Molecular pathogenesis of germinal center-derived B cell lymphomas

Summary
B cell lymphomas comprise a heterogeneous group of genetically, biologically, and clinically distinct neoplasms that, in
most cases, originate from the clonal expansion of B cells in the germinal center (GC). In recent years, the advent of novel
genomics technologies has revolutionized our understanding of the molecular pathogenesis of lymphoid malignancies as a
multistep process that requires the progressive accumulation of multiple genetic and epigenetic alterations. A common
theme that emerged from these studies is the ability of lymphoma cells to co-opt the same biological programs and signal
transduction networks that operate during the normal GC reaction, and misuse them for their own survival advantage. This
review summarizes recent progress in the understanding of the genetic and epigenetic mechanisms that drive the
malignant transformation of GC B cells. These insights provide a conceptual framework for the identification of cellular
pathways that may be explored for precision medicine approaches.
diseases, we will focus on those that are most frequently
affected and have been functionally characterized in the
context of the normal biology of the GC. We refer readers to
INTRODUC TION other reviews for a comprehensive overview on the interaction
between the tumor cells and the microenvironment, 11 the role
of infectious agents,12 and the clinical implications for
The adaptive immune system has evolved to allow the
mechanism-based therapeutic approaches.13,14
effective recognition and response against a virtually unlimited
number of invading pathogens. These processes require the THE D OUBLE-EDG ED S WORD O F T HE
formation of germinal centers (GC), a specialized
microenvironment that ensures the generation and selection of GC R E AC TION
cells producing antibodies with high affinity for the antigen. 1–4
The GC reaction is thus critical for the establishment of Germinal centers are highly dynamic structures that form
humoral immunity. However, the unique and complex events transiently in secondary lymphoid organs upon encounter of a
that are associated with this reaction pose a significant risk to B cell with a foreign pathogen to enable the generation and
the genome of GC B cells, which have to endure high selection of clones with high affinity antibodies. 1,2,4,5 In 2007,
replication stress while undergoing multiple DNA breakage an elegant series of in vivo imaging studies demonstrated that,
and recombination events required by the antibody within these structures, B cells constantly recirculate between
diversification processes of somatic hypermutation (SHM) and two anatomically separate compartments15– 18: the DZ, which
class switch recombination (CSR).5 consists of rapidly proliferating cells (also known as
Additionally, GC B cells must have elevated epigenetic centroblasts) capable of dividing every 6-12 hours, and the LZ,
plasticity in order to rapidly reprogram their transcriptional which comprises more quiescent cells termed centrocytes,
networks while recirculating between the GC dark zone (DZ) admixed with resident accessory cells such as follicular
and light zone (LZ) compartments to assume distinct dendritic cells (FDC), follicular helper T (Tfh) cells, and
functional states. Therefore, it does not come as a surprise that macrophages (Figure 1).2,8 These two compartments
the majority of mature B cell lymphomas derive from the correspond to distinct functional states of the B lymphocytes.
clonal expansion of a GC B cell, as documented by multiple Specifically, the DZ is the site where GC B cells undergo
evidences that include the presence of somatically mutated secondary diversification of their antibody genes through the
immunoglobulin (IG) genes,6 the strong similarity of their gene process of SHM,19 a DNA remodeling event initiated by the
expression profiles with that of B cells in various phases of the activation induced cytidine deaminase (AID) enzyme 20,21 that
GC reaction,7,8 and the frequent association with genetic introduces single nucleotide substitutions, as well as small
lesions—ie, chromosomal translocations and aberrant SHM— deletions and duplications, in the variable region of the IG
that result from errors occurring during GC-specific DNA genes (IGV), the final goal being to increase the affinity of
remodeling events.9,10 More recently, next-generation their B cell receptor (BCR) for the antigen. 22,23 DZ B cells then
sequencing studies and functional genomics cease proliferation and move to the LZ, where they compete
analyses of lymphomas have refined this concept by for signals delivered by a limited number of resident Tfh cells
uncovering multiple genetic alterations, the consequence of that trigger positive selection. 8 Based on affinity for the
which is the dysregulation of epigenetic and transcriptional antigen, LZ B cells will either re-enter the DZ for iterative
programs that operate during distinct phases of the normal GC rounds of cell division and selection, which lead to affinity
reaction, while facilitating evasion from immune recognition maturationat the population level, exit the GC to become a
mechanisms. Indeed, the common theme of these studies has memory B cell or a plasma cell, or be eliminated by the default
been the realization that malignant cells co-opt the same GC apoptotic program. The LZ is also the site where B cells
signaling pathways and regulatory networks that are normally undergo CSR, an intrachromosomal deletional recombination
utilized by GC B lymphocytes to sustain their own growth and event that requires the activity of AID and confers distinct
survival. This review will summarize current knowledge about effector functions to antibodies with identical specificities. 24,25
the genetics and pathogenesis of the most common GC-derived Ultimately, cells will terminate the cyclic DZ-LZ re-entry
B-cell non-Hodgkin lymphomas, including Burkitt lymphoma process and be selected for survival and differentiation into
(BL), follicular lymphoma (FL), and diffuse large B cell long-lived memory B cells and plasma cells.25,26
lymphoma (DLBCL). The two distinct phases of GC development reflect discrete
transient states within the same B cell developmental stage,
which can be recognized to some extent in the transcriptional
signature of the various lymphoma subtypes, with BL being
more similar to a DZ B cell, and FL and DLBCL being more
related to a GC LZ cell.8
TR ANSCRIPTION F AC TOR N E TWORKS
Among the plethora of genes and biological programs WIRING T HE G C
identified to date as targets of genetic alterations in these

As mentioned, a common theme of recent genomic analyses
has been the realization that most genetic alterations associated
with B lymphoid malignancies converge on transcription factor
networks that are normally used by the GC B cell to sense
antigens and assume distinct functional states during the GC
reaction. This section focuses on selected proteins that, among
those implicated in the control of GC physiology, are most
commonly targeted by genetic lesions in lymphoma (see ref. 2
for a comprehensive review of the molecular switches that
coordinate the complex dynamics of the GC
reaction).
As a sequence specific DNA-binding transcription factor, the
MYC protein controls a broad range of cellular programs,
including proliferation, cell growth, energy metabolism,
telomerase maintenance, differentiation, and apoptosis, 27 its
target gene network being estimated to include ~15% of all
protein-coding genes as well as non-coding RNAs. 28 In
addition, MYC was shown to control DNA replication through
mechanisms that are independent of its transcriptional activity,
a property that may promote genomic instability by inducing
replication stress.29
During the GC reaction, MYC expression is tightly regulated
through a bimodal distribution pattern whereby the protein is
first induced in the GC founding B cells, shortly upon antigen
binding to the BCR, but is then transcriptionally repressed by
BCL6 (and most likely other factors) in the DZ B cell
population, before being re-induced in a small subset of LZ B
cells that are undergoing affinity-based positive selection
(Figure 2).30 Given its critical role in almost all proliferating
cells, the absence of MYC expression in the GC DZ population
has been a matter of confusion and represents a paradox that is
still not completely understood. 31 However, elegant in vivo
studies have shed light into the stage-specific role of MYC
during the GC response. In particular, it has been demonstrated
that upregulation of MYC expression in the LZ is required for
antigen selected B cells to re-enter the cell cycle and
recirculate to the DZ for additional rounds of proliferation,
SHM and selection.30 This process, known as “cyclic re-entry,”
is critical to sustain affinity maturation and to maintain the GC
reaction.8,30,32 Consistently, conditional deletion of MYC early
upon antigenic stimulation abrogates GC formation,32 and
inhibition of its activity specifically in the LZ leads to
dissolution of established GCs.30 Recent elegant work suggests
that, in LZ B cells, MYC is induced through BCR and CD40
ligation via NF-κB and
FOXO1 activation respectively
MEF2B and MEF2C
The myocyte-specific enhancer factor 2B (MEF2B) and
MEF2C proteins belong to an ancient family of transcription
factors involved in the regulation of multiple developmental
programs through interaction with specific transcriptional co-
factors. 34 Recent studies have uncovered critical roles for
these proteins in both normal and malignant GC B cells, where
MEF2B and MEF2C appear to play slightly distinct functions.
In particular, MEF2C, which is expressed at all stages of
mature B cell differentiation, was shown to regulate the
proliferation of GC B cells by functioning as a direct
transcriptional
effector of BCR signaling via the p38 MAPK pathway.35
Consistently, loss of MEF2C function caused reduced immune
responses to antigen and defective GC formation due to
impaired B-cell proliferation. 35 In contrast, MEF2B is
exquisitely expressed in the GC, where its transcription is
rapidly induced after T-cell dependent antigen activation,
slightly before the upregulation of BCL6. 36,37 This is in line
with the ability of MEF2B to directly bind promoter/enhancer
sequences in the BCL6 locus and transactivate its
expression.37,38 In addition, MEF2B is at the center of a broad
transcriptional network that includes, among other cis-
regulatory elements, the full set of GC-specific super-
enhancers, indicating an important role in
instructing the GC reaction, and particularly the DZ.39 In this
view, the modest effects on GC formation observed in vivo
upon single loss of Mef2b may suggest partially redundant
functions of Mef2b and Mef2c39, which can dimerize with
each other. In support of this hypothesis, co-deletion of these
two genes in GC B cells completely
abrogated GC formation
BCL6
Known as the “master regulator” of the GC reaction, BCL6 is
a potent transcriptional repressor that, within the mature B cell
lineage, is selectively expressed in GC B cells 40,41 and is
absolutely required for GC development. The specific signals
that drive upregulation of BCL6 following antigenic
stimulation are in large part unknown; however, multiple
transcription factors have been implicated in this process,
including IRF4,42 IRF8,43 and MEF2B.37 BCL6 orchestrates the
GC reaction by negatively modulating the expression of a
diverse set of genes involved in multiple biological programs,
which it recognizes through specific motifs in their promoter-
proximal ordistal sequences,44 recruiting distinct co-repressor
complexes.45,46
Functions critical to the GC B cell phenotype and controlled
by BCL6 include T-cell mediated B cell activation, 47,48 BCR
and CD40 signaling, 49,50 the induction of apoptosis (eg, by
suppression of BCL2),51
the sensing and response to DNA damage (eg, by suppression
of TP53, ATR, CHEK1),52–54 several cytokine and chemokine
signaling pathways,47,48 Notch2 signaling,55 and plasma cell
differentiation (via suppression of BLIMP1).56 Additionally,
BCL6 regulates gene transcription by repressing specific
microRNAs (eg, miR-155, which has inhibitory roles on
AID).57,58 As such, BCL6 is critical to establish the hyper-
proliferative status of DZ B cells, while allowing them to
tolerate the DNA breaks associated with SHM and CSR
without eliciting cell cycle arrest and apoptotic responses;
moreover, BCL6 prevents the transduction of receptor signals
that could lead to premature activation and differentiation prior
to the selection of high affinity B cell clones. Upon completion
of these processes, expression of BCL6 needs to be turned off
in order to license B cell exit from the GC; the mechanisms
responsible for this molecular switch have been partially
elucidated and include at least two signals that operate at the
translational and transcriptional level respectively: (a)
engagement of the BCR by the antigen acquired by FDCs, and
(b) activation of the CD40 receptor by the CD40 ligand present
on Tfh-cells. 49,50,59 By relieving the expression of its target
genes, downregulation of BCL6 will restore the ability of the
cell to respond to microenvironmental cues that lead to
termination of the GC reaction. The requirement of BCL6 for
the GC response was conclusively demonstrated by the lack of
GC formation and affinity maturation in Bcl6-deficient mouse
models,60,61 a phenotype also induced by deletion of a highly
interactive, GC-specific locus control region upstream of
Bcl6.62
FOXO1
FOXO1 is a member of the Fox-O family of Forkhead
transcription factors that plays critical roles at defined stage
transitions during B cell development.63 Within the GC,
FOXO1 is almost ubiquitously expressed in the DZ; this
pattern is in keeping with the low activity in this compartment
of PI3K-AKT signaling, a major negative regulator of FOXO1
that is only detectable in the GC LZ. 64 Indeed, FOXO1 is
necessary for the establishment and maintenance of GC
polarity, as well as for the DZ B cell phenotype. 64,65 The role
of FOXO1 in sustaining the DZ program entails its ability to
transactivate multiple genes involved in functions typical of
DZ B cells, such as the chemokine receptor CXCR4, cell
proliferation, and negative modulation of DNA repair, while
silencing signaling pathways characteristic of the GC LZ
program (eg, activation and differentiation), in part in
cooperation with BCL6.64,65 The importance of this dual
activity is demonstrated by the phenotype of GC-specific
Foxo1 null mouse models, which form apparently normal GCs
entirely composed of LZ B cells and devoid of DZ gene
programmes.64,65 Intriguingly, Foxo1-negative GC B cells
displayed normal SHM but defective affinity maturation and
CSR, suggesting that the AID-mediated SHM machinery can
be activated independently of the DZ program.64,65
E2A (TCF3)
The E2A transcription factor plays an instructive role in
normal B-cell development and GC formation by regulating
the transcription of several B-cell restricted
genes, including those encoding the immunoglobulin heavy
and light chains, the negative BCR regulator SHP1 (a key
contributor to the absence of BCR signaling in most DZ GC B
cells),66 and CCND3, which is required for the proliferative
expansion of GC B cells.67,68 Consistent with its pro-
proliferative function, E2A is expressed at higher levels in DZ
B cells8 and its deletion in vivo impaired the expansion of
GCs, although this gene appears to be dispensable during the
initial phases of antigen activation.69
The NF-κB-IRF4-BLIMP1 axis
Activation of the NF-κB signaling cascade downstream of
signals emanating from the BCR, CD40 receptor, and TLR, 70
represents acritical step in both the initiation and the
termination of the GC reaction (Figure 2). 42,71,72 In particular,
biochemical evidence and in vivo mouse models have allowed
to reconstruct a critical circuit that is triggered by
CD40:CD40L interaction in a subset of LZ B
cells and involves the upregulation of IRF4, a known NF-κB
target and a master regulator of terminal B cell
differentiation.71,72 At high concentrations, IRF4 can also
repress BCL6,59 a mechanism required for termination of the
mature GC programme and induction of the other plasma cell
master regulator, BLIMP1.73 BLIMP1, in turn, maintains the
plasma cell programme by suppressing BCL6 through a
negative feedback loop.56 Accordingly, a small subpopulation
of LZ B cells can be detected in the GC LZ by
immunofluorescence or immunohistochemistry analysis, which
displays nuclear translocation of the NFκB subunit RELA and
co-expression of IRF4 and BLIMP1, in the absence of
BCL6.49,74 Consistently, conditional GC-specific deletion of
RelA in mice blocks plasma cell differentiation, 75 analogous to
the phenotype observed in Irf4 or Prdm1-deficient mice
EPIGENETIC CONTROL OF THE GC
The highly dynamic nature of the GC reaction requires the
rapid and coordinated switching between discrete
transcriptional programs that contribute to cell fate
determination in response to signals delivered by the
microenvironment.76 GC B cells must therefore be able
to quickly remodel their epigenetic landscape. Indeed, recent
studies showed that, within the GC microenvironment, B cells
undergo massive reorganization of the genomic architecture
that encodes the GC B cell transcriptome. 62 This
reprogramming process requires the activity of dedicated
histone/chromatin modifying enzymes that catalyze the
deposition of specific histone marks associated with open or
close chromatin. The identification of recurrent somatic
mutations in the genes encoding for these very same enzymes
was a major, unanticipated finding of recent whole exome
sequencing efforts in GC B lymphoid malignancies.
EZH2
Enhancer of zeste homologue 2 (EZH2) is a SET-domain
Histone methyltransferase that silences gene transcription by
trimethylating the lysine 27 residue of histone H3
(H3K27me3) and recruiting the Polycomb Repressive
Complex-2 PRC2).77 Within the mature B cell lineage, EZH2
is expressed specifically in the GC, where it creates bivalent
promoters that control the transcription of genes involved in
the negative regulation of cell cycle (CDKN1A) and in
terminal differentiation (IRF4, PRDM1).78,79 In particular,
recent studies defined a positive feedback loop by which EZH2
enables the characteristic proliferative phenotype of GC B
cells by suppressing CDKN1A and releasing the expression of
E2F1.80 In vivo, loss of Ezh2 completely abrogated GC
formation, establishing this methyltransferase as a master
regulator of the GC.78,79
CREBBP/EP300
CREBBP and EP300 are ubiquitously expressed, pleiotropic
regulators of gene expression that catalyze the addition of
acetyl groups to specific lysine residues in both histone and
non-histone proteins.81 Of particular relevance to GC B cells,
CREBBP has been shown to oppose the proto-oncogenic
activity of BCL6 by two mechanisms: (a) the direct acetylation
of the BCL6 protein, which prevents the recruitment of
HDACs and thus impairs its trans-repressor function 82,83; and
(b) acetylation of H3K27 at the promoter/enhancer sequences
of virtually all BCL6 target genes, which facilitates their
transcription84,85 (Figure 3). Moreover, CREBBP-mediated
acetylation is an essential requirement for the activation of the
p53 tumor suppressor, which is itself a target of BCL6. 52,86 A
comprehensive characterization of the transcriptional network
modulated by CREBBP in the GC has been recently obtained
and revealed a preferential enrichment in GC LZ signature
genes,84,85 including components of the BCR and CD40
signaling cascade, master regulators of terminal differentiation,
the CIITA transactivator, multiple MHC-class II loci, and
additional genes involved in antigen
presentation/processing84,85,87 (Figure 3).
CREBBP therefore appears to be particularly important in the
decision making of B cells that exit the GC.
KMT2D
The KMT2D gene (also known as MLL2 or MLL4) encodes a
member of the SET1 family of histone methyltransferases that
facilitates transcription by predominantly mono-and di-
methylating
the lysine at position 4 of histone H3 (H3K4), two
modifications that mark competent enhancers.88 The genes
bound by KMT2D in GC B cells and negatively influenced in
expression upon its deletion play key
roles in B-cell physiology, including the positive regulation of
apoptosis, CD40 signaling, and the control of cell migration
and proliferation. 89,90 Intriguingly, loss of Kmt2d in CD19-Cre
conditional knock-out
models, that is, before antigen encounter by naive B cells,
leads to a significant expansion of the GC B cell population.
These data suggest a role for this methyltransferase in the
commitment of mature B cells to enter the GC reaction, as well
as at later stages of GC differentiation, and support current
models of FL pathogenesis inferring that KMT2D mutations,
frequently observed in FL and DLBCL, are early events in the
history of tumor evolution (see below).