Itk Activation Research Articles

Synthesis and characterization of soquelitinib a selective ITK inhibitor that modulates tumor immunity

ITK is a kinase involved in T cell activation, proliferation and differentiation. In mice, selective knock-out of the ITK gene produces Th1 skewing of T helper cell differentiation. Soquelitinib, a covalent ITK inhibitor, blocks ITK activity with greater than 100-fold selectivity compared to inhibition of a related kinase, RLK. We describe the chemistry and biologic effects of soquelitinib. In vitro studies with normal or malignant T cells demonstrated that soquelitinib suppresses Th2 cytokine production preferentially with relative sparing of Th1 cytokines. Soquelitinib inhibits the in vivo growth of several syngeneic murine tumors including those that do not express ITK. Treatment with soquelitinib leads to increased tumor infiltration of normal CD8+ cells that possess enhanced T effector function. Soquelitinib reduced expression of T cell exhaustion markers and was able to restore T effector function to exhausted cells. Pharmacologic selective ITK inhibition may represent a novel approach to cancer immunotherapy.

Novel PI(3)K-p85α/p110δ-ITK-LAT-PLC-γ2 and Fyn-ADAP-Carma1-TAK1 Pathways Define Reverse Signaling via FasL.

The role of FasL in initiating death signals through Fas is well characterized. However, the reverse signaling pathway downstream of FasL in effector lymphocytes is poorly understood. Here, we identify that FasL functions as an independent activation receptor in NK cells. Activation via FasL results in the production of LFN-γ, GM-CSF, RANTES, MIP-1α, and MIP1-β. Proximal signaling of FasL requires Lck and Fyn. Upon activation, FasL facilitates the phosphorylation of PI(3)K-p85α/p55α subunits. A catalytically inactive PI(3)K-p110δD910A mutation significantly impairs the cytokine and chemokine production by FasL. Activation of ITK and LAT downstream of FasL plays a central role in recruiting and phosphorylating PLC-γ2. Importantly, Fyn-mediated recruitment of ADAP links FasL to the Carmal/ Bcl10/Tak1 signalosome. Lack of Carma1, CARD domain of Carma1, or Tak1 significantly reduces FasL-mediated cytokine and chemokine production. These findings, for the first time, provide a detailed molecular blueprint that defines FasL-mediated reverse signaling.

Blockade of Tyrosine Kinase, LCK Leads to Reduction in Airway Inflammation through Regulation of Pulmonary Th2/Treg Balance and Oxidative Stress in Cockroach Extract-Induced Mouse Model of Allergic Asthma.

Asthma is one of the most common inflammatory diseases affecting the airways. Approximately 300 million individuals suffer from asthma around the world. Allergic immune responses in the asthmatic airways are predominantly driven by Th2 cells and eosinophils. Lymphocyte-specific protein tyrosine kinase (LCK) is a non-receptor tyrosine kinase which regulates several key intracellular events through phosphorylation of its substrates. Some of the intracellular signaling pathways activated by LCK phosphorylation help in differentiation of Th2 cells which secrete allergic cytokines that amplify airway inflammation. Therefore, this investigative study was designed to determine the role of LCK in a cockroach extract (CE)-induced airway inflammation murine model of allergic asthma. Further, the effect of a pharmacological LCK inhibitor, A-770041, on allergic airway inflammation and key intracellular pathways in CD4+ T cells was assessed. Our data exhibit that there is an activation of LCK during allergic airway inflammation as depicted by increased p-LCK levels in CD4+ T cells. Activated LCK is involved in the activation of ITK, PLC-γ, GATA3, NFkB, and NFATc1. Activated LCK is also involved in the upregulation of Th2 related cytokines, such as IL-4/IL-5/IL-13 and oxidative stress, and the downregulation of Treg cells. Furthermore, utilization of LCK inhibitor causes the reduction in p-LCK, PLC-γ, GATA3, and NFATc1 as well as Th2 cytokines and oxidative stress. LCK inhibitor causes upregulation of Treg cells in allergic mice. LCK inhibitor also caused a reduction in CE-induced airway inflammation and mucus secretion. Therefore, the inhibition of LCK signaling could be a fruitful approach to adjust allergic airway inflammation through the attuning of Th2/Treg immune responses. This study could lead to the design of newer treatment options for better management of allergic inflammation in asthma.

Reciprocal SH2-SH3 Domain Contacts between ITK Molecules Limit T Cell Receptor Signaling in Th2-type CD4+ T Cells

ABSTRACT The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4+ T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in Itk-deficient CD4+ T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells.

Targeting of the Tec Kinase ITK Drives Resolution of T Cell–Mediated Colitis and Emerges as Potential Therapeutic Option in Ulcerative Colitis

The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. We analyzed patients with inflammatory bowel disease (n= 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.

Targeting SLP76/ITK Signaling Interaction Separates GBHD from GVL in Allo-HSCT

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for relapsed hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also primarily mediated by the same donor T cells. Here we assessed the role of T cells with attenuated TCR-mediated ITK activation in mediating GVL vs GVHD effects after allo-HSCT. T cells from mice with genomic knock-in to express a tyrosine to phenylalanine mutation at position 145 (Y145F) of the adaptor protein SLP-76 display defective TCR-mediated activation of the downstream kinase ITK. Alloreactive T cells from SLP76 Y145F knock-in (KI) mice did not cause GVHD but preserved cytotoxicity, accompanied by upregulation of Eomesodermin (Eomes), which was necessary for GVL function. We developed a novel peptide that can specifically inhibit SLP76 and ITK interactions, resulting in decrease phosphorylation of PLCγ1, ERK and reduce cytokine production in human T cells. This peptide inhibited donor T cell-mediated GVHD while maintaining GVL effects. Altogether, our data suggest that inhibiting SLP-76/ITK interaction could be a therapeutic strategy to reduce GVHD while maintaining the beneficial GVL effects after allo-HSCT treatment.

HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane

The HIV-1 virulence factor Nef promotes high-titer viral replication, immune escape, and pathogenicity. Nef interacts with interleukin-2-inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk), two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages, respectively). Using a cell-based bimolecular fluorescence complementation assay, here we demonstrate that Nef recruits both Itk and Btk to the cell membrane and induces constitutive kinase activation in transfected 293T cells. Nef homodimerization-defective mutants retained their interaction with both kinases but failed to induce activation, supporting a role for Nef homodimer formation in the activation mechanism. HIV-1 infection up-regulates endogenous Itk activity in SupT1 T cells and donor-derived peripheral blood mononuclear cells. However, HIV-1 strains expressing Nef variants with mutations in the dimerization interface replicated poorly and were significantly attenuated in Itk activation. We conclude that direct activation of Itk and Btk by Nef at the membrane in HIV-infected cells may override normal immune receptor control of Tec-family kinase activity to enhance the viral life cycle.

ITK signaling differentiates GVT and GVHD after allogeneic bone marrow transplantation by regulating IRF-4, JAK/STAT and Eomesodermin expression

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for patients with hematologic malignancies. Donor T cells contained within the graft prevent tumor recurrence by exhibiting graft-versus-tumor (GVT) effects but also cause graft-versus-host disease (GVHD); therefore, novel treatment strategies are needed to maintain GVT while suppressing GVHD. We investigated the role of TCR-mediated ITK activation in mediating GVT vs. GVHD effects after allo-HSCT. We discovered that T cells from ITK−/−mice display a CD62Lhi CD122+ CD44hi Innate Lymphoid Cell-Like (ILCL) functional phenotype, and higher Eomes but not T bet expression compared to WT. We showed that T cells from ITK−/−mice reduced IRF-4, JAK1 and JAK2 and STAT3. T cells from ITK−/− mice displayed reduced cytokine production but showed preserved cytotoxicity after allo-HSCT. These cells also showed defective upregulation of the chemokine receptors CX3CR3, CXCR1, and CXCR6, which correlated with their reduced migration into GVHD target organs. The defective migration of ITK deficient T cells into GVHD target organs contributed to separation of GVHD and GVT effects, since ITK deficeint T cells cleared intravenously injected but not subcutaneously injected tumor cells in allo-HSCT mice. Moreover, pharmacological ITK inhibition attenuated GVHD and preserved GVT function by wild type CD8+ T cells. Together, our data suggest that ITK inhibition could be used as therapy after allo-HSCT to reduce GVHD while preserving the beneficial GVT effects by donor T cells

Membrane Binding of HIV-1 Accessory Protein Nef on Sparsely-Tethered Bilayer Lipid Membranes: An Spr Study

Human Immunodeficiency Virus-1 Nef is an accessory protein essential for the progression of HIV-1 infection by aiding viral production by down-regulating defense mechanisms of the infected cell to help evade immune response. Nef has been shown to interact with Src- and Tec-family kinases at the plasma membrane of infected cells, leading to constitutive kinase activation. For example, blocking Interleukin-2-inducable T-cell Kinase (Itk) activity with pharmacological inhibitors has been shown to result in decreased viral spread. Evidence also shows that Nef dimerizes on the membrane as it recruits kinases. However, the exact mechanism of this interaction between Nef and the kinases, or indeed between those two classes of proteins and the membrane, is not much understood. We used Surface Plasmon Resonance to study the binding of Nef on sparsely-tethered bilayer lipid membranes (stBLMs) in preparation for future studies in complex with kinases. Nef exhibits two distinct binding modes, one fast and one slow, which we attribute to two different membrane binding sites on Nef. The fast binding mode is likely that of the expected binding mechanism for Nef; insertion into the membrane of a myristoyl tail, whereby the core region of the protein is distant from the membrane surface. The second binding mode has Nef in much closer proximity to the membrane, with a much higher surface coverage. This in turn blocks the fast binding mode in subsequent concentration additions. For a given concentration, the binding rate is far higher when that concentration is added directly to a neat bilayer, rather than reached through a series of concentration additions. Previous Neutron Reflectometry (NR) characterization of membrane-bound Nef showed a concentration-dependent conformation change, consistent with this interpretation.

A switch in nucleotide affinity governs activation of the Src and Tec family kinases

The Tec kinases, closely related to Src family kinases, are essential for lymphocyte function in the adaptive immune system. Whilst the Src and Abl kinases are regulated by tail phosphorylation and N-terminal myristoylation respectively, the Tec kinases are notable for the absence of either regulatory element. We have found that the inactive conformations of the Tec kinase Itk and Src preferentially bind ADP over ATP, stabilising both proteins. We demonstrate that Itk adopts the same conformation as Src and that the autoinhibited conformation of Src is independent of its C-terminal tail. Allosteric activation of both Itk and Src depends critically on the disruption of a conserved hydrophobic stack that accompanies regulatory domain displacement. We show that a conformational switch permits the exchange of ADP for ATP, leading to efficient autophosphorylation and full activation. In summary, we propose a universal mechanism for the activation and autoinhibition of the Src and Tec kinases.

ITK is a magnesium sensor during T cell activation

Abstract Interleukin-2-Inductible T-cell Kinase (ITK), a member of the TEC family of kinases, serves a vital role in the activation and differentiation of T-cells. Following stimulation of the T cell receptor (TCR) and phosphorylation by LCK, ITK phosphorylates PLCy1 and initiates a transduction pathway that results in the release of intracellular Ca2+ stores and the transcription of IL-2. Although ITK’s role in this signal transduction pathway is known, there is still much to be characterized about the regulation of ITK activity. Previous studies have indicated that Mg2+ serves a key role as a second messenger in T lymphocyte activation and that attenuated intracellular Mg2+ levels inhibit the phosphorylation of PLCy1 and impair Ca flux. We propose that ITK serves as a sensor for the Mg2+ flux required for optimal T cell activation. Kinase assays show that ITK activity is enhanced by Mg2+ and suggest a model where two Mg2+ ions occupy the catalytic site. Molecular modeling also suggests that ITK possesses two Mg2+ binding sites in the catalytic domain. We are currently studying the effect of Mg2+binding on ITK kinase activity via site directed mutagenesis and are in the process of generating crystal structures of ITK complexed with Mg2+. Finally, we propose that this ‘second site’ Mg2+ is a general feature of kinases and may highlight the importance of divalent cations in kinase regulation.

When inhibitors don't mimic knockouts

Immunology The T helper 2 (TH2) subset of lymphocytes releases cytokines implicated in the pathogenesis of asthma, a process that requires the kinase ITK. ITK-knockout mice are resistant to airway inflammation, suggesting that ITK inhibitors might be used to treat human asthma. However, Sun et al. found that an ITK-specific inhibitor aggravated disease symptoms in a mouse model of asthma. The airways of these mice had more T cells and higher levels of cytokines that are typically released by TH2 lymphocytes. Thus, targeting ITK activity in asthma patients may exacerbate disease. Sci. Signal. 8 , ra122 (2015).

Abstract 5496: Effect of BTK inhibitors on differentiation of human monocyte-derived dendritic cells

Abstract Dendritic cells (DCs) are primary antigen-presenting cells (APCs), which process tumor antigen and present it on the cell surface to the T cells of the adaptive immune system. However, immature DCs facilitate tolerance toward cancer cells. Bruton's tyrosine kinase (BTK) is highly expressed in B cells, monocytes, macrophages and dendritic cells and plays crucial roles in differentiation and activation of myeloid cells. Dendritic cells in Btk−/− mice were reported to have more mature phenotype and stronger in vitro and in vivo T cell stimulatory ability than wild-type DCs. However, the functions of BTK in human DC differencation and maturation were not well characterized. In this study, we revealed the effect of BTK inhibition with ibrutinib, an FDA approved drug for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), in human monocyte derived dendritic cells (moDCs). We show that BTK was expressed in monocytes and moDCs. BTK activity was regulated during moDCs differencation and BTK inhibitors blocked GM-CSF/IL4 induced activation of BTK. LPS instead of CD40L treatment increased BTK phosphorylation. It was noted that the percentage of CD11c+ MHC IIlow subsets, which represented the immature DCs, was significantly reduced upon ibrutinib treatment during moDC differencation. Meanwhile, ibrutinib dose-dependently increased proportion of CD11c+ MHC IIhigh cells, which represent more mature DCs. We also tested BGB-3111, a more selective BTK inhibitor without ITK, TXK, EGFR, HER2 and JAK3 activity in the same assay. We observed increased MHC II level during differentiation of human moDCs with BGB-3111 treatment. These results support combination strategies for BTK inhibitors with other cancer immunotherapy agents. Citation Format: Zhijian Sun, Dongping Zhou, Lusong Luo. Effect of BTK inhibitors on differentiation of human monocyte-derived dendritic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5496. doi:10.1158/1538-7445.AM2015-5496

Allele-sensitive mutant, Itkas, reveals that Itk kinase activity is required for Th1, Th2, Th17, and iNKT-cell cytokine production.

Itk(-/-) mice exhibit defects in the activation, development, and function of CD4(+) and CD8(+) T cells and iNKT cells. These and other defects in these mice make it difficult to uncouple the developmental versus functional requirement of Itk signaling. Here, we report an allele-sensitive mutant of Itk (Itkas) whose catalytic activity can be selectively inhibited by analogs of the PP1 kinase inhibitor. We show that Itkas behaves like WT Itk in the absence of the inhibitor and can rescue the development of Itk(-/-) T cells in mice. Using mice carrying Itkas, we show using its inhibitor that Itk activity is required not only for Th2, Th17, and iNKT-cell cytokine production, but also surprisingly, for Th1 cytokine production. This work has important implications for understanding the role of Itk signaling in the development versus function of iNKT cells, Th1, Th2, and Th17 cells.

Reduced MCMV Δm157 viral clearance in the absence of TSAd.

The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice.

SRC Homology 2 Domain-containing Leukocyte Phosphoprotein of 76 kDa (SLP-76) N-terminal Tyrosine Residues Regulate a Dynamic Signaling Equilibrium Involving Feedback of Proximal T-cell Receptor (TCR) Signaling

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.

Correction: In Silico Modeling of Itk Activation Kinetics in Thymocytes Suggests Competing Positive and Negative IP4Mediated Feedbacks Increase Robustness

Correction: In Silico Modeling of Itk Activation Kinetics in Thymocytes Suggests Competing Positive and Negative IP₄Mediated Feedbacks Increase Robustness

In Silico Modeling of Itk Activation Kinetics in Thymocytes Suggests Competing Positive and Negative IP4 Mediated Feedbacks Increase Robustness

The inositol-phosphate messenger inositol(1,3,4,5)tetrakisphosphate (IP4) is essential for thymocyte positive selection by regulating plasma-membrane association of the protein tyrosine kinase Itk downstream of the T cell receptor (TCR). IP4 can act as a soluble analog of the phosphoinositide 3-kinase (PI3K) membrane lipid product phosphatidylinositol(3,4,5)trisphosphate (PIP3). PIP3 recruits signaling proteins such as Itk to cellular membranes by binding to PH and other domains. In thymocytes, low-dose IP4 binding to the Itk PH domain surprisingly promoted and high-dose IP4 inhibited PIP3 binding of Itk PH domains. However, the mechanisms that underlie the regulation of membrane recruitment of Itk by IP4 and PIP3 remain unclear. The distinct Itk PH domain ability to oligomerize is consistent with a cooperative-allosteric mode of IP4 action. However, other possibilities cannot be ruled out due to difficulties in quantitatively measuring the interactions between Itk, IP4 and PIP3, and in generating non-oligomerizing Itk PH domain mutants. This has hindered a full mechanistic understanding of how IP4 controls Itk function. By combining experimentally measured kinetics of PLCγ1 phosphorylation by Itk with in silico modeling of multiple Itk signaling circuits and a maximum entropy (MaxEnt) based computational approach, we show that those in silico models which are most robust against variations of protein and lipid expression levels and kinetic rates at the single cell level share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains at the plasma membrane. This identifies MaxEnt as an excellent tool for quantifying robustness for complex TCR signaling circuits and provides testable predictions to further elucidate a controversial mechanism of PIP3 signaling.

Inhibitors of BTK and ITK: State of the New Drugs for Cancer, Autoimmunity and Inflammatory Diseases

BTK and ITK are cytoplasmic tyrosine kinases of crucial importance for B and T cell development, with loss-of-function mutations causing X-linked agammaglobulinemia and susceptibility to severe, frequently lethal, Epstein-Barr virus infection, respectively. Over the last few years, considerable efforts have been made in order to develop small-molecule inhibitors for these kinases to treat lymphocyte malignancies, autoimmunity or allergy/hypersensitivity. The rationale is that even if complete lack of BTK or ITK during development causes severe immunodeficiency, inactivation after birth may result in a less severe phenotype. Moreover, therapy can be transient or only partially block the activity of BTK or ITK. Furthermore, a drug-induced B cell deficiency is treatable by gamma globulin substitution therapy. The newly developed BTK inhibitor PCI-32765, recently renamed Ibrutinib, has already entered several clinical trials for various forms of non-Hodgkin lymphoma as well as for multiple myeloma. Experimental animal studies have demonstrated highly promising treatment effects also in autoimmunity. ITK inhibitors are still under the early developmental phase, but it can be expected that such drugs will also become very useful. In this study, we present BTK and ITK with their signalling pathways and review the development of the corresponding inhibitors.