B cell receptor signaling in chronic lymphocytic leukemia

The lymphatic tissue microenvironment stages BCR activation in CLL

CLL cells proliferate in distinct microanatomical tissue sites called “proliferation centers” or “pseudofollicles”, a hallmark finding in CLL histopathology[1]. In these areas, CLL cells are in intimate contact with accessory cells, such as monocyte-derived nurselike cells (NLC)[2, 3], T cells[4], and mesenchymal stromal cells[5], which, together with matrix factors, constitute the CLL microenvironment[6]. Proliferation of CLL cells in tissues accounts for daily birth rate of approximately 0.1 to 2% of the entire clone, as demonstrated by deuterated “heavy” water (²H₂O) labeling in patients[7]. The lymphatic tissues are the apparent principal site of BCR activation for normal and malignant B cells. BCR activation can be induced by antigen or can be ligand-independent (“tonic” BCR signaling), and triggers a cascade of signaling events that normally cause B cell selection, proliferation, differentiation, and antibody production[8]. Thereby, BCR signaling allows for the expansion of selected, foreign antigen-specific B cells, and deletion of unwanted, self-reactive B cells[9]. In B cell malignancies, such as CLL[10–13] and diffuse large cell B cell lymphoma (DLBCL)[14], BCR signaling plays a critical role in pathogenesis, even though the mechanisms of BCR stimulation are heterogeneous and to some degree controversial[15–19]. Antigen-dependent and -independent BCR activation are two fundamentally different mechanism of BCR pathway activation which exist in B cell lymphomas. For example, there can be activating mutations in the BCR pathways, such as 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[14, 16]. In CLL, on the other hand, BCR pathway activation does not appear to involve activating pathway mutation and therefore can be viewed as antigen-dependent, resulting from BCR ligation via antigens (auto-antigens and/or microbial antigens) that are present in the microenvironment. Recent studies raise an additional target of BCR recognition, i.e., the binding of an intrinsic IGHV motif[15, 20]. There are several sets of indirect data suggesting the importance of BCR signaling in CLL; the most compelling are comparative gene expression profiling (GEP) data that revealed BCR signaling as the most prominent pathway activated in CLL cells isolated from areas of proliferation within the lymphatic tissues[21]. In this review we discuss mechanisms of BCR activation in CLL and the potential for targeting BCR signaling with novel inhibitors of BCR-associated kinases BTK, SYK, and PI3Kδ.

BCR activation in the CLL: mechanism and relationship with prognostic markers

The BCR is composed of a ligand binding moiety, the antigen-specific surface membrane immunoglobulin (smIg), paired with the signal transduction moiety, Ig-α/Ig-β heterodimers (CD79A, CD79B, see Figure 1). Engagement of the BCR by antigen induces membrane movement and aggregation of BCR components that lead to phosphorylation of ITAMs in the cytoplasmic tails of CD79A and CD79B. The latter is accomplished by Lyn and other Src family kinases (Fyn, Blk), which in turn activate SYK, BTK, and PI3Ks (Figure 1). BCR oligomerization and BCR microcluster growth[22] activate downstream pathways, including calcium mobilization, MAP kinases and RAS activation, activation of phospholipase Cγ, protein kinase C β (PKCβ), and CARD11, causing recruitment of BCL10 and MALT1 into a multiprotein “CBM” complex that activates IκB kinase (IKK), thereby initiating NF-kB signaling[14, 16]. Collectively, these signaling events promote B cell survival and proliferation [16].

Several prognostic markers in CLL, such as IgV_H mutational status[23, 24], ZAP-70[25, 26] and CCL3[27] are associated with (auto)antigen binding and function of the BCR, suggesting a relationship between enhanced BCR signaling and worse prognosis. Also, BCRs in CLL patients are characterized by a biased usage of IGHV and IGLVκ/λ genes, which differ from those of normal B cells. Oftentimes, specific IGHVs partner with specific IGHD-Js and specific IGLVs with specific IGLJs, leading to remarkably similar, “stereotyped” HCDR3s and smIgs[28, 29]. These data support the concept of antigen-driven selection and expansion of CLL clones, and they suggest that recurrent binding of restricted sets of antigenic epitopes are linked to the selection of those normal B-cells clones that enter the CLL pathogenetic process[11, 30–32]. More evidence for the importance of BCR signaling in CLL comes from comparative GEP data that revealed BCR signaling as the most prominent pathway activated in CLL cells isolated from lymphatic tissues[21]. Along the same lines, cells from those patients with the worse outcome (e.g., U-CLL) display GEPs identifying activation of genes downstream of the BCR[33].

BCR signaling in CLL

Normal B lymphocytes depend on the presence of an intact BCR signal transduction pathway for survival[75], and this is likely due to a combination of “tonic” (antigen-independent) and antigen-induced BCR signaling[75, 76]. These same two types of BCR signaling impact on the survival and growth on malignant B-cell clones in CLL and in other non-Hodgkins lymphomas (NHLs), although the relative contributions of these types of signals in these two classes of B-cell lymphoproliferative disorders is debatable. Two findings favor tonic stimulation being dominant in CLL: [1] unmanipulated primary CLL B cells display phosphorylated Lyn[77], Syk[78], ERK[79] and subunits of NFkB[80]; and [2] changes in IgM glycosylation after surrogate antigen engagement, which retards endocytosis, extends surface membrane display, and might allow interactions with lectins in/on the stroma [47]. However it remains unclear if each of these is a primary event or secondary to ongoing BCR engagement by autoantigens present in the microenvironment or on the cells themselves. Finally, an experimental model using transgenic over-expression of MYC leads to a CLL-like disease that appears independent of antigen[81]. Although this model generates a disease of CD5⁻ murine B cells that originate in the spleen and subsequently populate lymph nodes and bone marrow, it and related models[81] emphasize the cooperation of tonic and antigen-induced BCR signals and gene overexpression (MYC) in the development of CLL versus NHL. In opposition to tonic signaling being dominant in CLL is the absence[82] or rare presence[83] of mutations in BCR signaling components, which may enhance tonic or antigen-induced BCR-mediated signals, as have been identified in DLBCL[14]. Furthermore, the strong bias in IGHV gene use[34] and the association of certain discrete IGHVs with specific IGHD and IGHJ segments[28, 29] (“stereotyped BCRs”) in CLL mediate against a global enhancement in BCR signaling that is independent of antigen-BCR engagement and selection.

Regarding the latter, polyreactive BCRs from U-CLL patients can recognize various autoantigens and other environmental or microbial antigens[37–40, 42–47]. For example, CLL BCRs can bind cytoskeletal non-muscle myosin heavy chain IIA and vimentin, as well as the Fc-tail of IgG, ssDNA, or dsDNA, LPS, apoptotic cells, insulin and oxidized LDH. In addition, microbial antigens, such as on bacteria and fungi, can be targeted. For example, Hoogeboom et al. recently described a small subset of M-CLL patients expressing IGHV3–07 with short third complementary determining regions of the IG heavy chain variable domain (HCDR3) sequences (designated “V3–7Sh”) with high-affinity for a major antigenic determinant of yeasts and filamentous fungi, β-(1,6)-glucan[41]. Interestingly, β-(1,6)-glucan also promotes the proliferation of V3–7Sh CLL cells, suggesting that BCR stereotypy in CLL results from antigen selection and affinity maturation and that ubiquitous antigens, such as β-(1,6)-glucan and auto antigens, could promote the expansion of certain CLL clones via antigen/pathogen-specific BCR signaling. Thus, (auto)antigen recognition, binding, and subsequent signaling through smIg likely leads to B-cell survival and proliferation in human CLL.

In addition, a recent study demonstrated an interesting and unexpected form of auto-reactive BCR activation in CLL[15]. When CLL BCRs were expressed by retroviral gene transfer in mouse cells lacking endogenous components of the BCR, binding of the HCDR3 to an epitope in the second framework region (FR2) led to induction of Ca⁺⁺ signaling (Figure 2A). This in vitro finding could relate also to the presence of phosphorylated Lyn and Syk and subunits of NFkB seen in CLL cells[77, 80, 84], although it does not appear to account for clinical differences between M-CLL and U-CLL, as the same process occurred with BCRs of both CLL subsets[15], or for the lack of CLL cell proliferation in the absence of external BCR stimulation[41]. Along the same lines, Binder et al. reported an alternative epitope for CLL BCR self-recognition located in FR3 of Igs[20]. Thus, it appears that recognition of structural Ig determinants may play a key role in BCR signaling in CLL. This feature appears to be characteristic of Igs made by CLL cells and not by tumor cells from other B-cell lymphoproliferative disorders[15], and is consistent with the ability of CLL BCRs to bind Ig components and act like “rheumatoid factors”[37–40]. This also supports the notion that the development of CLL involves the selection of normal B-cell clones with distinct BCR structures that often bind limited types/classes of autoantigenic epitopes[30].

The two types of autoantigen-mediated BCR signaling in CLL appear to have common and distinct features[17]. For example, both result from autorecognition: one binding non-Ig determinants of exo- or autoantigens that are likely multimeric in nature such as on microbes or apoptotic cells, and the other reacting with mono/oligomeric autoepitopes existing on adjacent or in the same Ig(s). The two signal-initiating events, however, probably differ as well. Reactivity with Ig autoepitopes could occur in all members of the clone on a continuous basis, because the cell carries the targets of smIg binding and consequent BCR activation with it (its smIg). This would in effect be “constitutive” signaling, but not because of potential pathway mutations as can occur in NHL but rather as the result of ongoing BCR crosslinking. In contrast, reactivity with non-Ig epitopes would likely occur most often and most effectively at sites where these autoantigens exist or are made (e.g., the tissue microenvironment where cellular apoptosis or molecular catabolism occurs). For these reasons, it is unlikely that all members of a CLL clone would be involved in this type of binding and hence stimulation; rather this would occur more readily for those clonal members residing in solid tissues and possibly more often in certain types of solid tissues (e.g., lymph nodes > bone marrow)[21]. Hence this form of CLL cell signaling would be recurrent, albeit intermittent for an individual cell within the clone. Finally, the consequences on CLL cell function of these two types of autoantigen binding might differ. For non-Ig binding events, which could be of higher affinity because of their multimeric nature and could be of longer duration if they occurred in solid tissues, this would more likely lead to both survival and growth signals. For Ig binding, which would be of a more oligo/monomeric nature and continuous, this might lead to a “tolerogenic” signal and anergy, which could nevertheless result in longer cell survival[85].

Thus, CLL cells could receive both continuous and intermittent BCR-mediated signals that would facilitate cell survival and expansion. The relative frequency that the latter occurred would likely be one of the factors determining the birth rates of individual clones, and hence their pathogenicity. The proposed sequence of (initial) antigen-driven CLL B-cell expansion, with clonal selection and evolution of exo-antigen-independent clones that can persist and subsequently expand, is an interesting hypothesis to integrate these two different models[30, 32, 35, 86, 87].

Targeting BCR signaling in CLL

For therapeutic purposes, BCR signaling could be abrogated on different levels, for instance by antigen deprivation, by interference with antigen binding, or by disruption of BCR downstream signaling. The promiscuity of U-CLL BCRs for binding to different antigens, and the ubiquitous presence of many of the BCR ligands in the CLL microenvironment complicate the first two approaches, which at this time are not being pursued. In contrast, targeting of BCR signaling has become a field of major interest and explosive drug development. The lead compounds in this arena in CLL are kinase inhibitors that target the BCR-associated kinases BTK, PI3Kδ, and SYK (Table 2). Development of therapeutic kinase inhibitors has largely focused on ATP-competitive compounds that target the ATP binding site of protein kinases[88]. However, key challenges in the development of such kinase inhibitors have been relatively poor selectivity, and binding site competition due to high concentrations of endogenous ATP substrate. Covalent, irreversible inhibitors, such as the BTK inhibitor ibrutinib, are an alternative, because they exhibit high selectivity, prolonged pharmacodynamics, and potency in overcoming endogenous ATP competition[89]. A largely restricted expression of these enzymes in B cells confers target selectivity that is geared towards CLL B cells, but it is currently unknown how normal B cells are affected by use of these novel agents. Moreover, BTK, PI3Kδ, and SYK are expressed by other hematopoietic cells, albeit generally at lower levels, and therefore non-B cell effects of these kinase inhibitors (which can be beneficial and/or result in side-effects) might occur. Finally, we need to keep in mind that the specificity of a kinase inhibitor is never absolute, and, to make things even more complex, BTK, PI3Kδ, and SYK participate in signaling involved in diverse pathways. This complexity makes data interpretation about mechanism of actions and clinical responses to these drugs difficult. For example, the BCR kinase inhibitor-associated lymphocytosis in CLL due to CLL cell redistribution appears to be a class effect of each of these novel drugs[70, 90, 91] and could be interpreted as a non-BCR “off-target” effect, given that BCR blockade is not expected to result in such dramatic interference with retention/homing mechanisms. Indeed, BTK, PI3Kδ, and SYK are components of chemokine- and integrin receptor-signaling in normal B cells and CLL cells[69, 92, 93], and hence this phenomenon actually can be considered an “on-target” non-BCR effect.

Concluding remarks

After decades of exciting, fruitful research about BCR structure and signaling and their relation to prognosis, the CLL field recently transformed due to the success of early-stage clinical trials with kinase inhibitors designed to target BCR signaling kinases (BTK-, PI3Kδ-, and SYK-inhibitors). In addition, a series of research projects have shed new light onto the mechanisms of BCR activation in CLL. These discoveries will impact CLL therapy in a broad sense, with an expected major push for these new, more targeted therapeutic agents, along with a deeper understanding about the role of BCR signaling in CLL pathophysiology. As with any major discovery, these new concepts challenge us with new questions. For example, the clinical response to this class of kinase inhibitors in CLL initially is puzzling, given that it is characterized by an early resolution of enlarged lymph nodes and organs (i.e. spleen), accompanied by lymphocytosis due to redistribution of tissue-resident CLL cells into the blood during the first few months of therapy. This unexpected finding points away from the BCR and toward functions of these same kinases in pathways outside the BCR. At the same time, and more in line with BCR signaling inhibition, these kinase inhibitors also thwart CLL cell proliferation and possibly survival, although we are only beginning to learn about the relative contribution of each of these functional aspects of BCR-associated kinases for CLL cells. Collectively, BCR activation and signaling in CLL has become one of the most exciting translational fields in biomedicine and, in a broader sense, a role model for translational cancer research. The clinical findings now need to be taken back to the bench in order to better define the critical mechanism(s) of action of this fascinating new class of drugs.