Kinase Btk Research Articles

Aberrant glycosylation of anti-SARS-CoV-2 spike IgG is a prothrombotic stimulus for platelets

AbstractA subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in severely ill patients with COVID-19 are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesized that platelets might be susceptible to activation by anti–severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies and might contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike immunoglobulin G enhanced platelet-mediated thrombosis on von Willebrand factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcγRIIA, and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by the therapeutic small molecules R406 (fostamatinib) and ibrutinib, which inhibit tyrosine kinases Syk and Btk, respectively, or by the P2Y12 antagonist cangrelor.

Btk Inhibitors: A Medicinal Chemistry and Drug Delivery Perspective.

In the past few years, Bruton’s tyrosine Kinase (Btk) has emerged as new target in medicinal chemistry. Since approval of ibrutinib in 2013 for treatment of different hematological cancers (as leukemias and lymphomas), two other irreversible Btk inhibitors have been launched on the market. In the attempt to overcome irreversible Btk inhibitor limitations, reversible compounds have been developed and are currently under evaluation. In recent years, many Btk inhibitors have been patented and reported in the literature. In this review, we summarized the (ir)reversible Btk inhibitors recently developed and studied clinical trials and preclinical investigations for malignancies, chronic inflammation conditions and SARS-CoV-2 infection, covering advances in the field of medicinal chemistry. Furthermore, the nanoformulations studied to increase ibrutinib bioavailability are reported.

Bletinib ameliorates neutrophilic inflammation and lung injury by inhibiting Src family kinase phosphorylation and activity.

Background and PurposeNeutrophil overactivation is crucial in the pathogenesis of acute lung injury (ALI). Bletinib (3,3′‐dihydroxy‐2′,6′‐bis(p‐hydroxybenzyl)‐5‐methoxybibenzyl), a natural bibenzyl, extracted from the Bletilla plant, exhibits anti‐inflammatory, antibacterial, and antimitotic effects. In this study, we evaluated the therapeutic effects of bletinib in human neutrophilic inflammation and LPS‐mediated ALI in mice.Experimental ApproachIn human neutrophils activated with the formyl peptide (fMLP), we assessed integrin expression, superoxide anion production, degranulation, neutrophil extracellular trap (NET) formation, and adhesion through flow cytometry, spectrophotometry, and immunofluorescence microscopy. Immunoblotting was used to measure phosphorylation of Src family kinases (SFKs) and downstream proteins. Finally, a LPS‐induced ALI model in male BALB/c mice was used to investigate the potential therapeutic effects of bletinib treatment.Key ResultsIn activated human neutrophils, bletinib reduced degranulation, respiratory burst, NET formation, adhesion, migration, and integrin expression; suppressed the enzymic activity of SFKs, including Src, Lyn, Fgr, and Hck; and inhibited the phosphorylation of SFKs as well as Vav and Bruton's tyrosine kinase (Btk). In mice with ALI, the pulmonary sections demonstrated considerable amelioration of prominent inflammatory changes, such as haemorrhage, pulmonary oedema, and neutrophil infiltration, after bletinib treatment.Conclusion and ImplicationsBletinib regulates neutrophilic inflammation by inhibiting the SFK‐Btk‐Vav pathway. Bletinib ameliorates LPS‐induced ALI in mice. Further biochemical optimisation of bletinib may be a promising strategy for the development of novel therapeutic agents for inflammatory diseases.

A Phase I Study Of The Oral Btk Inhibitor ONO-4059 In Patients With Relapsed/Refractory and High Risk Chronic Lymphocytic Leukaemia (CLL)

IntroductionThe B-cell receptor (BCR) pathway plays a central role in signal transduction pathways that regulate survival, activation, proliferation and differentiation of B-lineage lymphoid cells. Bruton's tyrosine kinase (Btk) is a critical kinase in BCR signal transduction and recent studies support that targeting Btk is effective in the treatment of B-cell malignancies. ONO-4059 is a highly potent and selective oral Btk inhibitor with an IC50 in the sub-nmol/L range that has demonstrated anti-tumour activity in several pre-clinical models (Yasuhiro T et al, AACR 2013). MethodsThis Phase I study was initiated to determine the safety, tolerability, dose-limiting toxicity (DLT), pharmacokinetics and pharmacodynamics of ONO-4059 given as monotherapy to patients with relapsed/refractory, high risk CLL for whom no therapy of higher priority was available. In this safety-driven, dose-escalating 3+3 design, ONO-4059 was administered as an oral, daily dose (flat dose) given continuously initially for up to 6 months, with the option of additional dosing up to 2 years. We present the safety and efficacy data on 16 evaluable patients, median age 67 yrs (range 40-77), with high risk cytogenetics (17p [38% (6/16)] or 11q [19% (3/16)], 12/16 displaying unmutated IgVH status, 8/16 displaying TP53 mutation), median baseline tumour burden 4,534mm2 [461-14,492 mm2]. Patients had received a median of 3 prior therapies [2-7], including fludarabine (16/16) and rituximab-containing therapy (13/16). Baseline median haematology parameters included hemoglobin 11.3 g/dL [8.5 -15.2], WBC 56.02 x 109/L [2.06-394.74], platelets 95.5 x 109/L [23-173], lymphocytes 50.6 x 109/L [0.5-390], neutrophils 2.46 x 109/L [0.5-11.1]. Patients were treated with ONO-4059 at doses ranging from 20mg-320mg (cohorts 1-5) and the study is currently ongoing with additional dose escalation cohorts to be completed. ResultsONO-4059 was found to be well tolerated, with no dose limiting toxicities (DLTs). A total of 15 ONO-4059-related Grade 1 (n=10) and 2 (n=5) adverse events were reported in 8 out of 16 patients, including one event of diarrhea (G2) and two events of rash (G1, G2). ONO-4059-related G3 or G4 haematological toxicities were infrequent and independent of dose; neutropenia (n=3 [G4 x1, G3 x 2]), febrile neutropenia (n=1 [G3]) occurring in 2 patients at the 20mg dose level only. Only one Grade 3 ONO-4059-related non-haematological event (pyrexia) was reported. Infection rate was low with only one event of bronchitis reported (G2, related). Three ONO-4059-related SAEs were reported in two patients (febrile neutropenia [G3] and pyrexia [G3] in one patient [20 mg]; rash [G2] in another [80 mg]). The pharmacokinetics of ONO-4059 reflects rapid absorption and elimination, a half-life of ∼6 hours, a dose dependent increase in exposure with no accumulation of ONO-4059 exposure and low inter- or intra-patient variability; with Btk occupancy in peripheral blood (as measured by phosphorylated Btk) being maintained for at least 24 hours across all dose levels.Responses have occurred across all dose levels. All patients experienced rapid reductions in lymphadenopathy observed early in treatment, between D1-D28 of the first cycle, accompanied by an increase in absolute lymphocyte count in some, but not all patients. Best overall response rate according to IWCLL criteria (including modified PR with lymphocytosis) was 70% [based on CT-scan and P/E for 7/10 evaluable patients]; with 2 PR, 5 PR with lymphocytosis (For 7 responding patients, median reduction of lymph nodes was 67% [Range 61%-79%]), 2 SD, 1 patient PD (possible Richter's). All patients showed an improvement in haematological parameters after ≥3 months on treatment. Patients (ONO-4059 20mg [1pt] and 40mg [1pt] respectively) who had grade 3 thrombocytopenia at study baseline had a significant improvement in platelet count (>100 x 109/L) with ONO-4059 treatment. To date, 15 of 16 patients remain on treatment with a median progression-free survival of 99 days [Range 8-268].In conclusion, ONO-4059 is a highly potent and selective oral Btk inhibitor with a favourable safety profile along with promising efficacy in this heavily pre-treated population, with responses observed in relapsed, refractory and 17p deleted patients. Disclosures:Salles:Janssen: Honoraria; Gilead: Honoraria; Celgene: Honoraria. Karlin:Janssen: Honoraria; Celgene: Honoraria. Morschhauser:ONO Pharma: Honoraria; Roche: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria. Dyer:ONO Pharma: Honoraria, Research Funding. Hutchinson:ONO Pharma: Research Funding. Fegan:ONO Pharma: Honoraria. Cartron:ONO Pharma: Honoraria. Knurowski:ONO Pharma: Consultancy. Saunders:ONO Pharma: Consultancy; Pharmacyclics: Consultancy. Wright:ONO Pharma: Consultancy. Honda:ONO Pharma: Employment. Mazur:ONO Pharma: Consultancy. Yoshizawa:Ono Pharma: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment. Birkett:Ono Pharma UK: Employment.

Abstract 1480: Comparative cancer cell panel profiling of kinase inhibitors approved for clinical use from 2018 to 2020

Abstract Kinases are the major anticancer drug target class of the 21st century with nearly sixty small molecule kinase inhibitors approved for clinical use in the first two decades. While there are more than 500 kinases encoded by the human genome, currently approved inhibitors act primarily through approximately twenty different targets. Key to the success of kinase inhibitor therapy has been the simultaneous development of biomarker assays to enable the selection of patients most likely to respond. Predictive drug response biomarkers can be identified with cancer cell panel profiling, which is the parallel testing of compounds on a large panel of cancer cell lines. By correlating drug sensitivity with genomic information of the cell lines, strategies for patient stratification or drug repurposing have been developed [1-3]. In two earlier studies [2,3], we compared the kinase selectivity and the cellular inhibition profiles of all kinase inhibitors approved by the FDA until May 2018. Here, we will present the cancer cell panel profiling of all twenty small molecule kinase inhibitors approved since then. Several of these inhibitors act through well-known targets, such as ALK (lorlatinib), BRAF (encorafenib), EGFR (dacomitinib) and MEK1 (binimetinib, selumetinib). Others act through kinases for which no small molecule inhibitors have been approved before, such as CSF1R (pexidartinib), FGFR (erdafitinib, pemigatinib), c-MET (capmatinib), RET (selpercatinib, pralsetinib) and TRK (larotrectinib, entrectinib). All compounds were profiled on a panel of 102 cancer cell lines, known as Oncolines, representing a wide range of solid tumors and hematological malignancies, and harboring mutant and wild-type versions of many major cancer driver genes [3]. To determine similarities in the mechanisms underlying the anti-proliferative activity of the inhibitors, their IC50 fingerprint in the cell proliferation assays were compared by hierarchical clustering [4]. Compounds that act through the same primary kinase clustered together in this analysis. Exceptions were investigated further by profiling of additional cell lines, representing cancer gene alterations that were not present in the 102 cell line panel, such as FLT3 mutation and TRK-gene fusions, which occur in relatively small cancer patient populations. To compare the genomic targeting of kinase inhibitors acting on the same biochemical target, the cancer cell lines were classified as either “mutant” or “wild-type” for specific cancer gene alterations and were grouped per cancer gene. The relationship between drug sensitivity and cancer gene mutation status was examined. Detailed genomic biomarker analyses and comparative profiling results of novel BTK, EGFR, FGFR, FLT3 and TRK kinase inhibitors will be presented.[1] Mol Cancer Ther 2017;26:2609-17; [2] PLoS ONE 2014;9:e92146; [3] Mol Cancer Ther 2019;18:470-81; [4] Mol Cancer Ther 2016;15:3097-109 Citation Format: Jeffrey J. Kooijman, Wilhelmina E. van Riel, Martine B. Prinsen, Jelle Dylus, Jeroen A. de Roos, Yvonne Grobben, Nicole Willemsen-Seegers, Michelle Muller, Jos de Man, Yugo Narumi, Yusuke Kawase, Rogier C. Buijsman, Suzanne J. van Gerwen, Guido J. Zaman. Comparative cancer cell panel profiling of kinase inhibitors approved for clinical use from 2018 to 2020 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1480.

CG '806, a Novel Pan-FLT3/BTK Multi-Kinase Inhibitor, Induces Cell Cycle Arrest, Apoptosis or Autophagy in AML Cells Depending on FLT3 Mutation Status

Gain-of-function mutations of the fms-like tyrosine kinase 3 (FLT3) play a pivotal role in hematopoietic malignancies (Gilliland et al., 2002). Therefore, FLT3 kinase inhibitors are important component in the treatment of acute myeloid leukemia (AML). However, relapse or resistance to these inhibitors are frequent because of secondary acquired mutations in FLT3. Recently, we reported on a novel small molecular multi-kinase inhibitor, CG'806, which showed promising effects in AML harboring FLT3-internal-tandem-deletion (ITD) mutations, tyrosine kinase domain (TKD) mutations, or both by inhibiting FLT3/Bruton's tyrosine kinase (BTK)/aurora kinase (AuroK) activation. We observed an impressive inhibition of leukemic cell proliferation (i.e., IC50s at sub-nanomolar or low nanomolar levels, Zhang, et al., AACR Hematological Malignancies, 2017).To further characterize the mechanisms underlying this anti-leukemia effect, we investigated the impact of CG'806 on cell cycle progression. Measuring BrdU incorporation by flow cytometry, CG'806 triggered profound G1 cell cycle arrest in FLT3-ITD-mutated cells. Interestingly, CG'806 triggered G2/M phase arrest in FLT3 wild type (WT) cells. Immunoblotting demonstrated that the G1 arrest was mediated by downregulation of signaling associated with p-FLT3-ITD, the p-AKT/p-mTOR/cyclin D1/p-Rb axis, and that of cyclin B1/A2, cdk1/cdc2, and cdk4/6 in FLT3-ITD-mutated cells. This was not observed in FLT3 WT cells. In addition, c-Myc, a primary regulator of the G1/S transition, was downregulated in FLT3-mutated but not in WT cells treated with CG'806. In support of this finding, knockdown of c-Myc with siRNA increased the G1-arrested population in FLT3-mutated cells, suggesting a critical role of c-Myc in the CG'806-induced G1 arrest. However, only suppression of p-BTK and p-AuroK but not p-FLT3, and no modulation of G1 arrest-related proteins was observed in FLT3 WT cells.Interestingly, we observed autophagy induction in FLT3 WT cells but induction of apoptosis in FLT3 mutated cells after exposure to low nanomolar CG'806 for 24 hours as evidenced by modulated LC3-II and cleaved-caspase 3 levels. Inhibiting autophagy with 3-methyladenine (3-MA) partially reversed CG'806-induced G2/M arrest in FLT3 WT cells, suggesting that autophagy induction may also be involved in G2/M arrest besides suppression of AuroK and BTK in FLT3 WT cells.Next, we investigated if nucleoside analogues and intercalating agents enhance activity of CG'806, and observed that combination of CG'806 with cytarabine or idarubicin profoundly enhanced pro-apoptotic effects.Conclusions: CG'806 exerts profound suppression of cell proliferation by arresting cell cycle progression at G1 phase in FLT3-mutant AML cells which is associated with inhibition of mutant FLT3 and downstream p-AKT/p-mTOR/cyclin D1/p-Rb axis. CG'806 exerts a G2/M arrest in FLT3 WT cells associated with inhibition of aurora and BTK kinases and induction of autophagy. CG'806 sensitized AML to standard chemotherapeutic agents-mediated cytotoxicity. Taken together, these data support the development of CG'806 for AML patients with FLT3-ITD, FLT3-ITD with additional TKD mutations, as well as FLT3-WT. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Lgr5 Functions As Negative Regulator of Wnt Signaling in B Cells and Is Critical for Self-Renewal of Normal and Transformed B Cells

Background and significance: The identification of leukemia-initiating cells (LIC) in myeloid leukemias allowed for focused therapeutic approaches to prevent drug-resistance and relapse. In B cell leukemia and lymphoma, LIC occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B cell development (Le Viseur 2008). While self-renewal of HSCs and other organ-stem cells is regulated by a developmental hierarchy, self-renewal in the B cell lineage is induced by positive selection events, i.e. induced by antigen-receptor (BCR) signaling when B cells bind to their cognate antigen.Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple organs, however its expression levels are very low in hematopoietic cells.Results: Unexpectedly, B cells upregulate Lgr5-expression by >20-fold, (i) when they express the pre-BCR for the first time and (ii) when they encounter antigen for the first time in germinal centers. Engagement of BCR signaling on B cell lymphomas strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway.In addition, in patients with pediatric or adult ALL, or B cell chronic lymphocytic leukemia (CLL), higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. For these reasons, we studied the function of Lgr5 during normal B cell development and malignant transformation. Conditional ablation of Lgr5fl/fl alleles during earliest stages of B cell development (Mb1-Cre) resulted in a >100-fold reduction of absolute B cell numbers. In addition, inducible deletion of Lgr5 in cultured pre-B and mature B cells caused rapid cell death, demonstrating that Lgr5 function is an essential requirement for B cell development. Most studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, Cre-mediated deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear b-catenin. Our genetic experiments in leukemia models demonstrated that LGR5 is critical for leukemia-initiation and survival. Cre-mediated deletion of Lgr5 and b-catenin accumulation abolished colony forming capacity and the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of b-catenin resulted in rapid cell death in B cells.Conclusion: Lgr5 is widely established as a marker of organ stem cells with self-renewal capacity. In the absence of the developmental stem cell hierarchy in B cells, we conclude that Lgr5 expression is a biomarker of positive selection in B cells. Lgr5-expression and positive selection is induced by BCR-engagement (e.g. by antigen) or oncogenic mimics of BCR signaling in B cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear b-catenin and enables B cell survival through negative regulation of Wnt-signaling. DisclosuresNo relevant conflicts of interest to declare.

Daratumumab Mitigates B-Cell Receptor Signaling and Enhances Antitumor Activity of Ibrutinib in Chronic Lymphocytic Leukemia (CLL) Cells

Introduction: B-cell receptor (BCR) signaling is a central driver of proliferation in chronic lymphocytic (CLL) cells. BCR engagement leads to recruitment of several intracellular kinases such Lyn, Syk and BTK, that activate ERK, NFkB and induce CLL cell proliferation. While this pathway is effectively targeted with the BTK inhibitor, ibrutinib, various combination treatment strategies that result in greater tumor cell killing are under investigation. On B-cells, the BCR interacts with the CD19 and CD38 cell surface antigens; where the later amplifies intracellular pro-survival signal transmission through the BCR. CD38 is a multifunctional protein, which possess both enzymatic and receptor functions. As such, we hypothesized that concurrent targeting of CD38 and BTK would lead to enhanced mitigation of BCR signaling as well as direct cell death than use of either agent alone.Materials: CD19+ cells were isolated from CLL patients (n=30, collected under an IRB-approved protocol) by magnetic bead sorting. JVM13 (CD38+) and MEC1 (CD38-) cell lines were also used. CD38 expression was measured by flow cytometry with quantification of MFI and surface antibody binding (sAbc). Drug-induced antibody-dependent cellular cytotoxicity (ADCC) in target Calcein AM labeled CLL cells was assessed by co-culturing them with healthy donor PBMCs as effectors (effector: target [E:T] ratio, 50:1) and complement dependent cytotoxicity (CDC) was measured using complement-containing serum from a healthy donor. Antibody dependent cellular phagocytosis (ADCP) was measured by co-culturing calcein AM labeled CLL cells with healthy human macrophages at a 2:1 ratio. Apoptosis by annexin-V/PI dual staining along with mitochondrial outer membrane potential (MOMP) using MitoProbeTMDilC1(5) were assessed by flow cytometric analysis. Immunoblotting was performed using antibodies against Lyn, Syk, BTK, PLCγ2, ERK1/2, AKT, Ikβα, NFAT2 and their phosphorylated isoforms along with GAPDH followed by densitometry analysis.Results: Using a cutoff of 30% (gated CD38 expressing cells), we observed 26.6% (n=8) of CLL pts. to be CD38+ and 73.3% of pts. to be CD38- (n=22). As some pts. deemed CD38- had high CD38 MFI or sAbc, we applied an interquartile range (IQR with 95% confidence interval) for CD38 MFI; re-classifying pts. as CD38lo (56.75 - 199) vs. CD38hi (199 - 355). Using this system, specific-lysis of CLL cells from Dara-mediated ADCC was 23.74±2.55% vs. 14.17±1.27, CDC: 15.93±1.68% vs. 12.29±0.88, ADCP: 8.630.96% vs. 9.50±0.97% and apoptosis: 32.42±3.42 vs. 23.65±1.82 in CD38hi vs. CD38lo pts., respectively. Apoptosis was accompanied by a significant shift in MOMP [21.65±7.90% (CD38hi) vs. 32.52±7.42% (CD38lo), p<0.05]. In CLL cells treated with the combination of Ibr + Dara (ID), ADCC increased to 54.21±4.09% vs. 37.66±2.16; CDC: 34.35±2.47% vs. 28.88±1.51%, ADCP 25.56±2.30% vs. 16.69±1.97%, apoptosis: 61.63±3.14% vs 62.06±3.41% and MOMP: 66.22±3.68% vs 71.13±3.31%: in CD38hi vs. CD38lo pts., respectively. Next we examined the effect of Dara +/- Ibr on BCR signaling in CD38hi and CD38lo CLL cells. In CD38hi cells, single agent Dara significantly decreased phosphorylation Lyn, Syk, BTK and PLCγ2 as well as ERK1/2, AKT, Ikβα and NFAT; the ID combination decreasing these further (except Ikβα and NFAT2) (p<0.01). In CD38lo cells, Dara significantly downregulated phosphorylation of Lyn and PLCγ2, however, insignificant changes were noted in all other proteins. In ID combination treated CD38lo cells, Lyn, Syk, BTK, PLCγ2 and AKT were further decreased.Conclusions: Herein, we provide first evidence on the ability of Dara to modulate BCR signaling and associated proteins in primary CLL cells and that this can be further augmented with Ibr. Dara-induced protein changes were more prominent in CD38hi cells; accompanied by higher % apoptosis vs. that observed in CD38lo cells, yet the ID combination significantly enhanced mitochondrial-mediated apoptosis in both CD38hi and CD38lo cells. This indicates that the combination can potentially overcome lower induction of apoptosis and ADCC in CD38lo cells treated with single agent Dara or Ibr. Studies on how Dara and the ID combination induce direct programmed cell death (independent of modulating BCR signaling) are underway. Overall, our investigations provide rationale for clinical testing of Ibr combined with Dara in CLL. DisclosuresParikh:Pharmacyclics: Research Funding; Pharmacyclics: Honoraria; AstraZeneca: Honoraria. Ding:Merck: Research Funding. Sher:LAM Therapeutics, Inc: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Pharmacyclics: Research Funding; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

Ibrutinib As a Possible Therapeutic Strategy for Epstein-Barr Virus-Positive Lymphoma/Leukemia

Epstein-Barr virus (EBV) is associated with many types of lymphoproliferative disorders. EBV-coded latent membrane protein 2A (LMP2A) functions in promoting B cell survival partly through Bruton's tyrosine kinase (Btk) -dependent signaling pathway. In the present study, we investigated whether a Btk inhibitor, Ibrutinib, shows therapeutic efficacy for EBV-related lymphoma. Ibrutinib has been already approved as a therapeutic drug for several B cell malignancies, such as chronic lymphocytic leukemia. Hence, once it shows efficacy, Ibrutinib could be quickly moved to clinical usage.First, we examined the effects of Ibrutinib on B cells transformed by EBV infection in vitro, called lymphoblastic cell lines (LCLs). We cultured LCLs in the presence or absence of Ibrutinib and analyzed cell survival using flow cytometry. As a result, Ibrutinib treatment decreased survival rate of the LCLs compared with a vehicle (Fig. A). Indeed, Btk phosphorylation was detected in the LCLs by western blot analysis. Collectively, Btk signal inhibition by Ibrutinib is probably lethal for EBV-LCLs. LCLs are classified as latency type III and have a polyclonal nature like lymphoproliferative disorders in post-transplant and HIV-associated immune suppressed individuals. Hence, Ibrutinib possibly be applicable to treatment for such disorders. To examine whether Ibrutinib is effective in vivo, we are now planning to administer Ibrutinib to EBV-infected humanized mice, which can be used as a model for EBV-related lymphoproliferative disorders (Yajima et al., J. Infect. Dis. 2008).Further, we obtained peripheral blood mononuclear cells (PBMCs) from EBV+ aggressive NK cell leukemia (ANKL) patient. Approximately 90% of the PBMCs were positive for NK cell marker CD56 and hence probably be leukemia cells. First, we examined in vitro effects of Ibrutinib on the PBMCs. The PBMCs were cultured in the presence or absence of Ibrutinib, and survival rate was analyzed using flow cytometry. Results showed that Ibrutinib treatment decreased survival of CD56High cells (Fig. B). Next, we examined Btk phosphorylation in the PBMCs using western blot. Unexpectedly, even total Btk was not detected. This suggests Ibrutinib-induced lethality of the ANKL cells is dependent on other pathways. Ibrutinib could target other kinases than Btk including those reported to be expressed in NK cells, such as ITK, TXK, and JAK3. Hence, it is possible that Ibrutinib decreases the ANKL cells through inhibiting these kinases.Next, we tried to establish a mouse model of ANKL xenograft for in vivo assays. We transplanted the PBMCs from ANKL patient into immunodeficient NOG mice after X-ray irradiation. Around 2 weeks after the transplantation, a large number of the transplanted PBMCs were detected at least in spleen and bone marrow. These ANKL cells in spleen and bone marrow could be successively transplanted into other NOG mice. After transplantation, the recipient mice gradually decreased their body weight and died in several weeks. Now, we are analyzing these mice in detail and are investigating the effects of Ibrutinib administration on the survival of these mice.Collectively, we found that Ibrutinib inhibits survival of EBV+ LCLs and ANKL cells in vitro . This raises a possibility that Ibrutinib can be used for EBV-related lymphoproliferative disorders and EBV+ ANKL, although we should confirm the effectiveness of Ibrutinib in vivo . [Display omitted] DisclosuresKotani:Janssen Pharma: Research Funding.

Inhibition of Btk Does Not Block Platelet Activation By GPVI: Studies with Ibrunitib, Acalabrutinib and Patients with X-Linked Agammaglobulinaemia

Background : Ibrutinib and acalabrutinib, inhibitors of the protein tyrosine kinase Btk, are used to treat B cell malignancies. They bind irreversibly to Cys481 of Btk preventing autophosphorylation and phosphorylation of other substrates including PLCγ2. Patients treated with ibrutinib have increased bleeding events which correlate with inhibition of platelet aggregation by the collagen and fibrin receptor GPVI. However, patients treated with acalabrutinib, and patients with mutations in their Btk gene (X-linked Agammaglobulinaemia: XLA), do not bleed excessively.Aims: We sought to investigate this discrepancy between the two Btk inhibitors and patients with XLA.Methods: Aggregometry, tyrosine phosphorylation and Ca2+ mobilisation were performed in washed platelets. Aggregometry was also performed with platelet rich plasma (PRP) from patients taking ibrutinib 420mg daily, acalabrutinib 100mg twice daily, a control treatment and XLA patients.Results: We have compared the effect of the irreversible inhibitors of Btk, ibrutinib, acalabrutinib, and platelets from patients with XLA on activation by a high concentration (10 mg/ml) of the GPVI agonist collagen receptor peptide (CRP). Loss of activation of Btk, as verified by western blotting for autophosphorylation (Y223), and phosphorylation of PLCγ2 on tyrosines 759 and 1217, had no effect on aggregation to CRP. 10-20 fold higher concentrations of ibrutinib and acalabrutinib blocked aggregation to CRP. Inhibition was independent of Tec inhibition since (i) it was reversible on washout; (ii) there was no further reduction in PLCγ2 phosphorylation; (iii) concentrations of ibrutinib and acalabrutinib that blocked phosphorylation of Tec did not block aggregation.Conclusions: Our results confirm that ibrutinib blocks GPVI-induced platelet aggregation in vitro and ex vivo but show that, contrary to current understanding, this is due to an unidentified off-target effect. We hypothesise that reducing the dose of ibrutinib given to patients would abolish its off-target effects and therefore reduce bleeding. This is supported by the fact that patients with XLA, and those taking acalabrutinib, do not experience abnormal bleeding.Acknowledgment: this work was supported by the British Heart Foundation. DisclosuresNo relevant conflicts of interest to declare.

Differential Use of the Signaling Molecules BTK and PLC-γ in Mast Cells Stimulated Via the Receptors FcεRI and KIT

Mast cells belong to the myeloid lineage of the hemopoietic system, are localized to the tissue-environment interphase, and execute regulatory and protective roles in both innate and adaptive immunity. However, mast cells also are involved in pathophysiological processes ranging from allergic diseases to autoimmune diseases and tumors. Two receptors expressed on the surface of mast cells and prominently involved in physiology and pathophysiology of mast cell biology are the high-affinity receptor for IgE (FcεRI) and the receptor tyrosine kinase KIT (a.k.a. CD117), the receptor for stem cell factor (SCF). Whereas antigen (Ag)-triggered activation of the IgE-bound FcεRI results in immediate release of preformed mediators (degranulation) and production of arachidonic acid metabolites and cytokines/chemokines, SCF-induced mast cell activation is only causing the latter. On the other side, SCF is inducing mast cell proliferation and chemotaxis, two responses that are not caused by FcεRI activation. Intriguingly, the signal transduction downstream of these receptors involves activation of the same signaling pathways and engagement of comparable sets of signaling proteins, raising the question, how differential mast cell effector functions are controlled by these receptors. Therefore, it is essential to learn more about the molecular pathways leading to FcεRI and KIT-mediated activation of mast cells. In the presented work, primary murine mast cells were stimulated either with Ag via the IgE-bound FcεRI or with SCF via its receptor KIT and differential signal transduction was analyzed. We put our focus on two central signaling elements in mast cells, the cytosolic tyrosine kinase BTK and its target, phospholipase C-γ, and also addressed differences between using suboptimal and optimal ligand concentrations. Cell responses like degranulation, proinflammatory cytokine production, activating protein phosphorylation, and intracellular calcium mobilization were analyzed making use of mast cells from wild-type and transgenic (knock-out) mice as well as selective pharmacological inhibitors. Interestingly, marked differences were found with respect to pathway activation, importance of particular signaling proteins, and induction of effector responses. Ag-triggered degranulation is thought to be dependent on BTK/PLC-γ-mediated calcium mobilization and thus it was unexpected to observe only a weak effect of PLC-γ inhibition. On the other side, SCF-induced calcium mobilization was strictly dependent on PLC-γ, however, only a minor involvement of BTK could be observed. In combination with the additional results obtained in our study, future analyses of differential pathway organization by multiprotein assemblies as well as kinetic control mechanisms appear to be important. Detailed knowledge of these mechanisms will help to precisely promote beneficial and strictly control detrimental mast cell responses. DisclosuresNo relevant conflicts of interest to declare.

Resistance Mutations to BTK Inhibitors Originate From the NF-κB but Not From the PI3K-RAS-MAPK Arm of the B Cell Receptor Signaling Pathway.

Since the first clinical report in 2013, inhibitors of the intracellular kinase BTK (BTKi) have profoundly altered the treatment paradigm of B cell malignancies, replacing chemotherapy with targeted agents in patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström’s macroglobulinemia. There are over 20 BTKi, both irreversible and reversible, in clinical development. While loss-of-function (LoF) mutations in the BTK gene cause the immunodeficiency X-linked agammaglobulinemia, neither inherited, nor somatic BTK driver mutations are known. Instead, BTKi-sensitive malignancies are addicted to BTK. BTK is activated by upstream surface receptors, especially the B cell receptor (BCR) but also by chemokine receptors, and adhesion molecules regulating B cell homing. Consequently, BTKi therapy abrogates BCR-driven proliferation and the tissue homing capacity of the malignant cells, which are being redistributed into peripheral blood. BTKi resistance can develop over time, especially in MCL and high-risk CLL patients. Frequently, resistance mutations affect the BTKi binding-site, cysteine 481, thereby reducing drug binding. Less common are gain-of-function (GoF) mutations in downstream signaling components, including phospholipase Cγ2 (PLCγ2). In a subset of patients, mechanisms outside of the BCR pathway, related e.g. to resistance to apoptosis were described. BCR signaling depends on many proteins including SYK, BTK, PI3K; still based on the resistance pattern, BTKi therapy only selects GoF alterations in the NF-κB arm, whereas an inhibitor of the p110δ subunit of PI3K instead selects resistance mutations in the RAS-MAP kinase pathway. BTK and PLCγ2 resistance mutations highlight BTK’s non-redundant role in BCR-mediated NF-κB activation. Of note, mutations affecting BTK tend to generate clone sizes larger than alterations in PLCγ2. This infers that BTK signaling may go beyond the PLCγ2-regulated NF-κB and NFAT arms. Collectively, when comparing the primary and acquired mutation spectrum in BTKi-sensitive malignancies with the phenotype of the corresponding germline alterations, we find that certain observations do not readily fit with the existing models of BCR signaling.

Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

CD45-dependent regulation of B cell receptor signaling in atherosclerosis

Abstract Atherosclerosis is an inflammatory disease of the large and medium size arteries that is characterized by deposition of oxidized lipids within the vessel. While specific antigens are not well characterized, data show that the immune response is involved in atherogenesis. The role of B cells in atherosclerosis is B cell specific: FO B cells are proatherogenic, while B1 and MZ B cells serve as protective. B cell development, activation, and differentiation is dependent on B cell receptor signaling while important costimulatory molecules, like CD45, assist in the initial propagation of signal. To date, it is unknown how regulation of BCR signaling by CD45 impacts activation and functions of B cells in atherosclerosis. We took advantage of transgenic mice that express low levels of CD45 (CD45L/L) and examined the phenotype of CD45L/L B cells. We demonstrate that low expression of CD45 leads to attenuated BCR-induced B cell activation via the reduced Ca2+ flux and phosphorylation of ERK, BTK, and SYK kinase, while favoring MZ and B1 B cell differentiation in homeostatic conditions. To test the effects of B cells expressing low levels of CD45 in atherosclerosis, we performed adoptive transfer of CD45L/L or WT B cells into B cell-deficient atherosclerotic prone mice. After 23–28 wks of western diet feeding, the CD45L/L vs WT B cell recipients had reduced lesion formation accompanied by increased MZ B cells and decreased germinal center formation. This suggests that attenuation of BCR signaling through CD45 affects B cell subset differentiation and activation and highlight a potential role of BCR-related signaling in regulation of immunity in atherosclerosis.

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions—the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton’s tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions.

Aromatic Rings as Molecular Determinants for the Molecular Recognition of Protein Kinase Inhibitors.

Protein kinases are key enzymes in many signal transduction pathways, and play a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. However, aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Over the past three decades, thousands of protein kinase inhibitors (PKIs) with varying molecular frames have been developed. Large-scale data mining of the Protein Data Bank resulted in a database of 2139 non-redundant high-resolution X-ray crystal structures of PKIs bound to protein kinases. This provided us with a unique opportunity to study molecular determinants for the molecular recognition of PKIs. A chemoinformatic analysis of 2139 PKIs resulted in findings that PKIs are “flat” molecules with high aromatic ring counts and low fractions of sp3 carbon. All but one PKI possessed one or more aromatic rings. More importantly, it was found that the average weighted hydrogen bond count is inversely proportional to the number of aromatic rings. Based on this linear relationship, we put forward the exchange rule of hydrogen bonding interactions and non-bonded π-interactions. Specifically, a loss of binding affinity caused by a decrease in hydrogen bonding interactions is compensated by a gain in binding affinity acquired by an increase in aromatic ring-originated non-bonded interactions (i.e., π–π stacking interactions, CH–π interactions, cation–π interactions, etc.), and vice versa. The very existence of this inverse relationship strongly suggests that both hydrogen bonding and aromatic ring-originated non-bonded interactions are responsible for the molecular recognition of PKIs. As an illustration, two representative PKI–kinase complexes were employed to examine the relative importance of different modes of non-bonded interactions for the molecular recognition of PKIs. For this purpose, two FDA-approved PKI drugs, ibrutinib and lenvatinib, were chosen. The binding pockets of both PKIs were thoroughly examined to identify all non-bonded intermolecular interactions. Subsequently, the strengths of interaction energies between ibrutinib and its interacting residues in tyrosine kinase BTK were quantified by means of the double hybrid DFT method B2PLYP. The resulting energetics for the binding of ibrutinib in tyrosine kinase BTK showed that CH–π interactions and π–π stacking interactions between aromatic rings of the drug and hydrophobic residues in its binding pocket dominate the binding interactions. Thus, this work establishes that, in addition to hydrogen bonding, aromatic rings function as important molecular determinants for the molecular recognition of PKIs. In conclusion, our findings support the following pharmacophore model for ATP-competitive kinase inhibitors: a small molecule features a scaffold of one or more aromatic rings which is linked with one or more hydrophilic functional groups. The former has the structural role of acting as a scaffold and the functional role of participating in aromatic ring-originated non-bonded interactions with multiple hydrophobic regions in the ATP binding pocket of kinases. The latter ensure water solubility and form hydrogen bonds with the hinge region and other hydrophilic residues of the ATP binding pocket.

Lactobacillus delbrueckii Protected Intestinal Integrity, Alleviated Intestinal Oxidative Damage, and Activated Toll-Like Receptor-Bruton's Tyrosine Kinase-Nuclear Factor Erythroid 2-Related Factor 2 Pathway in Weaned Piglets Challenged with Lipopolysaccharide.

Oxidative stress is increasingly being recognized as a player in the pathogenesis of intestinal pathologies, and probiotics are becoming an attractive means of addressing it. The present study investigated the effects of dietary supplementation with Lactobacillus delbrueckii (LAB) on intestinal integrity and oxidative damage in lipopolysaccharide (LPS)-challenged piglets. A total of 36 crossbred weaned piglets (Duroc × Landrace × Large Yorkshire) were randomly divided into three groups: (1) non-challenged controls (CON), (2) LPS-challenged controls (LPS), and (3) 0.2% LAB (2.01 × 1010 CFU/g) + LPS treatment (LAB + LPS). On the 29th day of the experiment, the LPS and CON groups were injected intraperitoneally with LPS and saline at 100 ug/kg body weight, respectively. The results show that the LPS-induced elevation of the serum diamine oxidase (DAO) level and small intestinal crypt depth (CD) were reversed by the dietary addition of LAB, which also markedly increased the ileal expression of tight junction proteins (occludin, ZO-1, and claudin-1) in the LPS-challenged piglets. Furthermore, LAB supplementation normalized other LPS-induced changes, such as by decreasing malondialdehyde (MDA) in both the serum and intestinal mucosa and 8-hydroxy-2-deoxyguanosine (8-OHdG) in the jejunal mucosa, increasing glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in both the serum and intestinal mucosa, and increasing glutathione (GSH) and superoxide dismutase (SOD) in the jejunal mucosa. LAB also activated Toll-like receptor (TLR)–Bruton’s tyrosine kinase (Btk)–nuclear factor erythroid 2-related factor 2(Nrf2) signaling pathways in the intestine, suggesting that it plays a vital role in the ameliorative antioxidant capacity of weaned piglets. In summary, LAB increased intestinal integrity by improving the intestinal structure and tight junctions while enhancing antioxidant functions via the activation of the TLR–Btk–Nrf2 signaling pathway.

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.

Bruton's agammaglobulinemia tyrosine kinase (Btk) regulates TPA‑induced breast cancer cell invasion via PLCγ2/PKCβ/NF‑κB/AP‑1‑dependent matrix metalloproteinase‑9 activation

Bruton's agammaglobulinemia tyrosine kinase (BTK) is an important cytoplasmic tyrosine kinase involved in B-lymphocyte development, differentiation, and signaling. Activated protein kinase C (PKC), in turn, induces the activation of mitogen-activated protein kinase (MAPK) signaling, which promotes cell proliferation, viability, apoptosis, and metastasis. This effect is associated with nuclear factor-κB (NF-κB) activation, suggesting an anti-metastatic effect of BTK inhibitors on MCF-7 cells that leads to the downregulation of matrix metalloproteinase (MMP)-9 expression. However, the effect of BTK on breast cancer metastasis is unknown. In this study, the anti-metastatic activity of BTK inhibitors was examined in MCF-7 cells focusing on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MCF-7 cells. The expression and activity of MMP-9 in MCF-7 cells were investigated using quantitative polymerase chain reaction analysis, western blotting, and zymography. Cell invasion and migration were investigated using the Matrigel invasion and cell migration assays. BTK inhibitors [ibrutinib (10 µM), CNX-774 (10 µM)] significantly attenuated TPA-induced cell invasion and migration in MCF-7 cells and inhibited the activation of the phospholipase Cγ2/PKCβ signaling pathways. In addition, small interfering RNA specific for BTK suppressed MMP-9 expression and cell metastasis. Collectively, results of the present study indicated that BTK suppressed TPA-induced MMP-9 expression and cell invasion/migration by activating the MAPK or IκB kinase/NF-κB/activator protein-1 pathway. The results clarify the mechanism of action of BTK in cancer cell metastasis by regulating MMP-9 expression in MCF-7 cells.

Bleeding by Bruton Tyrosine Kinase-Inhibitors: Dependency on Drug Type and Disease.

Simple SummaryBruton tyrosine kinase (Btk) is expressed in B-lymphocytes, myeloid cells and platelets. Since the launch of the first in class Btk-inhibitor (BTKi) ibrutinib in 2013, the list of indications and further drug candidates has expanded greatly. BTKi are not only used to treat patients with B-cell malignancies and in development against various autoimmune diseases, but they have been also proposed as novel antithrombotic drugs and been tested in patients with severe COVID-19. The number of BTKi approved or in clinical studies is rapidly increasing. Although X-linked agammaglobulinemia (XLA) patients with Btk deficiency do not show impaired hemostasis, bleeding events are frequently observed upon treatment with many but not all BTKi. This review describes twelve BTKi approved or in clinical trials. By focusing on their pharmacological properties, targeted disease, bleeding side effects and actions on platelets it attempts to clarify the mechanisms underlying bleeding. Moreover, specific platelet function tests in blood are described which will help to estimate the probability of bleeding side effects of newly developed BTKi.Bruton tyrosine kinase (Btk) is expressed in B-lymphocytes, myeloid cells and platelets, and Btk-inhibitors (BTKi) are used to treat patients with B-cell malignancies, developed against autoimmune diseases, have been proposed as novel antithrombotic drugs, and been tested in patients with severe COVID-19. However, mild bleeding is frequent in patients with B-cell malignancies treated with the irreversible BTKi ibrutinib and the recently approved 2nd generation BTKi acalabrutinib, zanubrutinib and tirabrutinib, and also in volunteers receiving in a phase-1 study the novel irreversible BTKi BI-705564. In contrast, no bleeding has been reported in clinical trials of other BTKi. These include the brain-penetrant irreversible tolebrutinib and evobrutinib (against multiple sclerosis), the irreversible branebrutinib, the reversible BMS-986142 and fenebrutinib (targeting rheumatoid arthritis and lupus erythematodes), and the reversible covalent rilzabrutinib (against pemphigus and immune thrombocytopenia). Remibrutinib, a novel highly selective covalent BTKi, is currently in clinical studies of autoimmune dermatological disorders. This review describes twelve BTKi approved or in clinical trials. By focusing on their pharmacological properties, targeted disease, bleeding side effects and actions on platelets it attempts to clarify the mechanisms underlying bleeding. Specific platelet function tests in blood might help to estimate the probability of bleeding of newly developed BTKi.