Best Practice & Research Clinical Haematology 29 (2016) 40e53

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Haematology
journal homepage: www.elsevier.com/locate/beha

The chronic lymphocytic leukemia

microenvironment: Beyond the B-cell receptor
Michael Y. Choi*, Manoj Kumar Kashyap 1, Deepak Kumar 1
Moores Cancer Center, UCSD-Moores Cancer Center, La Jolla, 92093-0820, CA, USA

a b s t r a c t
Keywords:
Chronic lymphocytic leukemia Malignant B cells accumulate in the peripheral blood, bone marrow,
Microenvironment and lymphoid organs of patients with chronic lymphocytic leukemia
Stroma (CLL). In the tissue compartments, CLL shape a protective microen-
Nurse-like cell vironment by coopting normal elements. The efficacy of drugs that
Mesenchymal stromal cell target these interactions further underscores their importance in the
CXCR4
pathogenesis of CLL. While the B cell receptor (BCR) pathway clearly
CXCL12
Chemokine
plays a central role in the CLL microenvironment, there is also
Wnt rationale to evaluate agents that inhibit other aspects or modulate
the immune cells in the microenvironment. Here we review the
main cellular components, soluble factors, and signaling pathways of
the CLL microenvironment, and highlight recent clinical advances.
As the BCR pathway is reviewed elsewhere, we focus on other as-
pects of the microenvironment.
© 2016 Elsevier Ltd. All rights reserved.

Introduction

The dependence of cancer cells on a protective niche is an old concept, at least dating back to
Stephen Paget and his 1889 “seed and soil” hypothesis [1]. Since then, the formation of a tumor
microenvironment that promotes growth and allows cancer cells to evade apoptosis or immune sur-
veillance has emerged as a central hallmark of cancer [2]. It is clear that cancersdincluding the he-
matologic malignanciesdare far from homogenous populations of malignant cells. In some cases, non-

* Corresponding author. Moores Cancer Center, UCSD-Moores Cancer Center, La Jolla, 92093-0820, CA, USA. Fax: þ1 858
534 5620.
E-mail addresses: mychoi@ucsd.edu (M.Y. Choi), mkashyap@ucsd.edu (M.K. Kashyap), dkumar@ucsd.edu (D. Kumar).
1
Fax: þ1 858 534 5620.

http://dx.doi.org/10.1016/j.beha.2016.08.007
1521-6926/© 2016 Elsevier Ltd. All rights reserved.
 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53 41

transformed cells account for more than half of the mass or cell population of primary tumors and
metastases [3]. These surrounding elements have been increasingly characterized in recent years, and
include tumor-associated macrophages, tumor-infiltrating lymphocytes, and other stromal cells, which
collectively provide various cytokines or chemokines to recruit and sustain the cancer cells. Another
emerging aspect of the tumor microenvironment is that cancer cells not only derive benefit from it, but
also are able to evade the immune cells within it. The clinical relevance of this is further underlined by
the emergence of “immuno-oncology” drugs that shown efficacy in a variety of tumors, including
melanoma and non-small cell lung cancer.
The tumor microenvironment is particularly relevant in chronic lymphocytic leukemia (CLL).
Although the malignant B-cells from patients with CLL have some intrinsic genetic features that
promote their accumulation such as mutations of TP53 or ATM [4], they are also highly dependent on
external factors for survival and proliferation. Evidence is abundant that the bone marrow and sec-
ondary lymphoid organs are privileged niches in which CLL cells are protected, highlighted by the
following:

 The common observation that CLL cells accumulate in vivo, but undergo spontaneous apoptosis
in vitro, even in conditions that support the growth of other human B-cell lines [5].
 The formation of “pseudofollicles” (also known as proliferation centers) in the secondary lymphoid
organs, in which CLL are more likely express KI-67 or have paraimmunoblast or prolymphocyte
morphology, compared to the quiescent circulating cell population [6]. These pseudofollicles may
account for a turnover of 1%e2% of the entire clone [7].
 The distinct gene expression profile of cells retrieved from the lymphoid organs versus the circu-
lation, including activation of genes in the B-cell receptor (BCR) signaling pathway [8].
 The clinical efficacy of drugs that inhibit BCR signaling and disrupt lymphocyte trafficking to the
lymphoid organs.

The BCR pathway has been the focus of recent attention, particularly due to the clinical efficacy of
the drugs that inhibit it. The BCR pathway is certainly central to the CLL microenvironment and has
reviewed thoroughly [9,10]. Therefore, this article will focus more on other aspects of the CLL micro-
environment. We will review the [1] The main cellular constituents and the bidirectional effects be-
tween them and the CLL cells [2]; The chemokine signals that recruit CLL cells [3]; Downstream effects
on CLL cells aside from the BCR pathway; and [4] Therapeutic strategies that may allow further
refinement in the treatment of patients with CLL.

What are the constituents of the CLL microenvironment?

The non-transformed elements of the microenvironment that promote the homing, retention, and
proliferation of the CLL cells have been increasingly characterized, and include stromal cells, monocyte
lineage nurse-like cells (NLC), and T-cells (Fig. 1).
Nurse-like cells (NLCs) are monocyte-derived cells similar to tumor-associated macrophages. NLCs
were first reported as an in vitro phenomenon: when co-cultured with CLL cells, monocytes sponta-
neously differentiate into NLCs, which can then promote the survival and proliferation of the CLL cells
[11]. They have also been identified in vivo in the lymph nodes and bone marrow of patients with CLL,
and correlate with leukemic lymphocyte viability [12]. The mechanism by which CLL cells induce NLC
differentiation may be through activation of the receptor for advanced glycation end-product (RAGE)-
toll like receptor 9 (TLR9) pathway by release of high mobility group box 1 (HMGB1) from the CLL cells
[13]. NLCs have high levels of expression of genes for adhesion molecules, and interact with CLL cells
through secreted chemokines and cytokines, including B-cell activating factor (BAFF) and a
proliferation-inducing ligand (APRIL); as well as CD14 [14,15]. Collectively, this results in higher
expression of important anti-apoptotic genes in cultured CLL lymphocytes such as BCL2, SURVIVIN,
BCL2A1, and XIAP [16]. NLCs have proved to be a useful in vitro model for evaluation of drugs that target
the interaction of CLL cells and the microenvironment [16,17].
42 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53

Fig. 1. Different modalities of interaction between CLL and the microenvironment. The CLL-B cells are dependent on the pro-survival
signals provided through the interaction of leukemic B cells with nurse like cells (NLCs), endothelial, and dendritic cells. The
leukemic cells form a nexus with microenvironment, which facilitates leukemia cell survival, proliferation, homing and tissue
retention. The nexus between leukemic B cells and bone marrow stromal cells (BMSCs) or NLCs is regulated by chemokine, che-
mokine receptors, kinases, and adhesion molecules expressed by leukemic or cells constituting the microenvironment. The cross-
talk between leukemic B cells and the microenvironment is initiated by different receptors after binding to their respective li-
gands e.g. chemokine receptors, BCR, CD38, CD40 and TLRs (toll like receptors). These receptoreligand interactions of CLL micro-
environment lead to a number of events regulating cell cycle, apoptosis, proliferation, and migration of leukemic cells. The NLCs
express chemokine's like CXCL12 and CXCL13, as compared to BMSCs, which exclusively CXCL12. These cell types of microenvi-
ronment (NLCs & BMSCs) attract leukemic B cells via interaction of CXCR4/5 receptors, which express significantly higher levels in
CLL as compared to normal B cells (NBC). Among other chemokine receptors are CXCR3 and CCR7 present on leukemic B cells and
involved in lymphatic tissue homing. NLCs express some of the proteins belonging to TNF family e.g. BAFF and APRIL. By secreting
these molecules NLCs pro-survival signals to leukemic B CLL cells by binding to their respective receptors i.e BCMA, TACI, and BAFF-R.
The receptor VLA-4 (CD49d) express on the surface of leukemic cells and interact/cooperate with other chemokine receptor via it's
respective ligands expressed in MSCs (VCAM-1 and fibronectin) for facilitating cellecell adhesion. The receptor ligand interaction of
CD38-CD31, which expressed by NLCs and MSCs, leads to induction phosphorylation of ZAP-70. Further, the recruitment of ZAP-70
and other kinases to the activated BCR complex increases the capability of leukemic B cells for antigen response. The stimulation of
the BCR complex (BCR and CD79a,b) induces activation of downstream signaling via recruitment of Syk, BTK and PI3Ks and other
signaling pathways, leading to leukemic cell survival and proliferation. CCL3, CCL4, and CCL22 chemokine secretion by the leukemic
 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53 43

Mesenchymal stromal cells (MSC) can be thought of as the “feeder” layer for normal hematopoietic
progenitor cells. In the marrow, they are referred to as bone marrow stromal cells (BMSCs), and
contribute to normal bone marrow architecture. MSCs are commonly found in the secondary lymphatic
tissues of CLL patients [18]. Similar to NLCs, they constitutively secrete chemokines to recruit CLL cells
into the tissue environments. Upon direct contact with CLL cells, they induce up-regulation of factors
that promote CLL cell survival and proliferation, including ZAP70 and CD38 [19], and also appear to
promote cell survival and drug resistance by increasing glutathione synthesis and glycolysis [20].
Marrow stromal cells also express high levels of Wnt5a, which can promote the survival of CLL cells by
signaling through the ROR1 pathway [21]. In vitro, there is spontaneous migration of some CLL cells
beneath the MSC layer, termed pseudoemperipolesis [22]. There is also cross-talk between the cells:
MSCs are activated by contact with CLL cells, with induction of protein kinase C beta II (PKCbII)
expression and NF-kB pathway activation [23]. CLL cell supernatants also activate BMSCs through the
platelet-derived growth factor receptors (PDGFRs) [24]. Furthermore, it has been found that CLL-
derived exosomal protein and microRNA induce an inflammatory phenotype in the target cells,
including MSCs, enhancing proliferation, migration and secretion of inflammatory cytokines. Co-
injection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice
[25].
CLL cells are also in intimate contact with T cells in the microenvironment. While they play a role in
providing critical regulatory signals within the tissue niches [26], the CLL T-cells are also notable for
their functional defects. They show evidence of chronic activation and “exhaustion”, with increased
expression of the exhaustion markers CD244, CD160, and PD1, with expansion of a PD1þBLIMP1HI
subset [27]. This results in impaired cytotoxicity, granzyme packaging, immune synapse formation
with antigen presenting cells, and migration. The CLL. Co-culture of CLL cells will induce similar defects
in gene expression in normal donor T-cells, indicating that this T-cell exhaustion is due to the CLL cells.
CLL cells express high levels of PD-1 ligand [28]. Patients will CLL also have increased CD4þCD25hi
regulatory T-cells (Tregs), potentially induced by increased expression of CD27 and CD200 on CLL cells
[29,30]. Expression of the inhibitory receptor, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4;
CD152) on those and other CLL T cells have been noted. Inhibition of CTLA-4 or the PD-1/PD-L1 axis may
therefore be of clinical interest to disrupt the immune privileged state of the CLL microenvironment
[28].
Natural killer cells (NK cells) in the CLL microenvironment are also characterized as having
reduced ability to lyse leukemia cell lines associated with a lack of cytoplasmic granules [31]. NK cells
from CLL patients also show defective actin polymerization and impaired immunological synapse
formation, comparable to the cytoskeletal dysfunction seen in CLL T cells. CLL cells may also inhibit NK-
cell by direct contact. Human leukocyte antigen G (HLA-G) molecule overexpression in the plasma of
CLL patients induces NK-cell apoptosis and impairs NK-cell mediated cytotoxicity [32]. The NK-cell
defect appears to be of clinical significance, correlating with disease stage, MBL vs. CLL, and time to
treatment, implying a protective effect of NK cells [33].
Myeloid-derived suppressor cells (MDSCs) have been more recently identified and play a role in
the defective T and NK cells of the CLL microenvironment. These cells have an aberrant myeloid
phenotype: CD14(þ), lacking HLA-DR expression. Increased numbers of MDSCs have been identified in
patients with untreated CLL, and function in suppressing T-cell function in the microenvironment. They
suppress T cells and induce regulatory T cells, with the proposed mechanism being increased indo-
leamine 2,3-dioxygenase (IDO) activity. CLL cells induce MDSCs from healthy donor monocytes sug-
gesting bidirectional crosstalk between CLL-cells, MDSCs, and TRegs [34].
Additional cellular elements in the CLL microenvironment include endothelial cells and follicular
dendritic cells (FDCs), which are essential for tissue homing and CLL retention to tissues. Adhesion to
microvascular endothelial cells promotes CLL survival, activation and drug resistance. These in-
teractions appear to be primary through binding to integrins [35]. In addition, CD44 on CLL cells

B cells may also contribute to recruitment of immune cells (T cells and monocytes) for related interactions. Among other molecules is
CD40, which is present on leukemic B cells and can interact with T cells expressing CD40L that are preferentially found in the
proliferation centers of CLL. The expression of PD1 receptor by the T cells is also of interest as the respective ligand PD-L1 is present
on the leukemic B cells and it more prominent in leukemic splenocytes as compared to cells in circulation.
44 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53

appears to function as a receptor for interaction with follicular dendritic cells, as well as hyaluronic
acid. Ligation of CD44 results in up-regulation of myeloid cell leukemia 1 (MCL1), a member of the BCL2
family of anti-apoptotic proteins, and also interacts with other surface proteins, including CD38;
CD49d; MMP-9; and ZAP-70 [36]. A humanized monoclonal antibody (mAb), RG7356, is specific for
CD44 has direct cytotoxic activity for CLL cells and could overcome the prosurvival influence marrow
stromal cells in co-culture or stimulation with HA or BCR ligation [36]. Other agents that interfere with
CD44 signaling are in development [37]. CD27 also appears to influence CLL binding to stroma. CD27
expression is correlated with ZAP-70 expression, and is by acutely elevated upon BCR cross-linking, and
correlates with functional capacity to adhere to stromal cells. Antibody blockade of CD27 impaired CLL
binding to stroma, implicating CD27 in cellecell interactions with the lymphoid tissue microenvi-
ronment [38].

How are CLL cells recruited to traffic to the microenvironment?

The nexus of chemokine and their receptors plays a very crucial role in the mobilization, trafficking,
and homing of the cells. The constitutive secretion of chemokines by the stromal cells from the
lymphatic tissues guide the CLL-B cells to the microenvironment compartment [39]. The summary of
major chemokine receptor and ligands is in Tables 1 and 2. The microenvironment constituents,
including NLCs, secrete CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL19 and CCL21. CLL cells in turn
express receptors at high levels while in the peripheral blood, including CXCR4, CXCR5, and CCR7. The
interaction between ligand and receptor leads to chemotaxis of CLL cells into the tissue microenvi-
ronment [40] and down-regulation of surface CXCR4. The regulation of CXCR4 by CXCL12 promotes the
continued trafficking of cells between the stromal and blood compartments, and can also be used to
identify cells that have recently left or are returning to the stroma [41]. The leukemic B cells also secrete
other chemokines, such as CCL3, CCL4, CLL17, CCL22, IL-4, and IL-8, which may act as chemoattractants
for monocytes or other lymphocytes [42].
CLL-B cell migration into the lymph nodes also involves interaction high endothelial venules (HEVs),
which are increased in number in CLL lymph nodes compared to normal ones. The leukemic cells bind
to HEVs in a series of events in adhesion involving rolling, sticking and crawling of the CLL B cells on the
endothelium. L-selectin (lymphocyte homing receptor) or CD62L is the factor which regulates the
binding of CLL B cells with HEV walls [43].
Leukemic cell entry into bone marrow and lymphoid organs moves through different barriers, from
the endothelial surface toward endothelial junctions [44]. The ability for adhesion and move across
endothelial barriers depends on dynamic adhesive interactions via integrins VLA-4, LFA-1, and Mac-1
[45]. Integrins are glycoproteins that mediate cell-to-cell and cell-to-matrix adhesion in different cell
types. The a4b1 integrin VLA-4 (CD49d) is a receptor for fibronectin (FN) and vascular cell adhesion
molecule-1 (VCAM-1/CD106), expressed on cytokine-activated endothelium and expressed on
monocytes, lymphocytes, and other hematopoietic cells. VLA-4 integrins are involved in lymphocyte
trafficking and homing and it cooperate with chemokine receptors in CLL cell adhesion to stromal cells,
which eventually cross talk with CD38. VLA-4 integrins therefore play a key role for adhesion of CLL
cells to stromal cells and extracellular matrix, and provide a rationale to further exploration and tar-
geting of this molecule in CLL.

How are CLL cells activated by the microenvironment?

CLL cells that hone to the marrow encounter and interact with accessory cells. Gene expression
studies of CLL cells isolated from the lymph nodes and the marrow and other functional studies have
evaluated the intracellular pathways that are activated in the CLL cells, and showed BCR pathway and
nuclear factor-kappa B pathway activation [8]. CLL cells in the microenvironment also have activation
of receptors of the TNF family, such as the transmembrane activator and calcium modulator and
cyclophilin ligand interactor (TACI), B cell maturation antigen (BCMA), and B celleactivating factor
(BAFF) receptor.
The BCR is a complex consisting of antigen-specific membrane bound immunoglobulin (Ig) and
accessory molecules. Upon ligation, the BCR complex recruits and activates intracellular kinases and
 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53 45

Table 1
Chemokine receptors and cell surface proteins.

Receptor HGNC approved Ligands Cell type Expression in CLL Function Reference
gene symbol interacting
(name) with CLL cells

BAFFR TNFRSF13C (tumor BAFF NLC CLL cells express Involved in cell [77,78]
necrosis factor BAFFR and can survival
receptor bind to BAFF,
superfamily, upon ligation, it
member 13C) can promote CLL
cell survival
in vitro.
BCMA TNFRSF17 (tumor APRIL NLC BCMA expression Involved in cell [79]
necrosis factor is significantly survival
receptor higher on CLL
superfamily, cells than on
member 17) normal B cells
TACI TNFRSF13B (tumor APRIL NLC CLL cells exhibit Involved in cell [80]
necrosis factor variable TACI survival
receptor expression, with
superfamily, the majority of
member 13B) cases displaying
low to
undetectable
TACI
CD38 PECAM1 (platelet/ CD31 NLC Enzymatic Involved in [81]
endothelial cell activity enhances adhesion,
adhesion molecule the growth and proliferation and
1) trafficking of CLL survival
cells
CXCR3 CXCR3 (chemokine CXCL4, NLC Consistently Act as regulators [12]
(C-X-C motif) CXCL4L1, expressed on CLL of cell
receptor 3) CXCL9, CXCL10, cells proliferation,
CXCL11 survival and
migration, Th1
response,
angiostasis,
leukocyte
recruitment,
inflammation,
integrin
activation,
cytoskeletal
changes, and
chemotactic
migration
CXCR4 CXCR4 (chemokine CXCL12 NLC/BMSC Approximately Chemotaxis, [22];
(C-X-C motif) five-fold higher hematopoiesis,
receptor 4) expression in lymphopoiesis,
CLL-B cells as cell migration
compared to and survival,
normal B cells angiogenesis, and
organogenesis
CCR7 CCR7 (chemokine CCL19, CCL21 Stromal cells Expressed Upon binging of [82]
(C-C motif) significantly the CCR7 to it's
receptor 7) higher in CLL as ligands CCL19/21,
compared to B it can lead to
lymphocytes in migration of CLL
lymph nodes cells across the
vascular
endothelium
CXCR5 CXCR5 (chemokine CXCL13 Follicular High expression Chemotaxis, B [83,84]
(C-X-C motif) dendritic cells in CLL cell migration,
receptor 5) (FDC)/NLC regulated
(continued on next page)
46 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53

Table 1 (continued )

Receptor HGNC approved Ligands Cell type Expression in CLL Function Reference
gene symbol interacting
(name) with CLL cells

lymphocyte
homing, Th2
response,
organogenesis
(cooperative with
the CCR
receptor), and
CLL positioning
within lymphoid
follicles
ETAR EDNRA (endothelin ET-1 Endothelial Survival and drug ET-1/ETAR axis [85]
receptor type A) cells resistance important for CLL
endothelial cells
interaction
contributes to
have role in
nursing and
protective niche
in infiltrated
tissues
ROR1 ROR1 (receptor Wnt5a BMSC Exclusively [21,86]
tyrosine kinase-like expressed in CLL
orphan receptor 1) cells only
PD-1 PDCD1 PD-L1 CD4þ T, CD8þ PD-L1 expressed T-cell [27,28,87]
(programmed cell T cells by leukemic B dysfunction,
death 1) cells impaired
immune synapse
formation
RAGE AGER (advanced HMGB1 NLC Ubiquitous Involved in NLC [13]
glycosylation end expression in CLL differentiation
product-specific
receptor)
VCAM-1 VCAM1 (vascular VLA-4 BMSC High expression CLL cell homing [88]
cell adhesion in CLL to BM and
molecule 1) adhesion
Syk SYK (spleen non-receptor NLC It leads to Important for [89]
tyrosine kinase) cytoplasmic amplification of survival and
tyrosine upstream maintenance of
kinases signaling of BCR leukemic B cells
via ITAM
phosphorylation.
CD40 CD40 (CD40 CD40L CD4þ T cells Survival, CCL17 CD40L/CD40 [90]
molecule, TNF and CCL22 interactions
receptor chemokine profound effects
superfamily secretion on leukemic B
member 5) cells and on the
cells of
hematopoietic
and non-
hematopoietic
compartments

adapter proteins, including spleen tyrosine kinase (Syk), which enhances the generation of second
messengers, and phosphorylation of multiple downstream molecules, including Bruton's tyrosine ki-
nase (Btk) [46] and phosphoinositol-3 kinase (PI3K) and PI3K. These activate downstream targets,
including Akt, which signals downstream through mammalian target of rapamycin (mTOR), NF-kB,
RAS, MAP kinase, or other factors [47]. The interaction between PI3K and Btk, and the shared down-
stream signaling between the BCR and chemokine receptors highlight the complex interactions and
 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53 47

Table 2
Chemokines.

Chemokine HGNC Receptor Cell type Expression in Function Reference

approved gene interacting CLL
symbol (name) with CLL cells

CCL3 CCL3 CCR1 and CCR5þ After BCR Inflammation, [91]

(chemokine CCR5 regulatory T triggering and recruitment and
(C-C motif) cells, nurse-like NLC coculture, activation of
ligand 3) cellscells (NLCs) higher in ZAP- polymorphonuclear
70(þ) CLL, Syk- leukocytes, activated B
dependent cells: recruitment of T
cells for T-cell/B-cell
interactions
CCL4 CCL4 CCR5 The activated Functioning Same as CCL3 [92]
(chemokine CLL cells similarly to
(C-C motif) express and CCL3, CCL4
ligand 4) secrete CCL4 in expression in
response to normal B cells
either co- is induced by
culture with BCR triggering
NLC or upon and CD40
BCR ligand
stimulation
CCL22 CCL22 CCR4 CLL22 was not Expressed in Recruitment of Tregs [92]
(chemokine detected in CLL CLL after CD40
(C-C motif) cells, but it was ligation
ligand 22) detected in
cells co-
cultured with
NLCs
CXCL9 CXCL9 CXCR3 NLCs Reported as co- Possible role in CXCL9 [93]
(chemokine expressed in in the trafficking of CLL
(C-X-C motif) CLL with CXCR3 cells
ligand 9) suggesting a
role in
autocrine
mechanism of
action

overlap between these pathways [48]. Unlike some cases of diffuse large B-cell lymphoma, there is not
an activating mutation of the BCR pathway in CLL. The microenvironment may also upregulate other
factors, including micro-RNA 155 (miR-155), that enhance BCR activation [49].
The microenvironment also influences other pathways in the CLL cells that complement the BCR
pathway activation. BAFF and APRIL secreted by NLCs or stromal endothelial cells bind BAFF and APRIL
receptors on CLL cells, and activate survival and differentiation pathways, including upregulation of
CD40 ligand (CD40L) [14]. BAFF may also contribute to resistance to anti-CD20 antibody therapy, which
may be overcome by a neutralizing BAFF antibody, Belimumab [50]. CLL cells also express several Toll-
like receptors (TLRs), including TLR-7 and TLR-9, and proliferate in response to TLR7 agonists [51].
Microenvironmental interleukin-6 may induce CLL cell tolerance to TLR7 agonists through miR-17 and
miR-19a [52]. There is also evidence that the Notch, Wnt, and Hedgehog anti-apoptotic signaling
pathways mediate the pro-survival effects of the CLL microenvironment, and can be overcome by drugs
that inhibit those pathways [21,53].
The BCL-2 family of anti-apoptotic proteins is also modulated by the microenvironment. Co-culture
of CLL cells with stroma results increased levels of MCL1 [54] and other BCL-2 family member proteins
[55]. Functionally, this results in decreased BH3-peptide induced mitochondrial depolarization, a
measure of “apoptotic priming” [56]. Similar changes are seen in CLL cells taken from the bone marrow
versus the peripheral blood. This effect was reversed with treatment with the PI3K delta-isoform in-
hibitor CAL-101 (GS1101), indicating that the BCL-2 upregulation is due at least in part to signaling
48 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53

through the chemokine or BCR pathways. Similarly, inhibition of the BCL-2 pathway can overcome the
stroma-mediated support of CLL cell viability [57].
Some of the effects of the CLL microenvironment may be mediated through post-translational
modifications, including modulation of alternative splicing (AS). Upon in vitro co-culture with stro-
mal cells, AS in CLL cells can shift the balance from the anti-apoptotic to the apoptotic forms of certain
genes of the BCL2 family, including MCL1, BFL1, and BCL-xL [55]. Drug-induced expression of BCL-x(S),
a pro-apoptotic isoform, has also been described, further supporting that alternative splicing at the Bcl-
x locus appears to have a role in the regulation of CLL cell survival [58]. Recently, FD-895 and
Pladienolide-B macrolides were tested in vitro where these compounds led to splice modulation of the
MCL1 pro- (short) and anti-apoptotic forms [59]. Spliceostatin (SSA), another spliceosome inhibitor
tested in CLL which was able to modulate MCL1 isoforms, but was able to overcome the tumor
microenvironment protection after combination with Bcl-2/Bcl-xL antagonists ABT-263 or ABT-199
[60].

Therapeutic implications

The high degree to which CLL depend on the microenvironment has made that interaction an
attractive target for therapy. Small molecule drugs such as ibrutinib or idelalisib that inhibit compo-
nents of the BCR and chemokine signaling pathways have been effective [61]. This also provides
rationale to target other components of the pathway. Another strategy of targeting the CLL microen-
vironment may include interfering with the binding of chemokines with their cell surface receptors. It
also appears that the immunomodulatory drug, lenalidomide, acts by interfering with the CLL
microenvironment. Finally, emerging drugs that inhibit T-cell costimulatory signals, can potentially
reverse the T-cell exhaustion that is induced by the CLL cells, and overcome the immune privilege of
the CLL microenvironment. A summary of different small molecules and antibodies targeting the CLL
microenvironment is presented in Table 3.
Direct inhibition of CXCR4 signaling may also be an effective strategy to debulk CLL cells. Blockage of
CXCR4 has been shown to overcome stromal cell mediated protection of CLL cells, and therefore
sensitize CLL cells to chemotherapy or immunotherapy agents [62,63]. Several agents that inhibit
CXCR4 are in development, including small molecule antagonists (Plerixafor, AMD3100), NOX-A12, an
RNA oligonucleotide that neutralizes CXCL12, and monoclonal antibodies to CXCR4 (Ulocuplumab/
BMS-936564 and PF-06747143). Both antibodies induce in vitro cytotoxicity in nanomolar concen-
trations in CLL alone or co-cultured with stromal cell support [64,65]. Ulocuplumab has been evaluated
in a phase I clinical trial in combination with bendamustine and rituximab. There was downregulation
of CXCR4 and the mobilization of CLL cells, with concurrent decreases in the lymphadenopathy [66].
The Raf-1/MEK/ERK1/2 pathway is also active in CLL cells. Previously, we and others have
demonstrated that sorafenib is an inhibitor of this pathway and inhibits CLL cell survival in vitro [17,67].
A small molecule inhibitor, MEKi-1 has also been reported to induce apoptosis of CLL cells and over-
come survival support from stromal co-culture [68].
Lenalidomide has also been evaluated as a potential CLL therapy, with clinical activity as a single
agent, and in combination with rituximab [69,70]. It is generally thought that its main mechanism of
action in CLL is inhibition of the microenvironment and modulation of cytokine expression [71],
though a cereblon-dependent direct effect on CLL cell proliferation has also been observed [72].
In vitro, lenalidomide can overcome the survival support of NLCs for CLL cells. This has been asso-
ciated with increased IL-10 secretion by NLCs, and impaired CLL cell chemotaxis [73]. Notably,
lenalidomide can rescue normal T-cell function, including immune synapse formation and migration
[74]. This has been observed both in vitro, and clinically as a correlate to a clinical trial of consoli-
dation therapy after chemoimmunotherapy [75]. Similarly, blockade of the programmed cell death 1
(PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint may effectively overcome the
dysfunctional immune system of the CLL microenvironment. PD-L1 blockade in the TCL1 mouse
model (which has aberrance PD-L1 expression similar to CLL) resulted in prevention of the T-cell
exhaustion, restoration of T-cell cytotoxicity and immune synapse formation, and prevention of CLL
development [28]. The immune checkpoint inhibitors, including nivolumab and pembrolizumab,
have shown efficacy, including durable responses, in relapsed and refractory patients with
 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53 49

Table 3
Different small molecules and antibodies targeting the CLL microenvironment.

Target Name Notes Trial (if any) Reference

Bruton's tyrosine Ibrutinib FDA Approved [61]

kinase (BTK) (Imbruvica)
ACP-196 Phase III
(NCT02477696)
AVL-292 Development halted in 2015. Phase I (NCT01692184) [94]
(CC-292)
PI3K Idelalisib Inihibits PI3Kd isoform FDA Approved [95]
(Zydelig)
Duvelisib Inhibits PI3K-d and g isoforms Phase I [96]
SAR245408 Pan-PI3K inhibitor Pilot study
(S08)
Dual mTOR SAR245409 Phase II [97]
and PI3K (S09) (NCT01403636)
SYK Fostamatanib Showed activity in relapse/ Phase II [98]
refractory cases of CLL in 54.5%
of the cases.
PRT318 Pre-clinical Studies [99]
P505-15 Pre-clinical Studies [99]
Multikinase Sorafenib Blocks phosphorylation of ERK Phase II (NCT00303966, [17]
and MEK NCT01510756), both
terminated
SRC kinase Dasatinib Inhibits Abl and Src. Trials Phase II Trial [100]
showed a ~20% partial response NCT00438854, Phase II
in relapsed refractory CLL (NCT01051115)
patients as a single agent, and
17% in combination with
fludarabine.
Immunomodulation Lenalidomide Inhibits NLC and stromal Phase II, III [70,72]
support and CLL cell
proliferation in vitro.
Development in CLL paused in
2014.
CXCR4 Ulocuplumab Monoclonal antibody targeting Phase I [65]
(BMS-936564) CXCR4.
PF-06747143 Monoclonal antibody targeting Pre-clinical [64]
CXCR4.
Plerixafor (Mozobil) Small molecule inhibitor of Phase I (NCT00694590,
CXCR4. CLL trials were in NCT01373229)
combination with either
rituximab or lenalidomide
PD1 Pembrolizumab Phase II recruiting
(NCT02332980)

lymphoma, particularly Hodgkin lymphoma [76]. Clinical trials with the agents have commenced for
patients with CLL.

Summary

The microenvironment is central to CLL pathogenesis, including recruitment of cells to nodal en-
vironments, stimulation of proliferation and survival of CLL cells, and blunting of immune surveillance
by other immune cells. Therapies like ibrutinib and idelalisib that target elements of the microenvi-
ronment have been effective. Future studies will investigate inhibiting other aspects of the microen-
vironment, including the T-cell exhaustion that results in immune privilege. Finally, the anti-apoptotic
signals derived from the microenvironment may promote the persistence of minimal residual disease.
Continued refinement in the therapeutic strategies to target the microenvironment may ultimately
allow us to gain deeper remissions for our patients with CLL.
50 M.Y. Choi et al. / Best Practice & Research Clinical Haematology 29 (2016) 40e53

Practice points

 CLL cells are recruited to and nourished by non-malignant cells and elements of the tumor
microenvironment.
 Therapies that inhibit the interaction between CLL cells and the microenvironment, such as
agents that inhibit B-cell receptor associated kinases, have been shown to be effective and
well tolerated.
 Other elements of the CLL microenvironment, such as chemokine receptors or other pro-
survival pathways, may also be potential targets for novel therapies.

Research agenda

 Characterization of the network of elements and signals that comprise the CLL microenvi-
ronment and evaluation of drugs that modulate them may further improve treatment options
and outcomes for patients with CLL.
 In addition to the drugs that inhibit B-cell receptor associated kinases, drugs are in devel-
opment that target chemokine receptors and Wnt receptors. These agents may be of
particular research interest in those cases that develop resistance to agents like ibrutinib and
idelalisib, or in combination with those agents.
 The CLL microenvironment is also characterized by immune privilege and T-cell exhaustion.
Further research and clinical testing of the emerging immune-oncology drugs for the treat-
ment of patients with CLL is needed.

Conflict of interest

MYC receives research funding from Abbvie and Angstrom Pharmaceuticals, and serves on speakers
bureau or advisory boards for Abbvie and Gilead. MKK receives research funding from Bristol Meyers
Squibb and Pfizer. DK declares no potential conflict of interest.

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