figure 1A). ChIP was performed using a SOX11 specific
antibody (supplemental Figure 1). ChIP-enriched DNA sequences
were amplified and hybridized to a high-resolution DNA tiling array
spanning promoter regions of the whole human genome. These
experiments identified 5842 significant enriched peaks belonging to
1133 unique genes (SOX11-bound genes) that were consistently
enriched in immunoprecipitated material across all 3 independent
Z138 replicates after subtracting the corresponding nonspecific
immunoprecipitated material in JVM2 (supplemental Table 2).
The promoter region of SOX11 was not identified in this analysis.
We have reported that SOX11 expression is associated with active
histone marks.
19
We have now observed a significant reduction in the
enrichment of the activating histone mark H3K4me3 at the SOX11
promoter upon SOX11 silencing (supplemental Figure 2), suggesting
that SOX11 may indirectly regulate its own expression by epigenetic
mechanisms.
Gene ontology (GO) term enrichment analysis showed several
biological processes overrepresented in the SOX11-bound genes, in-
cluding transcription, embryonic and neuronal development, cell growth
and proliferation, cell death and apoptosis, cell cycle, hematopoiesis,
and hematological system development and function (Figure 1B).
Differential gene expression profiling after SOX11 silencing
To determine the transcriptional programs regulated by SOX11,
we generated a MCL cellular model by silencing SOX11
expression (Figure 2A). We then compared the GEP of stably
transduced shSOX11 and shControl Z138 cells (Figure 2B and
Figure 1. ChIP-chip screening of SOX11-bound
target genes in MCL cell lines. (A) The expression
levels of SOX11 protein in different MCL cell lines
(JVM2, REC1, GRANTA519, JEKO1, HBL2, and Z138)
were detected by WB using an anti-SOX11 antibody
(H-290). a-Tubulin was used as a loading control. (B)
IPA functional annotation tool related to the high-
confidence SOX11-bound genes. The 10 most signif-
icant biological-process GO terms and their log P
values are shown.
BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12 SOX11 BLOCKS PLASMACYTIC DIFFERENTIATION IN MCL 2177
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supplemental Table 3). The top annotated functions of the
differentially expressed genes included lymphocyte activation
and differentiation, phosphorylation, cell cycle, immune system
development, and hematopoiesis (Figure 2C). A GSEA using
the genes differentially expressed in primary SOX11-positive and
SOX11-negative MCL confirmed that the GEP observed in our in
vitro model was representative of that observed in these primary
MCL (supplemental Table 4 and supplemental Figure 3).
Interestingly, among the significant differentially expressed
genes between shSOX11 and shControl cells, we observed
upregulation of key regulators of plasma cell differentiation and
pronounced downregulation of B-cell genes. To further characterize
the significance of these changes, we performed a GSEA using well-
defined gene signatures related to the different steps of the mature
B-cell and plasma cell differentiation programs (supplemental
Table 4).
21-24
These analyses showed that the GEP of Z138shCon-
trol cells was enriched for gene signatures related to the mature
B-cell program whereas Z138shSOX11 cells were enriched in
gene signatures related to the plasma cell differentiation program
(supplemental Table 5 and supplemental Figure 4). These findings
indicate that SOX11 silencing triggers a shift from the mature B cell
to a plasmacytic gene expression program in MCL cells.
SOX11 directly regulates the transcription of PAX5
To determine the direct targets transcriptionally regulated by
SOX11, we overlaid the list of SOX11-bound genes and those with
differential expression upon SOX11 knockdown and found 147
Figure 2. GEP upon SOX11 silencing. (A) SOX11 silencing in Z138 stably transduced with shSOX11.1, shSOX11.2, and shSOX11.3 was verified (upper panel) at the
protein level by WB using GAPDH as a loading control and (lower panel) at the transcriptional level by qRT-PCR. A scramble shControl and untransduced cells (Z138) are
also shown. Bar plot represents the mean 6SD of 3 independent experiments. (B) Heat map illustrating 366 significant differentially expressed genes (adjusted P,.05 and
log2 FC .0.7) in Z138 shSOX11.1 and shSOX11.3 compared with 2 Z138-shControl cells. Red indicates increased expression and green decreased expression relative to
the median expression level according to the color scale shown. (C) DAVID functional annotation of the 366 differentially expressed genes. The 11 most significant (P,.05)
biological process GO terms, their Pvalue, enrichment score, and gene count are shown. (D) Venn diagram depicting the overlap of 1133 SOX11-bound genes (orange
circle), 2790 significant differentially expressed genes (P,.05) upon SOX11 silencing (blue circle), and differentially expressed genes using more stringent statistical criteria
(P,.05 and log2 FC .0.7) (purple circle).
2178 VEGLIANTE et al BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12
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genes in common. From these 147 genes, 18 genes still overlapped
using more stringent statistical criteria (P,.05 and log2 fold
change .0.7) (Figure 2D and supplemental Table 6).
Interestingly, among these 18 genes we identified PAX5, an
essential transcription factor regulating B-cell identity in early
B-cell development
25
and late B-cell differentiation,
26
as one of
the highest-confident SOX11-bound genes (false discovery rate
[FDR] ,0.1 and peak score >0.5). We also found genes that
can contribute to SOX11 hematopoietic and B-cell activation
functions (MSI2 and HSPD1, respectively).
We then verified the direct binding of SOX11 to the regulatory
regions of the most significantly overlaid genes by ChIP-qPCR in
Z138 and GRANTA519 MCL cell lines. As expected, no binding
within these regulatory regions was detected in the SOX11-
negative JVM2 (Figure 3A). Downregulation of PAX5 in stable
SOX11-knockdown MCL cell lines was confirmed by qRT-PCR
and WB (Figure 3B-C, respectively). Upregulation of MSI2,
HSPD1, SUV39H2, and SEPT2 was also observed but to a much
lesser extent than PAX5, prompting us to focus on the SOX11-
PAX5 relationship.
To verify the transcriptional effect of SOX11 on the PAX5
promoter, the amplified SOX11 ChIPed-PAX5 fragment (supple-
mental Figure 5) was cloned in front of a minimal promoter luciferase
reporter and tested in transient cotransfections with SOX11 expres-
sion vector. The induction in luciferase activity was detected in the
coexpression with SOX11 but not with SOX4 or truncated SOX11
proteins lacking the HMG domain (DHMGSOX11) or the C-terminal
TAD domain (DTADSOX11) (Figure 3D), both of which are re-
quired for its transcriptional activity.
27,28
These findings demonstrate
the specificity of SOX11 in regulating the transcription of PAX5.
SOX11-knockdown MCL cell lines display early plasmacytic
differentiation
PAX5 is the critical transcription factor that determines and
maintains B-cell identity by activating the expression of B-cell–
specific genes and simultaneously repressing genes that promote
plasma cell differentiation.
25,26
The significant shift from a mature
B cell to a plasmacytic gene expression program identified by
GSEA in our SOX11-knockdown cells was highly suggestive of
Figure 3. SOX11 directly regulates PAX5 transcription. (A) Binding of SOX11 to regulatory regions of PAX5,MSI2,SEPT2,SUV39H2, and HSPD1 was confirmed by
ChIP-qPCR in different MCL cell lines. Results are shown as enrichment relative to input. (B) Levels of the indicated transcripts in Z138 and GRANTA519 quantified by qRT-
PCR after SOX11 silencing. Fold-change differences compared with control cells are shown. (C) Upper panel: Levels of SOX11 and PAX5 in Z138 and GRANTA519 cell lines
stably transduced with shControl, shSOX11.1, or shSOX11.3 determined by WB, using GAPDH as a loading control. Lower panel: Fold-change differences of SOX11 and
PAX5 protein expression levels between control and silenced cells. SOX11 and PAX5 expression levels were corrected by quantification of GAPDH expression levels. (D)
Luciferase assays in transient cotransfections of PAX5 enhancer-GL4.23 Luc with SOX11 full-length, the truncated SOX11 proteins (SOX11DHMG and SOX11DCtermTAD),
or SOX4 full-length expression vectors, in HEK293 cells. Upper panel: Schematic representation of the constructs and results of the WB experiment of SOX11 mutants are
shown. *Nonspecific bands. Bottom panel: Results are shown as fold induction relative to luciferase activity in cotransfection with the empty vector (pcDNA3). Bar plot
represents the mean 6SD of 3 independent experiments. The significance of difference was determined by paired ttest. HA, hemagglutinin.
BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12 SOX11 BLOCKS PLASMACYTIC DIFFERENTIATION IN MCL 2179
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a major effect of the direct modulation of PAX5 by SOX11 in these
tumor cells. These observations prompted us to further investigate
the expression of genes regulated by PAX5 in SOX11-knockdown
cells. As expected, several B-cell genes were downregulated (CD19,
VPREB3, SPIB,LCK, EBF1, LEF1, and IKZF3) in SOX11 knock-
down cells compared with control cells (Figure 4A).
The transcription factors BLIMP1, XBP1, and IRF4 are
critical regulators promoting plasma cell differentiation whereas
PAX5 inhibits plasma cell development by repressing BLIMP1
andXBP1expression.
26,29
We next examined the modulation of
these transcription factors in SOX11-knockdown MCL cells.
IRF4 was already expressed in control cells and SOX11
silencing did not modify its expression levels. On the contrary,
SOX11 knockdown resulted in a marked increase of BLIMP1
protein levels compared with control cells (Figure 4B and
supplemental Figure 6C). Consistent with BLIMP1 function as
a repressor of the B-cell program during plasma cell differen-
tiation,
30
we found decreased or undetectable expression of B-
cell surface markers CD20, CD24, IgD, and IgM in SOX11-
silenced cells compared with control cells. The plasma cell
marker CD38 was already expressed in control cells and only
marginally upregulated in SOX11 knockdown whereas expression
of CD138 was undetected in both control and silenced cell lines
(Figure 4C).
Full plasma cell differentiation includes synthesis and secretion
of immunoglobulins, and this secretory program is controlled by
the activated splicing form of XBP1. SOX11 knockdown slightly
increased the levels of XBP1(u) but the levels of the spliced form
of XBP1(s) did not change (Figure 4D).
Altogether, these findings indicate that SOX11 silencing in
MCL cell lines promotes the initial steps into plasmacytic
differentiation with downregulation of mature B-cell markers and
increasing levels of BLIMP1. However, the absence of XBP1
splicing and CD138 expression indicates that the full plasma cell
program is not completed.
31,32
SOX11 promotes tumor growth of MCL in vivo
To investigate the potential tumorigenic ability of SOX11 in vivo,
we studied the effect of SOX11 downregulation in a MCL-
xenotransplant model by inoculating CB17-SCID mice with
stably transduced Z138shSOX11 and shControl cell lines. Tumor
Figure 4. SOX11 activates B-cell–specific genes and represses plasma cell gene program. (A) Messenger RNA expression levels of the indicated genes were analyzed
by qRT-PCR in Z138 and GRANTA 519 stably tran sduced with shS OX11.1 or shSOX 11.3. Figure s hows the fold di fferences com pared with the co rrespondin g control
cells (6SD; n 53 technical replicates). Pvalues are shown. The significance of difference was determined by independent-samples ttest. (B) Upper panel : BLIMP1 and
IRF4 expression levels in Z138 and GRANTA519 stably transduced with shControl, shSOX11.1, or shSOX11.3 assessed by WB, using GAPDH as a loading control.
Lower panel: Fold-change differences of SOX11, BLIMP1, and IRF4 protein expression levels between control and silenced cells. SOX11, BLIMP1, and IRF4 express ion
levels wer e corrected by quan tificatio n of GAPDH expression levels. (C) Hist ograms showi ng the expressi on of B-cell and pl asma cell surfac e markers (CD20, CD24,
CD38, and CD138) and surface IgM and IgD measured by flow cytometry in shControl, shSOX11.1, and shSOX11.3 Z138 cell lines. Isotype control is shown in gray.
(D) XBP1 mRNA expression levels by RT-PCR in Z138shControl, shSOX11.1, or shSOX11.3. GADPH was used for the loading control.
2180 VEGLIANTE et al BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12
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growth was followed up, and at 23 days postinoculation (PI)
tumor size in control mice reached 8.8% of the body weight.
SOX11 silencing reduced tumor growth compared with SOX11-
positive control tumors, and significant differences in tumor size
were already achieved at day 16 PI (Figure 5A). Tumors isolated
from mice bearing shSOX11.1- and shSOX11.3-silenced cells
showed 73.1% and 54.9% reduction in tumor burden, respec-
tively, compared with SOX11-positive tumors (Figure 5B). We
also observed a similar significant reduction in tumor growth and
size, at day 23 PI, of the tumors derived from JEKO1shSOX11
and GRANTA519shSOX11 compared with their SOX11-positive
tumor counterparts, JEKO1shControl and GRANTA519shControl
(supplemental Figure 6A-B).
Concordant with the in vitro results, the in vivo SOX11-
silenced tumors showed SOX11 and PAX5 downregulation and
BLIMP1 upregulation compared with SOX11-positive tumors
(Figure 5C and supplemental Figure 6D). The Ki-67 proliferation
index and phosphohistone H3 were similar in control and silenced
tumors (supplemental Figure 7A). We did not observe significant
changes in cell density, proliferation, cycle phases, or viability after
SOX11 silencing in cell lines in culture (supplemental Figure 7B-E).
However, in vivo SOX11-silenced tumors had large necrotic areas
with high levels of activated cleaved caspase-3 that were minimal or
not observed in SOX11-positive tumors (Figure 5D and supplemen-
tal Figure 6D). These results suggest that SOX11 sustains the
B-cell differentiation program and tumor cell survival in vivo and
support the implication of SOX11 expression in the aggressive
behavior of these tumors.
SOX11 expression and B-cell differentiation program in
primary tumors
We next investigated whether SOX11 modulation of the mature
B-cell and early plasmacytic differentiation programs observed
in vitro also occurred in primary cells from SOX11-positive and
SOX11-negative MCL tumors.
We first investigated whether the expression of the SOX11-
bound genes was modulated in MCL. GSEA analysis showed
Figure 5. SOX11 silencing inhibits the growth of MCL tumors in SCID mice. (A) Z138 stably transduced with shSOX11.1, shSOX11.3, and shControl (10
7
cells/mouse)
were subcutaneously inoculated into the right flank of CB17-SCID mice. Tumor growth was measured using a caliper at the indicated days PI. Bar plot represents the mean 6
SD. (B) Tumor volume (mm
3
) of Z138shSOX11.1 (n 58), Z138shSOX11.3 (n 56), and Z138shControl (n 57) cells at day 23 PI into the lower dorsum of CB17-SCID mice.
(C) Macroscopic appearance (upper panel) and consecutive histological sections from representative shSOX11 and shControl tumors stained with hematoxylin and eosin
(H&E) and specific antibodies anti-human SOX11, PAX5, and BLIMP1 (340). (D) Histologic sections of representative shSOX11 and shControl tumors (upper panels, H&E
34 and 310). shSOX11 tumors show extensive necrotic areas that are minimal in shControl tumors. Necrotic areas are highlighted by the immunostaining for activated
caspase-3 (34). Lower panel: Density (percentage of necrotic areas vs total area of the histologic section) of necrotic areas in control and SOX11-silenced tumors delineated
by the presence of activated caspase-3–positive areas. Bar plot represents the mean percentage 6SD. Pvalues are shown. The significance of difference was determined by
independent-samples ttest.
BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12 SOX11 BLOCKS PLASMACYTIC DIFFERENTIATION IN MCL 2181
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a statistically significant enrichment of SOX11 high-confident
bound genes identified by ChIP-chip (supplemental Table 2) in
both Z138shControl and primary SOX11-positive MCL (supplemental
Figure 8), indicating that in these models SOX11 significantly
activates the expression of its target genes.
We then defined 2 gene sets related to SOX11 transcriptional
program extracted from our in vitro microarray data: SOX11-
upregulated genes (genes downregulated upon SOX11 silencing)
and SOX11-downregulated genes (upregulated upon knockdown)
(supplemental Table 4). Notably, we found that SOX11-positive
primary MCL had a GEP significantly enriched in SOX11-
upregulated genes (normalized enrichment score [NES] 52.46,
FDR ,10
24
). Conversely, SOX11-negative primary MCL presented
an enrichment in SOX11-downregulated genes (NES 522.10,
FDR ,10
24
) (Figure 6A).
These results suggest that the SOX11 transcriptional program
derived from our knockdown experiments is also observed in primary
tumors, prompting us to evaluate whether the modulation of B-cell
and plasmacytic differentiation programs also occurs in these primary
tumors.
We thus performed a GSEA with gene signatures of the different
steps of the mature B-cell and plasma cell differentiation programs
used in our previous analysis (supplemental Table 4). In line with the
in vitro observations, this analysis revealed that SOX11-positive
MCL were significantly enriched in B-cell vs plasmablast and PAX5
activated genes gene sets (NES 51.45, FDR 50.04 and NES 5
1.67, FDR 50.01, respectively) whereas SOX11-negative MCL
showed statistical significance in the plasmablast signature
23
and
XBP1 target genes (NES 522.06, FDR ,10
24
and NES 521.34,
FDR 50.1, respectively) (Figure 6B).
We then studied the same panel of surface B-cell markers in the
primary MCL cohort by flow cytometry. Concordant with the in
vitro studies, SOX11-positive tumors had stronger expression of
the mature B-cell marker CD24 and brighter IgM than SOX11-
negative MCL (Figure 6C-D).
SOX11-silenced tumors in xenografted experiments showed
increased apoptotic cells. We performed a GSEA using gene
signatures related to apoptotic pathways and observed that SOX11-
knockdown MCL cell lines were enriched in several apoptotic
gene signatures (Fas, apoptosis, and caspase pathways). The
Figure 6. MCL SOX11-negative primary tumors lose B-cell identity and gain in a plasmablast gene signature. (A-B) GSEA analysis on preranked lists. (A) Using our
customized gene sets described in “supplemental Materials and methods,” SOX11 upregulated and downregulated genes and primary MCL-SOX11
1
tumors are enriched for
SOX11-upregulated genes whereas primary MCL-SOX11
2
tumors are enriched in SOX11-downregulated genes. (B) MCL-SOX11
1
tumors are enriched in B-cell vs
plasmablast and PAX5 activated genes gene sets whereas MCL-SOX11
2
tumors are enriched in plasmablast signature and XBP1 target genes. NES and FDR are shown.
Statistical significance is considered when FDR ,0.1. (C) Analysis of CD24 and surface IgM expression in CD191CD51cells (MCL-SOX11
1
) or CD191CD5
2
cells (MCL-
SOX11
2
). Numbers inside the histograms indicate the percentage of positive cells above the isotype control. (D) Mean fluorescence intensity (MFI) of surface CD24 and IgM
expression in primary MCL tumors.
2182 VEGLIANTE et al BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12
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caspase-pathway gene signature was also significantly enriched in
SOX11-negative primary MCL (supplemental Figure 9). These
results suggest that SOX11 may regulate cell survival by
controlling the expression of apoptotic genes in MCL cells.
Primary MCL have a monotonous cell morphology lacking
the modulation toward plasmacytic differentiation observed in
other types of B-cell lymphomas.
1
Intriguingly, some rare cases
of MCL may show plasmacytic differentiation.
33
We postulated
that this differentiation would occur in SOX11-negative MCL.
In a review of our files, we found 2 MCL with plasmacytic dif-
ferentiation expressing IgM, kappa, and BLIMP1 (Figure 7A-
C). All tumor cells, including the cells with plasmacytic
differentiation, expressed strongcyclinD1butwereSOX11
negative (Figure 7D-E). These results reinforce the idea that
SOX11-negative MCL may modulate the mature B-cell and
early plasma cell differentiation programs in primary tumors
whereas the strong expression of SOX11 in conventional MCL
may block the cells in a mature B-cell stage, preventing their
further progress into differentiation.
Discussion
In this study, we have used a ChIP-chip approach to reveal the first
human genome-wide promoter analysis of SOX11 and identified
1133 unique genes as putative targets of this transcription factor in
MCL. Using GEP, we have also identified 366 genes whose
expression was modulated by silencing SOX11 in MCL cell lines.
GO analyses identified that genes regulated by SOX11 in these
lymphoid cells were involved in lymphocyte activation and
differentiation, phosphorylation, cell cycle, immune system de-
velopment, hematopoiesis, and lymphoid organ development as the
top annotated functions, but also in stem cell development, apoptosis,
and cell migration.
The role of different members of the SOX gene family, including
SOX11, as direct regulators of an expression program of early neural
lineage development has been recently recognized in a murine
model.
34
Our ChIP-chip study confirmed SOX11 binding to reg-
ulatory regions of similar genes involved in neuronal development
(BTG2, MEIS1, and NR2E1), but these genes were not expressed in
our lymphoid model, indicating the tissue-specific regulation of the
SOX11-bound genes. In our study, the main target genes modulated
by SOX11 were involved in immune system development and he-
matopoiesis, and PAX5,MSI2, and HSPD1 were among the most sig-
nificant genes directly regulated by SOX11.
The significant shift from a mature B cell to a plasmacytic gene
expression program identified by GSEA in our SOX11 knockdown
cells was highly suggestive of a major effect of the direct modu-
lation of PAX5 by SOX11 in these tumor cells. We have demon-
strated here that PAX5 is a direct target gene regulated by SOX11
and the constitutional expression of SOX11 in these tumors maintains
the downstream PAX5 program. Thus, several B-cell–specific genes
directly activated by PAX5 were downregulated in SOX11-
knockdown MCL cell lines whereas BLIMP1 was upregulated
upon SOX11 repression. The slight upregulation of XBP1(u) var-
iant and the surface phenotype of Z138shSOX11 cells confirmed
that SOX11 downregulation is able to initiate the terminal B-cell
differentiation process but is not sufficient to drive the full plasma
cell program.
SOX11 is constitutively overexpressed in the vast majority of
MCL.
14-16
However, recent studies have identified a subset of
SOX11-negative MCL
4
that differ from conventional SOX11-
positive tumors in their frequent hypermutated IGHV, leukemic,
nonnodal presentation and more indolent clinical behavior.
3-5,7
The
significant reduction on tumor growth of the SOX11-silenced cells in
the xenograft experiments is consistent with the indolent clinical
course of the nonnodal SOX11-negative primary MCL and high-
lights the implication of SOX11 expression in the aggressive
behavior of conventional MCL. Some studies have identified
Figure 7. Plasma cells in MCL tumors are SOX11 negative. (A) MCL with focal plasmacytic differentiation. Some cells show Dutcher bodies corresponding to the
accumulation of restricted immunoglobulins (arrow) (H&E; 360). (B-C) Tumor cells expressing restricted kappa light chain and BLIMP1 (360). (D) Tumor cells expressing
cyclin D1 (360). (E) SOX11 expression is negative in the tumor cells but positive in internal controls such as endothelial and histiocytic cells (360). Arrows point to tumor cells
with Dutcher bodies.
BLOOD, 21 MARCH 2013 xVOLUME 121, NUMBER 12 SOX11 BLOCKS PLASMACYTIC DIFFERENTIATION IN MCL 2183
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SOX11-negative MCL with aggressive behavior but virtually all
these cases have TP53 alterations.
6,7,35
In our study, we confirmed
that the gene signatures modulated upon SOX11 silencing in vitro
were also enriched in primary MCL, indicating that our cell line
model closely reflects the in vivo situation. Similarly, primary
SOX11-positive MCL showed a GEP characteristic of mature
B cells whereas negative tumors exhibited a relative shift to genes
distinctive of early plasmacytic differentiation steps. However, the
downregulation of SOX11 in both the in vitro model and primary
tumors is not sufficient to trigger a mature plasma cell program.
These results, together with our finding that the uncommon
plasmacytic differentiation in MCL appears to occur in SOX11-
negative tumors, reinforce the idea that these tumors may modulate
the mature B-cell and plasmacytic differentiation programs. Con-
versely, SOX11 overexpression in aggressive MCL may block the
cells in a mature B-cell stage, preventing their further differentiation.
The resistance of MCL to new treatments with proteasome inhibitors
has been linked to the development of plasmacytic differentiation,
suggesting that the findings in our study may also have implications
in the design of new therapeutic strategies.
36
The significance of blocking plasma cell differentiation program
as a relevant oncogenic mechanism in lymphoid neoplasias has been
previously observed. The impairment of terminal B-cell differen-
tiation caused by SOX11 overexpression in MCL may parallel the
forced expression of PAX5 by the t(9;14)(p13;q32) translocation
identified in some B-cell lymphomas,
37-39
and the inactivating
mutations of PRDM1 in diffuse large B-cell lymphomas.
40-42
In summary, our study reveals the global transcriptional network
and direct target genes regulated by SOX11 in MCL that may con-
tribute to the development and aggressive behavior of this tumor. Our
findings provide an improved understanding of the molecular mech-
anisms contributing to the pathogenesis of MCL and may have clinical
implications in the diagnosis of patients and selection of therapeutic
strategies more adapted to the molecular diversity of this tumor.
Acknowledgments
This work was supported by grants from the Ministerio de Econom´
ıa
y Competitividad (MINECO) (BFU2009-09235 and RYC-2006-
002110) (V.A.), (RYC-2009-05134) (P.P.-G.), (SAF08/3630) (E.C.),
and the Instituto de Salud Carlos III RTICC (2006RET2039)
(E.C.), (PS09/00060) (G.R.), Fondo Europeo de Desarrollo Regional,
Uni´
on Europea, Una manera de hacer Europa.
Authorship
Contribution: M.C.V. performed the ChIP experiments; J.P. performed
all in vitro experiments; P.P.-G. performed experiments with the MCL
primary cases; G.R. performed the in vivo study supervising; A.M. and
J.P. performed in vivo experiments; A.N. generated microarray data
from MCL primary tumors; G.C. performed statistical analysis; S.B.
provided information of the MCL patients; A.E. and H.S.-C. helped
with the ChIP-chip experiments; P.J., G.C., and J.I.M.-S. helped with
microarray data; L.H. and D.C. helped with the discussion; N.V.
performed fold-change analysis; E.C. performed immunohisto-
chemistry experiments, identified morphologically MCL tumors
with PC differentiation, analyzed data, and supervised experiments;
V.A. designed, performed, and supervised experiments, analyzed
data, and wrote the manuscript; and all authors discussed the results
and commented on the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Virginia Amador, Centre Esther Koplowitz
(CEK), C/Rossell´
o 153, Barcelona 08036, Spain; e-mail: vamador@
clinic.ub.es.
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