Journal of Cancer Therapy, 2013, 4, 485-491
http://dx.doi.org/10.4236/jct.2013.43A059 Published Online March 2013 (http://www.scirp.org/journal/jct)
485
Emerging Frontiers in Therapeutics of Diffuse Large B
Cell Lymphoma: Epigenetics and B Cell Receptor
Signaling
Soham Puvvada1, Lisa Rimsza2
1Department of Medicine, University of Arizona, Tucson, USA; 2Department of Pathology, University of Arizona, Tucson, USA.
Received January 23rd, 2013; revised February 27th, 2013; accepted March 6th, 2013
ABSTRACT
This review discusses the impact of gene expression profiling and sequencing discoveries on new therapeutic strategies
in Non-Hodgkin Lymphomas, particularly Diffuse Large B cell Lymphoma. Alterations in oncogenes, over-active sig-
naling pathways down-stream of the B cell receptor, and epigenetic gene mutations will be described. We will also re-
view new targeting strategies aimed at each of these aspects of cell biology encompassing BCL2, BTK, PKCβ, PI3K/
mTOR and HDAC inhibition. Specific new drugs in clinical trials and early trial results are included as well.
Keywords: BCR Signaling; Epigenetics
1. Introduction
Non Hodgkin Lymphomas (NHL) are the 7th leading
cause of cancer death in the United States [1]. Aggre-
ssive B subtypes of NHL include Diffuse Large B cell
Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL),
and Burkitts Lymphoma (BL). Of these, DLBCL is the
most common with approximately 30,000 new cases
each year and accounting for nearly 40% of all Non Ho-
dgkin Lymphomas [1]. Although the majority of cases
are curable with traditional anthracycline based regimens,
up to 40% of patients fail to achieve a remission or even-
tually relapse. This variability in response highlights the
underlying biological heterogeneity of DLBCL. In the
last decade, several new technologies have been deve-
loped for investigating lymphoma and other tumors. In
combination with known molecular techniques, the appli-
cation of gene expression profiling (GEP), and next ge-
neration sequencing have created a paradigm shift in our
understanding of DLBCL.
In a seminal publication, using a customized Lym-
phochip array, new molecular subtypes of DLBCL were
identified: Germinal Center B cell (GCB) type, and the
activated B cell (ABC) type resembling activated peri-
pheral blood B cells [2]. Subsequently, Primary Medi-
astinal (PMBL) was identified as a DLBCL type with a
better prognosis [3]. These three molecular subtypes have
different pathogenetic mechanisms with corresponding
prognostic implications despite similar clinical features
and risk stratifications by International Prognostic Index
(IPI) [4,5]. It was noted that the PMBL group had favor-
able clinical features and prognosis while the ABC sub-
type had the worst prognosis and response to anthracy-
cline based chemotherapy: CHOP (Cyclophosphamide,
Hydroxydaunorubicin/Adriamycin, Oncovin/Vincristine,
Prednisone) [3,6]. This has been further validated by se-
veral groups of investigators that used GEP to demon-
strate the clinical significance of the GCB versus ABC
distinction in R-CHOP (Rituxan-CHOP) treated patients
[5-7]. Further GEP and sequencing data have led to the
development of several new therapeutic strategies that at-
tempt to target the different subtypes of DLBCL.
2. Targeting Oncogenes
MYC and BCL2 are well characterized oncogenes in
Lymphoma, which impact proliferation and evasion of
apoptosis respectively. MYC translocations are detected
in B-cell lymphoma, unclassifiable with features inter-
mediate between DLBCL and BL as well as 10% of DL-
BCL. These usually involve non-Immunoglobulin trans-
location partners [8]. Many of these lymphomas are
“Double Hit” containing MYC translocations and trans-
locations or other abnormalities of BCL2 and/or BCL6.
BCL2 is translocated with the Immunoglobulin Heavy
chain gene in approximately 15% of DLBCL; Most of
these cases are of the GCB subtype (35%) [9]. Develop-
ing effective therapeutic strategies that target MYC has

Emerging Frontiers in Therapeutics of Diffuse Large B Cell Lymphoma: Epigenetics and B Cell Receptor Signaling 486
been elusive. Recently, it has been recognized that acetyl
lysine bromodomains on known MYC coactivator pro-
teins regulate its transcription [10]. Therefore, targeting
bromodomains is an attractive option. JQ1 is a novel
small molecule bromodomain inhibitor that downre-
gulates MYC transcription and transcription of MYC
dependent genes.When JQ1 was tested in multiple mye-
loma cell lines, it decreased proliferation and caused cell
cycle arrest [10]. JQ1 has been shown to decrease MYC
and IL7 receptor in high-risk Acute Lymphoblastic Leu-
kemia cell lines [11]. G-quadruplexes are secondary DNA
structures resulting from DNA folding. It has recently
been shown that MYC can form a G-Quadruplex. Quar-
floxin is a fluroquinolone derivative. It has been chemi-
cally modified so that it interacts with and stabilizes G-
quadruplexes [12]. It is currently in phase I clinical trials
in advanced solid tumors and lymphoma [13].
Oblimersen, an antisense nucleotide, was one of the
first agents targeting BCL2 in development. It was tested
in clinical trials in several solid tumors and hematologic
malignancies. Early phase clinical data were promising
in CLL but phase III data have been unconvincing, and
its future development remains uncertain [14]. Obatoclax
was developed as a small molecule with affinity to BCL-
2, BCL-XL, and MCL-1. Interestingly, obatoclax did not
cause thrombocytopenia which has served as an in vivo
marker of pharmacodynamics of BCL-XL inhibition [15]
Obatoclax has shown modest clinical activity in a phase I
combination with fludarabine and rituximab in relapsed
CLL with partial response(PR) of 54% with no complete
remissions [16]. In Small cell lung cancer, while early
phase data indicated responses, a randomized phase II
clinical trial in combination with chemotherapy did not
show a statistically significant benefit in overall response
rate (ORR) [17]. ABT-263 and ABT-199 are the next
generation of BCL2 inhibitors. ABT263 has been very
active in clinical trials with good response rates but th-
rombocytopenia has been a dose limiting toxicity. Pre-
clinical data have shown reproducible thrombocytopenia
caused by mitochondrial membrane permeabilization in
platelets thought to be mediated by BCLXL, and leading
to apoptosis and clearance by liver and spleen macro-
phages [18]. This is also suggested by relative resistance
of younger platelets to ABT 263 that have higher levels
of circulating BCLXL. This has been mitigated by the
development of ABT 199. A key feature of this drug is
that it lacks the high affinity binding to BCL-XL. This
decreases the amount of thrombocytopenia allowing for
higher dosing and correspondingly better response rates
[19]. It is currently undergoing phase I trials in CLL and
NHL. Additional agents in preclinical development in-
clude Sabutoclax which is a novel pan pro survival BCL2
family protein inhibitor currently in preclinical develop-
ment [20]. These agents are summarized in Table 1.
2.1. Targeting B Cell Receptor Signaling
Recently in both Chroniclymphocytic leukemia (CLL)
and particularly the ABC-DLBCL subtype, Chronic ac-
tive B cell receptor (BCR) signaling has been recently
identified as an important pathway. While it was known
that CARD11 mutations are present in 10% of ABC-
DLBCL, the oncogenic drivers associated with wildtype
CARD11 were unknown. Through RNAi screen data,
BCR signaling and Bruton’s Tyrosine Kinase (BTK)
were identified as key components of survival in ABC-
DLBCL [24]. Germline mutations in BTK are associated
with X-linked Agammaglobulinemia. It has a variable
phenotype characterized by recurrent bacterial infections
in affected males in the first two years of life [25]. More
than 600 different mutations in BTK have been reported,
and two thirds of mutations are premature stop codons,
splice defects, or frameshift mutations that interfere with
translational processing of the BTK transcript [26].
BCR signaling is mediated by CD79A and CD79B.
When an antigen occupies BCR, Srcfamily kinases pho-
sphorylate tyrosines in the ITAM motifs of CD79A and
CD79B [27]. This activates spleen tyrosine kinase (SYK)
by which in turn initiates a signaling cascade that in-
volves BTK, phospholipase Cγ, and protein kinase C β
(PKCβ). BTK forms a complex with B-cell linker (BL-
NK) and Phospholipase Cγ leading to the activation of
multiple pathways including Nuclear factor-kappaB (NF-
κB), Phosphatidylinositol 3-kinase (PI3K), Extracellular
signal-regulated kinase (ERK), Mitogen activated protein
kinase (MAP) and Nuclear factor of activated T cells
(NFAT) [27]. This process is distinct from tonic BCR
that promotes cell survival in mature B lymphocytes in
mouse models; in mouse cells, when the BCR was condi-
tionally ablated, all mature B cells died over a course of
2 weeks [28]. Implication of BTK in chronic active BCR
signaling, and discovery of targeted BTK inhibitors has
led to impressive clinical data. Recently, in an interim
analysis of a multicenter, Open-Label, Phase 2 study of
PCI32765 (targeted inhibitor to BTK) in ABC-DLBCL,
the ORR in relapsed refractory DLBCL was 40%. 60%
responses occurred in ABC-DLBCL tumors with CD79B
mutations but responses were also seen in 37% wildtype
CD79B suggesting that responses to PCI-32765 do not
require a BCR mutation [29].
PKCβ is a serine threonine kinase expressed by normal
and malignant B lymphocytes. It is required for BCR
survival signals including activation of NF-κB; it phos-
phorylates CARMA1 via IKK/TAK1. Enzastaurin is an
acyclic bisindolylmaleimide that was initially developed
as an adenosine triphosphate-competitive, selective inhi-
bitor of PKCβ [30]. The compound also modulates the
PI3K/AKT pathway in selected tumor models. In pre-
clinical studies, Enzastaurin induced apoptosis and inhi-

Emerging Frontiers in Therapeutics of Diffuse Large B Cell Lymphoma: Epigenetics and B Cell Receptor Signaling 487
Table 1. Targeting oncogenes in lymphoma.
Process Target
Small Molecule
Inhibitor
Development/
Response
Focal Adverse
Event Reference
JQ1:BET Bromodomain
Inhibitor
Preclinical:
Multiple myeloma
All
N/A [10,11]
Proliferation MYC
Quarfloxcin Ongoing Phase I N/A [13]
Oblimersen Phase I NHL: 1/21
CR Thrombocytopenia [21]
Obatoclax
Phase I CLL in
combination with
Fludarabine/
Rituximab: 54% PR.
Reversible mental status
changes. [22]
ABT 263
Phase 1: Relapsed
Refractory Lymphoid
Malignancies: 10/46
PR
Relapsed Refractory
CLL: 31% PR
Thrombocytopenia
Myelosuppression [19, 22]
Apoptosis BCL2
ABT 199
Ongoing first in
human Phase
CLL/NHL
N/A [23]
Sabutoclax Preclinical: CML
patient samples N/A [20]
bited the proliferation of DLBCL cell lines and xeno-
grafts at low micro molar doses [30]. In a randomized
phase II trial, Enzastaurin in combination with Rituxan-
CHOP (R-CHOP) was associated with an increased pro-
gression free survival (PFS) and complete response (CR)
rates [31]. It is currently in phase III trials: PRELUDE in
DLBCL [32]. Fostamatinib is a SYK inhibitor that is
orally bioavailable. In preclinical experiments, target in-
hibition of SYK abrogated BCR signaling and induced
apoptosis. When SYK was inhibited by R406 and R788
(Fostamatinib), it was associated with tumor regression
[33]. This led to a phase I/II trial of Fostamatinib in DL-
BCL. At a phase II dose of 200mg twice daily, the drug
was well tolerated but ORR of 23.5% was noted with one
only 1 CR [34]. The future of this drug in DLBCL re-
mains uncertain.
Another, downstream pathway activated by BCR is the
PI3K/AKT (Protein Kinase B)/mTOR (Mammalian tar-
get of Rapamycin) [35]. It is clinically significant in lym-
phoma and CLL.mTOR is serine-threonine kinase that is
present in two distinct complexes: mTORC1 and
mTORC2. mTORC2 phosphorylates AKT, SGK1(serum
and glucocorticoid-regulated kinase), PKC, and mTORC1
mediates cell growth and proliferation via the eIF4E (Eu-
karyotic translation initiation factor 4E) binding proteins
[36]. Everolimus is a rapamycin analog approved by the
FDA for Renal cell carcinoma. Everolimusinduced a G1
arrest in DLBCL cell lines. Further, increased cytotoxic-
ity was observed in rituximab sensitive cell lines [37]. It
was evaluated in a phase II trial of 77 patients NHL and
median age of 70. The predominant NHL subtypes en-
rolled on the trial included DLBCL, MCL, and Grade III
Follicular lymphoma (FL). The ORR was 30% with ma-
jority being PRs and three CRs. Grade III toxicities in-
cluded myelosuppression, hyperglycemia, and respiratory
Infections [38].
P110α, p110β, p110δ and p110γ mediate the activation
of the PI3K pathway via cell surface receptors. Of these,
the p110δ isoform is highly expressed inhematopoetic
cells, and required for cells to respond to BCR [39].
CAL-101/GS1101 was identified as a potent and selec-
tive inhibitor of p110δ. It blocked constitutive PI3K sig-
naling resulting in decreased phosphorylation of AKT
and induction of apoptosis [40]. Finally, it has been
shown that activated BCR also results in tyrosine phos-
phorylation of JAK-STAT via LYN. An in vitro phos-
phorylation assay demonstrated that LYN directly phos-
phorylates STAT3 [41]. Ruxolitinib is an oral selective
inhibitor of JAK1 and JAK2 that is FDA approved for
the treatment of Myelofibrosis. It is currently in a mul-
ticenter phase II study in DLBCL [42]. These agents are
summarized in Table 2.
2.2. Targeting Epigenetics
Post translational modification of histones via epigenetic
mechanisms is critical for maintaining integrity of chro-
matin and gene expression. Recently, several studies have
implicated epigenetic mechanisms in the development of
DLBCL. In a recent study that initially sequenced ge-
nomic DNA of 14 NHL and paired normal tissues, 717

Emerging Frontiers in Therapeutics of Diffuse Large B Cell Lymphoma: Epigenetics and B Cell Receptor Signaling 488
Table 2. Targeting the bcr signaling pathway in lymphoma.
Drug Target Disease Reference
PCI-32765/Ibrutinib BTK ABC-DLBCL [29]
Enzaustaurin PKC  DLBCL [30-32]
Fostamatinib SYK DLBCL [34]
Everolimus mTOR DLBCL [38]
GS1101 P110 CLL, Indolent NHL [40]
Ruxolitinib JAK-STAT DLBCL [42]
coding single nucleotide variants were identified affect-
ing 651 genes. These 14 NHL cases were then reana-
lyzed with another 113 samples including 83 DLBCL
with RNA sequencing; the tumor and matched normal
DNA from these cases were re-sequenced to confirm 109
genes with multiple somatic mutations. It was remark-
able that genes with roles for histone modification were
frequent targets for somatic mutations in DLBCL [43].
MLL2 (Myeloid/Lymphoid or mixed lineage leukemia
gene) showed the most significant number of nonsense
single nucleotide variants including 37% nonsense muta-
tions, 46% read frame altering indels, 8% point muta-
tions at splice sites, and 9% non-synonymous amino acid
substitutions. MLL2 was mutated in 59% DLBCL cell
lines, 32% patient samples and in none of the paired nor-
mal tissues. It is one of the six human H3K4 specific
methyl transferases. Tri methylated H3K4 (H3K4me3) is
epigenetically associated with promoters of actively tran-
scribed genes. These mutations are likely inactivating
since both alleles of the genes were affected by the mu-
tations resulting in loss of MLL2 activity. MLL2 muta-
tions were distributed amongst both ABC and GCB-DL-
BCL subtypes [43].
Histone acetylation is moderated by Histone acetyl-
transferases (HATs); it isan epigenetic mechanism for
keeping chromatin in an open, transcription ready state.
Histone Deacetylases (HDAC) keep the chromatin in ad-
eactelyated repressive state. MEF2 (Mycocyte enhancer
factor-2) proteins are a family of transcription factors
that can act as transcriptional coactivators or corepressors
of HAT and HDAC. Under normal intracellular calcium
levels, MEF2 is bound by type IIa HDACs [44]. In-
creased cytoplasmic calcium displaces the bound HDAC
allowing for competitive binding of the HATs CREB-
binding protein (CREBBP) and E1A binding protein
p300 (EP300). This enables transcription of MEF2 target
genes by acetylation of lysine residues on Histone H3
(H3K27). Somatic mutations of MEF2 that involved non
synonymous single nucleotide variantsare unique to FL
and GCB-DLBCL [43,45,46]. Mutations in HATs prin-
cipally CREBBP are one of the most frequent mutations
in DLBCL found in >30% of cases [45]. Thus, HDAC
inhibition is a promising clinical strategy in DLBCL.
Several HDAC inhibitors are in clinical trials for DL-
BCL. Panobinostat is currently undergoing phase II trials
as a single agent or in combination with rituximab or
Everolimus in DLBCL [47,48]. Vorinostat was well tol-
erated as a single agent in DLBCL but had suboptimal
responses. It is currently being evaluated in combination
with cytotoxic therapy in multiple trials [49]. The next
generation HDAC inhibitors in early clinical develop-
ment include Abexinostat, Bellinostat, and Rocilinostat
(Figure 1). In preclinical studies with CLL patient sam-
ples, Abexinostat resulted in cell death with physiologi-
cally achievable IC50. It was also found to be synergistic
with Bortezomib. Increase in Histone H3 acetylation and
P21 protein, a known cyclin dependent kinase inhibitor
up regulated by histone acetylation were also noted [50].
Abexinostatwas well tolerated in patients with relapsed
FL, and MCL. While the median PFS in MCL was 4
months, at a median follow up of 10.3 months the PFS
was 86% in FL patients [51]. A co-operative group trial
evaluated the efficacy of Bellinostat in relapsed refrac-
tory DLBCL patients with up to 5 prior chemotherapy
regimens. In this trial, 2 PRs were observed at 5 and 13
months after registration [52]. Rocilinostat is currently in
preclinical development, and it is synergistic with Bor-
tezomib in both DLBCL and Multiple Myeloma cell
lines [53,54].
BCL6 is an oncogene also involved with HAT repres-
sion and is frequently altered in DLBCL through trans-
locations and other mechanisms involving the 3q27 locus.
The protein is typically expressed in the GCB subtype of
DLBCL and is a potent therapeutic target in DLBCL. A
recombinant 120 amino acid peptide RI-BPI containing
the SMRTdomain inhibited transcriptional repressor ac-
tivity of BCL6 and had potent in vitro lymphoma effects
[55]. It was also noted that BCL6 repressed the expres-
sion of EP300 and its cofactor HLA-B associated tran-
script 3(BAT3). RI-BPI induced expression of p300 and
BAT3, resulting in acetylation of p300 targets including
p53 and Heat shock protein (hsp) 90. Induction of p300
and BAT3 was required for the antilymphoma effects of
RI-BPI, since specific blockade of either protein rescued
human DLBCL cell lines from the BCL6 inhibitor (Fig-
ure 1) [56]. RI-BPI has recently shown promising pre-
clinical synergy with small molecule inhibitors of BCL2,
NEDD8 activating enzyme and Bortezomib [57].
EZH2 is the catalytic subunit of the PRC2 (Polycomb
repressive complex 2) that enables methylation of histone
H2 on lysine 27 (H3K27), decreasing gene expression
and inhibiting transcription of dependent genes. EZH2
overexpression correlates with poor prognosis in Prostate
adenocarcinoma and Renal cell carcinoma. Somatic mu-
tations have been recently identified in the SET domain
of EZH2 occurring in FL and up to 22% GCB-DLBCL.
A mutation that results in the replacement of tyrosine to

Emerging Frontiers in Therapeutics of Diffuse Large B Cell Lymphoma: Epigenetics and B Cell Receptor Signaling 489
Figure 1. When the histones are deacetylated by Histone Deacetylases (HDAC), the chromatin is condensed into a closed
structure, and expression of target genes usually tumor suppressors is repressed. Histone acetylation by Histone Acetyl
transferases (HATs) permits transcription of target genes.
histidine on exome 15: Tyr641 was associated with in-
creased H3K27 tri-methylation(H3K27me3) [58]. EZH2
inhibitors are currently in preclinical development; GSK-
126 has been shown to decrease global H3K27me3 levels
and reactivate silenced PRC2 target genes thus inhibiting
the proliferation of EZH2 mutant DLBCL cell lines. It
also markedly inhibited the growth of EZH2 mutant DL-
BCL xenografts [58].
In summary, genomic technologies including gene ex-
pression profiling and next generation sequencing are
providing astonishing insights into the biology of lym-
phoma. It is apparent that multiple pathways are altered
and that lymphomas frequently use more than one stra-
tegy to their advantage. In fact, many different genetic
and epigenetic changes are present in any one case. Sub-
clones can also exist with yet additional changes reflect-
ing ongoing tumor heterogeneity [59]. In DLBCL, abnor-
malities of MYC, BCL2, the BCR and its downstream ef-
fector molecules, as well as epigenetic modifying genes
are all frequently altered. New drugs are being designed
to specifically target these abnormalities. As single agents,
these new drugs are currently under evaluation with early
promising results. Combinations of such targeted thera-
pies hold the potential of even more effective therapy.
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