This material may be protected by Copyright law (Title 17 U.S. Code)
`
`lncernacianal Reviewrnflmmunalagy, 31 :1 19- 132, 2012
`Copyright c lnforma Heal thcare USA, Inc.
`ISSN: 0883 -0185 print/ 1563-5244 online
`DOI: 10.3109/08830185.2012.664797
`
`informa
`
`healthcare
`
`Bruton Tyrosine Kinase (BTK) and Its Role in 8-cell
`Malignancy
`
`Joseph J. Buggy, PhD and Lau rence El ias, MD
`
`Pharmacyclics, Inc., Sunnyvale, California, USA
`
`BTK is a kinase that functions downstream of multiple receptors in various hematologic cell s. This
`review focuses on BTK-dependent pathways that are likely to be involved in maintaining the ma(cid:173)
`lignant phenotype in 8-cell lymphomas and leukemias. Survival of various 8-cell malignancies
`requires BTK-dependent signa ls from the 8-cell antigen receptor. Survival is also dependent on
`malignant cells homing to and interacting with lymphoid microenvironments, and these interac(cid:173)
`tions are also BTK-dependent due its role in signaling downstream of chemokine and innate im(cid:173)
`mune receptors. The potential for therapeutic targeting of BTK is currently being tested in clinical
`settings.
`
`Keywords 8-cell receptor, kinase, leukemia, lymphoma, PCl-32765, X-linked agammaglobuline(cid:173)
`mia
`
`The discovery and naming of Bruton tyrosine kinase (BTK) derives from the 1952
`description of the rare X-linked immunodeficiency (XLA) syndrome by Ogden Bru(cid:173)
`ton, a pediatrician then in the United States avy [1]. Patients with this condition had
`frequent severe infections in childhood and were noted to have virtually complete ab(cid:173)
`sence ofB cells and circulating immunoglobulins. Despite this severe abnormality, pa(cid:173)
`tients with this disease are currently able to lead relatively normal lives due to support
`with gamma-globulin \nfusions and antibiotics. BTK, the gene mutated in tl1is condi(cid:173)
`tion and recognized as essential for normal B-cell development, was first cloned and
`characterized in 1993 [2, 3j. Despite the apparent restriction of clinical features to B(cid:173)
`cell immunity in XLA, BTK is actually expressed and functional across all tile marrow
`derived (non-T) hematopoietic lineages [4-6]. The functional tolerance of BTK defi(cid:173)
`ciency in otl1er cell types is apparently attributable to robust redundancy of signaling
`and homeostatic pathways controlling essential functions. A furtlier surprising aspect
`is that despite BTK's function in normal B cells, pharmacologic inhibition of BTK does
`not replicate the clinical picture ofXLA; severe manifestations of the genetic deficiency
`are due to the role ofBTK in early differentiation at tile stage of pro- to pre-B cells. Xid
`[7] is a Bek functionaJJy deficient murine counterpart ofXLA, which is, however, phe(cid:173)
`notypically less severe than XLA. Thi difference is thought to be due to grea ter overlap
`in function with tile kinase TEC in mice than in humans. Xid mice have roughly 50%
`of normal B cells, lack the cos+ B-1 B-cell subpopulation, and have reduced serum lg
`levels [8- 10]. B cells isolated from these mice do not proliferate in respon e to anti -IgM
`treatment, and proliferation in respon e to LPS is reduced [11] . In addition, xid mice
`are not ab le to mount a thymus-independent-type lI (Tl-II) respon e to antigens, but
`do have normal T-dependent immune responses.
`
`Accepted 4 February 2012.
`Address correspondence to Joseph J. Buggy, PhD, Pharmacyclic , Inc., 995 East Arques Ave.,
`Sunnyvale, CA 94085, USA. E-mail: jbuggy@pcyc.cm
`
`119
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`

`

`120
`
`J. J. Buggy and L. Elias
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`Fostamat/nlb T
`
`GS-1101
`(CAL-101)
`
`FIGURE 1 A simplified B cell receptor signaling pathway. Upon antigen engagement of the BCR,
`the co-receptors CD79A and CD79B are phosphorylated by the tyrosine kinases LYN and FYN, which
`recruits the kinase SYK. Multiple proteins including Pl3K8, BLNK (not shown) and BTK are recruited
`to the membrane and form a multiprotein complex sometimes referred to as a signalosome. SYK
`then phosphorylates multiple substra~es including BTK and PI3K8. BTK phosphorylates and ac(cid:173)
`tivates PLCy, which.generates diacylglycerol (DAG) and inositol triphosphate (InsP3), which are
`necessary for the activation of protein kinase C (PKC). PKC phosphorylates IkB kinase (IKK) and
`this induces NF-K 8 activation. The targets of three drugs currently in clinical development (Fosta(cid:173)
`matinib, PCi-32765, and CAL-101) are shown.
`
`BTK is a member of the TEC family of nonreceptor tyrosine kinases. Its gene
`encodes a 659 amino acid protein that contains a single kinase domain and multiple
`protein-protein interaction domains: An NH2-terminal pleckstrin homology (PH)
`domain, which. binds to phosphatidylinositols during the process of membrane
`localization, is followed by Src homology 2 (SH2), Src homology 3 (SH3), and proline(cid:173)
`rich domains that regulate binding to other cellular signaling molecules [12). The
`expression of BTK is typically cytoplasmic, but it is known to translocate to the plasma
`membrane during the process of B-cell activation [13) through interactions of its
`PH domain with phosphatidylinositol-3,4,5-triphosphate (PIP3) generated by Pl3K
`(see Figure 1) [4]. Once localized to the membrane, BTK is phosphorylated by SYK
`or LYN on Y551, and BTK in turn phosphorylates and activates phospholipase-Cy
`(PLCy )[15), leading to Ca2+ mobilization [16) and activation of key pathways, in(cid:173)
`cluding that of mitogen-activated protein kinase (MAPK) [17), as well as activation of
`the inducible transcription factor NF-KB [18]. The active form also undergoes auto(cid:173)
`phosphorylation at Y223, but this step is of uncertain functional significance. Data
`from crystal structures of BTK, both alone and complexed with various inhibitors,
`have revealed specific conformational states associated with the active and inactive
`forms of the enzyme, and these insights continue to inform the structure-based
`design ofBTK-selective inhibitors [19-21).
`
`The Role of BTK in 8-cell Function and Receptor Signaling
`
`The generation and maintenance of B lymphocytes is controlled by biochemical sig(cid:173)
`nals transmitted by the B-cell antigen receptor (BCR). The expression of a functional
`BCR is required for survival during several stages of B-cell dev~lopment, but also in
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`International Reviews of Immunology
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`BTK and Cancer 121
`
`mature, resting B cells [22, 23). Conditional deletion of the BCR in the B cells of mice
`leads to rapid cell death; this result argues that there is a "tonic" signal that maintains
`cell viability (24]. The BCR is composed of two identical heavy-chain and light-chain lg
`peptides linked by disulfide bridges. The lg component binds to antigen presented by
`antigen-presenting cells, but also transmits lower level signals in the absence of anti(cid:173)
`gen engagement (25]. Depending on the developmental stage of the B cell, this signal
`may trigger B-cell proliferation, survival, or further differentiation. The lg portion as(cid:173)
`sociates with the BCR signaling proteins CD79A (Iga) and CD79B (Igp), which are sub(cid:173)
`ject to phosphorylation on immunoreceptor tyrosine-based activation motifs (ITAMs)
`by the SRC-family kinases. Phosphorylated ITAMs have extensive scaffolding interac(cid:173)
`tions with adapter molecules and kinases, including SYK kinase, which, along with
`FYN, is capable of phosphorylating and activating BTK [26, 27]. Genetic experiments
`in mice have demonstrated that BTK is limiting for the transmission of mitogenic sig(cid:173)
`nals from the BCR [ 10]. LYN is also active in this pathway but may have an inhibitory
`rather than stimulatory effect on signaling (28, 29].
`The BCR activates a variety of different signaling pathways in parallel [18]. The spe(cid:173)
`cific signaling pathway responsible for maintaining normal B-cell survival appears to
`require Pl3K [30], with downstream signaling by BTK being critical to regulation of
`apoptosis by NF-KB [31]. Direct BTK coupling of BCR signaling to NF-KB is evident
`from the lack of its activation by BCR in the xidmodel [32]. Activation of NF-KB alone
`does not, however, rescue the xid phenotype, suggesting that NF-KB is not the sole sur(cid:173)
`vival signal in normal B cells. BTK activates NF-KB by phosphorylating PKC,8, which
`in turn phosphorylates IKK [33], resulting in its dissociation from the NF-KB complex,
`allowing it to undergo nuclear translocation and function as a transcription factor. Re(cid:173)
`cent evidence suggests that BCR signaling may be amplified by a positive feedback
`loop involving the B-activating factor (BAFF) receptor BR3 (34]. This pathway can be
`activated by genes under control of the c-rel/p65 subunit of NF-KB, which is capable
`of augmenting BTK transcription [35).
`While BTK's role in signal transduction is best known as a key signaling molecule
`for the BCR, it is also functional in other receptor pathways (Table 1), including im(cid:173)
`munoglobulin Fe receptor signaling [36-38], and the G-protein coupled chemokine re(cid:173)
`_ceptors CXCR4 and CXCRS, essential for B-cell trafficking and tissue homing [39-41].
`Lymphocyte homing to and adhesion within peripheral lymph nodes and secondary
`lymphoid tissues are regulated by a complex molecular signaling pathway whereby
`chemokine binding to their receptors leads to the "inside-out" activation of cell sur(cid:173)
`face integrin proteins [ 42]. BTK activation is thought to occur following a direct interac(cid:173)
`tion with the chemokine receptor G protein subunits [43, 44], with BTK then mediating
`chemokine-controlled migration through PLCy2. The BCR itself has bee·n shown to
`regulate integrin a4,81 (VLA-4)-mediated adhesion to cellular substrates such as vas(cid:173)
`cular cell adhesion molecule-I (VCAM-1) and to fibronectin, and genetic knockdown
`of BTK inhibited this integrin function [45]. BTK has been shown to interact with sev(cid:173)
`eral members of the Toll-like receptor (TLR) family, including TLR8 and TLR9 [46-48].
`Stimulation of B cells with the TLR9 agonist CpG activates BTK and results in cellu(cid:173)
`lar proliferation, cytokine production, and activation of NF-KB; each of these effects
`are blocked in BTK-negative B cells [49, 50]. Most B-cell malignancies respond to CpG
`oligonucelotides by increasing proliferation and the expression of co-stimulatory and
`antigen-presenting molecules (51, 52].
`While the canonical BCR activation pathway has been most widely investigated, it
`should be noted that a large number of other signaling and scaffolding interactions of
`BTK have been reported but less widely studied, including the Wnt/ ,8-catenin [53] and
`JNK/SAPK pathways [54], the IL-6 signaling molecule gpl60 [55], and the transcription
`factor Bright [56]. Thus, the complexity ofBTK functioning is far from fully elucidated.
`
`

`

`(47], (48], (49], (50], (130]
`
`Cytokine secretion, immune
`
`activation
`
`B cells, macrophages, monocytes,
`
`Lipopolysaccharide, single stranded
`
`dendritic cells
`
`RNA, microbial nucleic acids
`
`(62], (63]
`
`Osteoclastogenesis
`
`Osteoclasts
`
`RANK ligand (RANKL)
`
`Collagen
`
`Platelets
`
`Von Willebrand Factor (VWF),
`
`Toll-like receptors 4, 8, 9
`
`(TLR4, 8, 9)
`
`Receptor activator of nuclear
`
`factor-KB (RANK)
`
`Glycoprote~n lb, VI
`
`(72], (73], (74]
`
`(66], (67], (68], (69], (103]
`
`(38], (128], (129]
`(40]
`(34]
`
`(22]
`
`Aggregation, agglutination
`
`secretion
`
`Degranulation, cytokine
`
`secretion
`
`Immune activation, cytokine
`Migration and homing
`B cell survival, self tolerance
`
`differentiation
`Proliferation and
`
`Mast cells, basophils
`
`neutrophils, dendritic cells
`
`B cells, monocytes, macrophages,
`B cells
`B cells
`
`IgE
`
`FcF.RI
`
`Immune complex
`SDF-1 (CXCL12), CXCL13
`B cell activating factor (BAFF)
`
`FcyR
`CXCR4 and CXCRS
`BAFF receptor (BR3)
`
`B cells
`
`B cell antigen receptor (BCR) Antigens
`
`Reference
`
`Biological process
`
`Cell type(s)
`
`Ligand
`
`Receptor
`
`TABLE 1 Cell surface receptors that signal through Btk
`
`IJ
`t,,J
`..l
`
`

`

`BTK and Cancer 123
`
`BTK in Non-lymphoid Lineages
`
`While the defect in B-cell development is the most salient feature of xid/XLA, ab(cid:173)
`normalities in other lineages are detectable. These appear to be of limited in vivo
`functional significance, presumably related to physiologic redundancies, including
`utilization of TEC as an alternative signaling molecule within specific cells as well
`as overlapping cellular functions. BTK has thus been demonstrated to participate
`in the regulation of multiple inflammatory effector functions, including nitric oxide
`induction, cytokine secretion, and bactericidal functions in macrophages [57] and
`neutrophilic chemotaxis, transmigration, adhesion, and maturation [58, 59]. BTK
`was shown to be activated by the hypoxia-induced mitogenic factor (HIMF) receptor,
`which stimulates bone marrow myeloid cell migration [60]. BTK was also shown
`to regulate E-selectin-mediated integrin activation and recruitment of neutrophils
`[58]. Polymorphonuclear neutrophil granulocytes (PMNs) from xid mice were shown
`to h~ve impaired recruitment to sites of inflammation [61), and treating mice with
`collagen-induced arthritis with the BTK inhibitor PCI-32765 resulted in near complete
`clearance of inflammatory infiltrates of affected joints [38).
`BTK is expressed in osteoclasts ( OC) and is important in their function and develop(cid:173)
`ment from monocytes. Although neither Xid mice nor XLA patients have bony abnor(cid:173)
`malities, monocytic QC precursor cells from Xid mice are defective in multinucleate
`osteoclast formation in response to RANKL (62). Btk/Tec knockout (KO)mice (but not
`Btk KO mice )have osteopetrotic bones associated with defective osteoclast formation
`(63). XLA patients have a similar defect in OC formation detectable in vitro, which may
`be compensated for in vivo by higher levels ofregulatory cytokines [64), thus masking
`clinical manifestations.
`In mast cells and basophils, BTK lies downstream of the high-affinity IgE recep(cid:173)
`tor (FceRI), and mediates signaling events that control the process of degranulation
`[65-68]. Btk knockout mice are known to have impaired mast cell degranulation fol(cid:173)
`lowing FceRI cross-linking (69). Mast cells may contribute to a microenvironment fa(cid:173)
`vorable for progression of a number of solid tumors and early evidence suggest that
`BTK could be a therapeutic target in such settings [70, 71]. BTK is expressed in platelets,
`and has been reported to be involved in signaling from both the GPVI collagen recep(cid:173)
`tor and GPIV von Willebrand complex receptor, both of which signal through FcRy
`and FYN, LYN, and SYK. In vitro impairment of collagen and shear stress-induced ag(cid:173)
`gregation due to deficient BTK activity have been reported, although no evidence has
`been noted of a clinical bleeding diathesis among XLA patients (72-74).
`Expression of BTK appears to be strongly restricted to hematopoietic lineages with
`the sole possible exception being reported expression by some colon tumors and cell
`lines [53). A number of studies have indicated an increased incidence of colon cancer
`in XLA patients (75, 76), but this is the only example reported of increased cancer risk
`in these patients. While evidence has been presented of a capability of BTK to act as a
`tumor suppressor [77], and an inhibitor or wnt ~-catenin signaling [53], a relationship,
`as suggested by Mohamed et al. (78], to altered intestinal flora and local inflammation
`due to deficient IgA might be a more likely basis for this clinical association.
`
`Targeting BTK in Cancer
`
`8-cell Lymphoproliferative Diseases
`There is a broad body of evidence pointing to an essential role of BCR and chemokine
`receptor signaling in the most common types of B-cell lymphoproliferative cancers,
`providing a general rationale for targeting BTK for treatment of these conditions.
`Normal B cells are under selective pressure to maintain expression of an appropriate
`
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`

`124
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`J. J. Buggy and L. Elias
`
`BCR, and this appears to also be true in malignant B cells. In B-<;:ell lymphoma, the
`emergence of BCR-negative variants is rare and was not found to be a tumor resis(cid:173)
`tance strategy even in patients continually treated with anti-idiotypic antibodies (79).
`Reciprocal chromosomal translocations (a hallmark of B-cell lymphoma) in which
`an oncogene is inserted into one of the lg loci typically occur on the nonproductively
`arranged lg locus. However, one would predict this event should happen equally as
`often on the expressed lg allele as the unexpressed lg allele. This presumably does
`not occur because if the translocation targeted the expressed lg allele, the resulting
`disruption of the BCR would force the malignant cell to die (80). Genetic knockdown
`of CD79A and CD79B using siRNA in lymphoma cell lines can lead to apoptosis,
`arguing that this signaling pathway is important, at least in certain cell lines (81).
`Cells from follicular lymphoma patients were shown to have enhanced BCR signaling
`compared to normal B cells [ 82].
`The mechanisms that control the homing and migration of normal B cells and
`myeloid cells are likely to be conserved in the malignant state and to control tumor cell
`homing, migration, and dissemination (83, 84). Indeed, the SDF-1 (CXCL12)/CXCR4
`axis has been shown to be important in solid tumor progression, angiogenesis, metas(cid:173)
`tasis, and survival and is a target for anti-cancer therapeutics [85). Both CXCR4 and
`CXCRS are widely expressed in different types of B-cell lymphoma and have been
`shown to mediate migration and homing [86-88). In CLL, BCR stimulation was shown
`to upregulate the expression of cell surface adhesion molecules and to increase CLL
`migration toward CXCL12 and CXCL13, and this migration was blocked by inhibit(cid:173)
`ing SYK kinase, a kinase directly upstream of BTK, with fostamatinib (89). Following
`BCR stimulation, CLL cells secrete cytokines and chemokines, including the T-cell
`chemokines CCL3 and CCU (90). The SDF-l(CXCL12)/CXCR4 interaction was also
`shown to be essential in the recruitment and retention of multiple myeloma cells in the
`bone marrow (91] and is also conserved in mantle cell lymphoma, where CXCL12 was
`shown to mediate chemotaxis [ 92]. A subgroup of patients with D LBCL has been iden(cid:173)
`tified where the tumor cells express a gene signature termed stromal-2, which suggests
`an association of the tumor with stromal cells (93). One component of the stromal-2
`signature is high levels of SDF-1, suggesting that SDF-1 may be an angiogenic factor
`and the CXCR4-BTK pathway could be a specific drug target in this population (94).
`
`BTK Inhibitors
`Inhibitors of BTK have become an area of substantial clinical interest. LFM-Al3 was
`described some years ago as a BTK inhibitor with an IC50 of 17 µM. in vitro (95),
`and has been widely used in preclinical studies as a probe for BTK involvement in
`various cellular functions. This compound, however, also inhibits PLK and JAK2 [96,
`97]. Dasatinib (Sprycel) is a multikinase BCR/ABL and Src family tyrosine kinase
`inhibitor that is marketed for the treatment of Philadelphia chromosome-positive
`(Ph+) CML and ALL and is actually quite potent as a BTK inhibitor, albeit not
`highly selective [98). PCl-32765 (ibrutinib) is a small molecule first-in-human, orally
`bioavailable, selective BTK inhibitor that is currently in phase 2 clinical development
`in patients with surface Jg+ B-cell malignancies. PCI-32765 is an irreversible inhibitor
`that binds covalently to a cysteine residue (Cys-481) near the BTK active site and
`inhibits the enzymatic activity of purified Btk with an IC50 of 0.5 nM [99, 100]. A
`fluorescently tagged derivative of PCI-32765 was shown to bind predominantly to
`a single band, corresponding to BTK, in B-cell lysates, suggesting a high degree
`of specificity within B cells [99). Few other kinases with significant affinity for the
`inhibitor also have homologous cysteines that can be irreversibly inhibited by
`PCI-32765 at similarly low concentrations. Covalent binding of PCI-32765 along with
`its short pharmacologic half-life (T1 °-ah= 1.5-2.7 h) (101) thus confers a high degree
`
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`BTK and Cancer 125
`
`of pharmacologic selectivity when the drug is administered once daily in vivo or for
`brief in vitro exposures. Another BTK-targeting irreversible inhibitor, AVL-292, also
`binds covalently to Cys-481 and is in phase lb clinical trials for B-cell malignancies
`[102). Like PCl-32765, AVL-292 was reported to result in Btk inhibition that persisted
`for 24 h despite levels of AVL-292 in plasma declining substantially by 8 h, consistent
`with an irreversible mechanism of action.
`The cellular selectivity of PCI-32765 was demonstrated by the observation that PCI-
`32765 inhibits antigen receptor signaling in B cells but not in T cells. In ex vivo stim(cid:173)
`ulation assays, PCI-32765 inhibited human BCR activation (IC50 < 10 nM) in B cells,
`but did not affect T-cell receptor activation [99). Consistent with the functional role of
`BTK in mast cells and basophils, it was determined that PCI-32765 fully inhibits de(cid:173)
`granulation following stimulation at the high-affinity IgE receptor [103]. In monocytes
`and macrophages, PCl-32765 inhibits the secretion of proinflammatory cytokines fol(cid:173)
`lowing stimulation at the FcyR by immune complex [38). Direct antitumor effects of
`PCI-32765 in vitro are readily demonstrable in a number of cell lines that express BTK,
`including the DLBCL lines OCI-LylO and TMD8 [104) and the MCL lines JVM-2 and
`MINO [105). Encouraging levels of clinical efficacy of PCI-32765 have been reported
`in CLL, DLBCL, MCL, Waldenstrom macroglobulinemia, and follicular and marginal
`zone lymphomas that were studied in the phase I trial [101), implying that BTK is func(cid:173)
`tionally relevant to a wide range oflymphoproliferative disorders. Interestingly, B-cell
`depletion has not been noted with patients treated with PCI-32765. The failure ofBTK
`inhibition in patients to replicate the XLA phenotype is surprising but it lends insight
`into normal BTK functioning, whereby the early developmental role at the pro-/pre(cid:173)
`B-cell stage is dissociable from the activating role in mature B cells. BTK functions pri(cid:173)
`marily as a scaffolding protein in early B development, while its kinase activity is most
`critical for mature B-cell activation [106].
`
`Chronic Lymphocytic Leukemia (Cll)
`CLL is a disease that can be subclassified into two broad subtypes, depending on the
`mutated (M) or unmutated (UM) state of the IgVH chain, with these states correspond(cid:173)
`ing to normal steps in the B-cell response to antigenic stimulation. The UM subgroup
`is more aggressive clinically and UM-CLL cells characteristically exhibit high BCR re(cid:173)
`activity. ZAP-70 (zeta-chain associated protein kinase-70), which is normally associ(cid:173)
`ated with the T-cell receptor [107) but is ectopically expressed in clinically aggressive
`CLL, itself augments signaling downstream of the BCR [108, 109]. In M CLL, the spe(cid:173)
`cific IgVi-i hypermutated sequences tend to occur in nonrandom and stereotypical pat(cid:173)
`terns among different patients, suggesting a functional role [110]. These observations
`support the view that BCR signaling is an essential patho-physiologic feature of CLL,
`which may be driven by engagement with microenvironmental antigens, or possibly
`in some instances by constitutive activation resulting from self-aggregation [111).
`An objective response rate of 70% to PCI-32765 ( 420 mg/day) has recently been re(cid:173)
`ported from a phase lb/II trial in CLL patients [112]. Clinical responses to PCI-32765
`are characterized by a rapid resolution oflymphadenopathy and/ or organomegaly, ac(cid:173)
`companied by a transient surge in peripheral blood lymphocyte counts, evidently due
`to mobilization of tissue-resident CLL cells into the blood. Thus, an additional 19% of
`patients attained the criteria for objective response based on lymph node measure(cid:173)
`ments but not peripheral blood criteria. Such mobilization has also been observed in
`other types oflymphoma studied, especially MCL [113). This observation points to the
`importance of microenvironmental interactions in the mechanism of action of BTK
`inhibitors, as well as other novel ag~nts that target BCR signaling. Models utilizing
`in vitro co-culture with cells or factors that are capable of providing microenviron(cid:173)
`mental signals, or in vivo models, may therefore be generally more informative in fully
`
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`

`126
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`J. J. Buggy and L. Elias
`
`explicating PCl-32765 anti-tumor effects. Thus, Herman et al. [114] recently reported
`CLL cellular inhibition of BTK by PCI-32765, resulting in CLL cell apoptosis and inhi(cid:173)
`bition of leukemia cell survival, in vitro in the presence of various BTK-activating ex(cid:173)
`ogenous stimuli (CD40L, BAFF, IL-6, IL-4, TNF-a, fibronectin, stromal cell contact).
`Consistent with a role for BTK downstream of TLR9, CpG oligonucleotide-induced
`proliferation of CLL cells was completely inhibited by the BTK inhibitor PCl-32765.
`PCl-32765 also significantly inhibits CLL cell survival, DNA synthesis, and migration in
`response to tissue homing chemokines ( CXCL12, CXCL13) [ 115]. Similar inhibitory ef(cid:173)
`fects of homing chemokine responsiveness have recently been reported in MCL cells.
`[113) PCl-32765 down regulated secretion of BCR-dependent chemokines (CCL3,
`CCL4) by the CLL cells, both in vitro and in vivo. In an adoptive transfer TCLI mouse
`model of CLL, PCl-32765 affected leukemia cell homing and disease progression. In
`this model, PCI-32765 caused a transient early lymphocytosis and profoundly inhib(cid:173)
`ited CLL progression, as assessed by weight, development of hepatosplenomegaly,
`and survival. [115)
`
`Diffuse Large 8-cell Lymphoma
`The cells of the clinically aggressive, activated B-cell (ABC) subtype of diffuse large B(cid:173)
`cell lymphoma (DLBCL) typically exhibit chronic active BCR signaling [104), which
`resembles that of an antigen-stimulated normal B cell [116). BTK was shown to play
`a crucial role in the activated pathway downstream of the BCR in these cells, as ge(cid:173)
`netic knockdown of BTK-induced cell death. Many cases of chronic active signaling in
`ABC DLBCL appear to be driven by mutations or deletions in the CD79A and CD79B
`ITAMs, which were not observed in other types of lymphomas. Eighteen percent of
`ABC DLBCL cases were found to have mutations of YI 96, which is the first tyrosine in
`the ITAM. These mutations appear to enhance BCR signaling by decreasing the nega(cid:173)
`tive autoregulatory influence of LYN. Approximately 10% of cases of ABC DLBCL have
`mutations of the CARDI I signaling adaptor, which bypass BCR signaling steps lead(cid:173)
`ing to NF-KB activation. PCI-32765 at concentrations as low as 3.12 nM was found to
`decrease viability of CARD 11 wild-type ABC (but not Germinal Center B) DLBCL cells
`in vitro. Some patients with DLBCL who have been treated in the PCI-32765 phase I
`trial (which included an ABC DLBCL expansion cohort) were reported to have rapid
`clinical responses, including some complete responses (CRs) [117).
`
`Mantle Cell Lymphoma
`A major role of BTK activation in mantle cell lymphoma (MCL) is strongly supported
`by the high level of clinical activity of PCI-32765 in this disease [118); however, the ba(cid:173)
`sis for increased BCR pathway signaling has not been as extensively investigated as
`in CLL and DLBCL. BCR signaling pathway phosphoproteins, assessed by phospho(cid:173)
`proteomic analysis, have been found to be abundantly represented in MCL cell lines
`[119]. A high incidence of PIK3CA gene amplification and mRNA expression has been
`reported in MCL cells, which could contribute to overactivity of the BCR signaling
`pathway [120).
`
`Multiple Myeloma
`Although BTK is markedly downregulated in normal plasma cells, it is highly ex(cid:173)
`pressed in the malignant cells from many multiple myeloma (MM) patients and
`some cell lines. Chauhan et al. [121) showed that BTK mRNA expression was 2.3-fold
`increased in 6 MM patient specimens compared to normal human marrow precur(cid:173)
`sors. Tai et al. [122) noted increased expression in 84% of primary myeloma sam(cid:173)
`ples and among some cell lines, especially ANBL-_6 and INA-6, both of which are
`IL-6 dependent. Barn et al. [123) reported BTK expression particularly among cases
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`BTK and Cancer 127
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`with specific gene expression profile signatures. The putative myeloma stem cell or
`cancer-regenerating cell as identified by Matsui [124) was found to be enriched in
`the CD138neg subfraction of both MM patient samples and cell lines. This functionally
`defined cell was further found to have phenotypic features generally consistent with
`a memory B-cell phenotype, including BCR component and associated marker pro(cid:173)
`teins, and small cell lymphoid morphology. BTK expression has been reported to be
`increased in such cell subfractions compared to the bulk population of MM cells [125).
`
`Acute lymphoblastic leukemia
`The cellular origin of most B-lineage acute lymphoblastic leukemia (ALL) cases corre(cid:173)
`sponds to early pro- or pre-B cells. This differentiation block appears in part to be re(cid:173)
`lated to absent or deficient pre-B-cell receptor signaling, typically including decreased
`BTK activity, which may be due to either decreased expression or expression of dom(cid:173)
`inant negative BTK splice variants (126). Thus, in leukemic cells at this stage of de(cid:173)
`velopment BTK can apparently function as a tumor suppressor, with functional defi(cid:173)
`ciency contributing to differentiation block and resistance to apoptosis [107). The spe(cid:173)
`cific molecular patterns differ in the various cytogenetically defined subtypes of ALL.
`In contrast to other ALL subtypes, t(9;22) leukemia has been reported to express an ac(cid:173)
`tive splice variant ofBTK, which is directly stimulated by the fusion product BCR-ABLI
`in the absence of a pre-BCR (127).
`
`CONCLUSIONS
`
`BTK plays a crucial role in B-cell development and function, as it is an essential sig(cid:173)
`naling molecule of the B-cell receptor. In addition to B-cell survival signaling, BTK
`is important in chemotaxis and adhesion, and in related functions of cells of non-B
`hematopoietic lineages. Its roles in mast cells, osteoclasts, and various inflammatory
`cells notably suggest a broad range of disease settings in which targeting BTK could
`be applicable. Although not a classical oncogene, BTK appears to be essential to the
`maintenance of the transformed phenotype of several malignancies and is clearly an
`important new target for cancer therapy. It is likely that much remains to be learned
`about the intricacies of BTK signaling and its range of cellular functions and full po(cid:173)
`tential as a clinical therapeutic target.
`
`Declaration of Interest
`
`Joseph J. Buggy is an employee and shareholder of Pharmacyclics, Inc. Laurence Elias
`is a paid consultant and shareholder o_f Pharmacyclics, Inc.
`
`REFERENCES
`[11 Bruton, O.C. (1952) Agammaglobulinemia. Pediatrics: 9;722-8.
`(21 Vetrie D, Vorechovsky I, Sideras P, et al. The gene involved in X-linked agammaglobulinaemia is a
`member of the src family of protein-tyrosine kinases. Nature. 1993;361:226-233.
`[3} Tsukada S, Saffran DC, Rawlings DJ et al. Deficient expression or a B cell cytoplasmic tyrosine ki(cid:173)
`nase in human X-linked agammaglobullnemia. Cell 1993;72:279-290.
`[4] Genevier HC, Hinshelwood S, Gaspar HB, et al. Expression of Bruton's tyrosine kinase protein
`within the B cell lineage. Eur J lmmunol. 1994;24:3100-3105.
`[5] Smith CI, Baskin B, Humire-Greiff P, et al. Expression of Bruton's agammaglobulinemia tyrosine
`kinase gene, BTK, is selectively down-regulated in T lymphocytes and plasma cells. J lmmunol.
`1994; 152:557-565.
`[6] de Weers M, Verschuren MCM, Kraakman MEM, et al. The Bruton's tyrosine kinase gene is
`expressed throughout B cell differentiation, from early precursor B cell stages preceding im(cid:173)
`munoglobulin gene rearrangement up to mature B cell stages. Eur J. lmmunol. 1993;23:3109-3114.
`
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`
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`
`(17)
`
`[7) Conley ME, Dobbs AK, Farmer OM et al. Primary B cell immunodeficiencies: comparisons and
`contrasts. Annu. Rev. Immunol. 2009;27:199-227.
`[8) Tsukada S, Rawlings DJ, Witte ON Role of Bruton's tyrosine kinase in immunodeficiency. Curr.
`Opin. Im