Tec Kinase Research Articles

Activation of the Tec Kinase ITK Controls Graded IRF4 Expression in Response to Variations in TCR Signal Strength.

TCR signal strength is critical for CD8+ T cell clonal expansion after Ag stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through the induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. In this study, we show that Ag potency regulates the kinetics but not the magnitude of NFAT1 activation in single mouse CD8+ T cells. Consequently, T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction, resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. We further show that the activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the function of ITK in TCR signal transduction that ultimately regulates IRF4 expression levels in response to variations in TCR signal strength.

Negative Regulation of Tec Kinase Alleviates LPS-Induced Acute Kidney Injury in Mice via theTLR4/NF-κB Signaling Pathway.

Tec kinase is an important mediator in inflammatory immune response that enhances the activity of neutrophils and macrophages. However, information on its function in lipopolysaccharide- (LPS-) induced acute kidney injury (AKI) is limited. This study is aimed at determining whether Tec kinase was a regulator in AKI. An AKI model in mice was successfully established using intraperitoneal LPS. Results showed that the serum levels of creatinine (Cr), blood urea nitrogen (BUN), and cystatin-C (Cys-C) increased after intraperitoneal LPS injection. Renal tissue sustained significantly severe injury as measured by pathological scores. Pretreatment with LFM-A13 improved the function of the kidney in mice and decreased the renal injury score. Enzyme-linked immunosorbent assay showed that LFM-A13 significantly reduced the release of IL-1β and TNF-α in mice exposed to LPS. LFM-A13 can evidently abrogate the expression of Tec protein, MyD88, TLR4, NF-κB p65, and Tec's phosphorylated protein as determined by Western blot. Immunohistochemistry analysis revealed that LFM-A13 markedly downregulated the expression of Tec kinase in renal tubular epithelial cells. In vitro, Tec kinase protein was expressed highly in NRK-52E cells after LPS exposure. Tec-siRNA also decreased IL-1β and TNF-α production and obviously abolished phospho-p65 and phospho-IκBα expression in NRK-52E cell stimulated by LPS; however, Tec-siRNA increased the IκBα level. Altogether, these data suggested that Tec kinase can be a modulating protein in AKI through TLR4/NF-κB activation.

SUN-LB56 Steroid and Sex Specific Responses of Neural Stem Cells to Prenatal Dexamethasone versus Betamethasone Administration

Synthetic glucocorticoids (sGCs) are widely administered to pregnant women for their anti-inflammatory, immunosuppressive and organ maturation properties. Worldwide, Dexamethasone (Dex) and Betamethasone (Beta) are the two most commonly administered prenatal sGCs to reduce morbidity and mortality associated with respiratory distress, intraventricular hemorrhage and necrotizing enterocolitis. Preterm administration of sGCs is associated with reduced birthweight and increased risk for hypertension, cardiovascular, metabolic, and neurological problems later in life. Adverse neurological outcome has been shown to depend on the type of sGCs used, the dose, timing of sGCs administration and sex. We have previously shown that the glucocorticoid receptor (GR) is expressed in the developing brain in stem and progenitor cells, neurons and glia from early developmental stages, and that prenatal Dex alters neural stem cell (NSC) biology and the developmental trajectory of the cerebral cortex, hypothalamus and adult behavior. To identify the molecular and cellular basis of the sex and steroid specific responses in the developing brain, we compared the consequence of Dex versus Beta exposure on embryonic cerebral cortical NSC biology. Murine NSC were isolated from the E14.5 cerebral cortex and exposed to 10-7 M Dex, 10-7 M Beta, or Vehicle for 4 or 24 hours and the immediate and long-term impact on transcription, proliferation and neuronal, glial and oligodendrocyte differentiation examined. Affymetrix complete genome transcriptional analyses reveal sex specific responses to Dex versus Beta within 4 hours. At >+/-1.5-fold change 548 genes were differentially regulated by Dex, 452 by Beta and 256 were altered by both Dex and Beta (P < 0.05). Distinct sex specific responses to Dex versus Beta were observed. At >+/-2-fold change 126 genes were significantly different in the Dex versus Beta female transcriptome, 146 in the male transcriptome with 18 genes unique to both male and female transcriptome. Ingenuity Pathway Analysis revealed that the most significantly altered pathway altered (Z score >2) with both sGCs is Inositol Phosphate metabolism. Cardiac hypertrophy, Tec kinase, and Th1 pathways were unique to Beta stimulation, whereas Melatonin, Neuropathic Pain and IL6 signaling pathways were specific to Dex stimulation. Both Dex and Beta significantly alter genes implicated in proliferation and differentiation as also described in other studies, therefore the biological response of NSC to sGCs stimulation was compared. Only Dex significantly decreased the rate of proliferation over a 72 hour. In-vitro differentiation studies reveal that both Dex and Beta reduced oligodendrocyte differentiation without altering neuronal differentiation when cells were exposed to sGCs as progenitors. However, when cells were exposed to sGCs during differentiation, Dex increased oligodendrocyte and neuronal maturation while Beta only increased oligodendrocyte differentiation. These results reveal gene targets, cellular pathways and processes that are differentially altered by prenatal Dex versus Beta exposure. Prenatal sGCs administration provides clear benefits for neonatal outcome, however, a detailed understanding of their targets in the brain is required to identify alternative sGCs drug regimens to reduce adverse neurological effects. Our finds may provide insights into the sex specific neurological outcomes observed in children exposed to sGCs in-utero.

5‐Aminoethylbenzimdazole Suppresses Lipopolysaccharide (LPS)/Interferon Gamma (IFNγ)‐Induced Inflammatory Responses in Macrophages

Tec family non‐receptor tyrosine kinases, including TEC, BTK, ITK/EMT/TSK, BMX and TXK/RLK, are expressed in a variety of immune cells. Previous studies have demonstrated that Tec kinases play pivotal roles in inflammatory disorders such as allergic asthma and atopic dermatitis. Several 5‐aminomethylbenzimdazoles (5‐AMBD) have been reported as highly potent inhibitors of ITK as well as other TEC kinases and showed strong activities on reducing IL‐2 and IL‐4 production. However, the impact of 5‐AMBD on macrophage‐mediated inflammatory responses remains unclear. The goal of this study was to determine the effect of 5‐AMBD on the inflammatory responses in macrophages stimulated with lipopolysaccharide (LPS) and interferon‐gamma (IFNγ). RAW 264.7 cells (a murine macrophage cell line) were stimulated with 10 ng/mL LPS and 1 ng/mL IFNγ for 10 min, then treated with various concentrations of 5‐AMBD. The cell viability and production of nitric oxide (NO) were measured 20 hr after treatment. The total RNA was extracted from cells treated for 4 hr, and then the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNFα), and interleukin 1 beta (IL‐1β) were evaluated using quantitative real‐time PCR. The impact of 5‐AMBD on the activation of nuclear factor kappa B (NF‐κB) was investigated using the RAW 264.7 cells stably expressing the NF‐kB luciferase reporter. 5‐AMBD dose‐dependently reduced the NO production with no significant toxicity to the macrophages. Treatment of 100 nM and 1 μM 5‐AMBD significantly inhibited the expression of iNOS by 50% and 95%, respectively. Meanwhile, 5‐AMBD (1 μM) also reduced the expression of IL‐1β and TNFα. Furthermore, we found that 5‐AMBD dose‐dependently diminished the activation of NF‐κB at 4 hr and 20 hr after treatment. These findings demonstrate that 5‐AMBD has a strong anti‐inflammatory activity in macrophages, and suggest that TEC kinases could be potential drug targets for inflammatory diseases.Support or Funding InformationWork supported by Small Pharmacy Awards for Research and Collaboration from the Presbyterian College School of Pharmacy.

Characterization of the signature of peripheral innate immunity in women with later-life major depressive disorder

The prevalence of depression in later life is higher in women than in men. However, the sex difference in the pathophysiology of depression in elderly patients is not fully understood. Here, we performed gene expression profiling in leukocytes of middle-aged and elderly patients with major depressive disorder, termed later-life depression (LLD) in this context, and we characterized the sex-dependent pathophysiology of LLD.A microarray dataset obtained from leukocytes of patients (aged ≥50 years) with LLD (32 males and 39 females) and age-matched healthy individuals (20 males and 24 females) was used. Differentially expressed probes were determined by comparing the expression levels between patients and healthy individuals, and then functional annotation analyses (Ingenuity Pathway Analysis, Reactome pathway analysis, and cell-type enrichment analysis) were performed.A total of 1656 probes were differentially expressed in LLD females, but only 3 genes were differentially expressed in LLD males. The differentially expressed genes in LLD females were relevant to leukocyte extravasation signaling, Tec kinase signaling and the innate immune response. The upregulated genes were relevant to myeloid lineage cells such as CD14+ monocytes. In contrast, the downregulated genes were relevant to CD4+ and CD8+ T cells.Remarkable innate immune signatures are present in the leukocytes of LLD females but not males. Because inflammation is involved in the pathophysiology of depression, the altered inflammatory activity may be involved in the pathophysiology of LLD in women. In contrast, abnormal inflammation may be an uncommon feature in LLD males.

Ingenuity pathway analysis of human facet joint tissues: Insight into facet joint osteoarthritis.

Facet joint osteoarthritis (FJOA) is a common degenerative joint disorder with high prevalence in the elderly. FJOA causes lower back pain and lower extremity pain, and thus severely impacts the quality of life of affected patients. Emerging studies have focused on the histomorphological and histomorphometric changes in FJOA. However, the dynamic genetic changes in FJOA have remained to be clearly determined. In the present study, previously obtained RNA deep sequencing data were subjected to an ingenuity pathway analysis (IPA) and canonical signaling pathways of differentially expressed genes (DEGs) in FJOA were studied. The top 25 enriched canonical signaling pathways were identified and canonical signaling pathways with high absolute values of z-scores, specifically leukocyte extravasation signaling, Tec kinase signaling and osteoarthritis pathway, were investigated in detail. DEGs were further categorized by disease, biological function and toxicity (tox) function. The genetic networks between DEGs as well as hub genes in these functional networks were also investigated. It was demonstrated that C-X-C motif chemokine ligand 8, elastase, neutrophil expressed, growth factor independent 1 transcriptional repressor, Spi-1 proto-oncogene, CCAAT enhancer binding protein epsilon, GATA binding protein 1, TAL bHLH transcription factor 1, erythroid differentiation factor, minichromosome maintenance complex component 4, BTG anti-proliferation factor 2, BRCA1 DNA repair-associated, cyclin D1, chromatin assembly factor 1 subunit A, triggering receptor expressed on myeloid cells 1 and tumor protein p63 were hub genes in the top 5 IPA networks (with a score >30). The present study provides insight into the pathological processes of FJOA from a genetic perspective and may thus benefit the clinical treatment of FJOA.

CD8+ T Cells Require ITK-Mediated TCR Signaling for Migration to the Intestine.

The Tec kinase IL-2–inducible T cell kinase (ITK) regulates the expression of TCR-induced genes. Itk−/− T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice. To examine the function of ITK in T cell migration to the intestine, we examined the number of gut T cells in Itk−/− mice and then evaluated their expression of gut-homing receptors. Combined with in vitro murine T cell stimulation and in vivo migration assay using congenic B6 mice, we demonstrated an essential role for ITK in T cell migration to the intestine in mice. Reconstitution of Itk−/− mouse CD8+ T cells with IFN regulatory factor 4 restored gut-homing properties, providing mechanistic insight into the function of ITK-mediated signaling in CD8+ T cell migration to the intestinal mucosa in mice.

Tec-1 kinase negatively regulates regenerative neurogenesis in planarians.

Negative regulators of adult neurogenesis are of particular interest as targets to enhance neuronal repair, but few have yet been identified. Planarians can regenerate their entire CNS using pluripotent adult stem cells, and this process is robustly regulated to ensure that new neurons are produced in proper abundance. Using a high-throughput pipeline to quantify brain chemosensory neurons, we identify the conserved tyrosine kinase tec-1 as a negative regulator of planarian neuronal regeneration. tec-1RNAi increased the abundance of several CNS and PNS neuron subtypes regenerated or maintained through homeostasis, without affecting body patterning or non-neural cells. Experiments using TUNEL, BrdU, progenitor labeling, and stem cell elimination during regeneration indicate tec-1 limits the survival of newly differentiated neurons. In vertebrates, the Tec kinase family has been studied extensively for roles in immune function, and our results identify a novel role for tec-1 as negative regulator of planarian adult neurogenesis.

Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib.

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cell-based occupancy assays that quantify the percentage of drug-inactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 ± 0.27 and 1.09 ± 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT: This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.

Ibrutinib, but not zanubrutinib, induces platelet receptor shedding of GPIb-IX-V complex and integrin αIIbβ3 in mice and humans

AbstractThe Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has proven to be efficacious in the treatment of B-cell chronic lymphocytic leukemia (B-CLL) and related diseases. However, a major adverse side effect of ibrutinib is bleeding, including major hemorrhages. The bleeding associated with ibrutinib use is thought to be due to a combination of on-target irreversible Btk inhibition, as well as off-target inhibition of other kinases, including EGFR, ITK, JAK3, and Tec kinase. In this study, we investigated the effects of ibrutinib vs zanubrutinib (a more selective Btk inhibitor) on platelet activation, glycoprotein expression, and thrombus formation. Ibrutinib, but not zanubrutinib, induced a time- and dose-dependent shedding of GPIb-IX complex and integrin αIIbβ3, but not of GPVI and GPV, from the platelet surface. The shedding of GPIbα and GPIX was blocked by GM6001 and TAPI-2, an ADAM17 inhibitor but not ADAM10 inhibitor. Ibrutinib but not zanubrutinib treatment of human platelets increased ADAM17 activation. Pretreatment of C57BL/6 mice with ibrutinib (10 mg/kg), but not zanubrutinib (10 mg/kg), inhibited ex vivo and in vivo thrombus growth over time. Platelets from ibrutinib-treated patients with CLL showed reduced GPIb-IX complex and integrin αIIbβ3 surface expression and reduced ex vivo thrombus formation under arterial flow, which was not observed in zanubrutinib-treated patients. In mice, ibrutinib, but not zanubrutinib, led to increased soluble GPIbα and soluble αIIb levels in plasma. These data demonstrate that ibrutinib induces shedding of GPIbα and GPIX by an ADAM17-dependent mechanism and integrin αIIbβ3 by an unknown sheddase, and this process occurs in vivo to regulate thrombus formation.

Lipid-targeting pleckstrin homology domain turns its autoinhibitory face toward the TEC kinases

The pleckstrin homology (PH) domain is well known for its phospholipid targeting function. The PH-TEC homology (PHTH) domain within the TEC family of tyrosine kinases is also a crucial component of the autoinhibitory apparatus. The autoinhibitory surface on the PHTH domain has been previously defined, and biochemical investigations have shown that PHTH-mediated inhibition is mutually exclusive with phosphatidylinositol binding. Here we use hydrogen/deuterium exchange mass spectrometry, nuclear magnetic resonance (NMR), and evolutionary sequence comparisons to map where and how the PHTH domain affects the Bruton's tyrosine kinase (BTK) domain. The data map a PHTH-binding site on the activation loop face of the kinase C lobe, suggesting that the PHTH domain masks the activation loop and the substrate-docking site. Moreover, localized NMR spectral changes are observed for non-surface-exposed residues in the active site and on the distal side of the kinase domain. These data suggest that the association of PHTH induces allosteric conformational shifts in regions of the kinase domain that are critical for catalysis. Through statistical comparisons of diverse tyrosine kinase sequences, we identify residues unique to BTK that coincide with the experimentally determined PHTH-binding surface on the kinase domain. Our data provide a more complete picture of the autoinhibitory conformation adopted by full-length TEC kinases, creating opportunities to target the regulatory domains to control the function of these kinases in a biological setting.

Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation.

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.

Role and mechanism of nonreceptor tyrosine kinase Tec in endotoxin/lipopolysaccharide-induced interleukin-8 production in human alveolar epithelial cells A549

Objective: To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549. Methods: Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 μg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 μmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, and 100 μmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 μmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 μg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test. Results: (1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P 0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 μmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 μmol/L LFM-A13+ LPS group and 100 μmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P 0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01). Conclusions: Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.

Discovery of Zanubrutinib (BGB-3111), a Novel, Potent, and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Aberrant activation of Bruton's tyrosine kinase (BTK) plays an important role in pathogenesis of B-cell lymphomas, suggesting that inhibition of BTK is useful in the treatment of hematological malignancies. The discovery of a more selective on-target covalent BTK inhibitor is of high value. Herein, we disclose the discovery and preclinical characterization of a potent, selective, and irreversible BTK inhibitor as our clinical candidate by using in vitro potency, selectivity, pharmacokinetics (PK), and in vivo pharmacodynamic for prioritizing compounds. Compound BGB-3111 (31a, Zanubrutinib) demonstrates (i) potent activity against BTK and excellent selectivity over other TEC, EGFR and Src family kinases, (ii) desirable ADME, excellent in vivo pharmacodynamic in mice and efficacy in OCI-LY10 xenograft models.

Abstract 13: Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib

Abstract Acalabrutinib (Calquence®) is a potent, selective, orally administered, covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma from the US FDA in October 2017. Profiling of acalabrutinib metabolites in human plasma revealed a late-eluting, +16 Da metabolite circulating at concentrations higher than parent drug. Metabolite regiochemistry could not be determined by mass spectrometry. In vitro metabolism and preparative HPLC was used to generate a pure sample of the metabolite for structural characterization by NMR. Confirmatory chemical synthesis revealed a pyrrolidine ring-opened ketone. The structure of the metabolite, designated ACP-5862, and a smaller -2 Da peak, identified as dehydropyrrolidine, M25, inferred a common carbinolamide intermediate in their genesis. Both metabolites retained the butynamide electrophile responsible for the inactivation of BTK. In vitro studies on the inhibition of BTK and related Tec and Src kinases revealed that ACP-5862 was active against BTK with similar selectivity and potency to acalabrutinib (Kaptein et al, 2019) This work then investigated the in vitro metabolism and drug transport features of acalabrutinib, and the metabolite ACP-5862, to establish the potential for clinical drug-drug interactions (DDI) via CYPs, UGTs and drug transporters. CYP reaction phenotyping indicated CYP3A4 was responsible for both the formation and further metabolism of ACP-5862. Km and Vmax values for the formation of ACP-5862 using rCYP3A4 were 2.78 μM and 4.13 pmol/pmol CYP/min, respectively. The in vitro intrinsic clearance of ACP-5862 was 23.6 μL/min/mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9 and CYP3A4 in vitro, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19, with no inhibition of CYP1A2, CYP2B6, or CYP2D6. Similarly, UGT1A1, UGT2B7, and aldehyde oxidase were not inhibited. Neither parent or ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of MDR1 and BCRP in vitro, but were not substrates of OATP1B1 or OATP1B3. Acalabrutinib was not a substrate of OAT1, OAT3, and OCT2. Based on static PK model calculations, acalabrutinib may cause a modest increase in exposure to coadministered BCRP substrates by inhibition of intestinal BCRP, but with no inhibition of BCRP at the systemic level. The PK of substrates of MDR1, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 are not likely to be altered by acalabrutinib or ACP-5862. These data were combined with clinical DDI data (Izumi et al, 2017) to simulate DDI in the presence of CYP3A inhibitors and inducers. PBPK models confirmed that acalabrutinib and ACP-5862 were not likely to perpetrate CYP2C8 or CYP3A4 mediated drug interactions (Zhou et al., 2019). Overall, acalabrutinib and major metabolite, ACP-5862 have a favorable drug interaction profile. Citation Format: Terry Podoll, Paul G. Pearson, Jerry Evarts, Tim Ingallinera, Hao Sun, Stephen Byard, Adrian J. Fretland, J. Greg Slatter. Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 13.

PF481 UPDATED SAFETY AND EFFICACY DATA IN A PHASE 1/2 TRIAL OF PATIENTS WITH WALDENSTRÖM MACROGLOBULINAEMIA (WM) TREATED WITH THE BRUTON TYROSINE KINASE (BTK) INHIBITOR ZANUBRUTINIB (BGB‐3111)

Background:Zanubrutinib, an investigational BTK inhibitor, achieves high plasma concentrations and sustained complete BTK occupancy in blood and lymph nodes, with greater selectivity for BTK vs other TEC and EGFR family kinases in biochemical assays, and favorable pharmacokinetic/pharmacodynamic properties in preclinical studies.Aims:To present updated zanubrutinib safety and efficacy data in patients (pts) with WM.Methods:This is a global, open‐label, multicenter, phase 1/2 study in pts with B‐cell malignancies with indication‐specific expansion cohorts. Pts received doses of zanubrutinib ranging from 40 mg once daily to the final recommended phase 2 dose of 160 mg twice daily or 320 mg once daily until disease progression (PD) or unacceptable toxicity. We report here on the 77 WM pts from the cohorts who had no prior BTK therapy. The primary endpoint is safety/tolerability. Secondary endpoints include response rate and PFS assessed by IRC.Results:As of September 16th, 2018, 77 pts with WM received zanubrutinib (Table). The most common (&gt;15%) adverse events (AEs) of any grade were upper respiratory tract infection (46%), contusion (30%), cough (20%), headache (18%), back pain (17%), diarrhea (17%), urinary tract infection (16%), and constipation (16%). Grade ≥3 AEs in ≥5% of pts were neutropenia (10%) anemia (8%), hypertension (5%), and basal cell carcinoma (5%). The most common serious AEs were cellulitis (5%), pneumonia (4%), pyrexia (3%), febrile neutropenia (3%), and atrial fibrillation (3%). Sixteen pts discontinued treatment, 8 due to AE, 4 due to PD, 1 due to investigator decision, and 3 others (radiation therapy [n = 1], not compliant with taking drug [n = 2]). Five fatal AEs occurred: abdominal sepsis, septic arthritis, scedosporium infection, bronchiectasis, and gastric adenocarcinoma, of which one was considered related to treatment (septic arthritis) Regarding AEs of special interest with BTK inhibitor therapy, 2 pts (3%) had major haemorrhage (1 with melaena and hemothorax, 1 with haemorrhagic cystitis) and 4 pts (5%) experienced atrial fibrillation or flutter, 1 of which was grade 3. Two pts (3%) had grade 3 diarrhea. Of the 73 efficacy evaluable pts (4 pts non‐evaluable due to having &lt;5 g/L IgM levels at baseline), 24 were treatment‐naïve and 49 were relapsed/refractory (Table; median follow‐up 23.9 mo [range, 4.4–45.7]). Based on modified IWWM‐6 (Owen) as assessed by IRC, the overall response rate (ORR; minor response [MR] or better) was 92% (67/73 pts), major response rate (MRR; partial response [PR] or better) 82%, and rate of very good PR (VGPR, ≥90% IgM reduction) or complete response (CR) was 42%. Consistent with prior reports of BTK inhibitors in WM, 26/57 pts (46%) with MYD88 L265P achieved VGPR or CR. Of the pts with known MYD88 WT WM, 7/8 (88%) responded to zanubrutinib, including 1 CR, 1 VGPR, 3 PR, and 2 MR. Median PFS has not been reached; PFS rate at 12 months was 90% (95% CI [80,95]) and at 24 months was 81% (95% CI [68,89]). 60 of 67 responding patients (MR or better) remained in response at 12 months and 58 of 67 pts at 24 months.Summary/Conclusion:These data demonstrate that zanubrutinib monotherapy is well tolerated and highly effective in the treatment of pts with WM. The notable CR/VGPR and 2 year PFS rates confirm this BTK inhibitor achieves both deep and durable responses. The results of a phase 3 study comparing zanubrutinib with ibrutinib are awaited.image

PS1159 POOLED ANALYSIS OF SAFETY DATA FROM MONOTHERAPY STUDIES OF THE BRUTON TYROSINE KINASE (BTK) INHIBITOR, ZANUBRUTINIB (BGB‐3111), IN B‐CELL MALIGNANCIES

Background:Zanubrutinib, a next generation BTK inhibitor, achieves high plasma concentrations and sustained complete BTK occupancy in blood and lymph nodes, with greater selectivity for BTK vs other TEC and EGFR family kinases in biochemical assays, and favorable pharmacokinetic/pharmacodynamic properties in preclinical studies.Aims:We conducted an analysis of pooled safety data from patients (pts) with non‐Hodgkin lymphoma (NHL), Waldenström's Macroglobulinemia (WM) and CLL/SLL in 6 ongoing zanubrutinib monotherapy studies.Methods:Patients received oral zanubrutinib at doses of 320 mg once daily or 160 mg twice daily. The analysis included frequency and severity of adverse events (AEs), AEs of interest (AESIs), and AEs leading to death, dose reduction or treatment discontinuation (d/c).Results:In total, 671 pts were included in this pooled analysis (data cutoff date 16 SEPT, 2018). The median age was 64 years (range, 20–90), 68.4% were male, and 45.6% were from study sites in China. The median duration of zanubrutinib exposure was 11 months (range, 0.1–46.9). AEs reported in ≥10% of pts were upper respiratory tract infection, absolute neutrophil count (ANC) decreased, diarrhea, rash, cough, contusion, anemia, platelet count decreased, urinary tract infection, hematuria, fatigue, and white blood cell count decreased. Grade (Gr) ≥3 AEs reported in at least 3% of pts were ANC decreased, anemia, neutropenia, pneumonia, platelet count decreased, lung infection, and hypertension. The most common serious AEs were pneumonia (5%) and lung infection (3.0%). AEs of special interest are shown in the Table. Infection rates were 12.5 events/100 person‐mo (Gr ≥3, 1.9 events/100 person‐mo); 39.9% of pts reported infection in the first 3 mo, 16.6% in mo 3–6, and 13.5% in mo 6–12. The most common bleeding events were contusion (17.0%) and hematuria (11.0%). Major hemorrhage (defined under Table) incidence was 0.21 events/100 person‐mo, most commonly upper gastrointestinal (GI) hemorrhage (4 pts), retinal hemorrhage and hematuria (2 pts each). The median time to 1st major hemorrhage was 62.5 days (range, 3–601). Among the 12 pts with atrial fibrillation/flutter, mosthad risk factors including hypertension (4 pts), cardiovascular disease (7 pts), hyperlipidemia (4 pts) and/or concurrent infection (4 pts). Second primary malignancies were reported in 53 pts, most commonly basal cell (24 pts, 83% from Australia [AUS]/New Zealand [NZL]) and cutaneous squamous cell (13 pts, all from AUS/NZL) carcinomas. AEs leading to death in &gt;1 pt were pneumonia (6 pts), septic shock (2 pts), unspecified (5 pts), and multiple organ dysfunction (2 pts). AEs led to zanubrutinib d/c in 10.0% of pts (10.5%, 9.9%, and 5.4% in pts with WM, NHL and CLL, respectively); 4.5% discontinued for treatment‐related AEs. Treatment‐related AEs leading to d/c in &gt;1 patient were pneumonia (4 pts), lung infection (4 pts) and thrombocytopenia (2 pts). The median time to d/c for AEs was 3.5 mo (range, 0.1–40.6). A total of 28 pts (4.2%) required ≥1 dose reduction for AEs.Summary/Conclusion:Zanubrutinib was generally well‐tolerated in pts with various B‐cell malignancies with &lt;5% d/c for treatment‐related AEs; dose reductions were uncommon. As previously reported (Tam, EHA 2018), AESIs such as atrial fibrillation and major hemorrhage were infrequent. These data demonstrated low, safety‐related treatment failure rates at doses of zanubrutinib associated with complete and sustained BTK inhibition in clinical trials.image

PF-06651600, a Dual JAK3/TEC Family Kinase Inhibitor.

PF-06651600 was developed as an irreversible inhibitor of JAK3 with selectivity over the other three JAK isoforms. A high level of selectivity toward JAK3 is achieved by the covalent interaction of PF-06651600 with a unique cysteine residue (Cys-909) in the catalytic domain of JAK3, which is replaced by a serine residue in the other JAK isoforms. Importantly, 10 other kinases in the kinome have a cysteine at the equivalent position of Cys-909 in JAK3. Five of those kinases belong to the TEC kinase family including BTK, BMX, ITK, RLK, and TEC and are also inhibited by PF-06651600. Preclinical data demonstrate that inhibition of the cytolytic function of CD8+ T cells and NK cells by PF-06651600 is driven by the inhibition of TEC kinases. On the basis of the underlying pathophysiology of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, alopecia areata, and vitiligo, the dual activity of PF-06651600 toward JAK3 and the TEC kinase family may provide a beneficial inhibitory profile for therapeutic intervention.

A negative role for the interleukin-2-inducible T-cell kinase (ITK) in human Foxp3+ TREG differentiation.

The Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively modulates regulatory T cell (TREG) differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in human primary naïve peripheral blood CD4 T cells increased Foxp3+ expression under both TREG and T helper priming conditions. TREG differentiated under ITK knockdown conditions exhibited enhanced expression of the co-inhibitory receptor PD-1 and were suppressive in a T cell proliferation assay. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and promoted Th1 differentiation. Lastly, a dual ITK/RLK Tec kinase inhibitor did not induce Foxp3 in CD4 T cells, but conversely abrogated Foxp3 expression induced by ITK knockdown. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but concomitant inhibition of other Tec family kinases may negate this effect.

Bruton's tyrosine kinase (Btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption

Osteoclasts are responsible for bone erosion in osteoporosis and rheumatoid arthritis (RA). Both Btk and Tec kinases have essential functions in osteoclast differentiation. Tirabrutinib is a highly potent and dual oral Btk/Tec inhibitor with an IC50 in the nmol/L range and significantly inhibits the M-CSF and RANKL-driven osteoclast differentiation. It was hypothesized that the in vitro activity of tirabrutinib could be demonstrated in mice bone resorption model. The RANKL model studies show that tirabrutinib significantly suppressed bone loss with the inhibition of serum TRAPCP5b and urinary CTX-1. Bone Mineral Density (BMD) loss in tirabrutinib-treated mice was 55% (P < .05), 87% (P < .001) and 88% (P < .001) for the 3, 10 and 30 mg/kg dose groups respectively.Btk and Tec are required for osteoclast differentiation and activation based on the genetic evidence obtained from Btk and Tec double deficient mice. Tirabrutinib may be a novel therapeutic target for bone diseases, such as osteoporosis and RA.