Tec Family Of Tyrosine Kinases Research Articles

Abstract 1043: Identification of the novel immune synapse-localized proteome for immuno-oncology using Microscoop-induced targeted photo-biotinylation

Abstract Although tens of immune synapse (IS) protein species are known, there remain many unknown IS-localized protein species. Understanding the proteome of the IS between a target cell and a lymphocyte is crucial for advancing immuno-oncology. The low abundance of ISs and the lack of a definitive enrichment marker have limited efficient proteomic profiling. Here, we used Microscoop™, an innovative system that integrates microscopy, machine learning, and targeted photo-biotinylation to enable precise and spatially specific enrichment of IS proteins, thereby facilitating proteomic discovery of the IS. We utilized Raji B cells as antigen-presenting cells (APCs) and induced IS formation with Jurkat T cells. The system first employed immunofluorescence imaging of CD3, a common IS marker in Jurkat T cells, and used a convolutional neural network-based deep learning algorithm to train the recognition of ISs from CMTPX-stained Raji B cells. Our automated system then successfully achieved spatially-targeted biotin-tagging of proteins at ISs through thousands of fields of view of imaging, deep learning-driven pattern generation, and photochemical labeling. Subsequent streptavidin pull-down and mass spectrometry analysis enabled the identification of IS-specific proteins. Remarkably, our spatial proteomic approach led to the isolation and identification of hundreds of different species at the IS interface, including proteins associated with key components of T-cell receptor (TCR) signaling pathways such as the TCR/CD3 complex, Src and Tec family tyrosine kinases, and pivotal NF-kB signaling proteins. Beyond these, we also identified a significant enrichment of proteins not previously associated with the IS. Our study not only illuminates previously unknown aspects of immune regulation at the IS interface, but also shedded light on proteins potentially important for immuno-oncology. These insights have significant implications for cancer research, particularly in understanding and manipulating immune responses for therapeutic purposes. Citation Format: Weng-Man Chong, Cecile Li, Chun-Kai Huang, Chantal Hoi Yin Cheung, Hsiao-Jen Chang, Chia-Wen Chung, Jung-Chi Liao. Identification of the novel immune synapse-localized proteome for immuno-oncology using Microscoop-induced targeted photo-biotinylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1043.

ITK Inhibitor Induces Dose-Dependent Th1 Skewing in Normal T Cells and Is Active in Refractory T Cell Lymphomas

Introduction Interleukin 2 inducible T cell kinase (ITK) is a TEC family tyrosine kinase expressed in T cells with an important role both in T cell receptor (TCR) signaling and T helper cell differentiation. Resting lymphocyte kinase (RLK), a closely related kinase, is redundant to ITK and is involved in the differentiation of naïve T cells into Th1 T cells. ITK-/- mice exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells that secrete IFNγ; whereas, double ITK/RLK knockout mice exhibit profound defects in T cell function. Th1 cells are needed for various cytotoxic T cell functions such as destruction of tumor cells. CPI-818 is a covalent inhibitor of ITK (KD 2.5nM) with > 100 fold selectivity over RLK and other TEC family kinases. CPI-818 is being evaluated in an ongoing phase 1 trial in patients (pts) with refractory T cell lymphomas (TCL). We now report that selective ITK blockade with CPI-818 induces Th1 skewing of normal T cells and demonstrates anti-tumor activity. Methods Normal human peripheral blood mononuclear cells (PBMC) were stimulated in vitro for 6 days with anti-CD3/CD28 in the presence of CPI-818. Th1 cells were measured by flow cytometry based on ratio of IFNγ:IL4 staining cells. Sezary cells were similarly stimulated in the presence of CPI-818. Peripheral blood and tumor samples were stained with antibodies to CD3, 4, 8, 183, 196, 197 and 45RA to enumerate Th1 and other T cell subsets using multicolor flow cytometry. Successive cohorts of TCL pts were treated in a phase 1 trial until disease progression with continuous oral dosing of CPI-818 100mg, 200mg, 400mg or 600mg given BID. Results T cell receptor stimulated PBMC from 12 healthy donors incubated in vitro with CPI-818 demonstrated Th1 skewing at concentrations of 0.1 to < 10uM (Figure 1). Maximal skewing was seen at 1uM; cell viability was unaffected but, proliferation was inhibited at 10uM. Similar studies using Th2 Sezary cells showed inhibition of proliferation with CPI-818 (0.5-2.5uM). Thirty-three pts with TCL with a median age of 62 yrs and median of 4 prior therapies were enrolled. Histologies included: PTCL-NOS (17), AITL (4), and CTCL (12). The most common (≥ 10%) AEs were nausea, chills, vomiting, fatigue, pyrexia, decreased appetite, pruritus, and rash. Treatment-related Grade 3+ AEs were: anemia (1), lymphocytosis (1), WBC decreased (1) and neutropenia (1). The swimmer plot (Figure 2) shows a dose-dependent relationship with an optimum dose of 200mg (N=10) producing 1 CR 25 mo; 1 PR 2+ mo; 1 nodal CR 16 mo (CTCL); 2 SD; 1 PD; 4 not yet evaluated; 6 pts remain on treatment. Pharmacokinetic and target occupancy studies for the 200mg dose showed mean plasma Cmax of 2.3uM (SD 1.9uM) and ITK target occupancy of >75%. Analysis of blood in 4 of 4 pts treated in this cohort showed increases in Th1 cells (CD3+/CD4+/CD8-/CD183+/CD196-) compared to baseline and increases in terminally differentiated T effector memory cells (TEMRA CD3+/CD4-/CD8+/CD197-/CD45RA+). Tumor biopsy from 1 patient taken during response demonstrated an increase in Th1 (0 to 40%) and TEMRA CD8+ T cells (2.83% to 18.6%). CPI-818 reduced blood eosinophils (Eos) in 3 of 3 pts in this cohort with elevated Eos count, consistent with inhibition of Th2 function. Conclusions This study demonstrates that selective inhibition of ITK, while sparing RLK, induces Th1 skewing. As expected, the effects on T cell differentiation and proliferation are dose dependent: lower doses induce Th1 skewing and higher concentrations in vitro and in vivo lead to inhibition of TCR signaling and proliferation. Tumor responses were observed in several patients receiving 200mg BID, which achieves drug plasma levels consistent with Th1 skewing observed in vitro. The increases in Th1 cells and TEMRA CD8+ cells suggest that anti-tumor activity is due to induction of a host anti-tumor response. This raises the possibility that immunomodulation with ITK blockade could be used for immunotherapy of other cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

Discovery of Reversible Covalent Bruton's Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib).

Bruton's tyrosine kinase (BTK), a Tec family tyrosine kinase, is critical in immune pathways as an essential intracellular signaling element, participating in both adaptive and immune responses. Currently approved BTK inhibitors are irreversible covalent inhibitors and limited to oncology indications. Herein, we describe the design of covalent reversible BTK inhibitors and the discoveries of PRN473 (11) and rilzabrutinib (PRN1008, 12). These compounds have exhibited potent and durable inhibition of BTK, in vivo efficacy in rodent arthritis models, and clinical efficacy in canine pemphigus foliaceus. Compound 11 has completed phase 1 trials as a topical agent, and 12 is in phase 3 trials for pemphigus vulgaris and immune thrombocytopenia.

Interleukin-2-inducible T-cell kinase (Itk) signaling regulates potent noncanonical regulatory T cells.

Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2‐inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of “noncanonical” CD4+CD25−FOXP3+ Tregs (ncTregs), as well as of “canonical” CD4+CD25+FOXP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft‐versus‐host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft‐versus‐leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk –/− ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders.

Itk Promotes the Integration of TCR and CD28 Costimulation through Its Direct Substrates SLP-76 and Gads.

The costimulatory receptor CD28 synergizes with the TCR to promote IL-2 production, cell survival, and proliferation; yet the obligatory interdependence of TCR and CD28 signaling is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 Y173 and PLC-γ1 Y783. In this study, we identified TCR-inducible, Itk-mediated phosphorylation of Gads Y45 in a human T cell line and in mouse primary T cells. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the dimerization-dependent binding of Gads to phospho-LAT. We provide evidence that Itk acts through SLP-76 and Gads to promote the TCR/CD28-induced activation of the RE/AP transcriptional element from the IL-2 promoter. Two Itk-related features of SLP-76, Y173 and a proline-rich Itk SH3 binding motif on SLP-76, were dispensable for activation of NFAT but selectively required for the TCR/CD28-induced increase in cytoplasmic and nuclear c-Rel and consequent RE/AP activation. We provide evidence that unphosphorylated, monomeric Gads mediates an RE/AP-directed inhibitory activity that is mitigated upon Gads dimerization and Y45 phosphorylation. This study illuminates a new, to our knowledge, regulatory module, in which TCR-induced, Itk-mediated phosphorylation sites on SLP-76 and Gads control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.

Discovery of potent and selective reversible Bruton’s tyrosine kinase inhibitors

Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-cell disorders. Here we disclose the discovery of a novel imidazo[4,5-b]pyridine series of potent, selective reversible BTK inhibitors through a rational design approach. From a starting hit molecule 1, medicinal chemistry optimization led to the development of a lead compound 30, which exhibited 58 nM BTK inhibitory potency in human whole blood and high kinome selectivity. Additionally, the compound demonstrated favorable pharmacokinetics (PK), and showed potent dose-dependent efficacy in a rat CIA model.

Design, synthesis and structure–activity relationship of indolylindazoles as potent and selective covalent inhibitors of interleukin-2 inducible T-cell kinase (ITK)

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays an important role in T cell signaling downstream of the T-cell receptor (TCR). Herein we report the discovery of a series of indolylindazole based covalent ITK inhibitors with nanomolar inhibitory potency against ITK, good kinase selectivity and potent inhibition of the phosphorylation of PLCγ1 and ERK1/2 in living cells. A computational study provided insight into the interactions between inhibitors and Phe437 at the ATP binding pocket of ITK, suggesting that both edge-to-face π-π interaction and the dihedral torsion angle contribute to inhibitors’ potency. Compounds 43 and 55 stood out as selective covalent inhibitors with potent cellular activity, which could be used as chemical tools for further study of ITK functions.

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.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%–92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14–21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.

Dynamics of the Tec-family tyrosine kinase SH3 domains.

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.

Ibrutinib Treatment Modulates T Cell Activation and Polarization in Immune Response

IL2-inducible T cell kinase (ITK) belongs to the Tec family of tyrosine kinases and is an important component of TCR-mediated signaling. ITK regulates T cell adhesion and migration, signaling and activation, Th17 polarization and Treg differentiation, which are all critical for immune responses in health and disease. Ibrutinib is a newly developed Bruton tyrosine kinase (BTK) inhibitor with outstanding clinical activity and tolerability in B-cell malignancies. Recently, it has been demonstrated that Ibrutinib as an irreversible inhibitor of both BTK and ITK that blocks downstream immune receptor activation. Given its critical role in T-cell signaling and activation, ITK is an appealing therapeutic target that can contribute to the pathogenesis of various immune disorders, which was our impetus for further studying Ibrutinib - a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility.Herein, we report that Ibrutinib inhibits T cell adhesion, proliferation and immunological synapse formation. We found Ibrutinib treatment reduced the homotypic aggregation of primary mouse T cells mediated by PMA and CD3-antibody at 0.5µM, and completely blocked aggregation at 5µM concentration. We investigated whether Ibrutinib treatment can prevent mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and found the inhibition of T cell proliferation by Ibrutinib is dose-dependent. With the increase of Ibrutinib concentration from 0.5µM to 1µM and 5µM, the frequency of dividing cells were greatly decreased for both CD4+ and CD8+ T cells; the proliferation was completely blocked at about 1µM concentration with majority T cells were still alive. To further investigate the role of Ibrutinib in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells using confocal microscope. We found that TCR was enriched proximal to the membrane and associated with the prominent cytoskeleton reorganization (FA) cluster in the after 30 min of TCR stimulation. In the presence of Ibrutinib (1µM), the predominant pattern was different with TCR and FA evenly distributed across the cell surface upon CD3-antibody simulation, similar as the control without any TCR stimulation. Thus, inhibition of ITK with Ibrutinib can prevents TCR signaling and activation.Although ablation of ITK have been shown to subvert Th2 immunity, thereby potentiating Th1-based immune responses, more recent published data demonstrated ITK is a positive modulator of IL-17A production, with reduced percentages of IL17A-producing cells in ITK-deficient CD4+ T cells. In addition, ITK has been implicated in the development and suppressor function of Treg. ITK-deficient CD4+ T cells can differentiate more efficiently into Treg cells and exhibit increased FoxP3 induction in mice. To determine the effect of Ibrutinib treatment in Th17 and Treg differentiation, naïve CD4+ T cells (CD62Lhi CD44lo CD25neg CD4+) from C57BL/6 mice were cultured with irradiated splenic APC from BALB/C mice in the presence of polarizing reagents (Th17 or Treg) for 5 days, then restimulated and after which Foxp3-, IL-17- and IFNγ-producing cells were analyzed using intracellular staining. We found Ibrutinib treatment (1μM) decreased the Th17-producing cells from 23% (PBS control) to 14% under Th17 culture condition, but increased FoxP3-producing cells from 43% (PBS control) to 57% under Treg culture condition. Furthermore, mRNA expression of Th17 (IL-17 and RORgt) genes was significant decreased while Treg (FoxP3) gene was significant increased in the presence of Ibrutinib. This is consistent with recently published data demonstrating that ITK-mediated signaling regulates the balance between Th17 and Treg in vitro and in vivo. Thus, inhibition of ITK with Ibrutinib can influence Th17 and Treg cell differentiation.In summary, our studies demonstrated that Ibrutinib treatment can: (1) inhibit primary mouse T cell aggregation and adhesion; (2) inhibit the proliferation of both CD4+ and CD8+ T cells; (3) prevent cytoskeleton reorganization, clustering and immune synapse formation mediated by TCR-activation and signaling; (4) decrease the number of Th17-producing cells and mRNA expression of Th17 (IL-17 and RORgt) genes under Th17 culture condition; (5) potentiate Treg differentiation and induces FoxP3 expression in vitro. DisclosuresNo relevant conflicts of interest to declare.

Critical role of ITK in mouse γ-herpes virus 68 infection as a model for human immunodeficiency (VIR1P.1140)

Abstract IL-2-inducible T cell kinase (ITK) is a Tec family tyrosine kinase predominantly expressed in T cells. In the absence of ITK, the magnitude of T cell receptor (TCR) signaling is greatly reduced, but not abolished, leading to an overall decrease in TCR signaling strength. Recently, various mutations in ITK were identified in patients suffering from a fatal lymphoproliferation due to a failure to control Epstein-Barr virus (EBV) infection. Interestingly, Itk-/- mice have been shown to mount protective responses to several acute virus infections. Thus, we hypothesized that ITK plays an essential role in long-term anti-viral immunity against latent γ-herpes virus infection. To study this, we infected WT and Itk-/- mice with mouse γ-herpes virus 68 (MHV68). Despite a reduced response at D7 post-infection, Itk-/- mice ultimately controlled MHV68 replication in the spleen by D14. After the initial response, both WT and Itk-/- mice showed no difference in their viral titers in the spleen at later time points. However, Itk-/- mice developed severe gastrointestinal pathology following LPS-mediated reactivation of latent MHV68, accompanied by a high viral titer in the intestine. Furthermore, the number of lymphocytes in the intestinal epithelium and lamina propria in naïve Itk-/- mice was substantially less than WT mice. Overall, these data suggest that ITK is essential for anti-viral immunity against γ-herpes virus infection in the gut following reactivation from latency.

Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

Property- and structure-guided discovery of a tetrahydroindazole series of interleukin-2 inducible T-cell kinase inhibitors.

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.

Targeting Interleukin-2-Inducible T-cell Kinase (ITK) in T-Cell Related Diseases.

IL2-inducible T-cell kinase (ITK), a member of the Tec family tyrosine kinases, is the predominant Tec kinase in T cells and natural killer (NK) cells mediating T cell receptor (TCR) and Fc receptor (Fc R) initiated signal transduction. ITK deficiency results in impaired T and NK cell functions, leading to various disorders including malignancies, inflammation, and autoimmune diseases. In this mini-review, the role of ITK in T cell signaling and the development of small molecule inhibitors of ITK for the treatment of T-cell related disorders is examined.

Role of Itk in Th17 mediated inflammation model hypersensitivity pneumonitis (IRC8P.491)

Abstract Hypersensitivity Pneumonitis (HP) is a lung disease caused by repeated inhalation of environmental antigens leading to inflammation, tissue scarring, and loss of lung function. This pathology is believed to be due to the increased IL-17A, a cytokine secreted primarily by Th17 cells that induces recruitment of inflammatory cells such as neutrophils. The thermophile Sacharopolyspora rectivigula (SR) causes HP, using a murine model of SR exposure; we study the molecular mechanism of this disease development. Using novel IL-17A-GFP reporter mice, our data suggests that the high levels of IL-17A induced in response to SR are produced in part by CD4+ T cells and not neutrophils. The Tec family tyrosine kinase Itk regulates T cell activation and cytokine production in conventional Th17 cells but Itk-/- mice still develop HP. Histology shows an increase in inflammatory cells in lung airways as well as decline of lung tissue architecture. Itk-/- lungs also have high levels of IL-17A mRNA expression, and an increase in the number of CD4+ IL-17A producing cells. However, Itk-/- mice lack IL-17A producing γδ T cells in the early stage of HP, which recovers in the later stage. This directly coincides with the emergence of IL-17A producing CD4+γδ T cells in the lungs of Itk-/- mice exposed to SR. We conclude that Itk regulated signals are not critical for the production of IL-17A in response to SR, and Itk may differentially regulate the production of IL-17A in different types of T cells.

The orally available Btk inhibitor ibrutinib (PCI-32765) protects against osteoclast-mediated bone loss

Bone-resorbing osteoclasts play an essential role in normal bone homeostasis, as well as in various bone disorders such as osteoporosis and rheumatoid arthritis. Previously we showed that the Tec family of tyrosine kinases is essential for the differentiation of osteoclasts and the inhibition of Btk is a promising strategy for the prevention of the bone loss in osteoclast-associated bone disorders. Here we demonstrate that an orally available Btk inhibitor, ibrutinib (PCI-32765), suppresses osteoclastic bone resorption by inhibiting both osteoclast differentiation and function. Ibrutinib downregulated the expression of NFATc1, the key transcription factor for osteoclastogenesis, and disrupted the formation of the actin ring in mature osteoclasts. In addition, genome-wide screening revealed that Btk regulates the expression of the genes involved in osteoclast differentiation and function in both an NFATc1-dependent and -independent manner. Finally, we showed that ibrutinib administration ameliorated the bone loss that developed in a RANKL-induced osteoporosis mouse model. Thus, this study suggests ibrutinib to be a promising therapeutic agent for osteoclast-associated bone diseases.

IL-2–inducible T-cell kinase modulates TH2-mediated allergic airway inflammation by suppressing IFN-γ in naive CD4+ T cells

Asthma is a predominantly TH2 cell-dominated inflammatory disease characterized by airway inflammation and a major public health concern affecting millions of persons. The Tec family tyrosine kinase IL-2-inducible T-cell kinase (Itk) is primarily expressed in T cells and critical for the function and differentiation of TH cells. Itk(-/-) mice have a defective TH2 response and are not susceptible to allergic asthma. We sought to better understand the role of Itk signaling in TH differentiation programs and in the development and molecular pathology of allergic asthma. Using a murine model of allergic airway inflammation, we dissected the role of Itk in regulating TH cell differentiation through genetic ablation of critical genes, chromatin immunoprecipitation assays, and house dust mite-driven allergic airway inflammation. Peripheral naive Itk(-/-) CD4(+) T cells have substantially increased transcripts and expression of the prototypic TH1 genes Eomesodermin, IFN-γ, T-box transcription factor (T-bet), and IL-12Rβ1. Removal of IFN-γ on the Itk(-/-) background rescues expression of TH2-related genes in TH cells and allergic airway inflammation in Itk(-/-) mice. Furthermore, small hairpin RNA-mediated knockdown of Itk in human peripheral blood T cells results in increased expression of mRNA for IFN-γ and T-bet and reduction in expression of IL-4. Our results indicate that Itk signals suppress the expression of IFN-γ in naive CD4(+) T cells, which in a positive feed-forward loop regulates the expression of TH1 factors, such as T-bet and Eomesodermin, and suppress development of TH2 cells and allergic airway inflammation.

Calmodulin boosts Itk activity to induce sustained calcium responses in T cells (P1117)

Abstract The Tec family tyrosine kinase Itk is an important transducer of T cell activating signals. Itk regulates the levels of two second messengers Ca2+ and diacylglycerol, which are required for initiating transcriptional programs important for T cell priming and differentiation. How T cells translate intracellular signaling cascades into distinct functional outcomes remains a fundamentally important question. We hypothesize that distinct mechanisms of Itk activation lead to Ca2+ patterns that result in divergent T cell responses. Itk activation requires recruitment via its Pleckstrin homology (PH) domain to its membrane ligand PIP3, which is generated by PI-3 kinase. Here, we report a role for Calmodulin (CaM) in sustaining Itk activity and Ca2+ responses. CaM is a downstream Ca2+ sensor that controls numerous cellular enzymes, including Calcineurin. We have identified a novel interaction between the Itk PH domain and CaM. NMR analyses revealed that CaM binds adjacent to the PIP3 binding pocket, leading us to hypothesize that CaM regulates PIP3 binding. CaM inhibition or genetic mutation of the CaM binding site resulted in decreased PIP3 association and Itk activation, resulting in less sustained Ca2+ responses. We propose a model in which initial Itk activation results in low Ca2+ increases that feedback to boost membrane recruitment of Itk via CaM. We speculate that T cell responses requiring sustained Ca2+ signals, such as T cell differentiation require this feedback loop.