Role of Bruton's tyrosine kinase in B cells and malignancies.

Background

Protein kinases represent classes of enzymes that catalyze phosphorylation of proteins and thereby alter their substrate’s activity or capacity to interact with other proteins. Kinase signaling pathways represent the most common form of reversible post-translational modifications that control many aspects of cellular function. Aberrant activation of protein kinases drive major hallmarks of malignancies, including alterations in cellular proliferation, survival, motility and metabolism, as well as angiogenesis and evasion of the anti-tumor immune response [ 1 , 2 ].

One such kinase that plays a crucial role in oncogenic signaling is Bruton’s tyrosine kinase (BTK), which is critical for the survival of leukemic cells in various B cell malignancies. BTK was initially shown to be mutated in the primary immunodeficiency X-linked agammaglobulinemia (XLA) and is essential at various stages of B lymphocyte development [ 3 , 4 ]. XLA is an inherited immunodeficiency disease originally described by the pediatrician Ogdon Bruton in 1952 and characterized by recurrent bacterial infections. Due to a severe block of B cell development in the bone marrow, XLA patients have very low numbers of B cells in the circulation and antibodies are almost completely absent in the serum. A milder phenotype of the disease is present in CBA/N mice, which harbor the loss-of-function mutation R28C BTK [ 5 , 6 ]. These mice, known as xid (X-linked immunodeficiency) mice, manifest only minor defects in B cell development in the bone marrow, but instead the differentiation and survival of mature peripheral B cells is severely impaired [ 7 – 10 ]. Importantly, BTK has received large interest since small-molecule inhibitors of this kinase have shown excellent anti-tumor activity in clinical studies [ 11 , 12 ]. In particular, the orally administered BTK inhibitor ibrutinib, which forms a covalent bond with a cysteine residue in the BTK active site, was also approved for first-line treatment of patients with chronic lymphocytic leukemia (CLL) and small lymphocytic leukemia (SLL) in 2016 [ 13 ].

Shortly after its discovery as the non-receptor tyrosine kinase defective in XLA [ 3 , 4 ], BTK was placed in the signal transduction pathway downstream of the B cell receptor (BCR). This receptor is expressed on the B cell surface and has the unique capacity to specifically recognize antigens due to hypervariable regions present in the immunoglobulin heavy (IGH) and light (IGL) chains that together form the BCR [ 14 ]. BTK is also involved in many other signaling pathways in B cells, including chemokine receptor, Toll-like receptor (TLR) and Fc receptor signaling. Expression of BTK is not restricted to B cells, as also cells of the myeloid lineage express BTK. In these cells, BTK acts also downstream of TLRs and e.g. the FcεR in mast cells [ 15 , 16 ] and the FcyRI in macrophages [ 17 , 18 ]. In addition, BTK is involved in various other pathways, including Receptor activator of nuclear factor-κB (RANK) in osteoclasts [ 19 ], collagen and CD32 signaling in platelets [ 20 ] and the NLRP3 inflammasome in macrophages and neutrophils [ 21 ]. Since myeloid cells are important components of the tumor microenvironment and particularly tumor-associated macrophages contribute to cancer progression [ 22 , 23 ], there is currently a considerable interest in BTK inhibition as an anti-cancer therapy not only in B cell leukemias but also in other hematological malignancies and solid tumors [ 24 – 27 ].

In this review, we describe the importance of BTK in multiple signaling pathways. We discuss the crucial function of BTK in different stages of normal B cell development. In addition, we discuss its role in oncogenic signaling in B cell malignancies associated with genetic events that result in increased BTK activity. We describe clinical benefits of targeting BTK with small molecule inhibitors in B cell malignancies. Finally, we discuss the effects of BTK inhibitors on tumor growth in solid malignancies in the context of the function of myeloid cells in the tumor environment.

BTK structure

BTK is one of the five members of the TEC family of non-receptor tyrosine kinases - along with tyrosine kinase expressed in hepatocellular carcinoma (TEC), interleukin-2-inducible T cell kinase (ITK), resting lymphocyte kinase (RLK) and bone marrow expressed kinase (BMX) - which are strongly conserved throughout evolution [ 28 ]. BTK, TEC and ITK are most similar and both contain five different protein interaction domains (Fig. 1a). These domains include an amino terminal pleckstrin homology (PH) domain, a proline-rich TEC homology (TH) domain, SRC homology (SH) domains SH2 and SH3, as well as kinase domain with enzymatic activity [ 28 , 29 ]. BTK is essentially cytoplasmic and is only transiently recruited to the membrane through interaction of its PH domain with phosphatidylinositol-3,4,5-triphosphate (PIP 3 ), which is generated by phosphatidylinositol-3 kinase (PI3K) (Fig. 1b ) [ 14 ]. BTK activation occurs in two steps upon its recruitment to the cell membrane. First, BTK is phosphorylated at position Y551 in the kinase domain by SYK or SRC family kinases [ 30 ]. Phosphorylation of BTK at Y551 promotes its catalytic activity and subsequently results in its autophosphorylation at position Y223 in the SH3 domain [ 31 ]. Phosphorylation at Y223 is thought to stabilize the active conformation and fully activate BTK kinase activity [ 32 ]. Nevertheless, a Y223F mutation did not significantly affect the function of BTK during B cell development in vivo, since B-cell specific transgenic expression of Y223F-BTK could still rescue the xid phenotype of Btk-deficient mice [ 33 ]. Therefore, the function of the Y223 BTK autophosphorylation site remains unclear in B cells and to date is unexplored in vivo in myeloid cells. Fig. 1 Domain structure of TEC kinase family members and key interacting partners of Bruton’s tyrosine kinase. a Schematic overview of the protein structure of BTK and other TEC kinase family members. Shown are five different domains, as explained in text, the Y223 autophosphorylation site, the Y551 phosphorylation site that activates BTK, and the C481 binding site of ibrutinib. b Schematic overview of the protein structure of key interacting partners of BTK. PH, pleckstrin homology; TH, TEC homology; BH, BTK homology; PRR, proline rich domain; SH2/SH3, SRC homology domains 2 and 3; Cys, cysteine-string motif

BTK in B cell receptor signaling

The IgM BCR is essential for survival of peripheral B cells [ 34 ]. In the absence of BTK B cells have a high rate of apoptosis, which correlates with strongly reduced BCR-mediated induction of the anti-apoptotic protein Bcl-xL [ 35 , 36 ]. Upon stimulation with anti-IgM, cell size enlargement and degradation of the cyclin inhibitor p27Kip1 occurs normally, indicating that BTK is not essential for several G1 events [ 37 ]. BTK-deficient B cells enter early G1, but not S phase of the cell cycle, because they fail to induce cyclin D2 expression [ 38 ]. Apart from B cell survival and proliferation, the BCR controls integrin α4β1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 (VCAM-1) and fibronectin via BTK [ 39 ].

BCR cross-linking activates four families of non-receptor protein tyrosine kinases and these are transducers of signaling events including phospholipase Cγ (PLCγ), mitogen-activated protein kinase (MAPK) activation, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) pathway components and activation of the serine/threonine kinase AKT (or protein kinase B, PKB).

The IgM BCR has a very short cytoplasmic domain and thus cannot signal directly, but associates with the disulphide-linked Ig-α/Ig-β(CD79a/CD79b) heterodimers. These transmembrane proteins contain immunoreceptor tyrosine based activation motifs (ITAMs) in their cytoplasmic domain (Fig. 2 ). BCR engagement by antigen induces ITAM phosphorylation by Src-family protein tyrosine kinases such as LYN, thereby creating docking sites for spleen tyrosine kinase (SYK)(Fig. 1b ) [ 40 ]. In addition, LYN and SYK also phosphorylate tyrosine residues in the cytoplasmic tail of the B-cell co-receptor CD19 and/or the adaptor protein B-cell PI3K adaptor (BCAP), which facilitates recruitment and activation of PI3K and the guanine nucleotide exchange factor VAV [ 41 , 42 ]. VAV further enhances enzymatic activity of PI3K through activation of RAC1, a member of Rho family of GTPases [ 43 ]. PI3K phosphorylates PIP2 to generate PIP3, which acts as a critical secondary messenger for activating downstream pathways. PIP3 interacts with the BTK PH-domain, resulting in its recruitment to the plasma membrane [ 44 ]. Fig. 2 Role of Bruton’s tyrosine kinase downstream of the B cell receptor. Signaling cascade showing important events downstream of B cell receptor (BCR). Antigen engagement by the BCR results in the formation of a micro-signalosome whereby BTK activates four families of non-receptor protein tyrosine kinases that transduce key signaling events including phospholipase Cγ, mitogen-activated protein kinase (MAPK) activation, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) pathway components and activation of the serine/threonine kinase AKT (PKB). In addition, BTK mediated signaling events are regulated by various phosphatases that can be recruited to the cell membrane, following crosslinking of inhibitory receptors, e.g., FcγRIIB that is exclusively expressed on B cells and signals upon immune complex binding. See text for details

In addition, Ig-α contains a conserved non-ITAM tyrosine residue, Y204, that upon activation by SYK recruits and phosphorylates the central B cell-linker molecule SH2-domain-containing leukocyte protein of 65 kDa (SLP65/BLNK) [ 45 ] (Fig. 2 ). Hereby, the adaptor molecule Cbl-interacting protein of 85 kD (CIN85) functions to oligomerize SLP65 and assembles intracellular signaling clusters for B cell activation [ 46 ]. SLP65 serves as a scaffold for various signaling molecules, including BTK and its substrate PLCγ2 [ 47 – 50 ]. In this micro-signalosome BTK is activated through Y551 phosphorylation by SYK or LYN and subsequently at Y223, as described above [ 30 – 32 ]. Fully activated BTK phosphorylates PLCγ2 at Y753 and Y759, which is important for its lipase activity [ 51 ]. Activated PLCγ2 hydrolyses PIP2 into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 regulates intracellular calcium levels and thereby activates nuclear factor of activated T cells (NFAT) transcription, via calcineurin and calmodulin. DAG mediates activation of protein kinase Cβ (PKCβ), which induces activation of several members of the MAPK family, including extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and other MAPK targets, such as Jun N-terminal kinase (JNK), p38, and NF-кB pathway components [ 52 ] (Fig. 2 ). Hereby, BTK links the BCR to NF-кB activation [ 53 , 54 ].

Another important branching point is induced more upstream in the BCR signaling cascade: in addition to BTK, PIP3 also interacts with PH-domain of AKT, resulting in its recruitment to the plasma membrane. Full activation of AKT requires phosphorylation at position T308, induced by 3-phosphoinositide-dependent protein kinase-1 (PDK1), and at S473, phosphorylated by mechanistic target of rapamycin (mTOR) complex 2 (See Ref [ 55 ] for an excellent review). Fully activated AKT then returns to the cytoplasm to enable a pro-survival signaling program that involves NFAT, forkhead transcription factors (FOXOs) and NF-кB-mediated pathways. Importantly, phosphorylation of AKT is positively regulated by BTK [ 56 ]. The BTK family member TEC, which can partly compensate for BTK [ 57 ], may on the other hand limit the capacity of BTK to activate AKT [ 58 ].

Upon activation in germinal centers (GCs), B cells can perform IGH chain class switching, by which it changes Ig expression from one isotype to another with different effector function, e.g. from IgM to IgG. In this process, the IGH constant (C) region is changed, but the variable (V) region remains the same. Interestingly, in contrast to IgM, the IgG BCR contains a cytoplasmic domain of considerable length with an Ig tail tyrosine (ITT) motif, which amplifies signaling [ 59 ]. SYK is required for ITT phosphorylation followed by recruitment of BTK through the adapter protein Grb2, leading to enhancement of IgG BCR-induced calcium mobilization. This amplification loop is thought to represent a cell-intrinsic mechanism for rapid activation of class-switched memory B cells.

Regulation of BTK activity and expression

Consistent with its crucial role in B cell differentiation, proliferation and survival, proper control of BTK activity is important for B cell homeostasis. Several mechanisms for its regulation have been identified to date.

The recruitment of BTK to the plasma membrane and its subsequent activation is regulated by various phosphatases that can be recruited to the cell membrane, similar to BTK. For example, the FcγRIIB is an inhibitory receptor that is exclusively expressed on B cells [ 60 ]. In contrast to the Igα/Ig-β ITAM motifs, FcγRIIB has immune tyrosine inhibitory motifs (ITIMs) in its cytoplasmic domain [ 61 , 62 ] (Fig. 2 ). The binding of IgG antibodies to FcγRIIB results in LYN-mediated phosphorylation of ITIMs and recruitment of protein phosphatases such as SH2-domain containing inositol polyphosphate 5’phosphatase-1 (SHIP1) [ 63 – 65 ]. SHIP1 catalyzes the dephosphorylation of PIP3 and thereby inhibits recruitment of PH-domain containing proteins, such as BTK and PLCγ2 to the cell membrane. As a result, the downstream increase in intracellular calcium levels is diminished. Another phosphatase, SH2 domain containing protein tyrosine phosphatase-1 (SHP1), has the capacity to dephosphorylate tyrosine on BTK [ 65 ]. SHP1 acts downstream of CD22, a lectin molecule, and the glycoprotein CD5, both of which are on the B cell surface and function as negative regulators of BCR signaling.

In addition, several negative regulators of BTK have been identified. The iBTK protein directly binds to the BTK PH domain and thereby inhibits its activity [ 66 ]; PKCβ phosphorylates BTK on residue S180 in TH domain, modulating its membrane localization [ 67 ]; microRNA-185 reduces BTK mRNA levels and thereby downregulates BTK expression [ 68 ]. Likewise, expression of other microRNAs, including miR-210 and miR-425, significantly reduce BTK expression [ 69 ]. In this context, it was shown that treatment of primary CLL samples with histone deacetylase (HDAC) inhibitors resulted in increased expression of these miRs and decreased BTK protein. On the other hand, BTK itself can initiate a proteasome-dependent positive autoregulatory feedback loop by stimulating transcription from its own promoter through a pathway involving NF-кB [ 70 ].

BTK in other signaling pathways

Chemokine receptors

These receptors are G-protein coupled receptors that consist of seven transmembrane spanning domains and intracellular hetero-trimeric G-proteins composed of α, β, and y subunits (Gα, Gβ, and Gy) [ 71 ]. The chemokine receptors CXCR4 and CXCR5 are expressed on B cells in different stages of their development and play important roles in trafficking, homing and homeostasis [ 72 ]. Chemokine binding to the extracellular domain of its receptor induces conformational changes that result in dissociation of Gα and Gβy subunits (Fig. 3a ). Both Gα and Gβy subunits can independently activate PI3K, which results in activation of BTK, AKT and MAPK dependent pathways [ 73 , 74 ]. In addition, both Gα and Gβy subunits can directly bind BTK via the PH and TH domain [ 74 , 75 ]. It has been shown that the Gα subunit directly stimulates the activity of BTK [ 76 ]. Due to its function downstream of chemokine receptors including CXCR4 and CXCR5, BTK is important for positioning of B cells in various lymphoid tissue compartments. This was first demonstrated by adoptive transfer experiments with BTK-deficient B cells, which exhibited impaired in vivo migration and homing to lymph nodes [ 77 ]. Fig. 3 Role of Bruton’s tyrosine kinase downstream of chemokine receptors, Toll-like receptors and activating Fcγ receptors. Signaling cascade showing important events downstream of ( a ) Chemokine receptors (e.g. CXCR4): upon chemokine binding to the extracellular domain Gα and Gβy subunits can independently activate PI3K, which results in activation of BTK, AKT and MAPK-dependent pathways. b Toll-like receptors: upon ligand recognition TLRs recruit different proteins including TIR, MYD88, IRAK1 and TIRAP/MAL, all of which interact with BTK and induce downstream activation of the transcription factor NF-κB. c Activating Fc receptors (e.g. FcγRI): Following FcγRI cross-linking, Src-kinases, SYK, PI3K-γ and BTK are activated. In contrast, inhibitory Fc-receptors (FcγRIIB) containing ITIM domains recruit phosphatases and reduce BTK activation (Fig. 2 ). See text for details

Toll-like receptors (TLRs)

These extracellular or intracellular pattern recognition receptors are characterized by leucine-rich repeats and Toll/interleukin-1 receptor (TIR) domains (Fig. 3b ). TLRs, expressed in B cells or myeloid cells, recognize structurally conserved molecules derived from bacteria and viruses. Upon activation most TLRs recruit the adaptor myeloid differentiation primary response 88 (MYD88) [ 78 ]. MYD88 activates interleukin-1 receptor-associated kinase1 (IRAK1), either on its own or in combination with an adaptor molecule, TIR domain containing adaptor protein (TIRAP, also known as MyD88 adapter-like (MAL)). BTK interacts with four different proteins downstream of TLR signaling including TIR, MYD88, IRAK1 and TIRAP/MAL) [ 79 – 81 ]. TLR signaling induces transcription factors including NF-кB, activator protein-1 (AP-1) and interferon regulatory factor 3 (IRF3), which results in activation, proliferation, antibody secretion, class switch recombination and pro-inflammatory cytokine production in B cells.

Fc receptor signaling

BTK is involved in signaling of both activating (ITAM-containing) and inhibitory (ITIM-containing) Fc-receptors, whose balance regulates several myeloid cell processes including activation, polarization and phagocytosis (Fig. 3c ) [ 60 , 82 ]. BTK is rapidly activated upon FcεRI cross-linking in mast cells [ 15 ]. In parallel to BCR signaling, following activating Fc-receptor cross-linking, SRC-kinases, SYK, PI3K-γ and BTK are activated [ 60 ]. In contrast, inhibitory Fc-receptors (FcγRIIB) containing ITIM domains recruit phosphatases and reduce BTK activation (see above).

BTK and B cell development in the bone marrow

Even before the gene involved in XLA was identified, X-chromosome inactivation studies showed that the defect in XLA patients was intrinsic to the B cell lineage and that myeloid cells had no developmental defects [ 83 , 84 ]. B cells are generated from hematopoietic stem cells in the bone marrow throughout life by the ordered rearrangement of IGH and IGL chain gene segments (Fig. 4 ). After productive recombination of the IGH V, D and J genes, the IGH μ protein is expressed on the cell surface in association with the two invariant surrogate light chain (SLC) proteins VpreB and λ5 [ 85 , 86 ],as the pre-BCR. Pre-BCR signaling marks a crucial checkpoint ( checkpoint 1 ) to test the functionality of the IGH μ protein (Fig. 4 ) [ 87 , 88 ]. To date, the mechanisms that initiate pre-BCR-mediated signaling are not fully resolved as both cell-autonomous and ligand-mediated signaling has been described [ 89 – 92 ]. An important function of pre-BCR signaling is to inhibit further IGH VDJ recombination, a phenomenon known as allelic exclusion [ 88 ]. Pre-BCR signaling leads to proliferation of pre-B cells and at the same time downregulation of SLC expression [ 88 ]. This is important for the exit of pre-B cells from the cell cycle to undergo the transition from large, cycling cells into small resting pre-B cells, in which IGL chain recombination occurs. In XLA patients B cell development is almost completely arrested at the pre-B cell stage. Although pre-B cells expressing intracellular IGH μ are present, they are small in size, indicating that BTK is essential for pre-BCR-dependent proliferation. BTK-deficient mice have only a mild pre-B cell defect, whereby pre-B cells show impaired developmental progression into immature B-cells [ 9 , 10 ]. Nevertheless, an almost complete block is only found in mice that are double-deficient for e.g. BTK and SLP65 or BTK and TEC [ 57 , 93 , 94 ]. Interestingly SLP65-deficient mice, which also have a mild arrest at the pre-B cell stage, develop pre-B cell leukemia resembling pre-B ALL in humans [ 93 , 94 ]. In this regard, BTK cooperates with SLP65 as a tumor suppressor independent of its kinase activity [ 95 , 96 ]. SLP65 also mediates downregulation of SLC expression [ 97 ]. Analyses in wild-type, BTK and SLP65 deficient pre-B cells demonstrated that pre-BCR signaling induces IGL κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions [ 98 ]. BTK and SLP65 are important for the induction of IGL chain germ-line transcripts that are associated with locus accessibility. Moreover, BTK-deficient mice exhibit a ~ 50% reduction of IGL κ chain usage [ 98 , 99 ]. Transcriptome analyses showed that BTK/SLP65deficient pre-B cells fail to efficiently upregulate many genes involved in IGL chain recombination, including Aiolos, Ikaros, Spib, Irf4, Oct2, polymerase-μ, and Mbp-1 [ 98 ]. Fig. 4 Stages of B cell differentiation and associated malignancies. Model of B cell development indicating different stages of B cell differentiation and important immune checkpoints where BTK plays a key role. Various B-cell malignancies are indicated, which are associated with abnormal BTK signaling at distinct stages of B-cell differentiation and activation. Note that the cellular origin of U-CLL is thought to be CD5 + mature B cells. Somatic hypermutation status of BCR and gene expression profiling indicates post-germinal center (GC) origin of M-CLL. See text for detailed information. CLP, common lymphoid progenitor; CSR, class switch recombination; FDC, follicular dendritic cell; SHM, somatic hypermutation

If IGL chain recombination is not productive or the resulting BCR is autoreactive ( checkpoint 2 ) (Fig. 4 ), developing B cells will undergo secondary IGL chain rearrangements, a process termed receptor editing [ 100 – 102 ]. Many autoreactive B cells are lost during development to the immature IgM + B cell stage (central B cell tolerance), but it has been estimated that ~ 40% of the newly formed B cells that leave the bone marrow have self-reactivity [ 92 ].

BTK and peripheral B cell development and activation

Immature B cells from the bone marrow migrate to the spleen, where selection and maturation is continued within the transitional B cell compartment containing T1 and T2 B cells. In mice, T1 B cells, but not T2 B cells, are very sensitive to BCR-mediated apoptosis, indicating that the T1 to T2 differentiation marks a peripheral tolerance checkpoint ( checkpoint 3 ) [ 103 , 104 ]. In the absence of BTK, T2 cells do not generate survival responses and peripheral B cells are reduced by ~ 50%. As a result, BTK-deficient B cells exhibit an impaired transition from IgM high IgD low into IgM low IgD high mature B cells. BTK-deficient mice lack the population of innate-like CD5 + B-1 cells, present in the peritoneal and pleural cavities and in small proportions in the spleen [ 7 – 9 ]. Consistent with the finding that these cells are important for IgM and IgG3 levels in the serum, in BTK-deficient mice IgM and IgG3 levels in serum are severely reduced, but the other isotypes are largely normal.

Marginal zone B cells are present in an area at the outermost portion of the white pulp in the spleen and are phenotypically defined as IgM hi IgD lo CD21 high CD23 low B cells that respond to polysaccharide antigens independently of T cell help (Fig. 4 ). BCR and NOTCH2 signaling determine whether T1 B cells expressing surface ADAM10 are committed to becoming MZ B cells in vivo in the spleen [ 105 , 106 ]. Although contradictory findings on the numbers of MZ B cells in BTK-deficient mice have been reported, it is clear that developing BTK-deficient MZ B cells have a selective disadvantage [ 107 , 108 ].

Upon antigen recognition, activated B cells may either go into an extrafollicular response or develop into GC B cells [ 109 , 110 ]. In the GCs B cells strongly proliferate and undergo somatic hypermutation (SHM) induced by activation induced cytidine deaminase (AID). GC B cells are selected involving follicular dendritic cells (FDCs) and T-follicular helper (T FH ) cells ( checkpoint 4 ) based on their antigen affinity [ 109 ]. Although BTK-deficient mice show normal T-cell dependent responses to model antigens, such as TNP-KLH [ 7 , 8 ], there is a significant reduction in GC B cell numbers in physiological models, e.g. influenza virus infection [ 108 ]. In this context, it is of note that mice expressing the constitutively active BTK mutant E41K fail to form GCs [ 111 , 112 ], whereas overexpression of wild-type BTK induces spontaneous GC formation [ 113 , 114 ]. Consequently, BTK-overexpressing mice develop autoimmunity involving B cell-induced disruption of T cell homeostasis [ 113 , 114 ].

BTK in B cell malignancies

BTK activity is crucial for survival and proliferation of leukemic B cells and for their interactions with cells in the tumor microenvironment. Below, we discuss the role of BTK in various B cell malignancies (Fig. 4 ).

CLL

This is the most common leukemia in the western world, primarily affecting the elderly, and is characterized by the accumulation of mature circulating IgM low CD5 + B cells [ 115 ]. Several genetic aberrations with prognostic value and impact on treatment decisions in CLL have been described. These include deletions of the chromosomal regions 17p13 (containing the TP53 tumor suppressor gene), 11q23 (containing DNA damage checkpoint protein ATM ), or 13q14 (miR-15a, miR-16-1), and trisomy of chromosome 12 [ 116 , 117 ]. Furthermore, > 80% of cases harboring del(17p) also carry TP53 mutations in the remaining allele [ 118 ]. Such patients with TP53 defects are classified as ‘high-risk’ and often respond poorly to therapy [ 119 ]. Moreover, a significant proportion of CLL patients carry a TP53 mutation in the absence of a 17p deletion [ 120 , 121 ].

On the basis of SHM status of IGHV, CLL can be grouped into mutated CLL (M-CLL) and unmutated CLL (U-CLL). M-CLL have a more favorable prognosis and are derived from post-GC B cells. The origin of U-CLL appeared less clear and several cellular origins of CLL were suggested, including MZ B cells, CD5 + B cells, and regulatory B cells [ 122 – 126 ]. Although initial gene expression profiling indicated that M-CLL and U-CLL were quite homogeneous and related to memory B cells derived from T cell-dependent and T-cell independent responses, respectively [ 123 ], more recent gene expression profiling studies have provided evidence for a different origin [ 124 ]. This study by Seifert et al. shows that U-CLL derives from unmutated mature CD5 + B cells. Moreover, it was concluded that M-CLL originate from a distinct and previously unrecognized post-GC B cell subset with a CD5 + CD27+ surface phenotype.

Several lines of evidence establish a role of chronic BCR-mediated signaling in CLL pathogenesis [ 127 ]. (i) Prognosis is correlated with the BCR SHM status [ 128 ]; (ii) The BCR repertoire is highly restricted [ 129 , 130 ], suggesting a role for antigenic selection in the initiation or progression of CLL. Antigens binding to CLL BCRs include self-antigens, such as non-muscle myosin IIA, vimentin, apoptotic cells and oxidized low-density lipoprotein [ 131 – 136 ], as well as foreign antigens (bacterial polysaccharides and β-(1,6)-glucan, a major antigenic determinant on fungi [ 132 – 137 ]); Interestingly, evidence was provided in mice that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with self-antigens [ 138 ]; (iii) CLL cells were reported to display cell-autonomous Ca 2+ mobilization in the absence of exogenous ligands, by virtue of recognizing a single conserved BCR-internal epitope in the IGHV second framework region [ 139 ]; very recently, it was found that the internal epitopes recognized by CLL BCRs from distinct subgroups are different [ 140 ]. Moreover, the avidity of the BCR-BCR interactions that can lead to receptor declustering influences the clinical course of the disease [ 139 , 140 ].

In line with chronic BCR-mediated signaling, CLL cells show constitutive activation of various BCR pathway associated kinases. Hereby, BTK is essential for constitutively active pathways implicated in CLL cell survival, including AKT, ERK and NF-кB, both in patient cells and mouse models [ 133 , 141 – 143 ]. CLL cells are thought to interact with the tissue microenvironment and lymph node resident CLL cells show gene expression signatures indicative of BCR activation [ 144 , 145 ]. Moreover, BTK is critical for BCR- and chemokine-controlled integrin-mediated retention and/or homing of CLL B cells in their microenvironment [ 146 ].

Mantle cell lymphoma (MCL)

This disease results from malignant transformation of B lymphocytes in the mantle zones surrounding GCs (Fig. 4 ) and has a remarkably biased BCR repertoire [ 147 ]. Approximately 85% of the patients harbor the hallmark chromosomal translocation t(11:14)(q13;32). This event juxtaposes the CCND1 gene to an enhancer in the Ig heavy chain locus [ 148 ], resulting in constitutively cyclin-D1 expression and abnormal proliferation. In a fraction of MCL patients lymphoma cells express the SOX11 transcription factor, which is associated with minimal Ig SHM, higher genetic instability and a more aggressive clinical course [ 149 , 150 ]. Primary MCL cells show strong expression and Y223-phosphoryation of BTK [ 151 ] and in a subset of patients constitutive phosphorylation of LYN, SLP65, SYK and PKCβ [ 152 , 153 ]. Similar to CLL, the tumor microenvironment plays an important role in MCL pathogenesis. BTK is essential for retention of MCL cells in lymphoid tissues, since BTK inhibition induces an egress of malignant cells into peripheral blood [ 154 ].

Waldenström’s Macroglobulinemia (WM)

This indolent B-cell malignancy is characterized by IgM-secreting lymphoma cells in the bone marrow. The majority of WM patients have a somatic leucine to proline substitution at position 265 of MyD88 (MyD88 L265P ) [ 155 ]. This activating mutation has also been reported in low frequencies in activated B-cell-like diffuse large B-cell lymphoma (14%–29%) (see below), primary central nervous system lymphoma (PCNSL; 33%), mucosa-associated lymphoid tissue (MALT) lymphoma (9%), and CLL (2.9%) [ 156 – 159 ]. The mutated MyD88 L265P protein binds phosphorylated-BTK and triggers NF-кB signaling [ 160 ]. In addition, ~ 30% of WM patients show the CXCR4 S338X somatic mutation, leading to enhanced CXCL12-triggered activation of AKT and ERK [ 161 ]. In this regard, CXCR4 and VLA-4 interactions have been shown to regulate trafficking and adhesion of WM cells to the bone marrow [ 162 ].

ABC-DLBCL

DLBCL is the most common form of B cell non-Hodgkin lymphomas (B-NHLs) representing ~ 30–40% of all cases. Patients most often present with a rapidly growing tumor in single or multiple, nodal or extranodal sites. Based on gene expression profiling, three major molecular subtypes have been identified: GC B-cell-like (GCB-DLBLCL), activated-B-cell-like (ABC-DLBCL) and primary mediastinal B-cell lymphoma (PMBL) [ 163 ]. Whereas GCB-DLBCL and ABC-DLBCL make up the majority of cases at roughly equal frequency, PMBL accounts for up to 10% of cases of DLBCL [ 164 ]. GCB-DLBCL tumors express many genes found in normal GC B cells and have typically switched to an IgG BCR, while gene expression in ABC-DLBCL, which are predominantly IgM + , resembles that of antigen-activated plasmablasts [ 165 , 166 ]. ABC-DLBCL has an inferior clinical outcome than GCB-DLBCL with a three-year overall survival of ~ 45% [ 167 ].

ABC-DLBCL are dependent on constitutive NF-кB signaling for their survival and proliferation [ 168 – 170 ]. Approximately 50% of ABC-DLBCL harbor mutations in CARD11 or other NF-кB pathway components, including the MyD88 L265P mutation [ 169 – 171 ]. In addition, ~ 20% of patients carry an activating mutation in CD79A/B. Consistent with a role of NF-кB downstream of the BCR (Fig. 2 ), it was found that knockdown of BCR components, CD79A/B and downstream signaling molecules, induced cell death in ABC-DLBCL lines with unmutated CARD11 [ 172 ]. Moreover, RNAi experiments demonstrated that ABC-DLBCL lines are dependent on MyD88 and its associated kinase IRAK1 for their survival in line with NF-kB function in the TLR pathway (Fig. 3b ). In addition, SYK amplification and deletion of PTEN, a phosphatase that dephosphorylated PIP 3 , are also selective genetic alterations identified in ABC-DLBCL [ 173 ].

In contrast to ABC-DLBCL, GCB DLBCLs do not acquire highly recurrent mutations in CD79A/B or NF-κB components. Whereas ABC-DLBCL frequently respond to BTK inhibition (see below), GC-DLBCL do not respond and exhibit tonic BCR signaling that does not affect their calcium flux, but acts primarily to activate AKT [ 174 ]. Accordingly, forced activation of AKT rescued GCB-DLBCL lines from knockout of the BCR or SYK and CD19, two mediators of tonic BCR signaling [ 174 ]. The importance of the oncogenic AKT/PI3K pathway in GCB-DLBCL is evident from the finding that in ~ 55% of patients the tumor suppressor phosphatase and tensin homolog (PTEN), a negative regulator of PI3K, is inactivated. The mechanisms of PTEN inactivation include mutation, deletion or amplification of the miR17–92 microRNA cluster that downregulates PTEN expression [ 175 , 176 ].

Primary CNS lymphoma (PCNSL), another DLBCL subtype, is an aggressive brain tumor that has a complete response rate of < 40% with methotrexate-based regimens and is subject to late recurrences. Patients showed mutations in the MYD88, CD79B and CARD11 genes in ~ 58%, ~ 41% and ~ 13% of cases, respectively [ 177 ].

Other B cell malignancies

The hallmark of follicular lymphoma (FL), the (14;18) translocation resulting in BCL2 overexpression, is found in up to ~ 85% of patients. The pathogenesis of FL is complex and involves additional cell-intrinsic genetic changes, frequently including mutations in histone-encoding genes (in ~ 40% of cases), the SWI/SNF complex or the interconnected BCR and CXCR4 chemokine receptor signaling pathways, as well as alterations within the FL microenvironment [ 178 ]. The importance of BCR and NF-κB signaling is underscored by the finding of recurrent mutations in the genes encoding C D22, SLP65/BLNK, PLCγ2, SYK, PKCβ, BCL10, the NF-κB p100 subunit and the deubiquitinating enzyme A20/ TNFAIP3, which is a negative regulator of NF-κB signaling. In addition, the HVCN1 gene (coding for a hydrogen voltage-gated proton channel that acts downstream of the BCR and is downregulated in proliferating B cells) is frequently mutated in FL. Interestingly, BTK mutations were found that suggest activation, e.g. the L528 W mutation in the kinase domain, which is associated with resistance to BTK inhibition in CLL (described below), and an in-frame deletion that also alters this amino acid and the adjacent C527. Moreover, two loss-of-function BTK mutations were identified, T117P and R562W, which are also found in XLA patients, but it remains unclear how these mutations contribute to FL pathogenesis [ 178 ].

In multiple myeloma (MM), a malignancy of plasma cells in the bone marrow, BTK was shown to be overexpressed, whereby BTK activated AKT signaling, leading to down-regulation of P27 expression and upregulation of key stemness genes [ 179 , 180 ]. MM cells originate from plasma cells, which do not express surface BCR, and rely for their survival and proliferation on signals from the microenvironment in the bone marrow. BTK may be critical in the MM microenvironment, in particular for secretion of cytokines and chemokines by osteoclasts [ 181 ].

Finally, BCR and TLR are thought to be key activation pathways in marginal zone lymphoma (MZL), often associated with chronic inflammation in the context of autoimmunity and/or infection [ 182 ], implicating BTK as a potential target. In this context, whole exome sequencing identified recurrent inactivating mutations in Kruppel-like factor 2 (KLF2) which impeded its capacity to suppress NF-κB activation. In addition, recurrent mutations in the TLR/NF-κB pathway were found, affecting e.g. the MYD88, TRAF3, CARD11, A20/TNFAIP3 and CARD11 genes [ 183 ].

The BTK inhibitor ibrutinib in clinical studies

Ibrutinib (PCI-32765) is an oral irreversible BTK inhibitor that covalently binds to cysteine at position 481 in the kinase domain and thereby blocks kinase activity [ 184 ]. As a result BTK has lost its kinase activity, but Y551 phosphorylation by SYK is not affected. The in vivo effect of ibrutinib was first confirmed in a mouse model of autoimmune disease and in dogs with spontaneous B-cell non-Hodgkin lymphoma, in which it induced objective clinical responses [ 185 ].

Efficacy of ibrutinib in a clinical study was first reported in patients with various relapsed/refractory B-cell malignancies, showing clinical safety and promising durable objective responses particularly in CLL and MCL [ 186 ]. Responding patients showed sustained reduction in lymphadenopathy, accompanied by transient rise in absolute lymphocyte count, a phenomenon known as lymphocytosis [ 186 ]. The next phase Ib/II multicenter trial, with a continuous ibrutinib regimen in relapsed/refractory CLL patients also showed lymphocytosis in the first weeks of treatment, but lymphocyte counts normalized or dropped below baseline after prolonged treatment [ 11 ]. Importantly, the overall response rate was ~ 71%, independent of clinical or genomic risk factors.

In a phase II study, patients with relapsed or refractory MCL were treated orally with ibrutinib, resulting in a response rate of ~ 68% [ 187 ]. It was subsequently demonstrated that Ibrutinib was also highly active and associated with durable responses in pretreated patients with Waldenström’s macroglobulinemia, whereby MYD88 and CXCR4 mutation status affected the response [ 188 ]. Ibrutinib very rapidly received breakthrough designation and was subsequently approved by the Food and Drug Administration (FDA) for the treatment of MCL, CLL and WM between November 2013 and January 2017.

In addition, ibrutinib has also been tested in other B cell malignancies. In line with the possible role of BTK in FL, 6 out of 16 (38%) relapsed/refractory FL patients show response upon ibrutinib treatment [ 186 ]. In a phase II study ibrutinib induced durable remissions in ~ 50% of the MZL patients [ 189 ]. In a phase I study the majority (77%) of patients with PCNSL show clinical responses to ibrutinib [ 177 ]. Table 1 summarizes the data from current clinical trials in various B-cell malignancies. Table 1 Clinical trials with BTK inhibitors in B cell malignancies Patient population Therapeutic regimen Phase Efficacy Ref R/R CLL Ibrutinib Ib/II ORR (71%), PR(20%) [ 11 ] R/R CLL Ibrutinib III ORR (63%) [ 248 ] TN CLL Ibrutinib Ib/II ORR (85%), CR(26%) [ 199 ] TN CLL Ibrutinib III ORR (86%), CR(4%) [ 13 ] R/R MCL Ibrutinib II ORR (68%), CR(21%) [ 187 ] R/R MCL Ibrutinib III ORR (72%), CR(19%) [ 249 ] R/R WM Ibrutinib II ORR(91%), Major response (73%) [ 188 ] R/R ABC-DLBCL Ibrutinib II ORR (37%) [ 196 ] R/R CLL Ibrutinib-Rituximab II ORR (95%), CR(8%) [ 250 ] R/R CLL Ibrutinib-bendamustine-rituximab III ORR (83%), CR(10%) [ 251 ] R/R MCL Ibrutinib-Rituximab II ORR (88%), CR(44%), PR(44%) [ 252 ] R/R CLL Acalabrutinib I/II ORR(95%) [ 12 ] R/R Acalabrutinib II ORR (81%), CR (40%), PR(41%) [ 219 ] R/R CLL ONO/GS-4059 I ORR(96%) [ 222 ] R/R MCL ONO/GS-4059 I ORR(92%) [ 222 ] R/R non-GCB DLBCL ONO/GS-4059 I ORR(92%) [ 222 ] R/R CLL BGB-3111 I ORR(90%) [ 221 , 253 ] R/R MCL BGB-3111 I ORR(80%) [ 253 ] R/R MZL Ibrutinib II ORR(51%) [ 254 ] R/R FL Ibrutinib I ORR(38%) [ 186 ] CLL Chronic Lymphocytic leukemia, MCL Mantle cell lymphoma, WM Waldenström’s Macroglobulinemia, ABC-DLBCL Activated B-cell Diffuse large B cell Lymphoma, MZL Marginal zone lymphoma, FL Follicular lymphoma, R/R relapsed or refractory, TN treatment-naïve, ORR overall response rate, CR complete response, PR partial response, Major response: complete response or at least 50% reduction in serum IgM levels

Several studies were performed to explain the therapeutic mode of action of ibrutinib. In CD40- or BCR-activated CLL cells, ibrutinib reduced survival by abrogating downstream pathways including ERK, PI3K and NF-кB [ 141 ]. Ibrutinib inhibited migration of CLL cells towards chemokines such as CXCL12 and CXCL13, suggesting that treatment inhibits homing and retention of malignant cells in their survival niches [ 77 ]. Ibrutinib was also found to reduce secretion of BCR-dependent chemokines CCL3 and CCL4 [ 142 ]. Another key effect was that it inhibited integrin α4β1-mediated adhesion of CLL cells to fibronectin and VCAM1 [ 146 ] and thus interaction with the tumor microenvironment [ 146 ]. Therefore, ibrutinib apparently works by a dual mechanism, by inhibiting intrinsic B cell signaling pathways to compromise their proliferation and survival as well as by disrupting tumor-microenvironment interactions. Importantly, both in CLL and MCL ibrutinib treatment induces a redistribution lymphocytosis, a transient rise of leukemic cells in the circulation and a concomitant rapid reduction of these cells at the affected tissue sites. In contrast to classical cytotoxic chemotherapy, ibrutinib does not cause tumor lysis syndrome, which is a common complication of cancer therapy because of metabolic disturbances when large numbers of tumor cells die quickly. Therefore, most likely the displacement of B cells from nurturing tissue niches because of inhibition of integrin-mediated retention of leukemic cells, is an important mechanism of action of ibrutinib, rather than robust inhibition of survival of malignant B cells [ 190 ]. As a result, leukemic cells undergo ‘death by neglect’, because their mobilization induces ‘homelessness’ (anoikis), a form of programmed cell death [ 191 , 192 ].

Despite impressive clinical success of ibrutinib, its curative potential in B cell malignancies is not established yet, as ibrutinib is often prescribed as life-long therapy. Importantly, continuous therapy may lead to selection or outgrowth of resistant clones, as described in a subset of patients who relapse upon ibrutinib therapy. Two important therapy-associated resistance mechanisms have been identified, involving BTK C481S mutation (the site of action of Ibrutinib) or activating mutations in PLCy2 (R665W, S707Y and L845F) [ 193 , 194 ]. Recently another BTK mutation, T316A in the SH2 domain, was described, as well as clonal evolution underlying leukemia progression in patients with ibrutinib-relapsed CLL [ 195 ]. In addition, missense mutation within the coiled-coil domain of CARD11 (R179Q) have been shown to promote BTK-independent activation of NF-κB and thus ibrutinib resistance in DLBCL, MCL and PCNSL [ 177 , 196 , 197 ]. Furthermore, an activating mutation in BTK (L528 W) that confers resistance to ibrutinib treatment has been found in CLL and FL [ 178 , 198 ].

In clinical trials the adverse events were mostly limited to grade 1 or 2 in severity, but in some cases side-effects led to discontinuation of the therapy [ 199 – 201 ]. Because ibrutinib treatment has a considerable high risk of bleeding in treated patients, concomitant anti-coagulation (~ 11%) and antiplatelet (~ 34) use is common and ~ 3% of the patients were reported to have major bleeding events [ 202 ]. Atrial fibrillation has been reported in up to 16% of patients taking ibrutinib, whereby stroke prevention poses a challenge because of the increased bleeding risk. Therefore, close monitoring is recommended, especially during the first 6 months of ibrutinib therapy [ 203 ]. Although the occurrence of atrial fibrillation might possibly be related to inhibition of the BTK-regulated PI3K/AKT pathway in cardiac myocytes [ 204 ], the mechanisms involved remain largely unidentified.

Three year follow-up of ibrutinib-treated CLL patients showed that prolonged treatment was associated with improvement in response quality (the ORR increased to > 90%) and durable remission, while toxicity including cytopenia, fatigue, and infection diminished. Moreover, progression remains uncommon [ 205 ]. Findings from the longest follow-up reported to date, evaluating up to 5 years of ibrutinib in CLL patients, show that it is relatively safe and effective, with ~ 89% of treatment-naïve and relapsed patients experiencing a response to the therapy [ 206 ].

Part of the toxicities and side effects of ibrutinib can be explained by its non-specific nature: ibrutinib is not an exclusive inhibitor of BTK and off-target inhibition includes kinases that contain a cysteine residue aligning with Cys-481 in BTK. These include other TEC-family kinases (ITK, BMX, TEC), as well as epidermal growth factor receptor (EGFR), T-cell X chromosome kinase (TXK) and Janus Kinase 3 (JAK3) [ 12 , 185 , 207 ]. In this context, it is of note that the bleeding risk in patients receiving ibrutinib was thought to relate to off-target inhibition of TEC [ 12 ]. BTK is expressed in platelets where it is important for signaling via the collagen receptor glycoprotein VI (GPVI); platelets from XLA patients display diminished aggregation, dense granule secretion and calcium mobilization in response to collagen and C-reactive protein [ 208 ]. Nevertheless, XLA patients do not have an increased risk of bleeding [ 209 ]. Findings by Bye et al. indicated that both BTK and TEC – although required for GPVI-mediated platelet aggregation – are redundant for platelet adhesion to collagen and thrombus formation [ 210 ]. Rather, ibrutinib but not the more selective BTK inhibitor acalabrutinib (see below) inhibits SRC family kinases that have a critical role in platelet function [ 210 ]. These findings explain why in contrast to ibrutinib, treatment with acalabrutinib was not associated with major bleeding events [ 12 ].

A recent systematic review of infectious events with ibrutinib in the treatment of B cell malignancies provided evidence for infection-related complications in ~ 50% of patients taking ibrutinib, whereby ~ 20% of patients developed pneumonia due to opportunistic pathogens [ 211 ]. Hereby, data suggest that these events may involve inhibition of both BTK and its closely related family member ITK. On the other hand, it was shown that ibrutinib treatment increased the in vivo persistence of both CD4 + and CD8 + activated T cells and diminished the immune-suppressive properties of CLL cells. As these effects were not seen with more specific BTK inhibitor acalabrutinib that lacks ITK inhibitory activity (see below), it was concluded that the T cell expansion is unlikely to be caused by BTK inhibition [ 212 ]. Rather, ibrutinib treatment of activated T cells diminishes activation-induced cell death by targeting ITK, a finding also reported in murine models of ITK deficiency. However, both inhibitors reduced the expression of the inhibitory co-receptors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) on T cells, as well as expression of the immunosuppressive molecules CD200, B- and T-lymphocyte attenuator (BTLA) and IL-10 by CLL cells [ 212 ]. Therefore, ibrutinib likely diminishes the immune-suppressive properties of CLL cells through both BTK-dependent and ITK-dependent mechanisms.

Inhibition of BTK and ITK with ibrutinib was shown to be effective in the prevention of chronic graft-versus-host (GvH) disease following allogeneic hematopoietic stem cell transplantation (SCT) in several mouse models [ 213 , 214 ]. Accordingly, also studies in patients with relapsed CLL following SCT support that ibrutinib augments the GvH versus-leukemia (GVL) benefit likely through ITK inhibition [ 215 ]. In particular, it was shown that ibrutinib selectively targeted pre–germinal B cells and depleted Th2 helper cells, whereby these effects persisted after drug discontinuation.

Taken together, these findings provide a rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers.

Ibrutinib in combination therapies and second generation BTK inhibitors

The finding of ibrutinib resistance, together with multiple modes of action and the microenvironmental dependence of B-cell malignancies, has fueled the development of novel combination strategies. With the aim to achieve deeper remissions within a short treatment time, many ibrutinib combination therapies are currently considered (Table 2 ). Hereby, ibrutinib treatment forces egress of malignant B cells out of their protective niches into circulation, where they become vulnerable to direct cytotoxic activity of either chemotherapy, an inhibitor of the pro-survival protein Bcl-2, or antibody mediated cytotoxicity (ADCC) of anti-CD20 antibody therapy. Table 2 Overview of Ibrutinib in combination therapies Combination Disease Model Rationale Effect Reference γ-secretase inhibitors (crucial protease in Notch signaling) CLL CLL patient cells NOTCH1 signaling is related to resistance to therapy in B-CLL. Combination therapy showed enhanced cytotoxicity and reduced CXCR4 expression and functions (response to SDF-1α) [ 255 ] Histone Deacetylase (HDACs) Inhibitor CLL - MCL cell line - mice engrafted with TCL-1 splenocytes HDACs increase transcription of miRNA that repress BTK HDAC induced increase in target miRNA and a decrease in BTK RNA; combination exhibited higher cytotoxicity than either drug alone; reduction of p-BTK and total BTK protein. [ 69 ] Anti-CD19 CAR T Cells (CART19) MCL MCL Xenograph model Efficient B cell depletion Long-term remission in 80–100% of mice (treated with CART19 only: 0–20% of mice) [ 245 ] Ethacridine (Poly(ADP-ribose) glycohydrolase inhibitor) AML SCID mice injected s.c. with OCI-AML2 cells Result of a drug screening High decrease of OCI-AML2 cell growth (more than with either drug alone). Suggested mechanism: increased intracellular ROS production in cells treated with combination. [ 256 ] ND-2158 (IRAK4 inhibitor) ABC-DLBCL - ABC-DLBCL cell lines OCI-Ly10 and TMD8 - OCI-Ly10 xenografts MYD88-IRAK4 signaling is important for ABC- DLBCL viability Combination was more effective than ND-2158 alone in inhibiting IKK activity, enhancing apoptosis, and blocking tumor growth in mice. [ 257 ] PU-H71 (Binds to tumor enhanced HSP90 complexes) ABC-DLBCL DLBCL cell lines (HBL-1 and TMD8) teHSP90 complexes are associated with tumor survival. PU-H71 disrupts teHSPP90 (but not house-keeping fractions associated with HSP90). Synergistic effect, with ~ 95% tumor growth inhibition; decreased NF-kB activity [ 258 ] TP-0903 (AXL inhibitor) CLL Patient CLL cells prior to or after ibrutinib therapy AXL contributes to oncogenic survival in CLL. TP-093 disrupts the activity of AXL; Induction of cell-death in a dose-dependent fashion [ 259 ] B-PAC-1 (pro-caspase activating compound) CLL B cells from patients on ibrutinib therapy B-PAC activates caspases dimers Induced cytotoxicity in leukemic cells [ 259 ] Carfilzomib (proteasome inhibitor) CLL Primary CLL patient samples MEC-1 and MEC2 cell lines Upregulation of pro-apoptotic transcription factor CHOP Combination showed an additive cytotoxic effect; Carfilzomib induced a pro-apoptotic response involving Noxa, MCL-1, Bax, and Bak and intrinsic and extrinsic caspase pathways [ 260 ] Selinexor (Exportin inhibitor) CLL Primary CLL patient samples Selinexor disrupts BCR signaling via BTK depletion Combination showed synergistic cytoxicity. Selinexor overcomes resistance to Ibrutinib (also in patient cells with C481S mutation) [ 261 ] Anti-PDL1 antibody (Negative regulator of T cell function) B cell lymphoma (A20) - BALB/c mice inoculated with A20 B cells - A20 B cells are resistant to Ibrutinib Blocking immune checkpoints can enhance the anti-tumor response Anti-PDL-1 treatment alone delayed tumor growth and slightly increased mouse survival Combination with anti-PDL-1 cured ~ 50% of the mice, delayed tumor growth and prolonged survival in the remaining mice, and increased IFN-γ producing T-cells [ 243 ] ABT-199 (BCL-2 antagonist) CLL Ex vivo samples from CLL patients on ibrutinib CLL samples show enhanced BCL-2 expression Ibrutinib enhances ABT-199 cytotoxicity, both in unstimulated and in αIgM-stimulated CLL cells from. ABT-199 action correlated with a decline in expression of anti-apoptotic MCL-1 [ 262 ] ABT-199 (BCL-2 antagonist) MCL CCMCL1 MCL cell line MCL cells show enhanced BCL-2 expression Combination results in decrease of p-BTK and p-AKT. Downregulation of both BCL2 and MCL1. ABT-199 and Ibrutinib target non-overlapping pathway s [ 263 ] Bortezomib (proteosome inhibitor) and lenalidomide chemotherapy MM Cells from MM patients and MM cell lines Blocking BTK to downregulate NF-kB activation and cell survival Ibrutinib increased the cytotoxicity of bortezomib and lenalidomide in both patient cells and cell lines [ 264 ] CpG (TLR9 ligand) B-cell lymphoma Murine pre-B cell (H11) and B cell lymphoma lines (BL3750, A20) CpG activates APCs and thereby induces T cell activation Combination of ibrutinib and intratumoral CpG resulted in tumor regression and resistance, whereby IFNy-producing CD4 and CD8 T are essential [ 265 ] Sudemycin D1 (spliceosome modulator) CLL Primary CLL cells (from SF3B1-unmutated and mutated cases) SF3B1 is frequently mutated in CLL, and correlates with aggressiveness Combination results in enhanced apoptosis of M-CLL and U-CLL. Effect is related to IBTK splicing. Sudemycin D1 downregulates anti-apoptotic MCL-1 through alternative splicing [ 266 ] BAY80–6946 (PIK3 inhibitor) INK128 (mTOR inhibitor) PCNSL - Xenograft model from CD79B-mutant biopsies CARD11 domain mutations increase the activity of the PIK3-mTOR axis In cell lines, cell death was induced with both combinations of drugs [ 177 ] Idelalisib (PI3K inhibitor) DLBCL - Cell lines. - Mouse TMD8 xenograft model PI3K is upstream regulator of NF-кB pathway. Cell lines: combination induced 50% apoptosis and inhibited signaling (more than either drug individually). Mouse xenograft: Significant tumor regression [ 267 ] 1 Idelalisib (PI3K inhibitor) MCL MCL cell lines A more robust blockage of BCR signaling Inhibition of BCR-stimulated integrin- mediated adhesion; stronger inhibition of adhesion compared to each drug alone [ 146 ] 1 1 In this study, also ONO/GS-4059, the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 and the AKT inhibitor MK-2206 were investigated CLL Chronic Lymphocytic leukemia, MCL Mantle cell lymphoma, AML Acute Myeloid Leukemia, ABC-DLBCL Activated B-cell Diffuse large B cell Lymphoma, MM Multiple Myeloma, PCNSL Primary central nervous system lymphoma

Side-effects associated with off-target kinase inhibition may limit the use of ibrutinib as therapeutic agent (as discussed above). Ibrutinib can antagonize rituximab-induced ADCC due to inhibition of its family member ITK in NK cells, further limiting its use in combination regimens [ 216 ]. Therefore, many efforts have focused on developing highly selective BTK inhibitors, of which three have reached advanced stages of clinical development [ 217 ].

Acalabrutinib (ACP-196)

This highly selective irreversible BTK inhibitor has significantly less off-target kinase activity [ 207 ]. Acalabrutinib also binds C481 and lacks irreversible targeting to alternative kinases, such as EGFR, ITK, TXK, SRC family kinases and JAK3. The first pre-clinical study in canine models of Non-Hodgkin B-cell lymphoma demonstrated enhanced in vivo potency compared to ibrutinib [ 218 ]. In a phase I/II clinical trial in patients with relapsed/refractory CLL the overall response rate was ~ 95% and in patients with del(17)(p13.1) this was 100%, with a median follow-up up ~ 14 months [ 12 ]. No dose-limiting toxicities, episodes of atrial fibrillation, or bleeding-related events have been reported to date. To investigate the superiority of either inhibitor, a phase III trial for direct comparison of ibrutinib with acalabrutinib in R/R CLL patients is currently ongoing ( NCT02477696 ). Additionally, in a phase II trial in patients with relapsed/refractory MCL, acalabrutinib induced an overall response of ~ 81% with ~ 40% patients achieving a complete response [ 219 ]. This led to accelerated FDA approval of acalabrutinib in MCL [ 220 ].

BGB-3111

Another selective inhibitor of BTK kinase activity with superior oral bioavailability and higher selectivity than ibrutinib is BGB-3111, which was shown to inhibit proliferation of several MCL and DLBCL cell lines. Due to weaker ITK inhibition, BGB-3111 was at least 10-fold weaker than ibrutinib in inhibiting rituximab induced ADCC. When 45 CLL patients were treated on a phase I/II study, therapy was well tolerated and was associated with a response rate of ~ 90% after a follow-up of 7.5 months and no cases of disease progression or Richter’s transformation [ 221 ] (see also Table 1 ).

Ono/GS-4059

In vivo efficacy of this compound was initially described in an ABC-DLBCL xenograft model and in vitro anti-proliferative effects in DLBCL, FL, MCL and CLL cell lines were described [ 222 ]. Early-phase clinical trial data in patients with several B-cell malignancies include clinical responses in patients with high-risk CLL genetics (Table 1 ).

Role of BTK in the tumor microenvironment

Inhibition of BTK has now also extended into the field of solid tumors, following insights into the role of BTK in various cells of the tumor microenvironment and in non-hematological tumor cells when ectopically expressed. An understanding of the diverse roles of BTK in non-lymphocytic cells will be pivotal in the development of novel treatment combinations for haematopoietic and solid tumors.

BTK is involved in TLR- and Fc-receptor mediated activation, maturation, migration and survival of myeloid cells [ 223 , 224 ]. However, the role of BTK identified is dependent on cell type investigated, the nature of activating stimuli, the model used (in vivo or in vitro) and the species investigated, i.e. mouse or human. Analyses in various mouse models and in vitro studies with myeloid cells from XLA-patients clearly implicate BTK in TLR4/8/9-signaling, and possibly others [ 79 , 225 – 227 ]. However, data are often conflicting, e.g. TLR8-induced IL-6 production by BTK-deficient DCs were reported to be impaired [ 226 ], enhanced [ 228 ], or unaffected [ 229 ]. Also TLR4/7/8-induced TNFα was reported to be reduced [ 226 , 229 ] or enhanced [ 228 ].

Of further relevance in the context of the tumor microenvironment is the polarization status of macrophages, with M1 macrophages displaying a pro-inflammatory anti-tumor phenotype and M2 macrophages being immunosuppressive [ 22 ]. Whereas one study indicated an M2-skewing of BTK-deficient macrophages [ 230 ], recently in a pancreatic cancer mouse model an M1-skewing of intratumoral macrophages was found following ibrutinib treatment [ 231 ]. In contrast, ibrutinib induced M1 to M2-skewing of nurse-like cells, which show properties of tumor-associated macrophages, accompanied by impaired phagocytosis, increased IL-10 production mediating pro-survival signals in CLL [ 232 ]. It remains unknown what causes these incongruences in BTK-dependent myeloid polarization, however it is conceivable that the different roles of BTK in a complex ecology of tumor-infiltrating cells and the limited specificity of ibrutinib contribute to the conflicting findings.

In solid tumors, chronic deposition of immune complexes foster carcinogenesis due to chronic inflammation, angiogenesis and M2 macrophage polarization in response to activating Fc-receptor ligation on myeloid cells [ 231 , 233 , 234 ]. Interestingly, inhibiting BTK during Fc-receptor stimulation of macrophages in vitro using Ibrutinib prevented M2-skewing [ 231 ].

Granulocytes and their immature immune-suppressive counterparts, myeloid derived suppressor cells (MDSC), are strongly implicated in tumor progression, rendering them important candidates for therapy [ 235 ]. Although loss of BTK in XLA neutrophils does not impair functional TLR responses [ 236 ], the numbers of circulating granulocytes are reduced in XLA-patients and BTK-deficient mice [ 237 – 239 ]. Moreover, BTK-deficient neutrophils manifest increased sensitivity to apoptosis, decreased maturation, differentiation, trafficking and impaired functionality including reactive oxygen species (ROS) production [ 238 – 241 ]. Likewise, ibrutinib treatment inhibited the generation, migration, TNFα and ROS-production of MDSCs both in vitro and in solid tumor mouse models [ 242 ]. Ibrutinib treatment partially alleviated MDSC-mediated CD8 + T-cell suppression and enhanced anti-PD-L1 therapy efficacy in a breast cancer model. BTK inhibition in granulocytes and MDSCs in solid tumors may therefore be important in the development of effective combination therapies.

BTK inhibition in solid malignancies

Ectopic BTK expression has been observed in various solid tumors, whereby evidence is accumulating for its involvement in oncogenesis [ 24 – 27 ]. These pre-clinical findings have led to the initiation of several early phase I/II clinical trials in which BTK inhibition monotherapy is evaluated in advanced ovarian, colorectal, prostate and brain cancer patients(Table 3 ). Table 3 Clinical trials with BTK-inhibitors in solid tumors BTK-inhibitor Tumor Type Treatment combination Phase Trial Status NCT# Ibrutinib Pancreatic Cancer nab-paclitaxel and gemcitabine ll/lll Ongoing, Not Recruiting NCT024B6668 Ibrutinib Pancreatic Cancer nab-paclitaxel and gemcitabine I Recruiting NCT02562898 Ibrutinib Renal/Urethelial/Gastric/Colorectal Cancer chemotherapy/small molecule Ib/II Recruiting NCT02599B24 Ibrutinib HER2/MYC + Oesophageal Cancer I Recruiting NCT02884453 Ibrutinib EGFR + Non-small Cell Lung Cancer (NSCLC) I/ll Ongoing, Not Recruiting NCT02321540 Ibrutinib NSCLC/Breast Cancer/Pancreatic Cancer Durvalumab (anti-PD-L1) I/ll Completed NCT02403271 Ibrutinib Pancreatic Neuro-endocrine tumors (pNET) /metastatic Carcinoid II Recruiting NCT02575300 Ibrutinib Stage IV Cutaneous Melanoma II Recruiting NCT02581930 Ibrutinib Metastatic Renal Cancer Nivolumab (anti-PD-L1) lb./ll Recruiting NCT02899078 Ibrutinib Localized Prostate Cancer I/II Recruiting NCT02643667 Acalabrutinib Advanced/Metastatic Pancreatic Cancer Pembrolizumab (anti-PD-L1) II Ongoing, Not Recruiting NCT02362048 Acalabrutinib Recurrent Ovarian Cancer Pembrolizumab (anti-PD-L1) II Ongoing, Not Recruiting NCT02537444 Acalabrutinib Glioblastoma lb/ll Recruiting NCT02586857 Acalabrutinib NSCLC Pembrolizumab (anti-PD-L1) II Ongoing, Not Recruiting NCT02448303 Acalabrutinib Head and Neck Squamous Cell Carcinoma (HNSCC) Pembrolizumab (anti-PD-L1) II Ongoing, Not Recruiting NCT02454179 Acalabrutinib Platinum-resistant Urethelial (Bladder) Cancer Pembrolizumab (anti-PD-L1) II Ongoing, Not Recruiting NCT02351739 ,

Also in BTK-negative solid tumors that do not express BTK, its inhibition may hold promise as multiple cell types in the tumor microenvironment are regulated by BTK. Inhibition of BTK in pre-clinical models of pancreatic cancer, breast cancer and BTK-negative colon cancer have shown only marginal improvement of survival as monotherapy, but when combined with chemo- or immunotherapy, survival was greatly enhanced [ 231 , 242 , 243 ]. This has sparked the emergence of several trials investigating the safety and efficacy of ibrutinib or acalabrutinib, in combination with conventional PD-1/PD-L1 checkpoint inhibition therapy (Table 3 ).

Given that ibrutinib shows off-target inhibition of JAK3, ITK and EGFR [ 185 , 207 ], it can be used to target oncogenic pathways other than BTK in tumor cells and as a T-cell modulator in combination immunotherapy [ 243 – 246 ]. Hereby, ibrutinib treatment increased cellular persistence and decreased expression of co-inhibitory surface molecules on Chimeric antigen receptor (CAR) T cells in models of CLL and MCL [ 245 , 246 ]. Whether in these studies ibrutinib acts on ITK in (CAR) T cells, on BTK in the malignant cells or other kinases remains undetermined. Paradoxically, inhibiting ITK in T cells may be efficacious in cancer, as this may enhance Th1-skewing of CD4 + T-cells and thereby improved memory formation and functionality of CD8 + T-cells, potentially leading to improved anti-tumor immunity [ 243 , 247 ]. These potentially beneficial off-target effects of ibrutinib may be lost in the highly specific BTK-inhibitors that are currently being evaluated.

Conclusions

Targeting of BTK, which has a central role in several signaling pathways in B cells, particularly the BCR, has shown impressive efficacy as therapeutic option for various B cell malignancies in clinical trials. Much progress has been made in recent years in defining the complex mechanisms of action of BTK inhibition. These involve intrinsic signaling pathways in leukemic cells that are central to cellular survival, proliferation and - most importantly - retention in supportive microenvironments. Moreover, BTK inhibition shows promise as a therapy that influences crucial immune cells in the tumor microenvironment. Because data from BTK-deficient or inhibitor-treated myeloid cells in the context of cancer are scarce, it is not clear whether BTK inhibition by e.g. ibrutinib is based on its specificity for BTK in particular myeloid cells and/or due to off-target effects in signaling pathways in CD4 + or CD8 + T cells. Of note, because in CLL ibrutinib treatment diminished the immunosuppressive properties of malignant cells through BTK-dependent and BTK-independent mechanisms (probably via ITK inhibition) [ 212 ], it will be interesting to observe whether the same level of anti-tumor efficacy is maintained by specific BTK inhibition alone. It is very well conceivable that for particular malignancies it may be advantageous to use BTK inhibitors that show additional specificity for related kinases.

Although the efficacy of BTK inhibition as a single agent therapy is strong, it has been shown that resistance may develop and now a broad range of studies focus on development of effective combination therapies to improve clinical responses. The identification of differences in efficacy and toxicity profiles between available BTK inhibitors awaits direct comparative studies. In this context, design of treatment strategies will depend on detailed analyses of clinical responses, resistance development, toxicity and quality of life for individual BTK inhibitors in combination therapies in relation to the various malignancies and patient subgroups.

Abbreviations

Activate B-cell diffuse large B-cell lymphoma

B cell receptor

Bruton’s tyrosine kinase

Chronic lymphocytic leukemia

Complete response

Diffuse large B cell lymphoma

Extracellular signal-regulated kinase

Germinal center

Immunoreceptor tyrosine-based activation motif

Immunoreceptor tyrosine-based inhibitory motif

Mantle cell lymphoma

Mutated chronic lymphocytic leukemia

Myeloid differentiation primary response 88

Nuclear factor of activated T cells

Nuclear factor kappa-light-chain-enhancer of activated B cells

Overall response rate

Overall survival

Primary central nervous system lymphoma

Programmed cell death protein 1

Phosphatidyl-inositol-3-kinase

Phosphatidylinositol-3,4,5-triphosphate

Phospholipase C γ

Partial response

SH2-domain containing inositol polyphosphate 5’phosphatase-1

Somatic hypermutation

SH2 domain containing protein tyrosine phosphatase-1

Spleen tyrosine kinase

Toll-like receptor

Unmutated chronic lymphocytic leukemia

Waldenström’s Macroglobulinemia

Funding

S.P.S is partly supported by Netherlands Organization for Scientific Research.

Authors’ contributions

All authors contributed to literature search and writing of the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Gross S Rahal R Stransky N Lengauer C Hoeflich KP Targeting cancer with kinase inhibitors J Clin Invest 2015 125 1780 1789 10.1172/JCI76094 25932675 PMC4463189

2. Hanahan D Weinberg RA Hallmarks of cancer: the next generation Cell 2011 144 646 674 10.1016/j.cell.2011.02.013 21376230

3. Vetrie D Vorechovsky I Sideras P Holland J Davies A Flinter F Hammarstrom L Kinnon C Levinsky R Bobrow M The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases Nature 1993 361 226 233 10.1038/361226a0 8380905

4. Tsukada S Saffran DC Rawlings DJ Parolini O Allen RC Klisak I Sparkes RS Kubagawa H Mohandas T Quan S Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia Cell 1993 72 279 290 10.1016/0092-8674(93)90667-F 8425221

5. Rawlings DJ Saffran DC Tsukada S Largaespada DA Grimaldi JC Cohen L Mohr RN Bazan JF Howard M Copeland NG Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice Science 1993 261 358 361 10.1126/science.8332901 8332901

6. Thomas JD Sideras P Smith CI Vorechovsky I Chapman V Paul WE Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes Science 1993 261 355 358 10.1126/science.8332900 8332900

7. Scher I The CBA/N mouse strain: an experimental model illustrating the influence of the X-chromosome on immunity Adv Immunol 1982 33 1 71 10.1016/S0065-2776(08)60834-2 6215838

8. Khan WN Alt FW Gerstein RM Malynn BA Larsson I Rathbun G Davidson L Muller S Kantor AB Herzenberg LA Defective B cell development and function in Btk-deficient mice Immunity 1995 3 283 299 10.1016/1074-7613(95)90114-0 7552994

9. Hendriks RW de Bruijn MF Maas A Dingjan GM Karis A Grosveld F Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage EMBO J 1996 15 4862 4872 8890160 PMC452224

10. Middendorp S Dingjan GM Hendriks RW Impaired precursor B cell differentiation in Bruton's tyrosine kinase-deficient mice J Immunol 2002 168 2695 2703 10.4049/jimmunol.168.6.2695 11884435

11. Byrd JC Furman RR Coutre SE Flinn IW Burger JA Blum KA Grant B Sharman JP Coleman M Wierda WG Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia N Engl J Med 2013 369 32 42 10.1056/NEJMoa1215637 23782158 PMC3772525

12. Byrd JC Harrington B O'Brien S Jones JA Schuh A Devereux S Chaves J Wierda WG Awan FT Brown JR Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia N Engl J Med 2016 374 323 332 10.1056/NEJMoa1509981 26641137 PMC4862586

13. Burger JA Tedeschi A Barr PM Robak T Owen C Ghia P Bairey O Hillmen P Bartlett NL Li J Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia N Engl J Med 2015 373 2425 2437 10.1056/NEJMoa1509388 26639149 PMC4722809

14. Hendriks RW Yuvaraj S Kil LP Targeting Bruton's tyrosine kinase in B cell malignancies Nat Rev Cancer 2014 14 219 232 10.1038/nrc3702 24658273

15. Kawakami Y Yao L Miura T Tsukada S Witte ON Kawakami T Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon fc epsilon RI cross-linking Mol Cell Biol 1994 14 5108 5113 10.1128/MCB.14.8.5108 7518558 PMC359029

16. Hata D Kawakami Y Inagaki N Lantz CS Kitamura T Khan WN Maeda-Yamamoto M Miura T Han W Hartman SE Involvement of Bruton's tyrosine kinase in FcepsilonRI-dependent mast cell degranulation and cytokine production J Exp Med 1998 187 1235 1247 10.1084/jem.187.8.1235 9547335 PMC2212237

17. Jongstra-Bilen J Puig Cano A Hasija M Xiao H Smith CI Cybulsky MI Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis J Immunol 2008 181 288 298 10.4049/jimmunol.181.1.288 18566394

18. Wang D Feng J Wen R Marine JC Sangster MY Parganas E Hoffmeyer A Jackson CW Cleveland JL Murray PJ Ihle JN Phospholipase Cgamma2 is essential in the functions of B cell and several fc receptors Immunity 2000 13 25 35 10.1016/S1074-7613(00)00005-4 10933392

19. Shinohara M Koga T Okamoto K Sakaguchi S Arai K Yasuda H Takai T Kodama T Morio T Geha RS Tyrosine kinases Btk and Tec regulate osteoclast differentiation by linking RANK and ITAM signals Cell 2008 132 794 806 10.1016/j.cell.2007.12.037 18329366

20. Oda A Ikeda Y Ochs HD Druker BJ Ozaki K Handa M Ariga T Sakiyama Y Witte ON Wahl MI Rapid tyrosine phosphorylation and activation of Bruton's tyrosine/Tec kinases in platelets induced by collagen binding or CD32 cross-linking Blood 2000 95 1663 1670 10688822

21. Ito M Shichita T Okada M Komine R Noguchi Y Yoshimura A Morita R Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury Nat Commun 2015 6 7360 10.1038/ncomms8360 26059659 PMC4490404

22. Noy R Pollard JW Tumor-associated macrophages: from mechanisms to therapy Immunity 2014 41 49 61 10.1016/j.immuni.2014.06.010 25035953 PMC4137410

23. Mantovani A Marchesi F Malesci A Laghi L Allavena P Tumour-associated macrophages as treatment targets in oncology Nat Rev Clin Oncol 2017 14 399 416 10.1038/nrclinonc.2016.217 28117416 PMC5480600

24. Kokabee L Wang X Sevinsky CJ Wang WL Cheu L Chittur SV Karimipoor M Tenniswood M Conklin DS Bruton's tyrosine kinase is a potential therapeutic target in prostate cancer Cancer Biol Ther 2015 16 1604 1615 10.1080/15384047.2015.1078023 26383180 PMC4846115

25. Grassilli E Pisano F Cialdella A Bonomo S Missaglia C Cerrito MG Masiero L Ianzano L Giordano F Cicirelli V A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation Oncogene 2016 35 4368 4378 10.1038/onc.2015.504 26804170 PMC4994017

26. Zucha MA Wu AT Lee WH Wang LS Lin WW Yuan CC Yeh CT Bruton's tyrosine kinase (Btk) inhibitor ibrutinib suppresses stem-like traits in ovarian cancer Oncotarget 2015 6 13255 13268 10.18632/oncotarget.3658 26036311 PMC4537012

27. Wei L Su YK Lin CM Chao TY Huang SP Huynh TT Jan HJ Whang-Peng J Chiou JF Wu AT Hsiao M Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes Oncotarget 2016 7 69961 69975 27564106 10.18632/oncotarget.11572 PMC5342527

28. Bradshaw JM The Src, Syk, and Tec family kinases: distinct types of molecular switches Cell Signal 2010 22 1175 1184 10.1016/j.cellsig.2010.03.001 20206686

29. Hyvonen M Saraste M Structure of the PH domain and Btk motif from Bruton's tyrosine kinase: molecular explanations for X-linked agammaglobulinaemia EMBO J 1997 16 3396 3404 10.1093/emboj/16.12.3396 9218782 PMC1169965

30. Rawlings DJ Scharenberg AM Park H Wahl MI Lin S Kato RM Fluckiger AC Witte ON Kinet JP Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases Science 1996 271 822 825 10.1126/science.271.5250.822 8629002

31. Park H Wahl MI Afar DE Turck CW Rawlings DJ Tam C Scharenberg AM Kinet JP Witte ON Regulation of Btk function by a major autophosphorylation site within the SH3 domain Immunity 1996 4 515 525 10.1016/S1074-7613(00)80417-3 8630736

32. Marcotte DJ Liu YT Arduini RM Hession CA Miatkowski K Wildes CP Cullen PF Hong V Hopkins BT Mertsching E Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases Protein Sci 2010 19 429 439 20052711 10.1002/pro.321 PMC2866269

33. Middendorp S Dingjan GM Maas A Dahlenborg K Hendriks RW Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity J Immunol 2003 171 5988 5996 10.4049/jimmunol.171.11.5988 14634110

34. Lam KP Kuhn R Rajewsky K In Vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death Cell 1997 90 1073 1083 10.1016/S0092-8674(00)80373-6 9323135

35. Anderson JS Teutsch M Dong Z Wortis HH An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis Proc Natl Acad Sci U S A 1996 93 10966 10971 10.1073/pnas.93.20.10966 8855292 PMC38267

36. Solvason N Wu WW Kabra N Lund-Johansen F Roncarolo MG Behrens TW Grillot DA Nunez G Lees E Howard M Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells J Exp Med 1998 187 1081 1091 10.1084/jem.187.7.1081 9529324 PMC2212200

37. Brorson K Brunswick M Ezhevsky S Wei DG Berg R Scott D Stein KE Xid affects events leading to B cell cycle entry J Immunol 1997 159 135 143 9200448

38. Glassford J Soeiro I Skarell SM Banerji L Holman M Klaus GG Kadowaki T Koyasu S Lam EW BCR targets cyclin D2 via Btk and the p85alpha subunit of PI3-K to induce cell cycle progression in primary mouse B cells Oncogene 2003 22 2248 2259 10.1038/sj.onc.1206425 12700661

39. Spaargaren M Beuling EA Rurup ML Meijer HP Klok MD Middendorp S Hendriks RW Pals ST The B cell antigen receptor controls integrin activity through Btk and PLCgamma2 J Exp Med 2003 198 1539 1550 10.1084/jem.20011866 14610042 PMC2194118

40. Rolli V Gallwitz M Wossning T Flemming A Schamel WW Zurn C Reth M Amplification of B cell antigen receptor signaling by a Syk/ITAM positive feedback loop Mol Cell 2002 10 1057 1069 10.1016/S1097-2765(02)00739-6 12453414

41. O'Rourke LM Tooze R Turner M Sandoval DM Carter RH Tybulewicz VL Fearon DT CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav Immunity 1998 8 635 645 10.1016/S1074-7613(00)80568-3 9620684

42. Okada T Maeda A Iwamatsu A Gotoh K Kurosaki T BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation Immunity 2000 13 817 827 10.1016/S1074-7613(00)00079-0 11163197

43. Inabe K Ishiai M Scharenberg AM Freshney N Downward J Kurosaki T Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation J Exp Med 2002 195 189 200 10.1084/jem.20011571 11805146 PMC2193613

44. Saito K Scharenberg AM Kinet JP Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk J Biol Chem 2001 276 16201 16206 10.1074/jbc.M100873200 11279148

45. Engels N Konig LM Heemann C Lutz J Tsubata T Griep S Schrader V Wienands J Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells Nat Immunol 2009 10 1018 1025 10.1038/ni.1764 19668218

46. Kuhn J Wong LE Pirkuliyeva S Schulz K Schwiegk C Funfgeld KG Keppler S Batista FD Urlaub H Habeck M The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation Sci Signal 2016 9 ra66 10.1126/scisignal.aad6275 27353366

47. Guo B Kato RM Garcia-Lloret M Wahl MI Rawlings DJ Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex Immunity 2000 13 243 253 10.1016/S1074-7613(00)00024-8 10981967

48. Su YW Zhang Y Schweikert J Koretzky GA Reth M Wienands J Interaction of SLP adaptors with the SH2 domain of Tec family kinases Eur J Immunol 1999 29 3702 3711 10.1002/(SICI)1521-4141(199911)29:11<3702::AID-IMMU3702>3.0.CO;2-R 10556826

49. Chiu CW Dalton M Ishiai M Kurosaki T Chan AC BLNK: molecular scaffolding through 'cis'-mediated organization of signaling proteins EMBO J 2002 21 6461 6472 10.1093/emboj/cdf658 12456653 PMC136961

50. Weber M Treanor B Depoil D Shinohara H Harwood NE Hikida M Kurosaki T Batista FD Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen J Exp Med 2008 205 853 868 10.1084/jem.20072619 18362175 PMC2292224

51. Kim YJ Sekiya F Poulin B Bae YS Rhee SG Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-gamma2 Mol Cell Biol 2004 24 9986 9999 10.1128/MCB.24.22.9986-9999.2004 15509800 PMC525462

52. Hashimoto A Okada H Jiang A Kurosaki M Greenberg S Clark EA Kurosaki T Involvement of guanosine triphosphatases and phospholipase C-gamma2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor J Exp Med 1998 188 1287 1295 10.1084/jem.188.7.1287 9763608 PMC2212492

53. Petro JB Rahman SM Ballard DW Khan WN Bruton’s tyrosine kinase is required for activation of IkappaB kinase and nuclear factor kappaB in response to B cell receptor engagement J Exp Med 2000 191 1745 1754 10.1084/jem.191.10.1745 10811867 PMC2193161

54. Bajpai UD Zhang K Teutsch M Sen R Wortis HH Bruton’s tyrosine kinase links the B cell receptor to nuclear factor kappaB activation J Exp Med 2000 191 1735 1744 10.1084/jem.191.10.1735 10811866 PMC2193152

55. Manning BD Toker A AKT/PKB signaling: navigating the network Cell 2017 169 381 405 10.1016/j.cell.2017.04.001 28431241 PMC5546324

56. Craxton A Jiang A Kurosaki T Clark EA Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt J Biol Chem 1999 274 30644 30650 10.1074/jbc.274.43.30644 10521450

57. Ellmeier W Jung S Sunshine MJ Hatam F Xu Y Baltimore D Mano H Littman DR Severe B cell deficiency in mice lacking the tec kinase family members Tec and Btk J Exp Med 2000 192 1611 1624 10.1084/jem.192.11.1611 11104803 PMC2193106

58. de Bruijn MJ Rip J van der Ploeg EK van Greuningen LW Ta VT Kil LP Langerak AW Rimmelzwaan GF Ellmeier W Hendriks RW Corneth OB Distinct and overlapping functions of TEC kinase and BTK in B cell receptor signaling J Immunol 2017 198 3058 3068 10.4049/jimmunol.1601285 28275136

59. Engels N Konig LM Schulze W Radtke D Vanshylla K Lutz J Winkler TH Nitschke L Wienands J The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module Nat Commun 2014 5 5456 10.1038/ncomms6456 25413232 PMC4263166

60. Nimmerjahn F Ravetch JV Fcgamma receptors as regulators of immune responses Nat Rev Immunol 2008 8 34 47 10.1038/nri2206 18064051

61. Amigorena S Bonnerot C Drake JR Choquet D Hunziker W Guillet JG Webster P Sautes C Mellman I Fridman WH Cytoplasmic domain heterogeneity and functions of IgG fc receptors in B lymphocytes Science 1992 256 1808 1812 10.1126/science.1535455 1535455

62. Muta T Kurosaki T Misulovin Z Sanchez M Nussenzweig MC Ravetch JV A 13-amino-acid motif in the cytoplasmic domain of fc gamma RIIB modulates B-cell receptor signalling Nature 1994 369 340 10.1038/369340a0 8183374

63. Bolland S Ravetch JV Inhibitory pathways triggered by ITIM-containing receptors Adv Immunol 1999 72 149 177 10.1016/S0065-2776(08)60019-X 10361574

64. Ono M Bolland S Tempst P Ravetch JV Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor fc(gamma)RIIB Nature 1996 383 263 266 10.1038/383263a0 8805703

65. Ono M Okada H Bolland S Yanagi S Kurosaki T Ravetch JV Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling Cell 1997 90 293 301 10.1016/S0092-8674(00)80337-2 9244303

66. Liu W Quinto I Chen X Palmieri C Rabin RL Schwartz OM Nelson DL Scala G Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein Nat Immunol 2001 2 939 946 10.1038/ni1001-939 11577348

67. Kang SW Wahl MI Chu J Kitaura J Kawakami Y Kato RM Tabuchi R Tarakhovsky A Kawakami T Turck CW PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization EMBO J 2001 20 5692 5702 10.1093/emboj/20.20.5692 11598012 PMC125669

68. Belver L de Yebenes VG Ramiro AR MicroRNAs prevent the generation of autoreactive antibodies Immunity 2010 33 713 722 10.1016/j.immuni.2010.11.010 21093320 PMC3687137

69. Bottoni A Rizzotto L Lai TH Liu C Smith LL Mantel R Reiff S El-Gamal D Larkin K Johnson AJ Targeting BTK through microRNA in chronic lymphocytic leukemia Blood 2016 128 3101 3112 27756747 10.1182/blood-2016-07-727750 PMC5201097

70. Yu L Mohamed AJ Simonson OE Vargas L Blomberg KE Bjorkstrand B Arteaga HJ Nore BF Smith CI Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-kappaB Blood 2008 111 4617 4626 10.1182/blood-2007-10-121137 18292289

71. Ritter SL Hall RA Fine-tuning of GPCR activity by receptor-interacting proteins Nat Rev Mol Cell Biol 2009 10 819 830 10.1038/nrm2803 19935667 PMC2825052

72. Okada T Ngo VN Ekland EH Forster R Lipp M Littman DR Cyster JG Chemokine requirements for B cell entry to lymph nodes and Peyer's patches J Exp Med 2002 196 65 75 10.1084/jem.20020201 12093871 PMC2194009

73. Servant G Weiner OD Herzmark P Balla T Sedat JW Bourne HR Polarization of chemoattractant receptor signaling during neutrophil chemotaxis Science 2000 287 1037 1040 10.1126/science.287.5455.1037 10669415 PMC2822871

74. Lowry WE Huang XY Protein G Beta gamma subunits act on the catalytic domain to stimulate Bruton's agammaglobulinemia tyrosine kinase J Biol Chem 2002 277 1488 1492 10.1074/jbc.M110390200 11698416

75. Tsukada S Simon MI Witte ON Katz A Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase Proc Natl Acad Sci U S A 1994 91 11256 11260 10.1073/pnas.91.23.11256 7972043 PMC45206

76. Bence K Ma W Kozasa T Huang XY Direct stimulation of Bruton's tyrosine kinase by G(q)-protein alpha-subunit Nature 1997 389 296 299 10.1038/38520 9305846

77. de Gorter DJ Beuling EA Kersseboom R Middendorp S van Gils JM Hendriks RW Pals ST Spaargaren M Bruton's tyrosine kinase and phospholipase Cgamma2 mediate chemokine-controlled B cell migration and homing Immunity 2007 26 93 104 10.1016/j.immuni.2006.11.012 17239630

78. Rawlings DJ Schwartz MA Jackson SW Meyer-Bahlburg A Integration of B cell responses through toll-like receptors and antigen receptors Nat Rev Immunol 2012 12 282 294 10.1038/nri3190 22421786 PMC3437941

79. Jefferies CA Doyle S Brunner C Dunne A Brint E Wietek C Walch E Wirth T O'Neill LA Bruton's tyrosine kinase is a toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by toll-like receptor 4 J Biol Chem 2003 278 26258 26264 10.1074/jbc.M301484200 12724322

80. Liu X Zhan Z Li D Xu L Ma F Zhang P Yao H Cao X Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk Nat Immunol 2011 12 416 424 10.1038/ni.2015 21441935

81. Gray P Dunne A Brikos C Jefferies CA Doyle SL O'Neill LA MyD88 adapter-like (mal) is phosphorylated by Bruton's tyrosine kinase during TLR2 and TLR4 signal transduction J Biol Chem 2006 281 10489 10495 10.1074/jbc.M508892200 16439361

82. Bournazos S, Wang TT, Ravetch JV. The role and function of Fcgamma receptors on myeloid cells. Microbiol Spectr. 2016;4 10.1128/microbiolspec.MCHD-0045-2016 PMC5240797 28087938

83. Fearon ER Winkelstein JA Civin CI Pardoll DM Vogelstein B Carrier detection in X-linked agammaglobulinemia by analysis of X-chromosome inactivation N Engl J Med 1987 316 427 431 10.1056/NEJM198702193160802 2880293

84. Puck JM Nussbaum RL Conley ME Carrier detection in X-linked severe combined immunodeficiency based on patterns of X chromosome inactivation J Clin Invest 1987 79 1395 1400 10.1172/JCI112967 2883199 PMC424401

85. Melchers F ten Boekel E Seidl T Kong XC Yamagami T Onishi K Shimizu T Rolink AG Andersson J Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells Immunol Rev 2000 175 33 46 10.1111/j.1600-065X.2000.imr017510.x 10933589

86. Hendriks RW Middendorp S The pre-BCR checkpoint as a cell-autonomous proliferation switch Trends Immunol 2004 25 249 256 10.1016/j.it.2004.02.011 15099565

87. Hendriks RW Bredius RG Pike-Overzet K Staal FJ Biology and novel treatment options for XLA, the most common monogenetic immunodeficiency in man Expert Opin Ther Targets 2011 15 1003 1021 10.1517/14728222.2011.585971 21635151

88. Herzog S Reth M Jumaa H Regulation of B-cell proliferation and differentiation by pre-B-cell receptor signalling Nat Rev Immunol 2009 9 195 205 10.1038/nri2491 19240758

89. Ohnishi K Melchers F The nonimmunoglobulin portion of lambda5 mediates cell-autonomous pre-B cell receptor signaling Nat Immunol 2003 4 849 856 10.1038/ni959 12897780

90. Ubelhart R Bach MP Eschbach C Wossning T Reth M Jumaa H N-linked glycosylation selectively regulates autonomous precursor BCR function Nat Immunol 2010 11 759 765 10.1038/ni.1903 20622883

91. ten Boekel E Yamagami T Andersson J Rolink AG Melchers F The formation and selection of cells expressing preB cell receptors and B cell receptors Curr Top Microbiol Immunol 1999 246 3 9 10396033 10.1007/978-3-642-60162-0_1

92. Wardemann H Yurasov S Schaefer A Young JW Meffre E Nussenzweig MC Predominant autoantibody production by early human B cell precursors Science 2003 301 1374 1377 10.1126/science.1086907 12920303

93. Jumaa H Mitterer M Reth M Nielsen PJ The absence of SLP65 and Btk blocks B cell development at the preB cell receptor-positive stage Eur J Immunol 2001 31 2164 2169 10.1002/1521-4141(200107)31:7<2164::AID-IMMU2164>3.0.CO;2-S 11449370

94. Jumaa H Bossaller L Portugal K Storch B Lotz M Flemming A Schrappe M Postila V Riikonen P Pelkonen J Deficiency of the adaptor SLP-65 in pre-B-cell acute lymphoblastic leukaemia Nature 2003 423 452 456 10.1038/nature01608 12761551

95. Middendorp S Zijlstra AJ Kersseboom R Dingjan GM Jumaa H Hendriks RW Tumor suppressor function of Bruton tyrosine kinase is independent of its catalytic activity Blood 2005 105 259 265 10.1182/blood-2004-07-2708 15331445

96. Kersseboom R Middendorp S Dingjan GM Dahlenborg K Reth M Jumaa H Hendriks RW Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in pre-B cells J Exp Med 2003 198 91 98 10.1084/jem.20030615 12835482 PMC2196076

97. Thompson EC Cobb BS Sabbattini P Meixlsperger S Parelho V Liberg D Taylor B Dillon N Georgopoulos K Jumaa H Ikaros DNA-binding proteins as integral components of B cell developmental-stage-specific regulatory circuits Immunity 2007 26 335 344 10.1016/j.immuni.2007.02.010 17363301

98. Stadhouders R de Bruijn MJ Rother MB Yuvaraj S Ribeiro de Almeida C Kolovos P Van Zelm MC van Ijcken W Grosveld F Soler E Hendriks RW Pre-B cell receptor signaling induces immunoglobulin kappa locus accessibility by functional redistribution of enhancer-mediated chromatin interactions PLoS Biol 2014 12 e1001791 10.1371/journal.pbio.1001791 24558349 PMC3928034

99. Dingjan GM Middendorp S Dahlenborg K Maas A Grosveld F Hendriks RW Bruton's tyrosine kinase regulates the activation of gene rearrangements at the lambda light chain locus in precursor B cells in the mouse J Exp Med 2001 193 1169 1178 10.1084/jem.193.10.1169 11369788 PMC2193329

100. Gay D Saunders T Camper S Weigert M Receptor editing: an approach by autoreactive B cells to escape tolerance J Exp Med 1993 177 999 1008 10.1084/jem.177.4.999 8459227 PMC2190958

101. Nemazee D Weigert M Revising B cell receptors J Exp Med 2000 191 1813 1817 10.1084/jem.191.11.1813 10839798 PMC2213535

102. Rolink A Grawunder U Haasner D Strasser A Melchers F Immature surface Ig+ B cells can continue to rearrange kappa and lambda L chain gene loci J Exp Med 1993 178 1263 1270 10.1084/jem.178.4.1263 8376934 PMC2191189

103. Petro JB Gerstein RM Lowe J Carter RS Shinners N Khan WN Transitional type 1 and 2 B lymphocyte subsets are differentially responsive to antigen receptor signaling J Biol Chem 2002 277 48009 48019 10.1074/jbc.M200305200 12356763

104. Levine MH Haberman AM Sant'Angelo DB Hannum LG Cancro MP Janeway CA Jr Shlomchik MJ A B-cell receptor-specific selection step governs immature to mature B cell differentiation Proc Natl Acad Sci U S A 2000 97 2743 2748 10.1073/pnas.050552997 10688906 PMC16000

105. Hammad H Vanderkerken M Pouliot P Deswarte K Toussaint W Vergote K Vandersarren L Janssens S Ramou I Savvides SN Transitional B cells commit to marginal zone B cell fate by Taok3-mediated surface expression of ADAM10 Nat Immunol 2017 18 313 320 10.1038/ni.3657 28068307

106. Gibb DR El Shikh M Kang DJ Rowe WJ El Sayed R Cichy J Yagita H Tew JG Dempsey PJ Crawford HC Conrad DH ADAM10 is essential for Notch2-dependent marginal zone B cell development and CD23 cleavage in vivo J Exp Med 2010 207 623 635 10.1084/jem.20091990 20156974 PMC2839139

107. Martin F Kearney JF Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk Immunity 2000 12 39 49 10.1016/S1074-7613(00)80157-0 10661404

108. Corneth OB Klein Wolterink RG Hendriks RW BTK signaling in B cell differentiation and autoimmunity Curr Top Microbiol Immunol 2016 393 67 105 26341110 10.1007/82_2015_478

109. Victora GD. SnapShot: the germinal center reaction. Cell. 2014;159:700–0. e701 10.1016/j.cell.2014.10.012 25417116

110. Oropallo MA Cerutti A Germinal center reaction: antigen affinity and presentation explain it all Trends Immunol 2014 35 287 289 10.1016/j.it.2014.06.001 24934509 PMC4174395

111. Dingjan GM Maas A Nawijn MC Smit L Voerman JS Grosveld F Hendriks RW Severe B Cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase EMBO J 1998 17 5309 5320 10.1093/emboj/17.18.5309 9736610 PMC1170858

112. Kersseboom R Kil L Flierman R van der Zee M Dingjan GM Middendorp S Maas A Hendriks RW Constitutive activation of Bruton's tyrosine kinase induces the formation of autoreactive IgM plasma cells Eur J Immunol 2010 40 2643 2654 10.1002/eji.201040521 20623551

113. Kil LP de Bruijn MJ van Nimwegen M Corneth OB van Hamburg JP Dingjan GM Thaiss F Rimmelzwaan GF Elewaut D Delsing D Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice Blood 2012 119 3744 3756 10.1182/blood-2011-12-397919 22383797

114. Corneth OB de Bruijn MJ Rip J Asmawidjaja PS Kil LP Hendriks RW Enhanced expression of Bruton's tyrosine kinase in B cells drives systemic autoimmunity by disrupting T cell homeostasis J Immunol 2016 197 58 67 10.4049/jimmunol.1600208 27226091

115. Ammann EM, Shanafelt TD, Wright KB, McDowell BD, Link BK, Chrischilles EA. Updating survival estimates in patients with chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL/SLL) based on treatment-free interval length. Leuk Lymphoma. 2017:1–7. 10.1080/10428194.2017.1349905 PMC5769448 28718694

116. Kil LP Yuvaraj S Langerak AW Hendriks RW The role of B cell receptor stimulation in CLL pathogenesis Curr Pharm Des 2012 18 3335 3355 10.2174/138161212801227041 22591389

117. Zenz T Eichhorst B Busch R Denzel T Habe S Winkler D Buhler A Edelmann J Bergmann M Hopfinger G TP53 mutation and survival in chronic lymphocytic leukemia J Clin Oncol 2010 28 4473 4479 10.1200/JCO.2009.27.8762 20697090

118. Gonzalez D Martinez P Wade R Hockley S Oscier D Matutes E Dearden CE Richards SM Catovsky D Morgan GJ Mutational status of the TP53 gene as a predictor of response and survival in patients with chronic lymphocytic leukemia: results from the LRF CLL4 trial J Clin Oncol 2011 29 2223 2229 10.1200/JCO.2010.32.0838 21483000

119. Robak P Robak T Novel synthetic drugs currently in clinical development for chronic lymphocytic leukemia Expert Opin Investig Drugs 2017 26 1249 1265 10.1080/13543784.2017.1384814 28942659

120. Malcikova J Smardova J Rocnova L Tichy B Kuglik P Vranova V Cejkova S Svitakova M Skuhrova Francova H Brychtova Y Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage Blood 2009 114 5307 5314 10.1182/blood-2009-07-234708 19850740

121. Zenz T Krober A Scherer K Habe S Buhler A Benner A Denzel T Winkler D Edelmann J Schwanen C Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up Blood 2008 112 3322 3329 10.1182/blood-2008-04-154070 18689542

122. Chiorazzi N Ferrarini M Cellular origin(s) of chronic lymphocytic leukemia: cautionary notes and additional considerations and possibilities Blood 2011 117 1781 1791 10.1182/blood-2010-07-155663 21148333 PMC3056635

123. Klein U Tu Y Stolovitzky GA Mattioli M Cattoretti G Husson H Freedman A Inghirami G Cro L Baldini L Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells J Exp Med 2001 194 1625 1638 10.1084/jem.194.11.1625 11733577 PMC2193527

124. Seifert M Sellmann L Bloehdorn J Wein F Stilgenbauer S Durig J Kuppers R Cellular origin and pathophysiology of chronic lymphocytic leukemia J Exp Med 2012 209 2183 2198 10.1084/jem.20120833 23091163 PMC3501361

125. Griffin DO Holodick NE Rothstein TL Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70 J Exp Med 2011 208 67 80 10.1084/jem.20101499 21220451 PMC3023138

126. DiLillo DJ Weinberg JB Yoshizaki A Horikawa M Bryant JM Iwata Y Matsushita T Matta KM Chen Y Venturi GM Chronic lymphocytic leukemia and regulatory B cells share IL-10 competence and immunosuppressive function Leukemia 2013 27 170 182 10.1038/leu.2012.165 22713648 PMC3742013

127. Muggen AF Singh SP Hendriks RW Langerak AW Targeting signaling pathways in chronic lymphocytic leukemia Curr Cancer Drug Targets 2016 16 669 688 10.2174/1568009616666160408145623 27055580

128. Damle RN Wasil T Fais F Ghiotto F Valetto A Allen SL Buchbinder A Budman D Dittmar K Kolitz J Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia Blood 1999 94 1840 1847 10477712

129. Agathangelidis A Darzentas N Hadzidimitriou A Brochet X Murray F Yan XJ Davis Z van Gastel-Mol EJ Tresoldi C Chu CC Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies Blood 2012 119 4467 4475 10.1182/blood-2011-11-393694 22415752 PMC3392073

130. Murray F Darzentas N Hadzidimitriou A Tobin G Boudjogra M Scielzo C Laoutaris N Karlsson K Baran-Marzsak F Tsaftaris A Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis Blood 2008 111 1524 1533 10.1182/blood-2007-07-099564 17959859

131. Hayakawa K Formica AM Colombo MJ Shinton SA Brill-Dashoff J Morse Iii HC Li YS Hardy RR Loss of a chromosomal region with synteny to human 13q14 occurs in mouse chronic lymphocytic leukemia that originates from early-generated B-1 B cells Leukemia 2016 30 1510 1519 10.1038/leu.2016.61 27055869 PMC4979312

132. Chen SS Batliwalla F Holodick NE Yan XJ Yancopoulos S Croce CM Rothstein TL Chiorazzi N Autoantigen can promote progression to a more aggressive TCL1 leukemia by selecting variants with enhanced B-cell receptor signaling Proc Natl Acad Sci U S A 2013 110 E1500 E1507 10.1073/pnas.1300616110 23550156 PMC3631671

133. Singh SP Pillai SY de Bruijn MJW Stadhouders R Corneth OBJ van den Ham HJ Muggen A van IW SE Kuil A Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling Oncotarget 2017 8 71981 71995 29069762 10.18632/oncotarget.18234 PMC5641105

134. Messmer BT Albesiano E Efremov DG Ghiotto F Allen SL Kolitz J Foa R Damle RN Fais F Messmer D Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia J Exp Med 2004 200 519 525 10.1084/jem.20040544 15314077 PMC2211936

135. Herve M Xu K Ng YS Wardemann H Albesiano E Messmer BT Chiorazzi N Meffre E Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity J Clin Invest 2005 115 1636 1643 10.1172/JCI24387 15902303 PMC1088018

136. Lanemo Myhrinder A Hellqvist E Sidorova E Soderberg A Baxendale H Dahle C Willander K Tobin G Backman E Soderberg O A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies Blood 2008 111 3838 3848 10.1182/blood-2007-11-125450 18223168

137. Hoogeboom R van Kessel KP Hochstenbach F Wormhoudt TA Reinten RJ Wagner K Kater AP Guikema JE Bende RJ van Noesel CJ A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi J Exp Med 2013 210 59 70 10.1084/jem.20121801 23296468 PMC3549718

138. Jimenez de Oya N De Giovanni M Fioravanti J Ubelhart R Di Lucia P Fiocchi A Iacovelli S Efremov DG Caligaris-Cappio F Jumaa H Pathogen-specific B-cell receptors drive chronic lymphocytic leukemia by light-chain-dependent cross-reaction with autoantigens EMBO Mol Med 2017 9 1482 1490 10.15252/emmm.201707732 28899929 PMC5666309

139. Duhren-von Minden M Ubelhart R Schneider D Wossning T Bach MP Buchner M Hofmann D Surova E Follo M Kohler F Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling Nature 2012 489 309 312 10.1038/nature11309 22885698

140. Minici C Gounari M Ubelhart R Scarfo L Duhren-von Minden M Schneider D Tasdogan A Alkhatib A Agathangelidis A Ntoufa S Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia Nat Commun 2017 8 15746 10.1038/ncomms15746 28598442 PMC5472768

141. Herman SE Gordon AL Hertlein E Ramanunni A Zhang X Jaglowski S Flynn J Jones J Blum KA Buggy JJ Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765 Blood 2011 117 6287 6296 10.1182/blood-2011-01-328484 21422473 PMC3122947

142. Ponader S Chen SS Buggy JJ Balakrishnan K Gandhi V Wierda WG Keating MJ O'Brien S Chiorazzi N Burger JA The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo Blood 2012 119 1182 1189 10.1182/blood-2011-10-386417 22180443 PMC4916557

143. Kil LP de Bruijn MJ van Hulst JA Langerak AW Yuvaraj S Hendriks RW Bruton's tyrosine kinase mediated signaling enhances leukemogenesis in a mouse model for chronic lymphocytic leukemia Am J Blood Res 2013 3 71 83 23359016 PMC3555194

144. Ramsay AD Rodriguez-Justo M Chronic lymphocytic leukaemia--the role of the microenvironment pathogenesis and therapy Br J Haematol 2013 162 15 24 10.1111/bjh.12344 23617880

145. Burger JA Gribben JG The microenvironment in chronic lymphocytic leukemia (CLL) and other B cell malignancies: insight into disease biology and new targeted therapies Semin Cancer Biol 2014 24 71 81 10.1016/j.semcancer.2013.08.011 24018164

146. de Rooij MF Kuil A Geest CR Eldering E Chang BY Buggy JJ Pals ST Spaargaren M The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia Blood 2012 119 2590 2594 10.1182/blood-2011-11-390989 22279054

147. Cheah CY Seymour JF Wang ML Mantle cell lymphoma J Clin Oncol 2016 34 1256 1269 10.1200/JCO.2015.63.5904 26755518

148. Bertoni F Rinaldi A Zucca E Cavalli F Update on the molecular biology of mantle cell lymphoma Hematol Oncol 2006 24 22 27 10.1002/hon.767 16402392

149. Meggendorfer M Kern W Haferlach C Haferlach T Schnittger S SOX11 overexpression is a specific marker for mantle cell lymphoma and correlates with t(11;14) translocation, CCND1 expression and an adverse prognosis Leukemia 2013 27 2388 2391 10.1038/leu.2013.141 23648671

150. Navarro A Clot G Royo C Jares P Hadzidimitriou A Agathangelidis A Bikos V Darzentas N Papadaki T Salaverria I Molecular subsets of mantle cell lymphoma defined by the IGHV mutational status and SOX11 expression have distinct biologic and clinical features Cancer Res 2012 72 5307 5316 10.1158/0008-5472.CAN-12-1615 22915760 PMC3763938

151. Cinar M Hamedani F Mo Z Cinar B Amin HM Alkan S Bruton tyrosine kinase is commonly overexpressed in mantle cell lymphoma and its attenuation by Ibrutinib induces apoptosis Leuk Res 2013 37 1271 1277 10.1016/j.leukres.2013.07.028 23962569

152. Pighi C Gu TL Dalai I Barbi S Parolini C Bertolaso A Pedron S Parisi A Ren J Cecconi D Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling Cell Oncol (Dordr) 2011 34 141 153 10.1007/s13402-011-0019-7 21394647 PMC3063577

153. Boyd RS Jukes-Jones R Walewska R Brown D Dyer MJ Cain K Protein profiling of plasma membranes defines aberrant signaling pathways in mantle cell lymphoma Mol Cell Proteomics 2009 8 1501 1515 10.1074/mcp.M800515-MCP200 19346216 PMC2709182

154. Chang BY Francesco M De Rooij MF Magadala P Steggerda SM Huang MM Kuil A Herman SE Chang S Pals ST Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients Blood 2013 122 2412 2424 10.1182/blood-2013-02-482125 23940282 PMC3790509

155. Treon SP Xu L Yang G Zhou Y Liu X Cao Y Sheehy P Manning RJ Patterson CJ Tripsas C MYD88 L265P somatic mutation in Waldenstrom's macroglobulinemia N Engl J Med 2012 367 826 833 10.1056/NEJMoa1200710 22931316

156. Pasqualucci L Trifonov V Fabbri G Ma J Rossi D Chiarenza A Wells VA Grunn A Messina M Elliot O Analysis of the coding genome of diffuse large B-cell lymphoma Nat Genet 2011 43 830 837 10.1038/ng.892 21804550 PMC3297422

157. Puente XS Pinyol M Quesada V Conde L Ordonez GR Villamor N Escaramis G Jares P Bea S Gonzalez-Diaz M Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia Nature 2011 475 101 105 10.1038/nature10113 21642962 PMC3322590

158. Ngo VN Young RM Schmitz R Jhavar S Xiao W Lim KH Kohlhammer H Xu W Yang Y Zhao H Oncogenically active MYD88 mutations in human lymphoma Nature 2011 470 115 119 10.1038/nature09671 21179087 PMC5024568

159. Montesinos-Rongen M Godlewska E Brunn A Wiestler OD Siebert R Deckert M Activating L265P mutations of the MYD88 gene are common in primary central nervous system lymphoma Acta Neuropathol 2011 122 791 792 10.1007/s00401-011-0891-2 22020631

160. Yang G Zhou Y Liu X Xu L Cao Y Manning RJ Patterson CJ Buhrlage SJ Gray N Tai YT A mutation in MYD88 (L265P) supports the survival of lymphoplasmacytic cells by activation of Bruton tyrosine kinase in Waldenstrom macroglobulinemia Blood 2013 122 1222 1232 10.1182/blood-2012-12-475111 23836557

161. Hunter ZR Xu L Yang G Zhou Y Liu X Cao Y Manning RJ Tripsas C Patterson CJ Sheehy P Treon SP The genomic landscape of Waldenstrom macroglobulinemia is characterized by highly recurring MYD88 and WHIM-like CXCR4 mutations, and small somatic deletions associated with B-cell lymphomagenesis Blood 2014 123 1637 1646 10.1182/blood-2013-09-525808 24366360

162. Ngo HT Leleu X Lee J Jia X Melhem M Runnels J Moreau AS Burwick N Azab AK Roccaro A SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia Blood 2008 112 150 158 10.1182/blood-2007-12-129395 18448868 PMC2435685

163. Iqbal J Shen Y Huang X Liu Y Wake L Liu C Deffenbacher K Lachel CM Wang C Rohr J Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma Blood 2015 125 1137 1145 10.1182/blood-2014-04-566778 25498913 PMC4326773

164. Dunleavy K Wilson WH Primary mediastinal B-cell lymphoma and mediastinal gray zone lymphoma: do they require a unique therapeutic approach? Blood 2015 125 33 39 10.1182/blood-2014-05-575092 25499450 PMC4281829

165. Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling Nature 2000 403 503 511 10.1038/35000501 10676951

166. Lenz G Nagel I Siebert R Roschke AV Sanger W Wright GW Dave SS Tan B Zhao H Rosenwald A Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma J Exp Med 2007 204 633 643 10.1084/jem.20062041 17353367 PMC2137913

167. Roschewski M Staudt LM Wilson WH Diffuse large B-cell lymphoma-treatment approaches in the molecular era Nat Rev Clin Oncol 2014 11 12 23 10.1038/nrclinonc.2013.197 24217204 PMC7709161

168. Davis RE Brown KD Siebenlist U Staudt LM Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells J Exp Med 2001 194 1861 1874 10.1084/jem.194.12.1861 11748286 PMC2193582

169. Ngo VN Davis RE Lamy L Yu X Zhao H Lenz G Lam LT Dave S Yang L Powell J Staudt LM A loss-of-function RNA interference screen for molecular targets in cancer Nature 2006 441 106 110 10.1038/nature04687 16572121

170. Compagno M Lim WK Grunn A Nandula SV Brahmachary M Shen Q Bertoni F Ponzoni M Scandurra M Califano A Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma Nature 2009 459 717 721 10.1038/nature07968 19412164 PMC2973325

171. Lenz G Davis RE Ngo VN Lam L George TC Wright GW Dave SS Zhao H Xu W Rosenwald A Oncogenic CARD11 mutations in human diffuse large B cell lymphoma Science 2008 319 1676 1679 10.1126/science.1153629 18323416

172. Davis RE Ngo VN Lenz G Tolar P Young RM Romesser PB Kohlhammer H Lamy L Zhao H Yang Y Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma Nature 2010 463 88 92 10.1038/nature08638 20054396 PMC2845535

173. Chen L Monti S Juszczynski P Ouyang J Chapuy B Neuberg D Doench JG Bogusz AM Habermann TM Dogan A SYK inhibition modulates distinct PI3K/AKT- dependent survival pathways and cholesterol biosynthesis in diffuse large B cell lymphomas Cancer Cell 2013 23 826 838 10.1016/j.ccr.2013.05.002 23764004 PMC3700321

174. Havranek O Xu J Kohrer S Wang Z Becker L Comer JM Henderson J Ma W Man Chun Ma J Westin JR Tonic B-cell receptor signaling in diffuse large B-cell lymphoma Blood 2017 130 995 1006 10.1182/blood-2016-10-747303 28646116 PMC5813722

175. Pfeifer M Grau M Lenze D Wenzel SS Wolf A Wollert-Wulf B Dietze K Nogai H Storek B Madle H PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma Proc Natl Acad Sci U S A 2013 110 12420 12425 10.1073/pnas.1305656110 23840064 PMC3725065

176. Lenz G Wright GW Emre NC Kohlhammer H Dave SS Davis RE Carty S Lam LT Shaffer AL Xiao W Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways Proc Natl Acad Sci U S A 2008 105 13520 13525 10.1073/pnas.0804295105 18765795 PMC2533222

177. Grommes C Pastore A Palaskas N Tang SS Campos C Schartz D Codega P Nichol D Clark O Hsieh WY Ibrutinib unmasks critical role of Bruton tyrosine kinase in primary CNS lymphoma Cancer Discov 2017 7 1018 1029 10.1158/2159-8290.CD-17-0613 28619981 PMC5581705

178. Krysiak K Gomez F White BS Matlock M Miller CA Trani L Fronick CC Fulton RS Kreisel F Cashen AF Recurrent somatic mutations affecting B-cell receptor signaling pathway genes in follicular lymphoma Blood 2017 129 473 483 10.1182/blood-2016-07-729954 28064239 PMC5270390

179. Yang Y Shi J Gu Z Salama ME Das S Wendlandt E Xu H Huang J Tao Y Hao M Bruton tyrosine kinase is a therapeutic target in stem-like cells from multiple myeloma Cancer Res 2015 75 594 604 10.1158/0008-5472.CAN-14-2362 25589346 PMC4384656

180. Gu C Peng H Lu Y Yang H Tian Z Yin G Zhang W Lu S Zhang Y Yang Y BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling Oncotarget 2017 8 56858 56867 28915637 10.18632/oncotarget.18096 PMC5593608

181. Tai YT Chang BY Kong SY Fulciniti M Yang G Calle Y Hu Y Lin J Zhao JJ Cagnetta A Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma Blood 2012 120 1877 1887 10.1182/blood-2011-12-396853 22689860 PMC3433091

182. Yan Q Huang Y Watkins AJ Kocialkowski S Zeng N Hamoudi RA Isaacson PG de Leval L Wotherspoon A Du MQ BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas Haematologica 2012 97 595 598 10.3324/haematol.2011.054080 22102703 PMC3347666

183. Clipson A Wang M de Leval L Ashton-Key M Wotherspoon A Vassiliou G Bolli N Grove C Moody S Escudero-Ibarz L KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype Leukemia 2015 29 1177 1185 10.1038/leu.2014.330 25428260

184. Pan Z Scheerens H Li SJ Schultz BE Sprengeler PA Burrill LC Mendonca RV Sweeney MD Scott KC Grothaus PG Discovery of selective irreversible inhibitors for Bruton's tyrosine kinase ChemMedChem 2007 2 58 61 10.1002/cmdc.200600221 17154430

185. Honigberg LA Smith AM Sirisawad M Verner E Loury D Chang B Li S Pan Z Thamm DH Miller RA Buggy JJ The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy Proc Natl Acad Sci U S A 2010 107 13075 13080 10.1073/pnas.1004594107 20615965 PMC2919935

186. Advani RH Buggy JJ Sharman JP Smith SM Boyd TE Grant B Kolibaba KS Furman RR Rodriguez S Chang BY Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies J Clin Oncol 2013 31 88 94 10.1200/JCO.2012.42.7906 23045577 PMC5505166

187. Wang ML Rule S Martin P Goy A Auer R Kahl BS Jurczak W Advani RH Romaguera JE Williams ME Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma N Engl J Med 2013 369 507 516 10.1056/NEJMoa1306220 23782157 PMC4513941

188. Treon SP Tripsas CK Meid K Warren D Varma G Green R Argyropoulos KV Yang G Cao Y Xu L Ibrutinib in previously treated Waldenstrom's macroglobulinemia N Engl J Med 2015 372 1430 1440 10.1056/NEJMoa1501548 25853747

189. Noy A de Vos S Thieblemont C Martin P Flowers CR Morschhauser F Collins GP Ma S Coleman M Peles S Targeting Bruton tyrosine kinase with ibrutinib in relapsed/refractory marginal zone lymphoma Blood 2017 129 2224 2232 10.1182/blood-2016-10-747345 28167659 PMC5399483

190. Burger JA, Wiestner A. Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nat Rev Cancer. 2018; 10.1038/nrc.2017.121 29348577

191. Mason JA Hagel KR Hawk MA Schafer ZT Metabolism during ECM detachment: Achilles heel of cancer cells? Trends Cancer 2017 3 475 481 10.1016/j.trecan.2017.04.009 28718402

192. Pals ST Kersten MJ Spaargaren M Targeting cell adhesion and homing as strategy to cure Waldenstrom's macroglobulinemia Best Pract Res Clin Haematol 2016 29 161 168 10.1016/j.beha.2016.08.019 27825462

193. Woyach JA Furman RR Liu TM Ozer HG Zapatka M Ruppert AS Xue L Li DH Steggerda SM Versele M Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib N Engl J Med 2014 370 2286 2294 10.1056/NEJMoa1400029 24869598 PMC4144824

194. Furman RR Cheng S Lu P Setty M Perez AR Guo A Racchumi J Xu G Wu H Ma J Ibrutinib resistance in chronic lymphocytic leukemia N Engl J Med 2014 370 2352 2354 10.1056/NEJMc1402716 24869597 PMC4512173

195. Kadri S Lee J Fitzpatrick C Galanina N Sukhanova M Venkataraman G Sharma S Long B Petras K Theissen M Clonal evolution underlying leukemia progression and Richter transformation in patients with ibrutinib-relapsed CLL Blood Adv 2017 1 715 727 10.1182/bloodadvances.2016003632 29296715 PMC5728051

196. Wilson WH Young RM Schmitz R Yang Y Pittaluga S Wright G Lih CJ Williams PM Shaffer AL Gerecitano J Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma Nat Med 2015 21 922 926 10.1038/nm.3884 26193343 PMC8372245

197. Wu C de Miranda NF Chen L Wasik AM Mansouri L Jurczak W Galazka K Dlugosz-Danecka M Machaczka M Zhang H Genetic heterogeneity in primary and relapsed mantle cell lymphomas: impact of recurrent CARD11 mutations Oncotarget 2016 7 38180 38190 27224912 10.18632/oncotarget.9500 PMC5122381

198. Maddocks KJ Ruppert AS Lozanski G Heerema NA Zhao W Abruzzo L Lozanski A Davis M Gordon A Smith LL Etiology of Ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia JAMA Oncol 2015 1 80 87 10.1001/jamaoncol.2014.218 26182309 PMC4520535

199. Coutre SE Furman RR Flinn IW Burger JA Blum K Sharman J Jones J Wierda W Zhao W Heerema NA Extended treatment with single-agent Ibrutinib at the 420 mg dose leads to durable responses in chronic lymphocytic leukemia/small lymphocytic lymphoma Clin Cancer Res 2017 23 1149 1155 10.1158/1078-0432.CCR-16-1431 28073846 PMC6317338

200. Brown JR, Hillmen P, O'Brien S, Barrientos JC, Reddy NM, Coutre SE, Tam CS, Mulligan SP, Jaeger U, Barr PM, et al. Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL. Leukemia. 2017; 10.1038/leu.2017.175 PMC5770586 28592889

201. Mulligan SP Ward CM Whalley D Hilmer SN Atrial fibrillation, anticoagulant stroke prophylaxis and bleeding risk with ibrutinib therapy for chronic lymphocytic leukaemia and lymphoproliferative disorders Br J Haematol 2016 175 359 364 10.1111/bjh.14321 27611114

202. Jones JA Hillmen P Coutre S Tam C Furman RR Barr PM Schuster SJ Kipps TJ Flinn IW Jaeger U Use of anticoagulants and antiplatelet in patients with chronic lymphocytic leukaemia treated with single-agent ibrutinib Br J Haematol 2017 178 286 291 10.1111/bjh.14660 28397242 PMC6084297

203. Wiczer TE Levine LB Brumbaugh J Coggins J Zhao Q Ruppert AS Rogers K McCoy A Mousa L Guha A Cumulative incidence, risk factors, and management of atrial fibrillation in patients receiving ibrutinib Blood Adv 2017 1 1739 1748 29296820 10.1182/bloodadvances.2017009720 PMC5728342

204. McMullen JR Boey EJ Ooi JY Seymour JF Keating MJ Tam CS Ibrutinib increases the risk of atrial fibrillation, potentially through inhibition of cardiac PI3K-Akt signaling Blood 2014 124 3829 3830 10.1182/blood-2014-10-604272 25498454

205. Byrd JC Furman RR Coutre SE Burger JA Blum KA Coleman M Wierda WG Jones JA Zhao W Heerema NA Three-year follow-up of treatment-naïve and previously treated patients with CLL and SLL receiving single-agent ibrutinib Blood 2015 125 2497 2506 10.1182/blood-2014-10-606038 25700432 PMC4400288

206. O'Brien SM, Furman RR, Coutre SE, Flinn IW, Burger J, Blum K, Sharman J, Wierda WG, Jones J, Zhao W, et al. Five-year experience with single-agent ibrutinib in patients with previously untreated and relapsed/refractory chronic lymphocytic leukemia/small lymphocytic leukemia. Blood. 2016;128

207. Herman SEM Montraveta A Niemann CU Mora-Jensen H Gulrajani M Krantz F Mantel R Smith LL McClanahan F Harrington BK The Bruton tyrosine kinase (BTK) inhibitor Acalabrutinib demonstrates potent on-target effects and efficacy in two mouse models of chronic lymphocytic leukemia Clin Cancer Res 2017 23 2831 2841 10.1158/1078-0432.CCR-16-0463 27903679 PMC5548968

208. Quek LS Bolen J Watson SP A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen Curr Biol 1998 8 1137 1140 10.1016/S0960-9822(98)70471-3 9778529

209. Futatani T Watanabe C Baba Y Tsukada S Ochs HD Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X-linked agammaglobulinaemia and carrier females Br J Haematol 2001 114 141 149 10.1046/j.1365-2141.2001.02905.x 11472359

210. Bye AP Unsworth AJ Desborough MJ Hildyard CAT Appleby N Bruce D Kriek N Nock SH Sage T Hughes CE Gibbins JM Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib Blood Adv 2017 1 2610 2623 29296914 10.1182/bloodadvances.2017011999 PMC5728643

211. Tillman BF, Pauff JM, Satyanarayana G, Talbott M, Warner JL. Systematic review of infectious events with the BTK inhibitor ibrutinib in the treatment of haematologic malignancies. Eur J Haematol. 2017; 10.1111/ejh.13020 29285806

212. Long M Beckwith K Do P Mundy BL Gordon A Lehman AM Maddocks KJ Cheney C Jones JA Flynn JM Ibrutinib treatment improves T cell number and function in CLL patients J Clin Invest 2017 127 3052 3064 10.1172/JCI89756 28714866 PMC5531425

213. Schutt SD Fu J Nguyen H Bastian D Heinrichs J Wu Y Liu C DG MD Pidala J Yu XZ Inhibition of BTK and ITK with Ibrutinib is effective in the prevention of chronic graft-versus-host disease in mice PLoS One 2015 10 e0137641 10.1371/journal.pone.0137641 26348529 PMC4562702

214. Dubovsky JA Flynn R Du J Harrington BK Zhong Y Kaffenberger B Yang C Towns WH Lehman A Johnson AJ Ibrutinib treatment ameliorates murine chronic graft-versus-host disease J Clin Invest 2014 124 4867 4876 10.1172/JCI75328 25271622 PMC4347242

215. Ryan CE Sahaf B Logan AC O'Brien S Byrd JC Hillmen P Brown JR Dyer MJ Mato AR Keating MJ Ibrutinib efficacy and tolerability in patients with relapsed chronic lymphocytic leukemia following allogeneic HCT Blood 2016 128 2899 2908 10.1182/blood-2016-06-715284 27802969 PMC5179333

216. Kohrt HE Sagiv-Barfi I Rafiq S Herman SE Butchar JP Cheney C Zhang X Buggy JJ Muthusamy N Levy R Ibrutinib antagonizes rituximab-dependent NK cell-mediated cytotoxicity Blood 2014 123 1957 1960 10.1182/blood-2014-01-547869 24652965 PMC3962169

217. Wu J Liu C Tsui ST Liu D Second-generation inhibitors of Bruton tyrosine kinase J Hematol Oncol 2016 9 80 10.1186/s13045-016-0313-y 27590878 PMC5010774

218. Harrington BK Gardner HL Izumi R Hamdy A Rothbaum W Coombes KR Covey T Kaptein A Gulrajani M Van Lith B Preclinical evaluation of the novel BTK inhibitor Acalabrutinib in canine models of B-cell non-Hodgkin lymphoma PLoS One 2016 11 e0159607 10.1371/journal.pone.0159607 27434128 PMC4951150

219. Wang M, Rule S, Zinzani PL, Goy A, Casasnovas O, Smith SD, Damaj G, Doorduijn J, Lamy T, Morschhauser F, et al. Acalabrutinib in relapsed or refractory mantle cell lymphoma (ACE-LY-004): a single-arm, multicentre, phase 2 trial. Lancet. 2017; 10.1016/S0140-6736(17)33108-2 PMC7864374 29241979

220. Acalabrutinib approved for MCL. Cancer Discov. 2018;8:OF6. 10.1158/2159-8290.CD-NB2017-158 29127086

221. Thompson PA Burger JA Bruton's tyrosine kinase inhibitors: first and second generation agents for patients with chronic lymphocytic leukemia (CLL) Expert Opin Investig Drugs 2018 27 31 42 10.1080/13543784.2018.1404027 29125406 PMC11784538

222. Walter HS Rule SA Dyer MJ Karlin L Jones C Cazin B Quittet P Shah N Hutchinson CV Honda H A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies Blood 2016 127 411 419 10.1182/blood-2015-08-664086 26542378 PMC4731845

223. Koprulu AD Ellmeier W The role of Tec family kinases in mononuclear phagocytes Crit Rev Immunol 2009 29 317 333 10.1615/CritRevImmunol.v29.i4.30 19673686

224. Mirsafian H Ripen AM Leong WM Chear CT Bin Mohamad S Merican AF Transcriptome profiling of monocytes from XLA patients revealed the innate immune function dysregulation due to the BTK gene expression deficiency Sci Rep 2017 7 6836 10.1038/s41598-017-06342-5 28754963 PMC5533715

225. Doyle SL Jefferies CA Feighery C O'Neill LA Signaling by toll-like receptors 8 and 9 requires Bruton's tyrosine kinase J Biol Chem 2007 282 36953 36960 10.1074/jbc.M707682200 17932028

226. Sochorova K Horvath R Rozkova D Litzman J Bartunkova J Sediva A Spisek R Impaired toll-like receptor 8-mediated IL-6 and TNF-alpha production in antigen-presenting cells from patients with X-linked agammaglobulinemia Blood 2007 109 2553 2556 10.1182/blood-2006-07-037960 17090647

227. Lougaris V Baronio M Vitali M Tampella G Cattalini M Tassone L Soresina A Badolato R Plebani A Bruton tyrosine kinase mediates TLR9-dependent human dendritic cell activation J Allergy Clin Immunol 2014 133 1644 1650 10.1016/j.jaci.2013.12.1085 24612681

228. Marron TU Martinez-Gallo M Yu JE Cunningham-Rundles C Toll-like receptor 4-, 7-, and 8-activated myeloid cells from patients with X-linked agammaglobulinemia produce enhanced inflammatory cytokines J Allergy Clin Immunol 2012 129 184 190 10.1016/j.jaci.2011.10.009 22088613 PMC3428022

229. Taneichi H Kanegane H Sira MM Futatani T Agematsu K Sako M Kaneko H Kondo N Kaisho T Miyawaki T Toll-like receptor signaling is impaired in dendritic cells from patients with X-linked agammaglobulinemia Clin Immunol 2008 126 148 154 10.1016/j.clim.2007.10.005 18271077

230. Ni Gabhann J Hams E Smith S Wynne C Byrne JC Brennan K Spence S Kissenpfennig A Johnston JA Fallon PG Jefferies CA Btk regulates macrophage polarization in response to lipopolysaccharide PLoS One 2014 9 e85834 10.1371/journal.pone.0085834 24465735 PMC3897530

231. Gunderson AJ Kaneda MM Tsujikawa T Nguyen AV Affara NI Ruffell B Gorjestani S Liudahl SM Truitt M Olson P Bruton tyrosine kinase-dependent immune cell cross-talk drives pancreas cancer Cancer Discov 2016 6 270 285 10.1158/2159-8290.CD-15-0827 26715645 PMC4783268

232. Fiorcari S Maffei R Audrito V Martinelli S Ten Hacken E Zucchini P Grisendi G Potenza L Luppi M Burger JA Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia Oncotarget 2016 7 65968 65981 10.18632/oncotarget.11782 27602755 PMC5323207

233. Andreu P Johansson M Affara NI Pucci F Tan T Junankar S Korets L Lam J Tawfik D DeNardo DG FcRgamma activation regulates inflammation-associated squamous carcinogenesis Cancer Cell 2010 17 121 134 10.1016/j.ccr.2009.12.019 20138013 PMC3082507

234. Pucci F Garris C Lai CP Newton A Pfirschke C Engblom C Alvarez D Sprachman M Evavold C Magnuson A SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions Science 2016 352 242 246 10.1126/science.aaf1328 26989197 PMC4960636

235. Coffelt SB Wellenstein MD de Visser KE Neutrophils in cancer: neutral no more Nat Rev Cancer 2016 16 431 446 10.1038/nrc.2016.52 27282249

236. Marron TU Rohr K Martinez-Gallo M Yu J Cunningham-Rundles C TLR signaling and effector functions are intact in XLA neutrophils Clin Immunol 2010 137 74 80 10.1016/j.clim.2010.06.011 20634142 PMC2941555

237. Farrar JE Rohrer J Conley ME Neutropenia in X-linked agammaglobulinemia Clin Immunol Immunopathol 1996 81 271 276 10.1006/clin.1996.0188 8938104

238. Fiedler K Sindrilaru A Terszowski G Kokai E Feyerabend TB Bullinger L Rodewald HR Brunner C Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia Blood 2011 117 1329 1339 10.1182/blood-2010-04-281170 21063022

239. Mangla A Khare A Vineeth V Panday NN Mukhopadhyay A Ravindran B Bal V George A Rath S Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses Blood 2004 104 1191 1197 10.1182/blood-2004-01-0207 15117762

240. Mueller H Stadtmann A Van Aken H Hirsch E Wang D Ley K Zarbock A Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways Blood 2010 115 3118 3127 10.1182/blood-2009-11-254185 20167705 PMC2858472

241. Volmering S Block H Boras M Lowell CA Zarbock A The neutrophil Btk Signalosome regulates integrin activation during sterile inflammation Immunity 2016 44 73 87 10.1016/j.immuni.2015.11.011 26777396 PMC5030078

242. Stiff A Trikha P Wesolowski R Kendra K Hsu V Uppati S McMichael E Duggan M Campbell A Keller K Myeloid-derived suppressor cells express Bruton's tyrosine kinase and can be depleted in tumor-bearing hosts by Ibrutinib treatment Cancer Res 2016 76 2125 2136 10.1158/0008-5472.CAN-15-1490 26880800 PMC4873459

243. Sagiv-Barfi I Kohrt HE Czerwinski DK Ng PP Chang BY Levy R Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK Proc Natl Acad Sci U S A 2015 112 E966 E972 10.1073/pnas.1500712112 25730880 PMC4352777

244. Gao W, Wang M, Wang L, Lu H, Wu S, Dai B, Ou Z, Zhang L, Heymach JV, Gold KA, et al. Selective antitumor activity of ibrutinib in EGFR-mutant non-small cell lung cancer cells. J Natl Cancer Inst. 2014;106 10.1093/jnci/dju204 PMC4200057 25214559

245. Ruella M Kenderian SS Shestova O Fraietta JA Qayyum S Zhang Q Maus MV Liu X Nunez-Cruz S Klichinsky M The addition of the BTK inhibitor Ibrutinib to anti-CD19 chimeric antigen receptor T cells (CART19) improves responses against mantle cell lymphoma Clin Cancer Res 2016 22 2684 2696 10.1158/1078-0432.CCR-15-1527 26819453

246. Fraietta JA Beckwith KA Patel PR Ruella M Zheng Z Barrett DM Lacey SF Melenhorst JJ McGettigan SE Cook DR Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia Blood 2016 127 1117 1127 10.1182/blood-2015-11-679134 26813675 PMC4778162

247. Dubovsky JA Beckwith KA Natarajan G Woyach JA Jaglowski S Zhong Y Hessler JD Liu TM Chang BY Larkin KM Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes Blood 2013 122 2539 2549 10.1182/blood-2013-06-507947 23886836 PMC3795457

248. Byrd JC Brown JR O'Brien S Barrientos JC Kay NE Reddy NM Coutre S Tam CS Mulligan SP Jaeger U Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia N Engl J Med 2014 371 213 223 10.1056/NEJMoa1400376 24881631 PMC4134521

249. Dreyling M Jurczak W Jerkeman M Silva RS Rusconi C Trneny M Offner F Caballero D Joao C Witzens-Harig M Ibrutinib versus temsirolimus in patients with relapsed or refractory mantle-cell lymphoma: an international, randomised, open-label, phase 3 study Lancet 2016 387 770 778 10.1016/S0140-6736(15)00667-4 26673811

250. Burger JA Keating MJ Wierda WG Hartmann E Hoellenriegel J Rosin NY de Weerdt I Jeyakumar G Ferrajoli A Cardenas-Turanzas M Safety and activity of ibrutinib plus rituximab for patients with high-risk chronic lymphocytic leukaemia: a single-arm, phase 2 study Lancet Oncol 2014 15 1090 1099 10.1016/S1470-2045(14)70335-3 25150798 PMC4174348

251. Chanan-Khan A Cramer P Demirkan F Fraser G Silva RS Grosicki S Pristupa A Janssens A Mayer J Bartlett NL Ibrutinib combined with bendamustine and rituximab compared with placebo, bendamustine, and rituximab for previously treated chronic lymphocytic leukaemia or small lymphocytic lymphoma (HELIOS): a randomised, double-blind, phase 3 study Lancet Oncol 2016 17 200 211 10.1016/S1470-2045(15)00465-9 26655421

252. Wang ML Lee H Chuang H Wagner-Bartak N Hagemeister F Westin J Fayad L Samaniego F Turturro F Oki Y Ibrutinib in combination with rituximab in relapsed or refractory mantle cell lymphoma: a single-centre, open-label, phase 2 trial Lancet Oncol 2016 17 48 56 10.1016/S1470-2045(15)00438-6 26640039

253. Tam C, Grigg AP, Opat S, Ku M, Gilbertson M, Anderson MA, Seymour JF, Ritchie DS, Dicorleto C, Dimovski B, et al. The BTK inhibitor, Bgb-3111, is safe, tolerable, and highly active in patients with relapsed/refractory B-cell malignancies: initial report of a phase 1 first-in-human trial. Blood. 2015;126

254. Noy A, de Vos S, Thieblemont C, Martin P, Flowers C, Morschhauser F, Collins GP, Ma S, Coleman M, Peles S, et al. Single-agent Ibrutinib demonstrates efficacy and safety in patients with relapsed/refractory marginal zone lymphoma: a multicenter, open-label, phase 2 study. Blood. 2016;128:1213.

255. Secchiero P Voltan R Rimondi E Melloni E Athanasakis E Tisato V Gallo S Rigolin GM Zauli G The gamma-secretase inhibitors enhance the anti-leukemic activity of ibrutinib in B-CLL cells Oncotarget 2017 8 59235 59245 10.18632/oncotarget.19494 28938632 PMC5601728

256. Rotin LE Gronda M MacLean N Hurren R Wang X Lin FH Wrana J Datti A Barber DL Minden MD Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism Oncotarget 2016 7 2765 2779 10.18632/oncotarget.6409 26624983 PMC4823070

257. Kelly PN Romero DL Yang Y Shaffer AL 3rd Chaudhary D Robinson S Miao W Rui L Westlin WF Kapeller R Staudt LM Selective interleukin-1 receptor-associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy J Exp Med 2015 212 2189 2201 10.1084/jem.20151074 26621451 PMC4689168

258. Goldstein RL Yang SN Taldone T Chang B Gerecitano J Elenitoba-Johnson K Shaknovich R Tam W Leonard JP Chiosis G Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma J Clin Invest 2015 125 4559 4571 10.1172/JCI80714 26529251 PMC4665772

259. Patel V Keating MJ Wierda WG Gandhi V Preclinical combination of TP-0903, an AXL inhibitor and B-PAC-1, a procaspase-activating compound with ibrutinib in chronic lymphocytic leukemia Leuk Lymphoma 2016 57 1494 1497 10.3109/10428194.2015.1102243 26440741 PMC4963009

260. Lamothe B Cervantes-Gomez F Sivina M Wierda WG Keating MJ Gandhi V Proteasome inhibitor carfilzomib complements ibrutinib's action in chronic lymphocytic leukemia Blood 2015 125 407 410 10.1182/blood-2014-07-585364 25573971 PMC4287645

261. Hing ZA Mantel R Beckwith KA Guinn D Williams E Smith LL Williams K Johnson AJ Lehman AM Byrd JC Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia Blood 2015 125 3128 3132 10.1182/blood-2015-01-621391 25838351 PMC4432007

262. Cervantes-Gomez F Lamothe B Woyach JA Wierda WG Keating MJ Balakrishnan K Gandhi V Pharmacological and protein profiling suggests Venetoclax (ABT-199) as optimal partner with Ibrutinib in chronic lymphocytic leukemia Clin Cancer Res 2015 21 3705 3715 10.1158/1078-0432.CCR-14-2809 25829398 PMC4537801

263. Zhao X Bodo J Sun D Durkin L Lin J Smith MR Hsi ED Combination of ibrutinib with ABT-199: synergistic effects on proliferation inhibition and apoptosis in mantle cell lymphoma cells through perturbation of BTK, AKT and BCL2 pathways Br J Haematol 2015 168 765 768 10.1111/bjh.13149 25284608

264. Rushworth SA Bowles KM Barrera LN Murray MY Zaitseva L MacEwan DJ BTK inhibitor ibrutinib is cytotoxic to myeloma and potently enhances bortezomib and lenalidomide activities through NF-kappaB Cell Signal 2013 25 106 112 10.1016/j.cellsig.2012.09.008 22975686

265. Sagiv-Barfi I Kohrt HE Burckhardt L Czerwinski DK Levy R Ibrutinib enhances the antitumor immune response induced by intratumoral injection of a TLR9 ligand in mouse lymphoma Blood 2015 125 2079 2086 10.1182/blood-2014-08-593137 25662332 PMC4375105

266. Xargay-Torrent S Lopez-Guerra M Rosich L Montraveta A Roldan J Rodriguez V Villamor N Aymerich M Lagisetti C Webb TR The splicing modulator sudemycin induces a specific antitumor response and cooperates with ibrutinib in chronic lymphocytic leukemia Oncotarget 2015 6 22734 22749 10.18632/oncotarget.4212 26068951 PMC4673195

267. Yahiaoui A Meadows SA Sorensen RA Cui ZH Keegan KS Brockett R Chen G Queva C Li L Tannheimer SL PI3Kdelta inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kdelta and BTK inhibitors PLoS One 2017 12 e0171221 10.1371/journal.pone.0171221 28178345 PMC5298344