Family Protein Tyrosine Kinases Research Articles

Development of REDX05194, a Novel, Potent and Selective Inhibitor of Bruton's Tyrosine Kinase (BTK) As a Potential Treatment for B-Cell Malignancies

The B-cell receptor (BCR) signaling pathway is required for the survival, activation, proliferation and differentiation of B-cells. Bruton's Tyrosine Kinase (BTK) is a member of the Tec protein tyrosine kinase family that has emerged as an attractive target for the treatment of B-cell malignancies due to the critical role it plays in BCR signaling. Redx Oncology has developed novel differentiated small molecule inhibitors of BTK, combining current best-in-class potency with distinct selectivity profiles, which are suitable for oral once daily dosing. Here we present REDX05194, the result of a successful lead optimization of our proprietary BTK inhibitor series.REDX05194 is a highly selective, covalent BTK inhibitor displaying subnanomolar binding affinity for BTK (0.39 nM) and nanomolar potency towards BTK in a biochemical assay (3.67 nM). In cell proliferation assays, REDX05194 showed significant in vitro potency against ABC-DLBCL cell lines inhibiting the growth of both TMD-8 (0.89 nM) and OCI-Ly10 (1.36 nM) cells. Analysis of BCR signaling in several lymphoma cell lines, including cell lines of ABC-DLBCL, MCL and FL origin, revealed that treatment with REDX05194 inhibits BTK autophosphorylation and downstream activation of PLCγ2. In human PBMCs, REDX05194 inhibited anti-IgM stimulated upregulation of the CD69 activation marker in CD19 positive B-cells. In addition, using a fluorescent probe that binds to BTK, occupancy of BTK in PBMCs has been demonstrated in response to increasing concentrations of REDX05194.To assess selectivity, 456 kinases were screened at 1 μM, confirming that REDX05194 does not significantly inhibit other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, REDX05194 was shown to have high selectivity versus structurally related cysteine-containing kinases such as ITK in binding assays, and EGFR as demonstrated in both binding and cellular assays. REDX05194 also has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile and solubility compared to competitor compounds.REDX05194 demonstrated in vivo efficacy in a mouse collagen-induced arthritis (CIA) model. At 10 mg/kg and 30 mg/kg QD, REDX05194 significantly improved all clinical readouts, including disease severity, compared to the vehicle group. Histological data showed that approximately 1/3 of the mice had no or minimal pannus infiltration and no bone resorption, or had bone resorption restricted to small areas. These findings demonstrate potential clinical efficacy and a dose response.In conclusion, REDX05194 is a highly selective and potent BTK inhibitor with proven efficacy in several lymphoma cell lines and human PBMCs and in vivo efficacy demonstrated in a mouse CIA model. DisclosuresNo relevant conflicts of interest to declare.

Abstract B190: Allosteric modulation of Src family kinases via SH3 domain displacement

Abstract The Src family of non-receptor protein-tyrosine kinases (SFKs) plays a crucial role in cancer progression by affecting multiple signaling pathways linked to tumor cell adhesion, invasion and survival. Recent work from our group and others has shown that individual Src-family members may have unique and in some cases opposing roles in the regulation of cell growth and differentiation. Discovery and development of SFK isoform-selective inhibitors and agonists is therefore essential for the most effective therapeutic targeting of this ubiquitous kinase family. Current SFK inhibitors target the ATP-binding site of the kinase domain or an allosteric pocket nearby. However, the highly conserved nature of the active site across the Src kinase family limits identification of isoform-selective compounds. One possible solution to this problem involves small molecule modulation of SFK activity via the regulatory SH3 and SH2 domains. SFK activity is tightly regulated by intramolecular interactions of the SH3 domain with the SH2:kinase linker and the SH2 domain with the phosphorylated C-terminal tail. Small molecules that either enhance or disrupt these interactions are anticipated to result in kinase inhibition or activation, respectively. Recently, we reported that binding of a short proline-rich peptide to the SH3 domains of several Src-family members is sufficient to induce kinase activation in vitro. This peptide, known as VSL12 (VSLARRPLPLP), was postulated to disrupt the regulatory SH3:linker interaction in the downregulated kinase. In the present study, we used VSL12 as probe to better understand the structural basis of kinase activation by SH3 displacement. We first compared the affinity and kinetics of the VSL12 peptide interaction with SFK SH3 domains vs. the near full-length kinase proteins using surface plasmon resonance. We found that VSL12 bound to the isolated SH3 domains of Src, Hck, Lyn and Fyn with similar micromolar potencies. Surprisingly, the KD values for VSL12 binding to the corresponding near-full-length kinases were in the low nanomolar range, suggesting that the peptide makes additional contacts with residues outside of the SH3 domain that result in the stable, active complex. This idea was tested further using differential scanning fluorimetry to explore the thermal stability of the protein:peptide complexes. Addition of VSL12 enhanced the thermal stability of a recombinant Src SH3-SH2-linker protein, consistent with increased molecular order. However, VSL12 decreased the thermal stability of near full-length c-Src, which may reflect conformational changes in the kinase domain that accompany kinase activation. Addition of the kinase domain inhibitor dasatinib restored thermal stability of the Src:VSL12 complex. Complexes of the Src SH3-SH2-linker protein with the VSL12 peptide readily formed crystals that diffract X-rays to 2.6 Å resolution, providing further support for the stabilization of the complex by VSL12 binding. Determining the structures of the Src SH3-SH2-linker protein and the near-full-length kinase in complex with VSL12 will provide fresh insight as to the mechanism of c-Src activation by SH3 domain displacement and aid in the design of potent and selective non-peptide allosteric modulators. Citation Format: Heather L. Rust, Jamie A. Moroco, John J. Alvarado, John J. Engen, Thomas E. Smithgall. Allosteric modulation of Src family kinases via SH3 domain displacement. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B190.

Biphasic effect of PTK7 on KDR activity in endothelial cells and angiogenesis

Protein tyrosine kinase 7 (PTK7) is a member of the defective receptor protein tyrosine kinase family which lacks catalytic activity. Expression of PTK7 is increased in various cancers but its role in carcinogenesis is not well understood. We previously showed that disruption of PTK7 function suppresses VEGF-induced angiogenic phenotypes in HUVECs and mice. Here, we investigated molecular mechanisms for modulating VEGF-induced physiological effects by PTK7. Treatment with a high concentration of extracellular domain of PTK7 (soluble PTK7; sPTK7) or knockdown of PTK7 inhibited VEGF-induced phosphorylation of kinase insert domain receptor (KDR) but did not inhibit phosphorylation of fms-related tyrosine kinase 1 (FLT-1) in HUVECs. PTK7, more specifically sPTK7, interacted with KDR but not with FLT-1 in HUVECs and HEK293 cells. In vitro binding assay showed that sPTK7 formed oligomers with the extracellular domain of KDR (sKDR) up to an approximately 1:3 molar ratio, and vice versa. sPTK7 at lower molar ratios than sKDR enhanced the binding of VEGF to sKDR. At the same or higher molar ratios, it reduced the binding of VEGF to sKDR. Increasing concentrations of sPTK7 or increasing levels of PTK7 expression first increased and then decreased VEGF-induced KDR phosphorylation, migration, and capillary-like tube formation of HUVECs, as well as in vivo angiogenesis. Taken together, our data demonstrates that PTK7 regulates the activity of KDR biphasically by inducing oligomerization of KDR molecules at lower concentrations and by surrounding KDR molecules at higher concentrations.

Identification of host proteins involved in rickettsial invasion of tick cells.

Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3'-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.

Anti-Myeloma Activity by the Combination of the JAK2 Inhibitor Ruxolitinib with Lenalidomide and Corticosteroids

Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV).Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs.Results:In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone.Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.

BONE Marrow Stromal CELL Paracrine Factors from PMF Patients, but NOT Healthy Donors, Protect JAK2V617F CELLS from Ruxotinib-Induced Apoptosis

Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The activating somatic mutation JAK2V617F is highly prevalent among patients with myeloproliferative neoplasias (MPNs), which has prompted the development of JAK2 inhibitors for patients with these malignancies.Ruxolitinib (INCB018424), an oral inhibitor of JAK1 and JAK2, induces marked and durable clinical benefits in patients with primary myelofibrosis (Verstovsek S et al. N Engl J Med 366:799-807, 2012) regardless of the presence of the JAK2V617F mutation.Ruxolitinib induces a dose-dependent suppression of phosphorylated signal transducer and activator of transcription 3 (STAT3), a marker of the JAK signaling pathway; however, after 12 cycles of therapy only a 13% mean suppression of JAK2V617F allele burden from baseline was observed in COMFORT-1 study. Therefore, we hypothesized that extracellular signals negate the effects of JAK2 inhibitors on malignant cells. To test our hypothesis, we devised a co-culture platform to test the interaction between the JAK2V617F cells and BM stromal cells. Ruxolitinib inhibited proliferation and induced marked apoptosis of the human JAK2V617F mutated SET2 (IC50 56nM) and UKE-1 (IC50 329nM) cell lines, but had no effect on wild-type JAK2 human mast MHC1.1 and MHC1.2 cells. However, the anti-proliferative effect of ruxolitinib was markedly attenuated when JAK2V617F mutated cells were co-cultured with either primary patient samples or immortalized PMF patient-derived human BM mesenchymal stromal cells (TM-R1 MSCs). Specifically, co-culturing JAK2V617F–positive cells with TM-R1 MSCs hampered the anti-proliferative and pro-apoptotic effect of ruxolitinib and inhibited its ability to dephosphorylate STAT3 and STAT5. However, when JAK2V617F–positive cells were co-cultured with MSCs from healthy donors the protective effect was not observed. Subsequent experiments demonstrated that the protective effect of the TM-R1 MSCs was due to a paracrine secretion of cytokines. The cytokine expression profile of supernatants from co-cultures of TM-R1 MSCs and JAK2V617F-positive cells significantly differed from those of supernatants from co-cultures of normal MSCs and JAK2V617F-positive cells (with or without ruxolitinib). Interleukin (IL)-6 was elevated in both co-cultures whereas basic fibroblast growth factor (b-FGF-basic p= 1.05E-17) and CXCL10/IP10 (p= 2.19E-4) were significantly lower in normal MSC/JAK2V617F supernatants. Furthermore, immunofluorescence staining demonstrated that nuclear factor-light chain-enhancer p-105 (p-NFK-β-105) was localized to the nucleus in TM-R1 MSCs, whereas in normal MSCs p-NFK-β-105 was found in the cytoplasm. Our results suggest that cytokines secreted by the bone marrow stromal play a significant role in protecting JAK2V617F–positive cells from JAK2 inhibitor therapy, highlighting the role of the bone marrow microenvironment not only in the pathogenesis of MPNs but also in resistance to JAK2–directed therapies. DisclosuresKantarjian:ARIAD, Pfizer, Amgen: Research Funding.

Suboptimal B-cell antigen receptor signaling activity in vivo elicits germinal center counterselection mechanisms.

Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.

Bruton's tyrosine kinase (Btk) is a useful marker for Hodgkin and B cell non-Hodgkin lymphoma.

Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases involved in B cell development and proliferation in neoplastic human lymphoid tissues. We used immunohistochemistry to evaluate a polyclonal anti-Btk antibody on formalin-fixed paraffin-embedded tissue blocks. The tested samples included normal lymphoid tissues, tissue samples of 395 different lymphomas and 14 malignant lymphoid cell lines. Btk was expressed more often in B cell lymphomas than in T cell lymphomas. This correlated well with the results obtained on B cell lymphoma cell lines, which strongly expressed Btk, in contrast to T cell lymphoma cell lines. More than 60% of myelomas expressed Btk. Among Hodgkin lymphomas, the nodular lymphocyte predominant variant was more often positive (14/16) than the classical variant (6/27). Only one out of three Hodgkin lymphoma-derived cell lines showed a few atypical large cells expressing Btk. Btk represents a useful marker to identify B cell non-Hodgkin lymphomas. Furthermore, Btk might help to distinguish the nodular lymphocyte predominant variant of Hodgkin lymphomas from the classical form. Finally, in view of the recently discovered therapeutic potential of Btk inhibitors in lymphoma, we report the pattern of expression of Btk in a large collection of different types of lymphoma.

The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module.

The vigorous response of IgG-switched memory B cells to recurring pathogens involves enhanced signalling from their B-cell antigen receptors (BCRs). However, the molecular signal amplification mechanisms of memory-type BCRs remained unclear. Here, we identify the immunoglobulin tail tyrosine (ITT) motif in the cytoplasmic segments of membrane-bound IgGs (mIgGs) as the principle signal amplification device of memory-type BCRs in higher vertebrates and decipher its signalling microanatomy. We show that different families of protein tyrosine kinases act upstream and downstream of the ITT. Spleen tyrosine kinase (Syk) activity is required for ITT phosphorylation followed by recruitment of the adaptor protein Grb2 into the mIgG-BCR signalosome. Grb2 in turn recruits Bruton’s tyrosine kinase (Btk) to amplify BCR-induced Ca2+ mobilization. This molecular interplay of kinases and adaptors increases the antigen sensitivity of memory-type BCRs, which provides a cell-intrinsic trigger mechanism for the rapid reactivation of IgG-switched memory B cells on antigen recall.

Induced pluripotent stem cell therapy ameliorates hyperoxia-augmented ventilator-induced lung injury through suppressing the Src pathway.

BackgroundHigh tidal volume (VT) mechanical ventilation (MV) can induce the recruitment of neutrophils, release of inflammatory cytokines and free radicals, and disruption of alveolar epithelial and endothelial barriers. It is proposed to be the triggering factor that initiates ventilator-induced lung injury (VILI) and concomitant hyperoxia further aggravates the progression of VILI. The Src protein tyrosine kinase (PTK) family is one of the most critical families to intracellular signal transduction related to acute inflammatory responses. The anti-inflammatory abilities of induced pluripotent stem cells (iPSCs) have been shown to improve acute lung injuries (ALIs); however, the mechanisms regulating the interactions between MV, hyperoxia, and iPSCs have not been fully elucidated. In this study, we hypothesize that Src PTK plays a critical role in the regulation of oxidants and inflammation-induced VILI during hyperoxia. iPSC therapy can ameliorate acute hyperoxic VILI by suppressing the Src pathway.MethodsMale C57BL/6 mice, either wild-type or Src-deficient, aged between 2 and 3 months were exposed to high VT (30 mL/kg) ventilation with or without hyperoxia for 1 to 4 h after the administration of Oct4/Sox2/Parp1 iPSCs at a dose of 5×107 cells/kg of mouse. Nonventilated mice were used for the control groups.ResultsHigh VT ventilation during hyperoxia further aggravated VILI, as demonstrated by the increases in microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 (MIP-2) and plasminogen activator inhibitor-1 (PAI-1) production, Src activation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and malaldehyde (MDA) level. Administering iPSCs attenuated ALI induced by MV during hyperoxia, which benefited from the suppression of Src activation, oxidative stress, acute inflammation, and apoptosis, as indicated by the Src-deficient mice.ConclusionThe data suggest that iPSC-based therapy is capable of partially suppressing acute inflammatory and oxidant responses that occur during hyperoxia-augmented VILI through the inhibition of Src-dependent signaling pathway.

Inhibition of TYK2 and JAK1 ameliorates imiquimod-induced psoriasis-like dermatitis by inhibiting IL-22 and the IL-23/IL-17 axis.

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM-10 μM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12-induced IFN-γ production and IL-22-dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

Inhibition of Src phosphorylation reduces damage to the blood-brain barrier following transient focal cerebral ischemia in rats.

The disruption of the blood-brain barrier (BBB) caused by cerebral ischemia determines the extent of injury and patient prognosis. Inhibitors of Src can markedly minimize the infarct size and preserve neurological function. The Src protein tyrosine kinase (PTK) inhibitor, PP2, protects the rat brain against ischemic injury, possibly through the reduction of vascular endothelial growth factor A (VEGFA) expression and the upregulation of claudin-5 expression, which preserves the integrity of the BBB. In this study, the expression levels of phosphorylated (p)-Src, VEGFA and claudin-5 were determined to investigate the changes occurring in the levels of these proteins and to determine the benefits of PP2 treatment following cerebral ischemia/reperfusion (I/R). Our study included a sham-operated group, an I/R group, a vehicle-treated group (V) and a PP2-treated group (PP2). We found that the rats in the PP2 group exhibited greater preservation of neurological function and reduced VEGFA and p-Src protein expression compared with the rats in the I/R and V groups. Moreover, the mRNA and protein levels of claudin-5 were markedly higher in the PP2 group than in the I/R group or the V group after 3 days of reperfusion. Immunofluorescence staining revealed that the co-localized immunostaining of fibrinogen and claudin-5 was reduced in the PP2 group, which suggests that the exudation of fibrinogen in this group was less than that in the I/R and V groups. Furthermore, the reduced co-localization of immunostaining of glial fibrillary acidic protein (GFAP) and claudin-5 indicated that the rats in the PP2 group had only a slight disruption of the BBB. These findings suggested that PP2 treatment attenuated the disruption of the BBB following ischemia and minimized the neurological deficit; these effects were associated with a decreased VEGFA expression and an increased claudin-5 expression. Members of the Src PTK family may be critical targets for the protection of the BBB following cerebral ischemia.

Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.

Alopecia areata (AA) is a common autoimmune disease resulting from damage of the hair follicle by T cells. The immune pathways required for autoreactive T cell activation in AA are not defined limiting clinical development of rational targeted therapies. Genome-wide association studies (GWAS) implicated ligands for the NKG2D receptor (product of the KLRK1 gene) in disease pathogenesis. Here, we show that cytotoxic CD8(+)NKG2D(+) T cells are both necessary and sufficient for the induction of AA in mouse models of disease. Global transcriptional profiling of mouse and human AA skin revealed gene expression signatures indicative of cytotoxic T cell infiltration, an interferon-γ (IFN-γ) response and upregulation of several γ-chain (γc) cytokines known to promote the activation and survival of IFN-γ-producing CD8(+)NKG2D(+) effector T cells. Therapeutically, antibody-mediated blockade of IFN-γ, interleukin-2 (IL-2) or interleukin-15 receptor β (IL-15Rβ) prevented disease development, reducing the accumulation of CD8(+)NKG2D(+) T cells in the skin and the dermal IFN response in a mouse model of AA. Systemically administered pharmacological inhibitors of Janus kinase (JAK) family protein tyrosine kinases, downstream effectors of the IFN-γ and γc cytokine receptors, eliminated the IFN signature and prevented the development of AA, while topical administration promoted hair regrowth and reversed established disease. Notably, three patients treated with oral ruxolitinib, an inhibitor of JAK1 and JAK2, achieved near-complete hair regrowth within 5 months of treatment, suggesting the potential clinical utility of JAK inhibition in human AA.

QSAR models for 1,2,4-benzotriazines as Src inhibitors based on Monte Carlo method

A series of 53 1,2,4-benzotriazines as inhibitors of the sarcoma family of protein tyrosine kinases have been studied. The Monte Carlo method has been used as a tool to build up the quantitative structure–activity relationships for appropriate inhibition activity. The QSAR models were calculated with the representation of the molecular structure by the simplified molecular input-line entry system. Three various splits into training and test sets have been examined. The statistical quality of all build models is very good. The best calculated model had following statistical parameters: r2 = 0.9843, q2 = 0.9831 for the training set and r2 = 0.9488, q2 = 0.9300 for the test set. The structural indicators (alerts) for the increase and decrease in the inhibition activity have been defined. The computer-aided design of new potential sarcoma inhibitor derivatives has been presented by using defined structural alerts.

Critical Role of Jak2 in the Maintenance and Function of Adult Hematopoietic Stem Cells

Jak2, a member of the Janus kinase family of nonreceptor protein tyrosine kinases, is activated in response to a variety of cytokines, and functions in survival and proliferation of cells. An activating JAK2V617F mutation has been found in most patients with myeloproliferative neoplasms, and patients treated with Jak2 inhibitors show significant hematopoietic toxicities. However, the role of Jak2 in adult hematopoietic stem cells (HSCs) has not been clearly elucidated. Using a conditional Jak2 knockout allele, we have found that Jak2 deletion results in rapid loss of HSCs/progenitors leading to bone marrow failure and early lethality in adult mice. Jak2 deficiency causes marked impairment in HSC function, and the mutant HSCs are severely defective in reconstituting hematopoiesis in recipient animals. Jak2 deficiency also causes significant apoptosis and loss of quiescence in HSC-enriched LSK (Lin(-)Sca-1(+)c-Kit(+)) cells. Jak2-deficient LSK cells exhibit elevated reactive oxygen species levels and enhanced p38 MAPK activation. Mutant LSK cells also show defective Stat5, Erk, and Akt activation in response to thrombopoietin and stem cell factor. Gene expression analysis reveals significant downregulation of genes related to HSC quiescence and self-renewal in Jak2-deficient LSK cells. These data suggest that Jak2 plays a critical role in the maintenance and function of adult HSCs.

Clinicopathologic features and prognostic implications of NOK/STYK1 protein expression in non-small cell lung cancer.

BackgroundThe expression of novel oncogenic kinase (NOK), a member of the protein tyrosine kinase (PTK) family, has been observed in several human malignancies including non-small cell lung cancer (NSCLC). However, the clinic relevance of NOK expression in NSCLC remains unclear.MethodsIn this study, NOK expression in tumor cells was assessed using immunohistochemical methods in 191 patients with resected NSCLC. The association of NOK expression with clinicopathological parameters, including the Ki-67 labeling index (LI), was also evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of NOK expression on survival.ResultsData showed that NOK was expressed in 75.4% and 14.1% of cancer lesions and corresponding adjacent non-cancerous tissue, respectively. Out of all the clinicopathological factors analyzed, NOK expression was significantly correlated with the grade of tumor differentiation (P = 0.035), pTNM stage (P = 0.020), lymphatic metastasis (P = 0.005) and high Ki-67 LI (P < 0.001). NOK positive NSCLC patients had a significantly shorter survival time (P = 0.004, Log-rank test) and the prognostic significance of NOK expression was apparent in squamous cell carcinoma patients (P = 0.022). Multivariate analysis indicated that NOK expression may be an independent prognostic factor in NSCLC (hazard ratio [HR], 1.731; P = 0.043).ConclusionsOur results indicate that NOK expression is of clinical significance and can serve as a prognostic biomarker in NSCLC.

Inhibition of food intake by PACAP in the hypothalamic ventromedial nuclei is mediated by NMDA receptors

Central injections of pituitary adenylate cyclase-activating polypeptide (PACAP) into the ventromedial nuclei (VMN) of the hypothalamus produce hypophagia that is dependent upon the PAC1 receptor; however, the signaling downstream of this receptor in the VMN is unknown. Though PACAP signaling has many targets, this neuropeptide has been shown to influence glutamate signaling in several brain regions through mechanisms involving NMDA receptor potentiation via activation of the Src family of protein tyrosine kinases. With this in mind, we examined the Src-NMDA receptor signaling pathway as a target for PACAP signaling in the VMN that may mediate its effects on feeding behavior. Under nocturnal feeding conditions, NMDA receptor antagonism prior to PACAP administration into the VMN attenuated PACAP-mediated decreases in feeding suggesting that glutamatergic signaling via NMDA receptors is necessary for PACAP-induced hypophagia. Furthermore, PACAP administration into the VMN resulted in increased tyrosine phosphorylation of the GluN2B subunit of the NMDA receptor, and inhibition of Src kinase activity also blocked the effects of PACAP administration into the VMN on feeding behavior. These results indicate that PACAP neurotransmission in the VMN likely augments glutamate signaling by potentiating NMDA receptors activity through the tyrosine phosphorylation events mediated by the Src kinase family, and modulation of NMDA receptor activity by PACAP in the hypothalamus may be a primary mechanism for its regulation of food intake.

Cardiomyocyte-specific expression of CRNK, the C-terminal domain of PYK2, maintains ventricular function and slows ventricular remodeling in a mouse model of dilated cardiomyopathy

Up-regulation and activation of PYK2, a member of the FAK family of protein tyrosine kinases, is involved in the pathogenesis of left ventricular (LV) remodeling and heart failure (HF). PYK2 activation can be prevented by CRNK, the C-terminal domain of PYK2. We previously demonstrated that adenoviral-mediated CRNK gene transfer improved survival and LV function, and slowed LV remodeling in a rat model of coronary artery ligation-induced HF. We now interrogate whether cardiomyocyte-specific, transgenic CRNK expression prevents LV remodeling and HF in a mouse model of dilated cardiomyopathy (DCM) caused by constitutively active Protein Kinase Cε (caPKCε). Transgenic (TG; FVB/N background) mice were engineered to express rat CRNK under control of the α-myosin heavy chain promoter, and crossed with FVB/N mice with cardiomyocyte-specific expression of caPKCε to create double TG mice. LV structure, function, and gene expression were evaluated in all 4 groups (nonTG FVB/N; caPKCε(+/−); CRNK(+/−); and caPKCε×CRNK (PXC) double TG mice) at 1, 3, 6, 9 and 12mo of age. CRNK expression followed a Mendelian distribution, and CRNK mice developed and survived normally through 12mo. Cardiac structure, function and selected gene expression of CRNK mice were similar to nonTG littermates. CRNK had no effect on caPKCε expression and vice versa. PYK2 was up-regulated ~6-fold in caPKCε mice, who developed a non-hypertrophic, progressive DCM with reduced systolic (Contractility Index=151±5 vs. 90±4s−1) and diastolic (Tau=7.5±0.5 vs. 14.7±1.3ms) function, and LV dilatation (LV Remodeling Index (LVRI)=4.2±0.1 vs. 6.0±0.3 for FVB/N vs. caPKCε mice, respectively; P<0.05 for each at 12mo). In double TG PXC mice, CRNK expression significantly prolonged survival, improved contractile function (Contractile Index=115±8s−1; Tau=9.5±1.0ms), and reduced LV remodeling (LVRI=4.9±0.1). Cardiomyocyte-specific expression of CRNK improves contractile function and slows LV remodeling in a mouse model of DCM.

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Focal adhesion signaling in heart failure

In this brief review, recent evidence is presented to indicate a role for specific components of the cardiomyocyte costamere (and its related structure the focal adhesion complex of cultured cardiomyocytes) in initiating and sustaining the aberrant signal transduction that contributes to myocardial remodeling and the progression to heart failure (HF). Special attention is devoted to the focal adhesion kinase family of nonreceptor protein tyrosine kinases in bidirectional signal transduction during cardiac remodeling and HF progression. Finally, some speculations and directions for future study are provided for this rapidly developing field of research.