In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function.

ABSTRACT: Bruton's tyrosine kinase (BTK) is an enzyme needed for B-cell survival, and its inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has, for more than 2 decades, generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using nonlymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas-mediated knockin mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and the morphology of lymphoid organs, as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control and mounted similar humoral immune responses as Y223F and wild-type animals. Collectively, our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase Cγ2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this posttranslational modification is dispensable for the function of BTK.

Ibrutinib Has Time-dependent On- and Off-target Effects on Plasma Biomarkers and Immune Cells in Chronic Lymphocytic Leukemia.

Ibrutinib is a covalently binding inhibitor of the B-cell receptor signaling-mediator Bruton's tyrosine kinase (BTK) with great efficacy in chronic lymphocytic leukemia (CLL). Common side effects like atrial fibrillation (AF), bleeding and infections might be caused by ibrutinib's inhibition of other kinases in non-B cells. Five-year follow-up of plasma biomarkers by proximity extension assay and immune cell numbers by flow cytometry during ibrutinib treatment revealed that 86 of the 265 investigated plasma biomarkers significantly changed during treatment, 74 of which decreased. Among the 12 markers that increased, 6 are associated with cardiovascular diseases and therefore potentially involved in ibrutinib-induced AF. Comparison between healthy donors and X-linked agammaglobulinemia (XLA) patients, who have nonfunctional BTK and essentially lack B cells, showed indicative changes in 53 of the 265 biomarkers while none differed significantly. Hence, neither B cells nor BTK-dependent pathways in other cells seem to influence the levels of the studied plasma biomarkers in healthy donors. Regarding immune cells, the absolute number of T cells, including subsets, decreased, paralleling the decreasing tumor burden. T helper 1 (Th1) cell numbers dropped strongly, while Th2 cells remained relatively stable, causing Th2-skewing. Thus, long-term ibrutinib treatment has a profound impact on the plasma proteome and immune cells in patients with CLL.

Comparative Analysis of BTK Inhibitors and Mechanisms Underlying Adverse Effects.

The cytoplasmic protein-tyrosine kinase BTK plays an essential role for differentiation and survival of B-lineage cells and, hence, represents a suitable drug target. The number of BTK inhibitors (BTKis) in the clinic has increased considerably and currently amounts to at least 22. First-in-class was ibrutinib, an irreversible binder forming a covalent bond to a cysteine in the catalytic region of the kinase, for which we have identified 228 active trials listed at ClinicalTrials.gov. Next-generation inhibitors, acalabrutinib and zanubrutinib, are approved both in the United States and in Europe, and zanubrutinib also in China, while tirabrutinib is currently only registered in Japan. In most cases, these compounds have been used for the treatment of B-lymphocyte tumors. However, an increasing number of trials instead addresses autoimmunity and inflammation in multiple sclerosis, rheumatoid arthritis, pemphigus and systemic lupus erythematosus with the use of either irreversibly binding inhibitors, e.g., evobrutinib and tolebrutinib, or reversibly binding inhibitors, like fenebrutinib. Adverse effects (AEs) have predominantly implicated inhibition of other kinases with a BTKi-binding cysteine in their catalytic domain. Analysis of the reported AEs suggests that ibrutinib-associated atrial fibrillation is caused by binding to ERBB2/HER2 and ERBB4/HER4. However, the binding pattern of BTKis to various additional kinases does not correlate with the common assumption that skin manifestations and diarrhoeas are off-target effects related to EGF receptor inhibition. Moreover, dermatological toxicities, diarrhoea, bleedings and invasive fungal infections often develop early after BTKi treatment initiation and subsequently subside. Conversely, cardiovascular AEs, like hypertension and various forms of heart disease, often persist.

BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib.

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the "gatekeeper" residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

Novel mouse model resistant to irreversible BTK inhibitors: a tool identifying new therapeutic targets and side effects.

Pharmacological inhibitors of Bruton tyrosine kinase (BTK) have revolutionized treatment of B-lymphocyte malignancies and show great promise for dampening autoimmunity. The predominant BTK inhibitors tether irreversibly by covalently binding to cysteine 481 in the BTK catalytic domain. Substitution of cysteine 481 for serine (C481S) is the most common mechanism for acquired drug resistance. We generated a novel C481S knock-in mouse model and, using a battery of tests, no overt B-lymphocyte phenotype was found. B lymphocytes from C481S animals were resistant to irreversible, but sensitive to reversible, BTK inhibitors. In contrast, irreversible inhibitors equally impaired T-lymphocyte activation in mice, mimicking the effect of treatment in patients. This demonstrates that T-lymphocyte blockage is independent of BTK. We suggest that the C481S knock-in mouse can serve as a useful tool for the study of BTK-independent effects of irreversible inhibitors, allowing for the identification of novel therapeutic targets and pinpointing potential side effects.

Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

Splice-correction strategies for treatment of X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Agammaglobulinemia: causative mutations and their implications for novel therapies.

Agammaglobulinemias are primary (inherited) immunodeficiencies characterized by the lack of functional B-cells and antibodies, and are caused by mutations in genes encoding components of the pre-B-cell or B-cell receptor, or their signaling pathways. The known genetic defects do not account for all agammaglobulinemic patients, suggesting that novel mutations underlying the disease remain to be found. While efficient, the current life-maintaining therapy with immunoglobulins and antibiotics is non-curative, prompting research into alternative treatment strategies that aim at rescuing the expression of the affected protein, thus giving rise to functional B-cells. These include gene therapy, which could be used to correct the defective gene or replace it with a functional copy. For a number of genetic defects, another alternative is to modulate the splicing of the affected transcripts. While these technologies are not yet ready for clinical trials in agammaglobulinemia, advances in genomic targeting are likely to make this option viable in the near future.

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.

BACKGROUND: The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. RESULTS: The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. CONCLUSION: Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.

Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT). In B cells, NF-kappaB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-kappaB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells.

Transcriptional pathways controlling the development of CD44(hi) memory phenotype (MP) T cells with "innate-like" functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44(hi), but not in CD44(lo), CD4(+) T cells. Transgenic expression of PLZF during T cell development and in CD4(+) and CD8(+) T cells induced a T cell intrinsic program leading to an increase in peripheral CD44(hi) MP CD4(+) and CD8(+) T cells and a corresponding decrease of naïve CD44(lo) T cells. The MP CD4(+) and CD8(+) T cells produced IFNgamma upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4(+) transgenic T cells showed reduced IL-2 and IFNgamma production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44(hi)CD62L(+) subset. Our data indicate that PLZF is a novel regulator of the development of CD44(hi) MP T cells with a characteristic partial innate-like phenotype.

Defective Toll-like receptor 9-mediated cytokine production in B cells from Bruton's tyrosine kinase-deficient mice.

Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll-like receptor 9 (TLR9) activation and the production of pro-inflammatory cytokines such as interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and IL-12p40. Our data show that Btk-deficient B cells respond more efficiently to CpG-DNA stimulation, producing significantly higher levels of pro-inflammatory cytokines but lower levels of the inhibitory cytokine IL-10. The quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL-12 family, IL-27, was significantly increased in Btk-deficient B cells after CpG-DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk-defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.

Distinct gene expression signature in Btk-defective T1 B-cells.

Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. Mutations in Btk give rise to the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (Xid) in mice. Recent studies have subdivided the mouse immature, or transitional, B-cells into two distinct subsets according to their respective surface markers. Transitional type 1 (T1) and transitional type 2 (T2) cells are also located in distinct anatomic locations. Based on a limited number of markers it has previously been reported that the earliest phenotypic sign of Btk deficiency is manifested at the T2 stage in mice. Here, we report on distinct genome-wide transcriptomic signature differences found in T1 B-lymphocytes from Btk-defective compared to normal mice and demonstrate that Btk deficiency is visible already at this stage.

Regulation of Bruton tyrosine kinase by the peptidylprolyl isomerase Pin1.

Bruton tyrosine kinase (Btk) is expressed in B-lymphocytes. Mutations in Btk cause X-linked agammaglobulinemia in humans. However, the mechanism of activation and signaling of this enzyme has not been fully investigated. We have here shown that the peptidylprolyl cis/trans isomerase (PPIase) Pin1 is a negative regulator of Btk, controlling its expression level by reducing its half-life, whereas the catalytic activity of Btk was unaffected. The negative regulatory effect of Pin1 was observed both in cell lines and in Pin(-/-) mice and was found to be dependent on a functionally intact Btk. This may constitute a feedback loop for the regulation of Btk. The target region in Btk was localized to the pleckstrin homology domain suggesting that interphase phosphorylation of serine 115 (Ser-115) in Btk is required, whereas mitosis phosphorylation of serine 21 (Ser-21) is critical. Accordingly, Pin 1 was shown to associate with Btk through binding to Ser-21 and -115, respectively, both of which lie in a classical Pin1-binding pocket. Using a phosphomitotic antibody, it was found that Btk harbors a bona fide MPM2 epitope corresponding to a phosphorylated serine or threonine residue followed by a proline. Our results indicate that the peptidylprolyl isomerase Pin1 interacts with Btk in a cell cycle-dependent manner, regulating the Btk expression level.

Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling.

Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B-lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full-length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype-phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering-RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA-, Xid-, and Btk-knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid- and Btk-KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C-missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

Gene expression profile of B cells from Xid mice and Btk knockout mice.

Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (Btk KO) and immunocompetent CBA mice were used in microarrays containing more than 12000 genes and expressed-sequence tags. We found 4515 common transcripts expressed in duplicate experiments in the three strains. Out of these, 38 were differentially expressed genes (21 were up-regulated >2-fold and 17 were down-regulated <-2-fold) between CBA and Btk defective (Xid or Btk KO) mice. Ten out of these genes were selected and quantitative real-time PCR was conducted for validation and further investigation. Real-time experiments correlated nicely with the microarray data. No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes whose expression differed (>2-fold) between the two strains. Moreover, when the 38 genes that differed between immunocompetent CBA mice and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant-negative form.

Functional interaction of caveolin-1 with Bruton's tyrosine kinase and Bmx.

Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82--101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.