Src Family Kinase Inhibitor Research Articles

SRC family kinase (SFK) inhibition reduces rhabdomyosarcoma cell growth in vitro and in vivo and triggers p38 MAP kinase-mediated differentiation.

Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.

Properties and functions of lactosylceramide from mouse neutrophils.

Lactosylceramide (LacCer), which is essential for many cellular processes, is highly expressed on the plasma membranes of human neutrophils and mediates innate immune functions. Less is known, however, about the properties and biological functions of LacCer in mouse neutrophils. This study therefore analyzed the properties of mouse neutrophil LacCer. LacCer was observed on the surface of these cells, with flow cytometry indicating that mouse neutrophil LacCer could be detected by the anti-LacCer mAb T5A7, but not by the anti-LacCer antibodies Huly-m13 and MEM-74. The molecular species of LacCer were nearly identical in mouse and human neutrophils, including C24:0 and C24:1 fatty acid chain-containing species, although the LacCer content in plasma membranes was ∼ 20-fold lower in mouse than in human neutrophils. Surface plasmon resonance analysis revealed that T5A7 bound to a lipid monolayer composed of LacCer, DOPC, cholesterol and sphingomyelin (molar ratio 0.1 : 10 : 10 : 1), whereas Huly-m13 did not. T5A7 induced neutrophil migration, which was abolished by inhibitors of Src-family kinases, PI-3 kinases, and trimeric G (o/i) proteins. T5A7 also inhibited phagocytosis of non-opsonized zymosans by neutrophils. Taken together, these findings suggest that in mouse neutrophils, (i) LacCer is expressed as LacCer-enriched microdomains in cell surface plasma membranes, (ii) these microdomains are recognized by T5A7 but not by other known anti-LacCer antibodies and (iii) LacCer is involved in cell migration and phagocytosis.

Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells

A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation.

Pharmacological Inhibition of Focal Adhesion Kinase Attenuates Cardiac Fibrosis in Mice Cardiac Fibroblast and Post-Myocardial-Infarction Models

Background: To investigate the role of focal adhesion kinase (FAK)-mediated signaling in hypoxia-induced cardiac fibroblasts (CFs) differentiation and cardiac fibrosis post-myocardial infarction (MI) on a mice model. Methods: CFs of neonatal C57BL/6 mice were treated under normoxic, hypoxic, or hypoxic+PP2 (known as a Src kinase family inhibitor) conditions. Gene expressions of FAK, alpha-smooth muscle actin (α-SMA) and collagen type I alpha 1 (Col1α1), or α-SMA and vimentin levels were performed by RT-PCR and immunofluorescence staining, respectively. Thirty mice were surgically treated into Sham (n=7) and MI (n=23) groups; and FAK inhibitor PF-562271 was given to six survivor MI mice (as PF group, from 15 survivors). Heart function and collagenous tissues were examined by echocardiography, as well as by Masson‘s trichrome and Sirius red staining, respectively. Type I collagen, FAK protein, mTOR, ERK1/2, AKT, P70S6K and phospho-FAK levels were also analyzed. Results: FAK inhibition with PP2 significantly decreased CFs differentiation and collagen synthesis under hypoxia treatment. In vivo, PF-562271 treatment resulted in fibrosis attenuation; however, deteriorated heart function of MI mice could not be significantly improved. PF-562271 may affect phospho-mTOR (p<0.05), phospho-ERK1/2 (p<0.01), phospho-AKT (p<0.001) and phospho-P70S6K (p<0.05) to exert its benefits. FAK can be activated either under hypoxia in CFs or MI in a mouse model to promote fibrosis. However, pharmacological inhibition of FAK can attenuate fibrosis response. Conclusion: This study provides novel evidence that FAK inhibition may become a promising pharmaceutical strategy to attenuate fibrosis post-MI.

Tamoxifen and Src kinase inhibitors as neuroprotective/neuroregenerative drugs after spinal cord injury.

Spinal cord injury (SCI) is a devastating condition that produces significant changes in the lifestyle of patients. Many molecular and cellular events are triggered after the initial physical impact to the cord. Two major phases have been described in the field of SCI: an acute phase and late phase. Most of the therapeutic strategies are focused on the late phase because this provides an opportunity to target cellular events like apoptosis, demyelination, scar formation and axonal outgrowth. In this mini-review, we will focus on two agents (tamoxifen and a Src kinase family inhibitor known as PP2) that have been shown in our laboratory to produce neuroprotective (increase cell survival) and/or regenerative (axonal outgrowth) actions. The animal model used in our laboratory is adult female rat (~250 g) with a moderate contusion (12.5 mm) to the spinal cord at the T10 level, using the MASCIS impactor device. Tamoxifen or PP2 was administered by implantation of a 15 mg pellet (Innovative Research of America, Sarasota, FL, USA) or by intraperitoneal injections (1.5 mg/kg, every 3 days), respectively, to produce a long-term effect (28 days). Tamoxifen and the Src kinase inhibitor, PP2, are drugs that in rats with a moderate spinal cord injury promote functional locomotor recovery, increase spared white matter tissue, and stimulate axonal outgrowth. Moreover, tamoxifen reduces the formation of reactive oxygen species. Therefore, these drugs are possible therapeutic agents that have a neuroprotective/regenerative activity in vertebrates with SCI.

Abstract IA11: The role of bone microenvironment in the lethal progression of prostate cancer

Abstract Lethal prostate cancer (PCa) is clinically characterized by bone-homing and bone- forming metastases, and bone is frequently the initial organ involved in castrate-resistant progression. These clinical observations led investigators to develop bone-tropic radiopharmaceuticals to treat men with advanced PCa. The ability of the bone-homing alpha emitter (RAD-223) to prolong progression free and overall survival establishes the therapeutic relevance of targeting bone 1. The long accepted paradigm of PCa progression has been dominated by the model of evolution from androgen dependence to androgen independence under the selective pressure of castration. Thus, Src family kinases (SFKs) were investigated experimentally and clinically for the treatment of putative “androgen independent” PCa, as SFKs play a role in both tumor growth and bone turnover. The roles of SFKs in migration, invasion, survival of tumor cells, and osteoclast and osteoblast function in tumor microenvironment were supported by numerous clinical and laboratory observations 2, 3. Despite promising phase I /II reports of dasatinib, a multikinase and potent SFK inhibitor, in men with advanced PCa 4, 5, a phase III trial failed to yield the expected survival advantage 6. The result of this trial highlights the difficulties in translating data from cell line models to biologically heterogeneous clinical PCa and in transitioning from phase I/II to phase III clinical trials. The efficacy of dasatinib was not observed likely due to persistent AR signaling, which remains the primary driver of progression in bone by the transition from paracrine to intracrine androgen signaling 7. Based on these considerations, we are testing the efficacy of an androgen biosynthesis inhibitor in combination with dasatinib to better understand the relevance of mechanism(s). A co–clinical investigational strategy is being applied to better define biomarkers to predict clinical efficacy. These studies also highlight the importance of preclinical work that utilizes models that better reflect the heterogeneity of PCa. This problem is partly overcome by patient- derived xenografts (PDX) 8, 9 10. These models reflect many aspects of PCa in bone and can be used to identify therapy targets and better identify both mechanisms of drug efficacy and development of resistance. Studies in PDX have led to identification of FGF signaling as a therapy target 11. Additional studies in PDX implicated the tumor microenvironment in the striking bone responses observed with cabozantinib 13. These observations led us to propose a model of prostate cancer progression founded on the hypothesis that cancer progresses from being an endocrine-driven cancer (Dihydrotestosterone-dependent) to paracrine/intracrine driven (microenvironment-dependent), and then finally to a cell autochthonous phase 12. Continued studies should lead to decisions on therapy treatment that are based on an understanding of markers that predict disease progression. This new paradigm will also result in more effective therapy combinations.

Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas.

EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread.

Dasatinib Is Effective in the Treatment of Mice Models with Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments.Methods, Results and Discussion:In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis.In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo.Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ASK1, a Novel Regulator of Thrombosis, Is Activated Downstream of G-Protein Coupledreceptors in Human Platelets

In the event of vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins such as collagen and Von Willebrand factor and activate to form a stable hemostatic platelet plug. Defects in the molecular mechanisms dictating platelet plug formation are responsible for numerous thrombotic disorders. Elucidating the signaling pathways and molecular mechanisms of platelet activation is paramount to the development of safer and more effective anti-thrombotic drugs. While it is known that MAP-Kinase signaling participates in platelet activation, it is unknown how MAP-Kinase signaling specifically mediates platelet activation. Our laboratory has identified the presence and activation of a MAP-Kinase Kinase Kinase known as Apoptosis Signal Regulating Kinase 1 (ASK1). We have demonstrated using an ASK1 knockout mouse model that ablation of ASK1 leads to a significantly increased (p = .0003) time of vessel occlusion associated with unstable thrombus formation following a carotid artery injury induced by 10% FeCl3. Furthermore, ASK1 knockout mice display protection from pulmonary thromboembolism induced by an intravenous injection of collagen and epinephrine. In order to determine the kinetics of ASK1 activation by physiological agonists, washed human platelets (4 x 108 platelets/mL) were treated with 0.1 U/mL of thrombin for 30”, 1’, 3’, 5’, and 8’. Robust activation of ASK1 by thrombin occurred as early as 30 seconds up until 5 min, after which ASK1 activation decreased sharply. Platelets treated with 100 µM of PAR1 (SFLLRN) or PAR4 (AYPGKF) peptides resulted in strong ASK1 activation, suggesting that both the PAR1 and PAR4 receptors lead to ASK1 activation. Inhibition of Src family kinases by PP2 or PI3K by wortmannin or Rho kinase by Y-27632 had no effect on thrombin-induced ASK1 activation. However, inhibition of PLC-β2, a mediator of platelet activation downstream of the PAR1/4 receptors, strongly inhibited ASK1 activation by thrombin. We next determined whether TxA2 generation was responsible for ASK1 activation by thrombin. Washed platelets were pre-treated with 1 mM aspirin to block TxA2 generation, followed by treatment with 0.1 U/mL of thrombin. It was found that blocking TxA2 generation eliminated ASK1 activation by thrombin at 30” and 1’, but not at a later time point, suggesting there may be an additional pathway contributing to ASK1 activation. The observation that TxA2 generation contributes to ASK1 activation by thrombin seemed to correlate with the finding that treatment of platelets with 1 µM of the TxA2 mimetic U46619, which activates the TP-α receptor, could also activate ASK1. We also determined whether ADP released from dense granules, which would activate the P2Y1 and P2Y12 receptors, leads to ASK1 activation. To test this, washed platelets were pre-treated with 1 U/mL of apyrase to hydrolyze secreted ADP. It was found that apyrase treatment completely eliminates ASK1 activation by thrombin, suggesting a strong dependency of thrombin-induced ASK1 activation on ADP release from dense granules. To further investigate this possibility, washed platelets were pre-treated with 50 µM of the P2Y1 antagonist MRS2179 or P2Y12 antagonist 2-MeSAMP, followed by treatment with 0.1 U/mL of thrombin. Antagonism of the P2Y12 receptor and not P2Y1 receptor severely diminished ASK1 activation by thrombin. This indicates that ASK1 activation by thrombin is also dependent on ADP released from dense granules and subsequent activation of the P2Y12 receptor. Surprisingly, collagen, a strong activator of platelets, was unable to activate ASK1 in washed platelets at a concentration of 2 µg/mL. Similarly, 2 µM epinephrine treatment also had no effect. However, when washed platelets were treated with 2 µg/mL collagen and 2 µM epinephrine together, a strong ASK1 activation was observed (p=.0012). This suggests the existence of a novel mechanism for ASK1 activation by simultaneous stimulation of the collagen receptors GPVI/α2β1 and epinephrine receptor α2A. The finding that ASK1 activation occurs downstream of TP-α, P2Y12, and possibly α2A receptors highlights the importance of ASK1 in regulation of these G-Protein Coupled Receptors in platelet activation. In conclusion, our data indicates ASK1 to be a key mediator in platelet activation and represents a novel target for anti-thrombotic drug therapy. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of Antileukemic Activity of the Multikinase Inhibitors Dasatinib and Ponatinib in Acute Lymphoblastic Leukemia (ALL) Harboring the E2A-PBX1 Fusion Gene

The development of small molecules targeting oncogenic signaling pathways as treatment for leukemias has focused on inhibitors of tyrosine kinases. Subtypes of acute leukemias harboring aberrant fusion genes that involve transcription factors rather than kinases have so far not been amenable to molecularly targeted therapies. The t(1;19) is a recurring translocation found in a small proportion of pediatric (5%) and adult (1-3%) ALL. We observed that leukemic cell growth of E2A-PBX1 positive ALL is inhibited by dasatinib (D) and ponatinib (P) at therapeutically achievable concentrations, but not by the ABL-kinase inhibitor nilotinib (N). The mechanism by which these multikinase inhibitors exert their antiproliferative activity is unclear, as the chimeric E2A-PBX1 fusion gene encodes a protein with transforming properties but has no known link to dysregulated kinase activity.As ALL cells with an E2A-PBX1 gene rearrangement express a functional pre-B cell receptor (pre-BCR), we investigated whether the anti-leukemic effect of D and P is mediated by inhibition of SRC family kinases (SFKs), known key mediators of pre-BCR signaling. In two different cellular models of t(1;19) ALL, i.e. two patient-derived ALL cells (PDLTC) maintained in long-term culture (denoted KR and RL, respectively) and the established t(1;19) cell lines 697 and RCH-ACV, D and P suppress SFK activity in a dose-dependent manner as determined by autophosphorylation of SFKs at Y416. However, 5-fold higher concentrations of D and P were needed to inhibit proliferation than to block SFK phosphorylation. To determine whether cooperation between SFKs and other kinases underlies the anti-proliferative effect of D and P, we examined the role of downstream components of the pre-BCR signaling pathway. Activity of SYK, Btk, PLCgamma2, CD79A and ZAP70 was only moderately reduced in response to high nanomolar concentrations of D and P. Pharmacological inhibition of BTK by ibrutinib, an irreversible Btk inhibitor clinically approved for CLL, dose-dependently inhibited proliferation of all t(1;19) ALL cells evaluated (10-50% at 0.25 µM, max. inhibition of 80% at 5 µM). Inhibition of SYK by the selective ATP-competitive Syk inhibitor fostamatinib had no growth suppressive effects on t(1;19) ALL cells. These results suggest that the antiproliferative effect of D and P involves cooperation between the proximal and distal components of the pre-BCR. To further elucidate the mechanisms underlying responsiveness of t(1;19) ALL cells to D and P and the possible involvement of pre-BCR signaling, we employed a proteomics screen in which the PDLTC KR and RL were exposed to D, N or DMSO, and differentially expressed proteins were identified by SILAC followed by mass spectrometry. With a threshold of 0.5 and 2-fold, 46 proteins were up- and 23 proteins were downregulated in the presence of 0.1 µM D in PDLTC KR cells, 11 proteins were up- and 35 down-regulated in PDLTC RL cells. Analysis of all differentially expressed proteins revealed deregulation of proteins participating in metabolism of proteins, nucleic acids or carbohydrates.To examine whether the dasatinib-induced changes in gene expression in t(1;19) ALL correlated with the proteomic profiles obtained by SILAC/MS, we compared the effects of D, N and DMSO on t(1;19) ALL by differential microarray-based gene expression analysis. In PDLTC KR cells, D exposure was associated with down-regulation of 7 genes associated with cell proliferation (NME1), DNA replication (POLR3G) and cell metabolism (SLC27A2). In PDLTC RL cells, 101 genes were down-regulated, predominantly genes involved in cell cycle progression (CHEK1), cell proliferation (BCAT1), apoptosis (PDCL3) and DNA-replication (ORC6L). Notably, several genes displaying the greatest changes in gene expression (e.g. FABP5) are involved in fatty acid metabolism. In both t(1;19) PDLTC (KR and RL), a significantly smaller number of genes was up-regulated in response to D, including USP18, P2RY14 and F13A1.Unraveling the mechanisms by which dasatinib and ponatinib exert their anti-proliferative activity in t(1;19) ALL may facilitate the rational development of SFK-inhibitor-based therapies in this ALL subtype. DisclosuresNo relevant conflicts of interest to declare.

Cytomegalovirus Infection Is Associated with Expansions of CD8 T Cells and Highly Oligoclonal Vdelta1 Gamma/Delta T Cells in Patients Treated with Dasatinib for Chronic Myelogenous Leukaemia

Abstract Dasatinib is a Src family kinase inhibitor and is widely used in the management of CML. It has been shown to have immunosuppressive effects in vitro and clinical use is associated with the development of CD8 T cell and Natural Killer cell expansions that can lead to lymphocytosis. Interestingly, increased lymphocyte counts have been shown to be associated with improved responses to dasatinib. We measured lymphocyte subsets in 94 patients with CML who were treated with either imatinib or dasatinib and were in at least a complete cytogenetic remission. 55 were treated with imatinib and 39 with dasatinib. The overall lymphocyte count was increased in dasatinib-treated patients, at 2.0 * 109/L versus 1.5 * 109/L. Furthermore, within the dasatinib –treated group, CMV seropositivity was associated with a marked increase in overall lymphocyte count within the dasatinib-treated cohort, at 2.3 * 109/L versus 1.7 * 109/L. CD8+ T cells were also increased within the dasatinib-treated cohort, and CMV seropositivity was associated with a marked further increase in cell count. Indeed, CMV seropositive dasatinib-treated patients had 60% higher CD8 T cell counts compared with imatinib treated CMV-seropositive patients, at 0.5 * 109/L versus 0.3*109/L (p = 0.006). A general linear model showed that both CMV seropositivity (p &lt; 0.001) and dasatinib treatment (p = 0.001) were both associated with increases in CD8 T cell numbers. The most marked changes were seen within the CD45RA+ CD27+ EM group and the CD45RA+ CD27- EM group of CD8 T cells. Here there were double the number of CD45RA+ CD27- CD8 T cell responses in dasatinib-treated CMV seropositive patients compared with those on imatinib (0.24 * 109/L versus 0.11 *109/L, p = 0.01), and a threefold increase in EM CD45RA- CD27- CD8 T cells (0.09 * 109/L versus 0.034 * 109/L, p = 0.05). Dasatinib-treated patients also showed increased functional CMV-specific T cell responses, as measured by IFNg release to CMV peptide pools, at 5.1% of CD8 T cells versus 1.4% (p = 0.019) gd T cell numbers were also increased in patients on dasatinib compared with imatinib. The median gd T cell count was 0.05 * 109/L versus 0.02 * 109/L (p = 0.005) and here CMV seropositivity was also associated with the highest gd T cell count. Further analysis of the gd T cell subset revealed that the majority of the gd T cells in the CMV-seropositive group expressed the atypical Vd1 T cell receptor, as opposed to the Vd2 TCR most commonly found in peripheral blood. The median Vd1 gd T cell count in dasatinib-treated CMV seropositive patients was six times that of those on imatinib, at 0.06 * 109/L versus 0.0097 * 109/L (p = 0.02). A general linear model showed that both dasatinib treatment (p = 0.013) and CMV seropositivity (p = 0.002) were associated with significant increases in the Vd1 gd T cell count. The predominant memory subset in those patients that had expansions was of the CD45RA+ CD27- TEMRA subset. The largest expansions of Vd1 gd T cells were seen in patients who were CMV IgM seropositive and here the Vd1 gd T cell count was threefold higher at 0.16 * 109/L compared with 0.05 * 109/L (p = 0.014). Sorted Vd1 gd T cells from these patients showed specificity for CMV-infected fibroblast monolayers as measured by IFNg release. RACE-PCR was then used to interrogate the T cell repertoire of the Vd1 gd T cell population which was found to be strikingly oligoclonal in all cases. Indeed, in one patient a single clone of Vd1 gd T cells made up 99% of the total Vd1 population. In addition to cellular immune response, the humoral CMV-specific immune response was also increased in patients on dasatinib. CMV seropositive patients on dasatinib had higher CMV-specific IgG titre than those on imatinib, at 20.1 IU/ml versus 16.1 IU/ml (p = 0.048). Furthermore, within the CMV IgG seropositive patients, 4 of 23 dasatinib-treated patients were CMV IgM seropositive at various timepoints on treatment, compared with only 1 of 38 of those on imatinib. CMV infection is known to lead to a marked increase in the number of cytotoxic lymphocytes in the blood of healthy individuals. These data reveal that this effect is augmented by the action of dasatinib, which may potentially act by increasing the level of subclinical viral reactivation. Interestingly, these cytotoxic populations include cells has been shown to have anti-leukaemia properties and may have a role in immune anti-tumour responses. Disclosures No relevant conflicts of interest to declare.

Management of Adverse Events Associated with Dasatinib during Early Periods of Therapy in the Treatment of Chronic Myeloid Leukemia -a Clinical Report of Daria-01 Study

AbstractBackground: Dasatinib is a novel kinase inhibitor of BCR-ABL and SRC family kinases that has shown promising anti-leukemic activity against chronic myeloid leukemia (CML). However, some patients experience treatment-related toxicity shortly after initiating dasatinib administration and are managed by drug interruption and/or dose reduction (altered treatment) which can limit the potential benefits of treatment.Methods: To determine the factors that influence compliance and the efficacy of dasatinib therapy, we conducted a phase 2 study of mid-term compliance and the effectiveness of dasatinib therapy in patients with chronic phase CML (CML-CP) (DARIA-01 Study). Treatment was administered until disease progression or intolerable toxicity, as determined by the treating physician. Dasatinib at 100 mg once daily was initially administrated. Dose interruption or reduction to 50 mg once daily was allowed after grade 2 or worse nonhematologic toxicity or grade 3 or worse hematologic toxicity. Trough plasma concentrations of dasatinib (Cmin) at steady state were assessed at 28 days of therapy.Results: In April 2014, 32 CML-CP patients were enrolled in the study, which included at least 6 months of follow-up. The median age was 53 years (range, 20–86). Twenty-four (75.0%) were previously untreated patients with CML-CP, and the remaining eight (25.0%) were switched from other tyrosine kinase inhibitors (imatinib, five patients; nilotinib, three patients) because of intolerance or resistance to these drugs. During the initial 28 days, 28 patients continued with 100 mg/day treatment, and four received doses of 50 mg/day. The incidence of altered treatment was 25% during the initial 3 months and 34% during the initial 6 months from dasatinib administration. Four patients who experienced dose reduction during the initial 3 months continued with 50 mg/day treatment thereafter at their request. Except one who experienced gastro-intestinal bleeding, all patients could continue dasatinib therapy. Age (>60 years) and Cmin divided by the given dasatinib dose (Cmin/D) were significantly correlated with a higher incidence of altered treatment during the initial 3 months (p = 0.0095) (p = 0.0083). Twenty-five (78%) patients achieved complete cytogenetic response (CCyR), and 13 (40%) achieved major molecular response at 6 months. The incidence of pleural effusion during the initial 28 days was significantly correlated with Cmin/D (p = 0.032) but was not related to the molecular response rate at 6 months (40% vs. 41%, p = 0.98).Conclusion: Current data demonstrate that patients treated with dasatinib who displayed a high Cmin/D (>1.0) value at day 28 and/or who were aged 60 and over were at a high risk for altered treatment during the early periods of therapy. Patients who experienced pleural effusion could be well managed by dose reduction and/or temporal interruption of dasatinib and could still achieve the preferable molecular response rate. Individualizing therapy by incorporating a pharmacokinetic assay would improve compliance and the efficacy of dasatinib therapy, particularly for elderly patients. DisclosuresMorita:Bristol: Honoraria.

Dasatinib Plus Smoothened (SMO) Inhibitor BMS-833923 in Chronic Myeloid Leukemia (CML) with Resistance or Suboptimal Response to a Prior Tyrosine Kinase Inhibitor (TKI): Phase I Study CA180323

Background: Dasatinib is a potent inhibitor of BCR-ABL and SRC-family kinases, but may not eradicate noncycling early progenitor cells (Konig, Cancer Res 2008). Hedgehog signaling is implicated in CML progenitor cell self-renewal (Dierks, Cancer Cell 2008; Long, J Exp Clin Cancer Res 2011), and may be inhibited by SMO antagonists (Zhao, Nature 2009). BMS-833923 is a potent orally-available SMO inhibitor (SMOi) with activity in selected solid tumors. In a phase 1 pharmacokinetic (PK) and pharmacodynamic study, we determine if addition of BMS-833923 to dasatinib improves control of CML.Methods: In study CA180323 (NCT 01218477), dasatinib plus BMS-833923 was evaluated using a “Rolling-Six” design. Patients (pts) with chronic phase (CP) CML and resistance or suboptimal response to any prior TKI or with either advanced phase (accelerated phase/blast phase [AP/BP]) CML or Ph+ acute lymphoblastic leukemia (Ph+ALL) resistant to imatinib or nilotinib were treated with dasatinib (100 mg/d in CML-CP, 140 mg/d in CML-AP/BP or Ph+ ALL). After 4 weeks, SMOi was added at dose levels (DLs) of 100 mg/d (DL1), 200 mg/d (DL2) or 50 mg/d (DL-1). Dose-limiting toxicities (DLT, definition also included grade 2 events requiring >7 days interruption) were defined in the first 4 weeks of combination therapy (weeks 5–8). For the purpose of SMO escalation or reduction, adverse events occurring at any time were also considered; pts discontinuing for disease prior to week 8 were replaced. PK was performed for both agents. Serial replating of marrow colony-forming cells (CFC), with fluorescence in situ hybridization (FISH) for BCR-ABL in secondary progeny, was performed to detect reduction of early Ph+ progenitors.Results: Twenty-seven pts were enrolled (19 CML-CP, 4 CML-AP/BP, 4 Ph+ ALL), of whom 5 discontinued before week 8 (4 with early progression and 1 ineligible), leaving 22 pts evaluable for DLT. DLT was reported in 2 of 12 pts at DL1, (1 CML-CP pt with confusion, somnolence, fatigue, nausea and 1 Ph+ ALL pt with anorexia, dysgeusia, asthenia), and 1 of 2 pts at DL2 (CML-CP pt with joint stiffness, muscle cramps, and peripheral sensory neuropathy). Although a maximum tolerated dose was not exceeded, frequent interruption and dose reduction led to exploration of DL-1; no DLT was experienced in 8 CML-CP pts at DL-1. Adverse events included previously reported toxicities of SMOi: dysgeusia (67%), alopecia (67%), anorexia or nausea with weight loss (42%), muscle spasms (33%), and fatigue (33%) were the most common, leading to discontinuation in 7 pts (32%). Both onset and resolution of alopecia were delayed >2 months, consistent with a stem cell effect. No drug-drug PK interactions were identified. Minor efficacy could be attributed to addition of SMOi; 3 of 10 pts with CML-CP resistant to dasatinib had prolonged (47–51 weeks) combination treatment and 1 attained complete cytogenetic response. No pt with CML-Adv or Ph+ALL showed benefit. Replating CFC assays with FISH on secondary progeny did not demonstrate preferential reduction of early BCR-ABL progenitors by addition of SMOi.Conclusions: Despite observation of expected toxicities due to SMO inhibition, no evidence of efficacy was shown for the combination. Extensive replating assays failed to demonstrate selective reduction of CML progenitors. Although SMOi was reasonably tolerable at a dose of 50 mg/d, the lack of efficacy and observed toxicities do not support use in early stage CML-CP. Translation of preclinical work on CML stem cell eradication to the clinical setting remains challenging. DisclosuresShah:ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding. Off Label Use: BMS-833923 is an investigational SMO inhibitor that was evaluated for safety and efficacy in this study.. Cortes:Teva: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding. Clarke:Bristol-Myers Squibb: CRO Other. Copland:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Travel funding Other. Strauss:Bristol-Myers Squibb: Employment. Talpaz:ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Sanofi: Research Funding; Pfizer: Research Funding; Incyte: Research Funding.

Platelet FcγRIIA Signaling Results in Ubiquitination and Cellular Translocation of Activated Syk

Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for αIIbβ3 outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunotyrosine activation motif (ITAM) signaling, such as FcγRIIA, GPVI and CLEC-2. We previously reported Syk ubiquitination following GPVI stimulation, and the role of c-cbl as the E3 ubiquitin ligase (Dangelmaier, Blood 2005). Ubiquitination is an important post-translational modification that modulates signal transduction by regulating the activity, subcellular localization or stability of proteins. We tested the hypothesis that ubiquitination participates in signaling, and examined ubiquitination of Syk downstream of platelet FcγRIIA activation.Using both washed human platelets and HEL cells, we observed ubiquitination of Syk upon FcγRIIA engagement by cross-linking IV.3 mAb (10 ug/ml) with goat anti-mouse Fab’2 (30 ug/ml). Both tyrosine phosphorylation and ubiquitination of Syk occurred within 15 sec, peaked by 1-3 min and decreased thereafter. The pattern of ubiquitination was consistent with 1 to 3 Ub molecules per Syk molecule. Ubiquitinated-Syk (Ub-Syk) was increased in the presence of PR-619, a deubiquitinating enzyme inhibitor, confirming ubiquitination of Syk. Ub-Syk associates with the cytoskeletal-rich platelet fraction, membrane skeleton fraction and with cytosolic fraction in detergent lysed platelets that were fractionated by lower g-forces (15,500 x g) and higher g-forces (100,000 x g). This suggests that Ub-Syk is translocated into all cellular compartments upon platelet activation. Ub-Syk was absent upon pre-treatment with Src-family kinase inhibitor PP2 (10 uM), but minimally affected in the presence of Syk inhibitor PRT318 (1 uM), in both platelets and HEL cells, as compared to DMSO treated control cells. Further, phosphorylation of c-cbl was inhibited strongly by PP2, but only slightly inhibited by PRT318, suggesting that ubiquitination of Syk depends on Src kinase activity. Of note, Ub-Syk was not degraded by the proteasome, since no accumulation of Ub-Syk was observed by pretreatment with proteasome inhibitors MG132 or Epoxomicin in either platelets or HEL cells, compared to control cells.In conclusion, Syk is ubiquitinated upon cross-linking platelet FcγRIIA and is translocated to all major subcellular compartments. Since ubiquitinated Syk is activation-dependent and not subject to proteasomal degradation, it likely serves as a novel adapter molecule for protein-protein interactions in mediating platelet activation via FcγRIIA. DisclosuresNo relevant conflicts of interest to declare.

284 INTRACELLULAR PATHWAYS MEDIATING DIRECT EFFECTS OF PROLACTIN AND GROWTH HORMONE ON BOVINE METAPHASE-II OOCYTES AGING IN VITRO

Maturation of mammalian oocytes coincides with various aging processes, which negatively affect the oocyte quality. Cellular and molecular changes in matured oocytes aging in vivo and in vitro are very similar, suggesting similarities in the underlying mechanisms. The goal of the present research was to study direct effects of prolactin (PRL) and growth hormone (GH) on bovine metaphase-II (M-II) oocytes aging in vitro and intracellular pathways mediating these effects. Cumulus-enclosed oocytes were matured for 20 h in TCM 199 containing 10% fetal calf serum (FCS), 10 µg mL–1 of porcine FSH, and 10 µg mL–1 of ovine LH. After IVM, oocytes were set free from cumulus and denuded oocytes (DO) were cultured for 44 h in the aging medium consisting of TCM 199 supplemented with 10% FCS in the absence of PRL and GH (Control), then in the presence of either 50 ng mL–1 of bovine PRL or 10 ng mL–1 of bovine GH or protein kinase inhibitors. The following inhibitors were added: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 20 µM), (2) U0126 (a MEK inhibitor, 20 µM), and (3) triciribine (an inhibitor of Akt kinase, 50 µM). At the end of culture, the state of the nuclear material of oocytes and embryos was evaluated by the method of Tarkowski. The number of oocytes undergone spontaneous parthenogenetic activation was determined by summarising the numbers of embryos cleaved and oocytes reached anaphase-II to pronucleus stages. The data from 4 replicates were analysed by ANOVA. During aging in the control medium, the rate of M-II oocytes with destructive changes of chromosomes (decondensation, clumping, fragmentation) increased from 20.3 ± 1.7 to 65.1 ± 3.5% (P &lt; 0.001) and was unaffected by PRL and GH. At the same time, the frequency of oocyte parthenogenetic activation (18.7 ± 4.9%) was reduced (P &lt; 0.001) by both PRL and GH (up to 6.0 ± 2.9 and 5.9 ± 2.4%, respectively). The inhibitor of Src-family tyrosine kinases PP2 eliminated (at least P &lt; 0.05) the supporting effects of PRL and GH on the meiotic arrest at M-II. The MEK inhibitor U0126 also abolished the effect of PRL (but not that of GH), increasing the frequency of the oocyte activation from 5.9 ± 2.3 to 21.6 ± 1.9% (P &lt; 0.01). Concurrently, both inhibitors did not affect the meiotic arrest in the control group. The inhibitor of Akt kinase triciribine did not influence the frequency of the oocyte activation in the PRL- and GH-treated groups. Meanwhile, triciribine decreased the rate of M-II oocytes with chromosome abnormalities in the control medium (from 71.2 ± 1.7 to 47.0 ± 2.7%; P &lt; 0.001). When added to the aging medium, PRL enhanced the triciribine effect, while GH suppressed it (P &lt; 0.01). Thus, PRL and GH can directly support the meiotic arrest at M-II in aging oocytes by activating MAP kinases or Src-family tyrosine kinases. Besides, GH may enhance the chromosome destruction in DOs through activation of Akt kinase.The research was supported by RFBR (No. 13-04-01888).

Cyclic stretch-induced TGF-β1 and fibronectin expression is mediated by β1-integrin through c-Src- and STAT3-dependent pathways in renal epithelial cells.

Extracellular matrix (ECM) proteins, including fibronectin, may contribute to the early development and progression of renal interstitial fibrosis associated with chronic renal disease. Recent studies showed that β1-integrin is associated with the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). However, the molecular events responsible for β1-integrin-mediated signaling, following UUO, have yet to be determined. In this study, we investigated the mechanism by which mechanical stretch, an in vitro model for chronic obstructive nephropathy, regulates fibronectin and transforming growth factor-β1 (TGF-β1) expression in cultured human proximal tubular epithelium (HK-2) cells. Mechanical stretch upregulated fibronectin and TGF-β1 expression and activated signal transducer and transcription factor 3 (STAT3) in a time-dependent manner. Stretch-induced fibronectin and TGF-β1 were suppressed by a STAT3 inhibitor, S3I-201, and by small interfering RNA (siRNA) targeting human STAT3 (STAT3 siRNA). Similarly, fibronectin and TGF-β1 expression and STAT3 activation induced by mechanical stretch were suppressed by the Src family kinase inhibitor PP2 and by transfection of HK-2 cells with a dominant-negative mutant of c-Src (DN-Src), whereas PP3, an inactive analog of PP2, had no significant effect. Furthermore, mechanical stretch resulted in increased β1-integrin mRNA and protein levels in HK-2 cells. Furthermore, neutralizing antibody against β1-integrin and silencing of β1-integrin expression with siRNAs resulted in decreased c-Src and STAT3 activation and TGF-β1 and fibronectin expression evoked by mechanical stretch. This work demonstrates, for the first time, a role for β1-integrin in stretch-induced renal fibrosis through the activation of c-Src and STAT3 signaling pathways.

Transmembrane adaptor protein PAG/CBP is involved in both positive and negative regulation of mast cell signaling.

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.

FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

CS-18 * Src FAMILY KINASES DIFFERENTIALLY INFLUENCE GLIOMA GROWTH AND MOTILITY

We have reported that tumor cells at the invasive front of human glioblastoma (GBM) xenografts exhibit increased activity of Src family kinases (SFKs). We also showed that increased invasion induced by treatment of GBM xenografts with bevacizumab can be suppressed by co-administration of the general SFK inhibitor dasatinib. Additionally, dasatinib significantly retards the growth of GBM cells in vitro. Here, we sought to identify specific SFKs that when targeted can account for the anti-tumorigenic activity of dasatinib. We used a targeted shRNA approach to initially validate and subsequently knockdown the expression of individual SFKs in the context of both conventional and patient derived xenograft cell lines in vitro and in orthotopic xenografts. We focused on Src, Yes, Fyn and Lyn, major SFKs expressed in the central nervous system. In vitro studies examined the conventional glioma cell lines LN229 and SF767, and the xenograft cell line GBM8, while in vivo studies focused on GBM8. All three cell lines express the four SFKs and respond with abrogated cell growth to treatment with dasatinib in vitro. The results revealed cell type specific differences in SFK activities; however, in general, Yes, Fyn and Src all promoted cell growth and migration to a differing degree, while Lyn had no effect in vitro. Surprisingly, Fyn or Src depletion had no effect on tumor growth in orthotopic GBM8 xenografts, while Yes knockdown significantly reduced tumor growth and extended survival. Unexpectedly, depletion of endogenous Lyn had the opposite effect, inducing rapid tumor growth in vivo and decreasing overall survival. The data are consistent with the lack of appreciable benefit of dasatinib treatment on tumor growth and overall survival in GBM8 xenografts, uncover a key tumor suppressive function of Lyn in GBM, and suggest interrogation of Lyn activity in GBM tumors prior to dasatinib therapy.