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The microenvironment in chronic lymphocytic leukemia (CLL) and other B cell malignancies: Insight into disease biology and new targeted therapies

Seminars in Cancer Biology, pages 71 - 81

Abstract

Over the last decade, the active role of the microenvironment in the pathogenesis of B cell lymphomas has been recognized, delivering signals that favor clonal expansion and drug resistance. We are only beginning to understand the complex cross talk between neoplastic B cells and the tissue microenvironment, for example in secondary lymphoid organs, but some key cellular and molecular players have emerged. Mesenchymal stromal cells, nurselike cells (NLC) and lymphoma-associated macrophages (LAM), in concert with T cells, natural killer cells and extracellular matrix components participate in the dialog with the neoplastic B cells. B cell receptor signaling, activation via TNF family members (i.e. BAFF, APRIL), and tissue homing chemokine receptors and adhesion molecules are important in the interaction between malignant B cells and their microenvironment. Disrupting this cross talk is an attractive novel strategy for treating patients with B cell malignancies. Here, we summarize the cellular and molecular interactions between B cell lymphoma/leukemia cells and their microenvironment, and the therapeutic targets that are emerging, focusing on small molecule inhibitors that are targeting B cell receptor-associated kinases SYK, BTK, and PI3Ks, as well as on immunomodulatory agents and T cell mediated therapies. Clinically relevant aspects of new targeted therapeutics will be discussed, along with an outlook into future therapeutic strategies.

Keywords: Chronic lymphocytic leukemia, CLL, Microenvironment, Nurselike cells, Stromal cells, CXCR4, CXCL12, B cell receptor, BCR, SYK, BTK, PI3Kδ, Chemokines, Chemokine receptors, T cells, NK cells.

1. Introduction to the microenvironment in CLL and selected other B cell lymphomas

The secondary lymphatic tissues are the principal site for expansion of normal mature B cells, ultimately leading to the generation of antigen-specific B cells and maturation into antibody-producing plasma cells. Normal B cell growth in germinal centers is based on antigen selection and clonal expansion, which is reinforced by antigen specificity, along with co-stimulatory signals from T cells and antigen-presenting cells (APC) [1] . Generally, the pathways responsible for the growth of antigen-specific normal B cells appear to be also functional in their malignant counterparts. CLL cells interact with different types of stromal cells, such as mesenchymal stromal cells [2] , monocyte-derived nurselike cells (NLC) [3], [4], and [5], as well as T cells [6] and [7], collectively referred to as the “microenvironment”, and they proliferate in the context of microanatomical tissue sites called proliferation centers (pseudofollicles), a hallmark histopathology finding in CLL [8] . Early evidence of microenvironment-dependency came from the notion that CLL cells normally undergo spontaneous apoptosis in suspension culture unless they are co-cultured with bone marrow stromal cells (BMSC) [3], [9], and [10] or NLC [3] . Microenvironment-dependence is also implied by the difficulty to establish CLL cell lines in the absence of EBV [11] . In follicular lymphoma (FL), a similar pattern of microenvironment-dependency has unfolded. The neoplastic B cells are also highly difficult to grow ex vivo, and microarray-based gene expression profiling (GEP) [12] and immuno-phenotyping of the accessory cells [13] revealed that the composition of the microenvironment has major impact on disease prognosis. The proposed mechanisms how the microenvironment impacts FL outcome include recruitment of lymphoma growth-promoting monocytes/macrophages and suppression of cytotoxic T cells. In the lymphatic tissues from FL patients, T cells display impaired formation of immunologic synapses, an immune evasion mechanism which can be restored by lenalidomide [14] . In diffuse large-cell B cell lymphoma (DLBCL), GEP and immunohistochemistry also revealed that the microenvironment significantly impacts disease prognosis. Based on GEP, DLBCL can be sub-classified into “germinal center B cell-like” (GCB) and “activated B cell-like” (ABC) DLBCL [15] . Further GEP analyses revealed two stromal signatures, the favorable stromal-1 signature reflecting infiltration by monocyte/macrophage lineage cells and extracellular-matrix deposition, while the unfavorable stromal-2 signature reflects high endothelial cell density [16] . Based on these concepts, CLL and other mature B cell malignancies are expected to be responsive to microenvironment-directed treatment approaches, provided that critical molecular pathways can be identified and targeted.

2. Tissue microenvironments in the bone marrow and secondary lymphoid organs

The BM and secondary lymphoid organs have entirely different, distinct microenvironments, supporting lymphocyte maturation and differentiation. B cell lymphopoiesis in the marrow results in the generation of B cells with functional antigen (Ag) receptors (BCRs). Mature B cells then migrate to secondary lymphoid organs where they are exposed to Ag within germinal centers (GC) of secondary lymphoid follicles [1] . The microenvironment of GC allows maturing B cells to interact with CD4+ T-cells for the necessary help upon Ag recognition and with specialized stromal cells (follicular dendritic cells/FDC) for the required quality control following affinity maturation [17] and [18]. Neighbor cells in the microenvironment are critical at all stages of B cell maturation, as they provide growth support signals and assistance in selection of Ag-specific B cells. Mesenchymal stromal cells are important during B lymphopoiesis in the marrow, as well as for B cell positioning and territoriality in GC. Monocyte-lineage cells, such as NLC, provide various growth-promoting signals and appear to be involved in BCR activation in the lymphatic tissues. T cells can either suppress or promote B cell expansion, and their function depends on activation status, T cell subset, and micro-anatomical location.

3. Mesenchymal stromal cells (MSC)

In the BM, stromal “feeder” cells maintain hematopoietic stem cells (HSC) in specialized “niches” which are close to the marrow vasculature (vascular niche) or to the endosteum (osteoblast niche) [19] . The importance of stromal cells for hematopoiesis was initially demonstrated in long-term BM cultures [20] and [21]. In vitro, CLL cells are attracted to BMSC, and the protective effects of BMSC require the close proximity between CLL and the stromal counterparts [3], [10], [22], and [23]. The high affinity of CLL cells for stromal cells is exemplified by a striking in vitro phenomenon termed pseudoemperipolesis [22] . Pseudoemperipolesis describes the spontaneous migration of a fraction of CLL cells beneath BMSC, which occurs within a few hours of co-culture. Pseudoemperipolesis describes symbiotic complexes of leukemia cells with their stromal cell component [24] and [25]. Co-culture systems of CLL cells with BMSC, typically BMSC cell lines, have been standardized [23] and represent a useful tool for studying CLL cell activation by BMSC, as well as stroma-mediated drug resistance. Intrinsic qualitative and quantitative abnormalities of CLL patient-derived primary BMSC have recently been characterized [26] , as well as the effects of more physiologic hypoxia present in the marrow microenvironment on BMSC function [27] . Interestingly, CLL cell activation by BMSC is bi-directional, and BMSC in turn also become activated by the CLL cells [28] . CLL cell supernatants activate platelet-derived growth factor receptors (PDGFRs) in BMSC [29] , and contact with CLL cells causes expression of protein kinase C (PKC)-βII and subsequent activation of NF-κB in BMSC [30] . Pro-survival effects of MSC also have been described in FL [31] , and this cell type appears to be generally present, to variable degrees in all types of B cell lymphomas [2] .

4. Nurselike cells (NLC) and lymphoma-associated macrophages (LAM)

NLC were named after thymic nurse cells that nurture developing thymocytes [3] . In vitro, NLC differentiate from blood monocytes co-cultured with CLL cells in high-density culture conditions after 7–14 days [3] . In vivo, NLC can be found in the spleen and lymphoid tissues of CLL patients [4] and [32], and the importance of NLC for CLL disease progression was highlighted in recent CLL animal models [33] and [34]. NLC attract CLL cells by secreting CXCL12 [3] and CXCL13 [4] and protect CLL cells from spontaneous or drug-induced apoptosis via CXCL12 [3] and [35], BAFF, APRIL [35] , CD31, plexin-B1 [36] , and activation of the BCR signaling cascade [37] . In other B cell malignancies, NLC are termed tumor-associated macrophages or lymphoma-associated macrophages (LAM). In classic Hodgkin's lymphoma, presence of a LAM GEP signature was associated with primary treatment failure and shorter survival [38] . Similarly, in DLBCL [16] and FL [12] , monocyte/macrophage GEP signatures impact the disease prognosis, indicating the LAM are a critical component in the microenvironment in these diseases. The molecular cross talk between monocyte-macrophages and malignant B cells in the tissue microenvironment is a new area of study and likely will guide us toward new targets. GEP has again provided us with insight into these complex interactions. In vitro GEP analyses revealed that NLC activate CLL cells in a different fashion than BMSC [37] and [39]. Specifically, BMSC induced a GEP pattern with prominent upregulation of the lymphoid proto-oncogene TCL1, paralleled by decreases of TCL1-interacting FOS/JUN [39] . In contrast, NLC induced a GEP response in CLL cells with characteristic induction of genes in the BCR- and NFκB pathways [37] that is strikingly similar to the GEP of CLL cells isolated from lymph nodes of CLL patients [40] . Several other genes of potential importance were also differentially up-regulated by BMSC (for example TNFRSF17, VPREB3, TNFSF10) and NLC (i.e. TNFRSF17, EGR2 and 3, MYCN), but their precise functions in the CLL microenvironment remain to be explored ( Fig. 1 ).

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Fig. 1 Cross talk between malignant B cells and nurselike cells (NLC)/lymphoma-associated macrophages (LAM) in (A) and mesenchymal stromal cells (MSC) in (B). (A) Malignant B cells, such as CLL cells, are activated in the tissues by NLC/LAM via TNF family members BAFF and APRIL, which promote B cell survival and proliferation. Activation of the BCR is another central mechanism of neoplastic B cell activation, but the precise mechanism of BCR activation are controversial and differ between lymphoma subtypes (see text). BCR activation leads to downstream activation of BCR-associated kinases Syk, BTK, and PI3Kδ, which can be targeted by small-molecule kinase inhibitors. Chemokines secreted by NLC/LAM attract B cells and confine them in close contact with these “feeder cells” via seven-transmembrane G-protein coupled chemokine receptors (CXCR4, CXCR5). (B) MSC also attract B cells via secretion of chemokines, and CXCL12, the ligand for the CXCR4 chemokine receptor, is the most prominent MSC-derived chemokine. Integrins expressed on the B cells, such as VLA-4 integrins (α4β1), facilitate the adhesion to the MSC, in cooperation with the chemokine receptors. MSC adhesion, in turn favors B cell survival, proliferation, and tissue retention (homing). Upon B cell contact, MSC also become activated, causing PKC-βII and NFκB activation [30] , indicating that these interactions are a true dialog.

5. T cells

The T-cell compartment is abnormal in CLL, with an increase in absolute numbers of peripheral blood T cells, particularly CD8+ T cells, with a fall in the CD4:CD8 ratio. Despite their increased numbers, these T cells show profound functional defects. CLL T cells show evidence of chronic activation, with upregulation of CD69, HLA-DR and CD57, downregulation of CD28 and CD62L, and expansions of oligoclonal T cells. These oligoclonal expansions are primarily restricted to populations with an activated CD57+ phenotype, suggesting a role for chronic antigen stimulation in their development. T cells exhibit features of “exhaustion”, with increased expression of the exhaustion markers CD244, CD160, and PD1, with expansion of a PD1+BLIMP1HI subset [41] . These T cells have functional defects in cytotoxicity, with impaired packaging of granzyme into vesicles and non-polarized degranulation. In contrast to virally induced exhaustion, CLL T cells showed increased production of interferon-γ and TNFα and increased expression of TBET, and normal IL2 production potentially protecting CLL cells from apoptosis.

T cells in CLL exhibit profound changes in their global gene expression profiles with alterations in expression of genes involved in cytoskeletal formation [42] . Similar defects in gene expression are induced in healthy T cells after co-culture with CLL cells, demonstrating that it is the leukemia cells that are inducing these changes. The altered expression of cytoskeletal genes results in functional defect in filamentous actin (F-actin) polymerization, with defective immunological synapse (IS) formation with antigen presenting cells (APCs) [43] . Functional screening assays identified that the proteins CD200, CD270 (Herpes virus entry mediator; HVEM), CD274 (Programmed death ligand-1; PD-L1), and CD276 (B7-H3) induced impaired IS formation in both allogeneic and autologous T cells [44] . Decreased actin polymerization also impairs integrin mediated migration, with altered RhoGTPase activation [45] . In addition to increased expression of inhibitory receptors in CLL, there is also an increase in CD4+CD25hi regulatory T-cells (Tregs) in CLL, which increases in advanced stage disease [46] . This increase may be mediated by increased expression on CLL cells of CD27 and CD200 [47] . Tregs express the inhibitory receptor, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4; CD152) and T cells from CLL patients have increased CTLA-4 expression. Therefore, it is likely that CTLA-4 signaling is yet another inhibitory pathway mediating T-cell dysfunction in CLL, in addition to the PD-1:PD-L1, CD160:HVEM and CD200:CD200R axes.

T cells also appear to be an integral part of cross talk within the microenvironment, providing critical regulatory signals within the tissue niches [48] . In turn, NLCs can contribute to the increased numbers of T-cells in CLL pseudofollicles [37] via BCR-dependent secretion of chemokines (CCL3, CCL4). This mix of tissue chemokines (CXCL12, CCL3, CCL4) likely contributes to co-localization of stromal cells, NLCs, and activated CD4+ T cells, that provide “sanctuary sites” for the tumor cells, protecting them from immuno-chemotherapy.

6. NK cells

Natural killer cells (NK cells) also have a functional defect in CLL, showing reduced ability to lyse leukemia cell lines associated with a lack of cytoplasmic granules [49] . This activity could be restored by the use of interleukin-2 (IL-2), which also resulted in increased granularity of the large granular lymphocyte subset [50] . The mechanism by which CLL cells downregulate NK-cell function is not known, although there is some evidence that it may involve soluble factors [51] and [52]. CLL cells may also inhibit NK-cell by direct contact, by their expression of the tolerogenic non-classical MHC class I molecule, HLA-G, or by expression of 4-1BB ligand [53] and [54]. Of note, NK cells from CLL patients show defective actin polymerization and impaired immunological synapse formation, comparable to the cytoskeletal dysfunction seen in CLL T cells [55] . The NK-cell defect appears to be of clinical significance, as higher NK-cell numbers were observed in patients with early stage disease and in those with mutated IGHV genes. Furthermore, for patients with a given Rai stage, a higher NK:CLL cell ratio was predictive of a longer time to treatment, implying a protective effect of NK cells [56] . In support of this, NK cells from patients with MBL have a higher cytolytic capacity than NK cells from CLL patients [57] .

7. Mechanism of malignant B cell trafficking and tissue homing

Normal B cell trafficking and function largely depends upon interactions between B cells and stromal cells [58] and [59]. For example, stromal cells in secondary lymphatic tissues constitutively secrete chemokines (CXCL12, CXCL13) that provide guidance for B cell positioning in lymph node compartments [58], [60], [61], and [62]. Lymphocyte trafficking and homing require the cooperation between chemokine receptors and adhesion molecules, such as integrins, CD44, and L-selectins, which are expressed on normal and malignant lymphocytes [63] . Integrins are highly versatile adhesion molecules; their adhesiveness can rapidly be regulated by the cells on which they are expressed, for example by chemokine receptor activation [63] . In B cell lymphomas/leukemia, the neoplastic B cells retain the capacity of their normal counterparts for trafficking and homing, as demonstrated in CLL and B cell acute lymphoblastic leukemia (ALL) [4], [22], and [64]. T and B lymphocytes express receptors for various chemokines, and their expression and function is modulated during lymphocyte differentiation and activation [65] . Circulating lymphocytes interact transiently and reversibly with vascular endothelium through adhesion molecules (selectins, integrins) in a process called rolling. Chemokines on the luminal endothelial surface activate chemokine receptors on the rolling cells, which triggers integrin activation [63] . These results in the arrest, firm adhesion, and transendothelial migration into tissues where chemokine gradients guide localization and retention of the cells [66] , collectively referred to as “homing”.

7.1. CXCR4 (CD184)

The chemokine receptor CXCR4 is expressed at high levels on the surface of peripheral blood CLL cells [22], [67], [68], and [69], and mediates CLL cell chemotaxis, migration across vascular endothelium, actin polymerization, and migration beneath and underneath CXCL12-secreting BMSC [22], [67], [70], and [71]. CXCL12 also has a pro-survival effect on CLL cells [3], [35], and [72] which is not surprising, given that CXCL12 initially was characterized as pre-B-cell growth-stimulating factor (PBSF) [73] . CXCR4 surface expression is regulated by its ligand CXCL12 (previously called stromal cell-derived factor-1/SDF-1) via receptor endocytosis [22] , with downregulation of surface CXCR4 on tissue CLL cells by CXCL12 present at high levels in the tissues. This characteristic can be used to distinguish tissue (lymphatic tissue- and marrow-derived) from blood CLL cells, which express low or high CXCR4 levels, respectively [22] and [40].

Proliferating, Ki-67+ CLL cells from marrow and lymphatic tissue displayed significantly lower levels of CXCR4 and CXCR5 than non-proliferating CLL cells [74] . In vivo deuterium (2H) labeling of CLL cells revealed that patients with higher CXCR4 expression on their CLL cells had delayed appearance of newly produced CD38+ cells in the blood, and increased risk for lymphoid organ infiltration and poor outcome [75] . These 2H studies also revealed intraclonal heterogeneity of CXCR4 expression, with an enrichment of CLL cells expressing lower CXCR4 surface levels in the CD38+/CD5bright fraction, along with increased 2H incorporation [75] . B cell antigen receptor (BCR) signaling results in down modulation of CXCR4 [76] and [77], along with enhanced chemotaxis toward CXCL12 and CXCL13, at least in our hands [76] . This may explain why ZAP-70+ CLL cells display increased chemotaxis and survival in response to CXCL12 when compared to ZAP-70-negative CLL cells [72] , given that ZAP-70 expression is associated with a higher responsiveness to BCR stimulation [78] . CD38+ CLL cell also display higher levels of chemotaxis [79] , and CD38 activation enhanced chemotaxis toward CXCL12, whereas a blocking anti-CD38 mAbs inhibited chemotaxis [80] . CXCR4 signaling in CLL cells is pertussis toxin-sensitive and induces calcium mobilization, activation of PI3 kinases [22] , p44/42 MAP kinases [3] , and serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) [81] . CXCR4 signaling can be inhibited by isoform-selective PI3 kinase inhibitors [82] , including CAL-101 [83] , and inhibitors of SYK [76] , and BTK [84] , leading to impaired CLL cell migration. Other malignant B cells, such as MCL [85] and FL [86] , also express CXCR4 and the available data suggest that CXCR4 function on these cells is similar to what has been published about CLL.

7.2. CXCR5 (CD185)

CXCR5 (CD 185) is the chemokine receptor for CXCL13, a chemokine that regulates lymphocyte homing and positioning within lymph follicles [87] . CXCR5 is expressed by mature B cells, a small subset of T cells, and skin-derived dendritic cells (reviewed in [88] ). CXCR5 gene deleted mice have defective primary follicles and germinal centers in the spleen and Payer's patches, and lack inguinal lymph nodes [89] . CXCL13 is constitutively secreted by stromal cells in B cell areas of lymphoid follicles [87] and [90]. CXCR5 induces recruitment of circulating naïve B cell to follicles [87] and [90] and is responsible for positioning within the germinal center [59], [61], [91], and [92]. In addition, it has been suggested that the primordial function of CXCL13 may be the recruitment of primitive B cells to body cavities for T-independent responses [93] . CLL and MCL cells express high levels of CXCR5 [4] and [85]. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis [71] , and prolonged activation of MAPK (ERK 1/2). In CLL, CXCR5 signals through Gi proteins, PI3-kinases, and p44/42 MAPK pathway [4] . CXCL13 mRNA and protein is expressed by NLC in vitro and in vivo [4] . These data suggest that CXCR5 plays a role in positioning and interactions between malignant B cells and CXCL13-secreting stromal cells, such as NLC/LAM in lymphoid tissues ( Fig. 2 ).

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Fig. 2 Interactions between malignant (CLL) B cells and accessory T cells. CLL cells can be activated in proliferation centers via activated, CD154+ CD4+ T cells [6] , which are in intimate contact with proliferating T cells [7] . These T-B cell interactions favor the survival and expansion of the malignant B cells. On the other hand, cytotoxic CD8+ T cells from CLL patients have an exhausted phenotype and fail to co-localize Granzyme B with CD107A, a prerequisite for successful establishing immune synapses [41] . Due to this defective cytotoxicity, T cells cannot effectively eliminate CLL cells.

7.3. Chemokines secreted by malignant B cells: CCL3, CCL4

CCL3 and CCL4 are chemoattractants for monocytes and lymphocytes [94] . CCL3 expression in normal B cells is induced by BCR triggering and CD40 ligand [95], [96], and [97], and repressed by BCL6 [98] . Activated CLL cells express and secrete CCL3/4 [37], [99], and [100] in response to BCR stimulation and in co-culture with NLC [37] . This BCR- and NLC-dependent induction of CCL3/4 is sensitive to inhibition of BCR-signaling, using SYK, BTK, and PI3K inhibitors [37], [76], [84], and [101]. CLL patients have elevated CCL3/4 plasma levels [37] and plasma levels of CCL3 were strongly associated with established prognostic markers and time to treatment. A multivariable analysis revealed that CCL3, advanced clinical stage, poor risk cytogenetics, and CD38 expression were independent prognostic markers in a cohort of 351 CLL patients [102] . The function of CCL3/4 in CLL remains poorly defined, but based upon the function of B cell-derived CCL3/4 in normal immune responses, increased CCL3/4 secretion by CLL cells may induce trafficking and homing of accessory cells, particularly of T cells and monocytes to CLL cells in the tissue microenvironments [37] and [103].

7.4. VLA-4 (CD49d) adhesion molecules in CLL

Integrins are a superfamily of heterodimeric glycoproteins, which mediate cell–cell and cell–matrix adhesion in various cell types. Integrins are categorized into subfamilies with members sharing a common β subunit pairing with a unique α subunit. β1 integrins are very late activation antigens (VLA) that have the same β1 subunit but various α chains (α 1 through 6) [104] . The α4β1 integrin VLA-4 (CD49d) is a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1/CD106), expressed on cytokine-activated endothelium. VLA-4 is expressed on lymphocytes, monocytes, and most other hematopoietic cells and plays a role in lymphocyte trafficking and homing as part of immune surveillance [65] . VLA-4 integrins cooperate with chemokine receptors in CLL cell adhesion to stromal cells [22] and [105], they cooperate with CD38 [106] , and their function can be inhibited by the BTK inhibitor ibrutinib [107] . Moreover, VLA-4 expression on CLL cells has prognostic impact [108] and [109], indicating the relevance of these interactions in vivo. Collectively, these studies indicate that VLA-4 integrins play a key role for adhesion of CLL and other leukemia cell to stromal cells and ECM, and provide a rationale to further explore and target this molecule in CLL.

8. Role of the BCR in cross talk between malignant B cells and the microenvironment

BCR activation is the quintessential example of a “conserved” B cell activation pathway, utilized by both normal and neoplastic B cells for survival and expansion [110], [111], and [112]. BCR signaling is critical for disease progression in CLL [113] and [114], MCL, the activated B cell-like (ABC) DLBCL subset [115] , and FL [116] and [117], based on gene expression profile (GEP) [40] and [118] and functional analyses of BCR activation and downstream signaling, as well as the clinical efficacy of kinase inhibitors that interfere with BCR signaling in these diseases [119], [120], and [121]. The importance of BCR activation by auto-antigen and/or microbial antigens versus ligand-independent (tonic) BCR activation is controversial and varies between lymphoma subtypes and among different clones of one disease entity, such as CLL [122] . The BCR is composed of a ligand binding moiety, the antigen-specific surface membrane immunoglobulin (smIg), and Ig-α/Ig-β hetero-dimers (CD79A, CD79B), the signal transduction moiety. Antigen binding to the BCR induces clustering of BCR components at the cell membrane, along with phosphorylation of ITAMs in the cytoplasmatic tails of CD79A and CD79B by Lyn and other Src family kinases (Fyn, Blk). This, in turn, activates SYK, BTK, and PI3Ks and downstream pathways, including calcium mobilization, MAP kinases and RAS activation, activation of phospholipase Cγ, protein kinase C β (PKCβ), CARD11, and NF-κB signaling [115] and [123]. Ligand-independent (also called “tonic”) BCR signaling or ligand (antigen)-dependent BCR activation are two distinct mechanisms which exist in B cell lymphomas. Mutations in the coiled-coil (CC) domain of CARD11 in DLBCL, gastric lymphoma, and primary central nervous system lymphoma, or activating mutations of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD79B and CD79A signaling modules of the BCR, as in ABC DLBCL are examples of ligand-independent BCR pathway activation [112], [115], and [123]. Unlike in DLBCL, CLL cells do not appear to contain activating BCR pathway mutations [124] . BCRs from CLL patients with unmutated immunoglobulin heavy chain variable gene segments (IGHVs) are polyreactive and can recognize various auto-antigens and other environmental or microbial antigens [125], [126], [127], and [128], such as cytoskeletal non-muscle myosin heavy chain IIA, vimentin, the Fc-tail of IgG (“rheumatoid factors”), ssDNA, or dsDNA, LPS, apoptotic cells, insulin and oxidized LDH [125], [127], [128], [129], [130], [131], and [132]. In addition, microbial antigens, such as bacterial and fungal antigens are recognized. A subset of M-CLL patients with mutated IGHVs expressing IGHV3-7 with short third complementary determining region of the IG heavy chain variable domain (HCDR3) sequences (designated “V3-7Sh”) has high-affinity binding to a major antigenic determinant of yeasts and filamentous fungi, β-(1,6)-glucan [133] , promoting the proliferation of V3-7Sh CLL cells. These findings indicate that antigen selection and affinity maturation play a role in clonal selection and expansion in CLL, similar to the role of H. pylori in MALT lymphoma pathogenesis. In addition, a recent study demonstrated binding of the HCDR3 to an epitope in the second framework region (FR2), inducing Ca2+ signaling as an additional form of auto-reactive BCR activation in CLL [134] . Along the same lines, Binder et al. reported an alternative epitope for BCR self-recognition in CLL, located in the framework region 3 of the variable region of Igs [135] .

9. TNF family members BAFF and APRIL

The B-cell-activating factor of the TNF family (BAFF, also known as BLyS) and a proliferation-inducing ligand (APRIL) are critical molecules for survival, proliferation, and differentiation of B cells and plasma cells. BAFF activates B cells by binding to the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R) receptors, whereas APRIL binds to TACI and BCMA, but not BAFF-R. CLL cells and B cells from other B cell lymphomas express the BAFF and APRIL receptors, and their activation has been linked to lymphoma cell survival and growth. Sites of B cell activation via these pathways appear to be the secondary lymphatic tissues, where NLC or LAM secretes BAFF and APRIL [35] .

10. Targeting of the microenvironment: BCR-associated kinases BTK, PI3Kδ, and SYK

10.1. Bruton's tyrosine kinase, BTK

BTK is a non-receptor tyrosine kinase of the Tec kinase family, and plays a central role in BCR signaling. BTK is primarily expressed in hematopoietic cells, particularly in B cells, but not in T cells or plasma cells [136] . BTK-deficiency due to mutations in BTK is the genetic basis for X-linked agammaglobulinemia (XLA) [137] and [138], a primary immunodeficiency characterized by low serum immunoglobulin levels and lack of peripheral B cells. Upon BCR activation, BTK becomes activated by other tyrosine kinases, such as Lyn and SYK, resulting in activation of transcription factors necessary for B-cell proliferation and differentiation [139] . In addition to its role in BCR signaling, BTK also is involved in signaling of other receptors related to B cell migration and adhesion, such as the CXCR4 and CXCR5 chemokine receptors and adhesion molecules (integrins) [140], [141], and [142].

Ibrutinib, previously called PCI-32765, is the first in human BTK inhibitor which binds specifically and irreversibly to a cysteine residue in the BTK kinase domain and inhibits BTK phosphorylation and its enzymatic activity [143] and [144]. Ibrutinib shows encouraging clinical activity in patients with B-cell malignancies, particularly in patients with CLL [119] and [120] and MCL [145] . Ibrutinib induces CLL cell apoptosis in the presence of CLL pro-survival factors (CD40L, BAFF, IL-6, IL-4, TNF-α, fibronectin, stromal cell contact) [146] . Ibrutinib also inhibits CLL cell survival and proliferation, leukemia cell migration toward tissue homing chemokines (CXCL12, CXCL13), and it downregulates secretion of BCR-dependent chemokines (CCL3, CCL4) by the CLL cells, both in vitro and in CLL patients receiving therapy with ibrutinib [84] . Inhibition of CLL cell migration and survival can possibly explain some of the characteristic clinical activity (CLL cell redistribution) of ibrutinib. Along the same lines, de Rooij and colleagues recently reported about ibrutinib's interference with CLL cell chemotaxis and integrin-mediated CLL cell adhesion [107] , suggesting that these BCR-independent actions of ibrutinib explain the redistribution of CLL cells from the tissues into the peripheral blood, characteristically seen during the first months of treatment with ibrutinib and other BCR signaling inhibitors [119], [147], and [148].

10.2. Phosphoinositide 3′-kinase delta, PI3Kδ

PI3Ks integrate and transmit signals from surface molecules, such as the BCR [149] , chemokine receptors, and adhesion molecules, thereby regulating cellular functions, such as cell growth, survival, and migration [150] . PI3Ks are divided into 3 classes (I through III). The class I kinases contain four isoforms designated PI3Kα, β, γ, and δ. While the PI3Kα and β isoforms are ubiquitously expressed and the PI3Kγ isoform has a particular role in T-cell activation, PI3Kδ expression is largely restricted to hematopoietic cells, where it plays a critical role in B-cell homeostasis and function [151] . Mice with inactivating PI3Kδ mutations have reduced numbers of B1 and marginal zone B cells, low levels of immunoglobulins, poor responses to immunization, and defective BCR and CD40 signaling, and can develop inflammatory bowel disease [151] and [152]. In CLL cells, PI3K are constitutive activated [153] , and high-risk CLL patients with unmutated IGHV genes (U-CLL) show overexpression of PI3K by quantitative polymerase chain reaction [154] . Furthermore, growth- and survival signals from the microenvironment, such as adhesion to stromal cells [155] , CXCR4 activation [22] , and BCR activation [156] cause PI3K activation in CLL cells.

Idelalisib, previously called GS-1101 and CAL-101, is a potent and highly selective PI3Kδ inhibitor, and represents the first PI3Kδ inhibitor in clinical use [157] . GS-1101 induces apoptosis in B-cell lines and primary cells from patients with different B-cell malignancies, including CLL [158] , mantle cell lymphoma and multiple myeloma [157] and [159]. GS-1101 also inhibits constitutive and CD40-, TNF-alpha-, fibronectin-, and BCR-induced PI3K activation [157], [158], and [159]. In patients receiving therapy GS-1101 there is an initial re-distribution of CLL cells from the tissues into the blood, along with a rapid lymph node size reduction and a transient lymphocytosis during the first weeks of treatment [160] , which is not explained by inhibition of pro-survival signaling. GS-1101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis) [101] . These in vitro results are corroborated by clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels, paralleled by a surge in lymphocytosis during GS-1101 treatment [101] . Therefore, it appears that GS-1101 has several mechanism of action, directly decreasing cell survival while reducing interactions that retain CLL cells in the tissue microenvironments.

10.3. Spleen tyrosine kinase, SYK

SYK belongs to the SYK/ZAP-70 family of nonreceptor kinases, and activates signaling pathways downstream of the BCR. Syk-deficient mice have severely defective B lymphopoiesis [161] and [162], with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-B-cell receptor signaling. Moreover, in vivo studies recently demonstrated that SYK is critical for survival and maintenance of mature normal and malignant B cells [161] and [163]. Besides their role in immune responses, SYK activation also modulate cell adhesion and chemotaxis of normal cells, such as B cells [164] and [165], suggesting that SYK participates in tissue homing and retention of activated B cells. R788 (fostamatinib disodium, FosD) is the only SYK inhibitor in clinical use to date. Fostamatinib, the clinically used oral formulation, is a prodrug that rapidly converts in vivo into the bioactive form called R406 [166] and [167]. Previous studies established that R406 is a relatively selective SYK inhibitor, although R406 also displayed activity against other kinases including Flt3, Jak, and Lck [167] . After encouraging results in a Phase I/II study in patients with relapsed B cell lymphomas, particularly in patients with CLL, where the objective response rate was 55% [147] , further development of this drug focused on rheumatoid arthritis (RA) [168] . Alternative SYK-specific inhibitors are under development and have demonstrated promising pre-clinical activity in CLL models [169] . Importantly, similarities in clinical response pattern of CLL patients to treatment with a SYK-, BTK-, or PI3Kδ inhibitors (transient lymphocytosis due to re-distribution, rapid lymph node shrinkage) suggest overlapping functions of these kinases in BCR signaling, B cell migration, and homing [170] .

10.4. Targeting the CXCR4-CXCL12 axis

CXCR4 antagonists initially were developed as new drugs for treatment of HIV-1 infection (reviewed in [171] ), where CXCR4 functions as a co-receptor for HIV-1 entry into T cells. However, their use in HIV-1 was abandoned because of lack of oral bioavailability and low efficacy. CXCR4 antagonists inhibit CLL cell activation by CXCL12 on functional and signaling levels, and they reverse, at least in parts, stomal cell-mediated drug resistance [81] . Several classes of CXCR4 antagonists are in clinical development, such as small modified peptide CXCR4 antagonists (BKT140), small molecule CXCR4 antagonists (AMD3100, now called plerixafor), antibodies to CXCR4 (MDX-1338/BMS 93656), and the CXCL12 antagonist NOX-A12. Plerixafor, a bicyclam, is a specific small molecule antagonist of CXCL12, inhibiting CXCL12-mediated calcium mobilization, chemotaxis, and GTP-binding, and it does not cross-react with other chemokine receptors [172] . Plerixafor causes the mobilization of various hematopoietic cells, including CD34-positive HSC, to the blood [173] and [174] and was approved by the FDA for administration together with G-CSF for mobilization of HSC in lymphoma and multiple myeloma patients. BKT140 is a high affinity inverse CXCR4 agonist, which finished phase I/II testing in multiple myeloma patients that undergo stem cell mobilization.

The preclinical data of plerixafor and BTK140 were the basis for a recent clinical trial in relapsed CLL patients, in which patients were treated with rituximab in combination with plerixafor. The goal of this proof-of-principle trial was to determine whether leukemia cells can be mobilized from the tissues, using a CXCR4 antagonist, and then targeted outside of the protective tissues. Data from this trial demonstrated a plerixafor dose-dependent mobilization of CLL cells from the tissues into the blood [175] .

10.5. CAR T cells

Although a large number of tumor associated antigens have been identified in CLL, it is difficult to generate autologous tumor antigen specific T cells in CLL. The use of the single chain variable fragment from an antibody molecule fused with an internal signaling domain such as CD3ζ, to form a chimeric antigen receptor (CAR) [176] , is able to overcome this difficulty. A number of phase I/II clinical trials are underway using anti-CD19 CAR T-cells for the treatment of B-cell malignancies, and impressive results have been observed in CLL [177], [178], [179], [180], and [181]. The addition of co-stimulatory domains, such as CD28, significantly improves the efficacy of CAR T-cells. Human anti-CD19 CARs T-cells containing the co-stimulatory domain CD137 (4-1BB) appear to be significantly more effective, show longer survival than cells expressing CARs containing the CD28 signaling domain, and were less likely to trigger induction of a “cytokine storm” and differentiation of regulatory T-cells [178] , and the CAR T cells express molecules associated with a “central memory” phenotype, important in maintaining robust and persistent anti-tumor immune responses. A correlative study that accompanied a clinical report of CAR T-cells in CLL, noted high expression of CD45RA, PD-1 and CD57 at day 169 post-infusion, which may reflect the emergence of T-cell exhaustion and incipient loss of function [179] . The potential of targeting of alterative tumor antigens, in particular those not expressed in normal tissues such as the onco-fetal antigen ROR1 [182] , other co-stimulatory approaches, and the engineering of other cell types with CARs, such as NK and NKT cells, means this remains an extremely exciting area of research.

10.6. Immune checkpoint blockade

Suppression of anti-tumor immunity by inhibitory pathways such as PDL1:PD1 appears to be an important mechanism underlying the failure of immune responses in CLL [183] , and blocking PD1:PDL1 interactions could restore T-cell effector responses. There is considerable interest in the potential for blockade of these “immune checkpoints” to enhance anti-viral and anti-tumor immunity [184] . Despite successes in solid tumors, clinical trials of these agents in hematological malignancies have been notably absent, despite the fact that these cancers are generally more “immunosensitive”. In a phase I study of an anti-PD1 antibody (CT-011; Pidilizumab) two of the three CLL patients that were enrolled showed evidence of stable disease [185] . Given the pre-clinical data highlighting the significance of the PD1:PDL1 axis in suppressing T-cell function in CLL, there is a strong rationale for clinical assessment of immune checkpoint blockade in this disease.

10.7. CD40 ligand gene therapy

CLL patients’ T-cells have reduced expression of CD154 (CD40L). CLL B cells have reduced expression of CD80 and CD86, and are functionally poor at antigen presentation. CD40 ligation of CLL cells upregulates expression of co-stimulatory molecules, and significantly improves their antigen presenting function [186] . Increased expression of CD40L in the CLL microenvironment can activate other B cells, even if they were not transfected. Clinical trials investigating the effects of infusions of autologous tumor cells transduced ex vivo with CD40L showed that this treatment was well tolerated even with intranodal injection and that patients responded [187] . Responses were also observed in patients with deletion of chromosome 17p [188] .

10.8. Lenalidomide

Lenalidomide is being evaluated in clinical trials in CLL where is shows clinical activity alone [189] and [190], in combination with rituximab [191] and as consolidation after immuno-chemotherapy [192] . Its mechanism of action in CLL appears primarily by enhancing anti-tumor immunity [193] . Lenalidomide treatment of both autologous T-cells and CLL cells results in repair of T cell defects suggesting that this may be a key component of this agent's activity in CLL [43] . Lenalidomide downregulates expression of T cell inhibitory molecules in CLL [44] and enhances T cell motility [45] . Enhancing immune cell function with immunomodulatory agents may therefore be useful to enhance T cell mediated responses such as vaccines or adoptive T cell transfer.

11. Conclusions and perspective

The microenvironment in CLL and other B cell malignancies has become an area of intensive basic and translational research. Critical pathways of cross talk between the B cells and the microenvironment have been identified and some of them already are targeted therapeutically (BCR signaling, CXCR4-CXCL12 axis). Inhibitors of BCR-associated kinases BTK (ibrutinib) and PI3Kδ (idelalisib) are the most advanced therapeutics for targeting the microenvironment in B cell malignancies, where these agents currently are tested in Phase 3 clinical trials in patients with CLL and MCL. These kinase inhibitors have unique, targeted mechanisms of action, promising clinical activity, and appear to lack major side effects, especially myelosuppression. They display unique clinical activities that are in parts explained by lymphoma growth inhibition due to interference with BCR signaling, and B cell redistribution due to interference with B cell homing mechanism. However, the exact mechanism of action, the potential benefit of combinations with conventional agents, and the durability of responses and potential resistance mechanism to these novel agents have not yet been defined. Despite these limitations, it is apparent that the microenvironment has become a rich source of insight into disease biology and for therapy. The microenvironment also will continue to shape our view of B cell malignancies as diseases of dialog between B cells and their tissue neighbors.

Conflict of interest

J.A.B. has received research funding from Gilead, Pharmacyclics, and Genzyme. J.G.G has received research funding from Celgene and honoraria from Roche, Pharmacyclics, and Celgene.

Acknowledgments

This manuscript was supported by a CLL Global Research Foundation grant, a Leukemia & Lymphoma Society Scholar Award in Clinical Research (both to JAB), a Cancer Prevention and Research Institute of Texas (CPRIT) grant (to JAB), and by funding from the NIH to the CLL Research Consortium (JAB and JGG).

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Footnotes

a Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

b Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London, UK

lowast Corresponding author at: Department of Leukemia, Unit 428, The University of Texas MD Anderson Cancer Center, PO Box 301402, Houston, TX 77230-1402, USA. Tel.: +1 713 563 1487; fax: +1 713 794 4297.

lowastlowast Corresponding author at: Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. Tel.: +44 020 7882 3804; fax: +44 020 7882 3891.