Y397 Phosphorylation Research Articles

Targeting The Wee1 Kinase With MK-1775 For Treatment Of Acute Myeloid Leukemia In The Down Syndrome Population

Approximately one seventh of pediatric acute myeloid leukemia (AML) patients also have Down Syndrome (DS), or constitutional trisomy 21. DS AML is considered to be a unique disease from non-DS AML, due in part to a high prevalence of somatic GATA1 mutations and a megakaryocytic phenotype (AMKL). In contrast to the non-DS population, DS AML is generally associated with a very favorable prognosis. However, treatment of this disease is not without complications. Adverse drug effects are significant within this population, especially with up to half of patients possessing congenital heart abnormalities. Furthermore, for those patients who suffer either an induction failure or relapse, prognoses are dismal (Taga T. et al., Blood, 120: 1810-1815, 2012) including the outcomes after stem cell transplant (Hitzler JK. et al., Biol Blood Marrow Transplant, 19:893-897, 2013). Therefore, less toxic and more efficacious therapies are needed for this population.Recently, chemotherapeutic targeting of cell cycle checkpoints has emerged as an exciting possibility, with many clinical trials underway. The wee1 kinase has been identified as a potential target in many malignancies, including leukemia. It is responsible for inhibiting CDK1 and CDK2 by adding phosphate groups at amino acid residue tyrosine 15 (Y15), which is necessary for subsequent checkpoint activation by checkpoint proteins (e.g. CHK1). The development of a wee1-specific inhibitor, MK-1775, has allowed investigations into its use alone or in combination with classical chemotherapeutics as treatment for many types of cancer, with favorable results thus far. However, there is some controversy with regards to the mechanism by which it enhances cell death.In this preclinical work we used DS AML cell lines to investigate the potential use of MK-1775, both alone and in combination with cytarabine, to improve outcomes in DS AML patients. The two cell lines, CMK and CMY, were derived from DS AMKL patients and both harbor GATA1 mutations. CMK was derived from a patient who relapsed after initial treatment and CMY was derived from a patient who experienced an induction failure. The CMY line displays significant resistance to multiple different classes of chemotherapy drugs including nucleoside agents (e.g. cytarabine) and anthracyclines. We found that MK-1775 alone was able to cause growth inhibition and apoptosis at clinically relevant doses in both cell lines, independent of sensitivity to cytarabine or other agents. This effect was greatly enhanced by the addition of cytarabine. Addition of 250nM MK-1775 was able to reduce the cytarabine IC50 9- and 45-fold in CMK and CMY cells, respectively. Combination indices (CI) for this combination ranged from 0.66-0.95 and 0.50-0.94 in CMK and CMY, respectively.The combination of MK-1775 and cytarabine was able to synergistically induce apoptosis as measured by annexinV/propidium iodide (PI) staining. Addition of MK-1775 was able to decrease inhibitory CDK1 and CDK2 Y15 phosphorylation induced by cytarabine treatment. Interestingly, treatment with MK-1775 increased DNA damage induced by cytarabine as measured by γH2AX signal on western blots. Furthermore, this damage was induced primarily in S-phase, as measured by γH2AX/PI dual-staining and flow-cytometry. Finally, to aid with the clinical translation of this work, the dose- and time-dependency of MK-1775 treatment on CDK1 and CDK2 Y15 phosphorylation was determined. Briefly, even low doses (100nM) of MK-1775 were able to reduce CDK1 and CDK2 Y15 phosphorylation after only four hours. However, this inhibitory phosphorylation was able to return after only 1 hour, suggesting that treatment with MK-1775 must be continued during cytarabine therapy.In conclusion, these data support the combination of MK-1775 and cytarabine for the treatment of DS AML, including possible relapsed/refractory cases which have a very poor prognosis. Combining MK-1775 with cytarabine was able to synergistically induce apoptosis in both cell lines, possibly by increasing DNA damage and decreasing inhibition of CDK1 and CDK2. Disclosures:No relevant conflicts of interest to declare.

Src Inhibitors: a Synergic Help for Pancreatic Neuroendocrine Tumors Treatment

Context Pancreatic neuroendocrine tumors (pNETs) are becoming high in prevalence and have started showing a more aggressive behavior. Current therapies for advanced pNETs are inadequate as many patients develop primary or secondary resistance to mTOR (mammalian target of rapamicin) inhibitors, in part due to the activation of escape routes such as the PI3K/AKT pathway. SFKs (Src Family of Kinases) are overexpressed in pNETs and have a central role in controlling cell growth, adhesion and migration, also by regulating the mTOR pathway and the activation of the epithelial growth factor receptor in pNETs. SFK inhibitors are already safely used in patients with other tumors but have never been tested on pNETs. Objective To evaluate the use of SFK inhibitors on pNETs both alone and in combination with mTOR inhibitors. Methods Different dosages of dasatinib and bosutinib were tested in two human pNET cell lines, a pancreatic carcinoid (BON-1) and a somatostatinoma (QGP-1), either alone or in combination with the mTOR inhibitor RAD001 (everolimus). Src activation was assessed by monitoring Y416 phosphorylation. mTOR activation was assessed by testing rp-S6 and 4E-BP1 phosphorylation. Escape pathways were investigated through Akt and eIF4E phosphorylation. MTT and colony formation assays were used to evaluate cell proliferation. Results Dasatinib and bosutinib are effective at inhibiting Src activation and, when administrated together with RAD001, escape pathways are not activated. Extremely low dosages of RAD001 reduce cell proliferation when administered in combination with dasatinib or bosutinib. Colony formation assays show a strong reduction in colony number. BON-1 cells respond better than QGP-1 cells to treatments. Conclusions SFK inhibitors are promising new drug options for pNETs, as they reduce tumor cell proliferation when used in combination with RAD001. They allow to reduce RAD001 dosage, which may lower the risk of developing adverse effects in patients. Additional studies on animal models should be pursued to better assess their potential use in clinical therapies.

Focal adhesion kinase autophosphorylation inhibition decreases colon cancer cell growth and enhances the efficacy of chemotherapy

Focal adhesion kinase (FAK) increasingly has been implicated in cancer growth and progression. 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) is a small molecule FAK inhibitor that blocks the Y397 autophosphorylation site. FAK inhibitor, Y15 decreased Y397 FAK in different colon cancer cells lines in a dose-dependent manner. In addition, Y15 decreased phosphorylated Src in SW480 and SW620 cells. Y15 decreased cell viability, increased detachment, and increased apoptosis in SW480 and SW620 cells in vitro. Combination of FAK inhibitor Y15 and Src inhibitor PP2 decreased colon cancer cell viability more effectively than each agent alone. In addition, when combined with 5-FU, oxaliplatin or 5-FU and oxaliplatin, colon cancer viability was decreased further, demonstrating that dual and triple therapy synergistically inhibits cell viability. In vivo, Y15 decreased subcutaneous SW620 tumor growth by 28%. Combination of oral Y15 with 5-FU/or oxaliplatin decreased tumor growth by 48% more effectively than each inhibitor alone. Finally, tumors treated with Y15 expressed less Y397 phosphorylation, Src phosphorylation and had greater apoptosis than controls. Thus, the small molecule FAK inhibitor, Y15, inhibits cell growth in vitro and in vivo and enhances the efficacy of chemotherapy, demonstrating that it can be an effective therapeutic inhibitor for treating colon cancer.

Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.

TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR

BackgroundEnhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.Methods18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 × 10mm cell culture dishes and were used from passages 10–14. 18Co cells were treated with TNF-α (8.3 ng/ml) and LPA (10 μM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors.ResultsExposure of 18Co cells to either TNF-α or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-α led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-α and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-α/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR.ConclusionSynergistic COX-2 expression induced by TNF-α and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-α promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer.

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.

Abstract 144: A20 Inhibits Pathologic Interferon-? Signaling in Smooth Muscle Cells to Prevent Atherosclerotic Vascular Disease

The pleiotropic cytokine Interferon-γ (IFNγ), best known for its anti-viral and immunomodulatory activities, also demonstrates pro-atherogenic effects. Implication of IFNγ in the pathogenesis of atherosclerosis results in part from the upregulation of a number of pro-inflammatory interferon stimulated genes (ISGs) in vascular smooth muscle cells (SMC). Hence, strategies to reduce IFNγ signaling are of particular interest to reduce or prevent pathologic vascular remodeling associated with atherosclerotic disease, for instance transplant arteriosclerosis. We discovered that the anti-inflammatory and vasculoprotective protein A20 inhibits IFNγ signaling and thus the expression of ISG in vitro in human coronary artery SMC, and in vivo in vascular allografts. The aim of this study was to unveil the mechanism(s) of A20 mediated inhibition of IFNγ signaling. Adenovirus-mediated overexpression of A20 in SMC significantly decreased mRNA and protein levels of signal transducer and activator of transcription 1 (STAT1), the key mediator of IFNγ signaling, as a result of lower transcription. Moreover, overexpression of A20 in SMC also efficiently inhibited activating Y701 phosphorylation of STAT1, through a mechanism possibly implicating A20 binding to STAT1, as confirmed by co-immunoprecipitation. As expected, overexpression of A20 also inhibited NF-κB activation in SMC, which is required for full-blown IFNγ responses. Indeed, we confirmed that inhibition of NF-κB by overexpression of its inhibitor, IκBα also quenched upregulation of ISGs in SMC, albeit without down-regulating STAT1. Overall, our results identify A20 as a potent inhibitor of IFNγ signaling in SMC through several non-mutually exclusive mechanisms. This novel function of A20 in SMC further qualifies its atheroprotective properties and supports the pursuit of A20-based therapies for the cure of atherosclerotic vascular disease.

Abstract 3368: Pre-clinical PK/PD/efficacy characterization of AZD1480, a JAK1/2 kinase inhibitor.

Abstract Constitutive activation of the transcription factor STAT3, manifest as Y-705 phosphorylation, is a feature of many hematologically-derived and solid tumor malignancies. Accumulating evidence indicates that JAK kinases are the key mediators of STAT activation in solid tumor models. AZD1480, an inhibitor of Janus kinases 1 and 2 (JAK1/2), is a bioavailable small molecule whose inhibition of the JAK-STAT signalling pathway may provide clinical benefit by suppression of the pleiotropic tumorigenic effects of constitutive STAT3 activation (tumor cell proliferation, motility and survival, tumor angiogenesis, tumor immune suppression). Here we present a pre-clinical population-based effect-compartment pharmacokinetic / pharmacodynamic (PK/PD) model to describe the onset, intensity, and duration of pSTAT3 inhibition as a function of unbound concentration and time in the MDAH-2774 ovarian xenograft mouse model. A mathematical tumor growth inhibition model capturing the relationship between systemic exposure to AZD1480 and tumor growth inhibition was built to provide a pharmacokinetic rationale for tumor growth inhibition. Furthermore, the pSTAT3 inhibition PK/PD relationship for AZD1480 was quantitatively integrated into a tumor growth inhibition model providing a mechanistic pharmacodynamic rationale for tumor growth inhibition pre-clinically. Taken together, these pre-clinical data recommend that increasing inhibition of pSTAT3 correlates with greater tumor growth inhibition and provide a PK/PD/Efficacy platform of evidence for clinical hypothesis testing. Citation Format: Patricia Schroeder. Pre-clinical PK/PD/efficacy characterization of AZD1480, a JAK1/2 kinase inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2013-3368 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3530: The role of IL-6 in ERα+ breast cancer and potential use for Siltuximab, an anti-IL-6 antibody, in ERα+ breast cancer treatment.

Abstract Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-alpha (ERα) positive breast cancer. High IL-6 serum levels are associated with poor prognosis in ERα+ breast cancer patients, and IL-6 related polymorphisms that lead to elevated IL-6 expression are associated with decreased overall survival (PMID 17261184, 12771987). Preclinical data suggest that in contrast to ERα negative breast cancer cell lines, ERα positive cell lines rarely produce autocrine IL-6, and are therefore dependent on paracrine IL6 produced by the microenvironment. Furthermore, when ERα positive cell lines are exposed to paracrine IL-6, phosphorylation of STAT3-Y705, increased growth rates and more aggressive tumor phenotypes are observed (PMID 17586727, 18974155, 19581928). We utilized 3D co-cultures and heterotypic xenograft models to investigate the ability of siltuximab, an anti-IL-6 antibody in clinical development, to attenuate paracrine IL-6 effects across a panel of 8 ERα positive breast cancer cell lines and heterotypic xenograft models. From a panel of 8 ERα postive breast cancer lines, 75% responded to recombinant human IL-6 (hIL-6) protein by phosphorylation of STAT3 (Ty705), but not AKT, MEK1/2, or ERK1/2. Siltuximab treatment blunted pSTAT3 induction in all IL-6 responsive ERα positive breast cancer lines but failed to reduce pSTAT3 phosphorylation in three ERα negative cell lines that produced autocrine IL-6. The 3D tumor growth assay demonstrated accelerated growth rates for ERα positive breast cancer lines in the presence of IL-6 or human mesenchymal stem cells (hMSC), which returned to baseline with siltuximab treatment. In addition, when hIL-6 was supplemented in vivo, the ERα positive tumor cell line, MCF-7 engrafted without the need for estrogen supplementation and tumor growthblunted with siltuximab treatment. When tumors were allowed to establish before treatment, siltuximab was able to induce tumor regressions in all treated animals (10/10). Tumor regression was associated with decreased mitotic counts in tumors. In addition to these experiments, we investigated STAT3 activation in a panel of primary patient-derived ERα positive breast cancer samples. 43% of samples demonstrated activated STAT3 as determined by Y705 phosphorylation. These same ERα positive breast cancer patients displayed an activated IL-6 network as assessed by an a priori 32-gene IL-6 network gene expression profile signature and was also associated with increased IL-6 serum levels. This activated IL-6 gene signature was also associated with ERα positive tumors and increased metastasis as assessed by positive node status in four independent breast cancer data sets. Taken together, these data suggest a key role for IL-6 in estrogen independent ERα positive breast cancer progression. Citation Format: Amy Axel, Tineke Casneuf, Peter King, JD Alvarez, Brett Hall, Kate Sasser. The role of IL-6 in ERα+ breast cancer and potential use for Siltuximab, an anti-IL-6 antibody, in ERα+ breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2013-3530

Abstract 14: The effect of Src family kinase inhibitors in non-metastatic clear cell renal cancer.

Abstract Approximately 8000 new cases of renal cancer are diagnosed each year, with an estimated five year survival rate of 50%. Treatment options are limited, however a potential target is a family of non-receptor tyrosine kinases, called Src family kinases (SFKs). SFKs have a role in multiple oncogenic processes and are associated with promoting metastases in solid tumours; however, research in renal cancer is limited. Previously, we reported that all eight SFKs were expressed in human renal clear cell carcinoma. Lyn, Blk, Lck, Fyn and Yes showed a significant increase in expression between stages T1 and T2 and a subsequent decrease between T2 and T3, suggesting SFKs increased to promote invasion and migration for metastatic spread. The aim of the current proposal is therefore to investigate if SFKs could be a therapeutic target for renal cancer. The prototypical SFK, Src was investigated for association with clinical outcome measures. High membrane Src expression was associated with improved disease specific survival (p=0.097). However, low expression of phosphorylated cytoplasmic FAK at Y861 a Src specific site, was associated with poor disease specific survival (p=0.001). This suggests that poor prognosis associated with FAK is due to a family member other than Src as all SFKs can phosphorylate this residue. The effect of SFKs inhibition on migration, apoptosis and proliferation in a non-metastatic renal cancer cell line, 786O was examined. Western blot analysis demonstrated that treatment of 786O with the SFK inhibitor, dasatinib, did not affect total levels of Src, but a dose dependent reduction in SFK activation was observed, as shown via Y416 phosphorylation and FAK phosphorylation at Y861. Cells also exhibited a dose dependent reduction in migration accompanied by an increase in apoptosis when treated with dasatinib for 48 hours. However, no effect on proliferation was observed. Treatment with Src siRNA to remove Src from the cells did not significantly influence the effects observed in response to dasatinib suggesting dasatinib acts via another SFK family member. Our data shows that activation measured via phosphorylation of the downstream marker FAK at Y861 was associated with poor prognosis and removing Src from 786O cells did not effect the actions of the Src inhibitor, dasatinib. This combined with our previous observation of SFKs expression increasing from stage T1 to T2 and decreasing from T2 to T3 lead us to conclude that SFKs are important for tumour cells migrating to the outer edge of the organ ready for metastasis then dissipate as the metastasis occurs and that this may be promoted by a SFK family member other than Src. However the specific family member responsible for these actions and the actions of dasatinib is still to be established. Citation Format: Antonia K. Roseweir, Tahir Qayyum, Robert Jones, Grenville Oades, Michael Aitchison, Joanne Edwards. The effect of Src family kinase inhibitors in non-metastatic clear cell renal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 14. doi:10.1158/1538-7445.AM2013-14

Abstract 4101: Thrombin stimulates glioblastoma cell proliferation, adhesion and in vivo tumor growth through Rap1 activation.

Abstract Rap1 is a Ras family GTPase that plays an important role in ERK1/2 activation, cell proliferation and adhesion. Little is known about the role of Rap1 in signaling through GPCRs. We report that thrombin coupling to the G-protein-coupled receptor PAR-1 in human glioblastoma cells induces a robust and sustained increase in Rap1 activation. This response is mediated through RhoA activation and phospholipase D (PLD) signaling. In addition to its role in cell proliferation, Rap1 mediates inside-out integrin activation thereby affecting cell adhesion. Accordingly, we tested the possibility that thrombin affects glioblastoma cell adhesion through Rap1. Thrombin treatment lead to a robust 4-5 fold increase in cell adhesion to fibronectin relative to control cells. Down-regulating either PLD1 or Rap1 with siRNA inhibited this response implicating PLD1 and Rap1 in thrombin stimulated cell adhesion. Thrombin was shown to increase cell adhesion through activation of β1 integrin, since a β1 neutralizing antibody blocked the ability of thrombin to increase cell adhesion to fibronectin. Thrombin treatment also increased phosphorylation of FAK at tyrosine 397 and blocking integrin activation with a β1 integrin neutralizing antibody significantly inhibited this response. FAK Y397 phosphorylation by thrombin was found to be Rap1 dependent, as it was abolished by down-regulating Rap1 expression via siRNA. We found that thrombin stimulation lead to a robust and sustained increase in ERK1/2 phosphorylation and glioblastoma cell proliferation. Down-regulation of Rap1 by siRNA inhibited thrombin stimulated ERK1/2 activation and cell proliferation in glioblastoma cells. Consistent with a connection between Rap1, integrin signaling and ERK1/2 activation, the β1 neutralizing antibody also significantly inhibited thrombin induced glioblastoma mitogenesis. Finally, we used a mouse xenograft model to assess the in vivo role of Rap1 in tumor cell growth. Lentiviral mediated Rap1 shRNA expression was used to inhibit Rap1 expression in glioblastoma cells. Nu/Nu mice were then injected with cells treated with lentiviral control scrambled or Rap1 lentiviral shRNA and tumor mass was assessed 22 days following injection. Rap1 knockdown in these cells had a profound effect, with tumor mass reduced by 90% relative to control. Rap1 gene expression was also decreased in these cells by 80% relative control. Taken together, our results suggest that thrombin induces glioblastoma cell proliferation through its ability to activate a Rho/Rap1/β1 integrin/ERK1/2 mediated pathway. These results also demonstrate a critical role for Rap1 in signaling via GPCRs in vitro and in glioblastoma tumor growth in vivo Citation Format: Jacqueline Sayyah, Dwayne Stupack, Joan Heller Brown. Thrombin stimulates glioblastoma cell proliferation, adhesion and in vivo tumor growth through Rap1 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2013-4101

Abstract 931: Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity.

Abstract Constitutively activated pY705-STAT3 is oncogenic and prevalent in many tumor types, and has been shown to be driven in large part by constitutive autocrine and paracrine cytokine signaling. JAK kinases are critical mediators of cytokine signaling, and JAK1 has been shown to play an essential role downstream of IL-6 family cytokines, the upregulation of which has been documented in many tumor types. A JAK1 selective inhibitor should provide a therapeutic means of suppressing Y705 phosphorylation of STAT3 without incurring the hematological toxicity associated with inhibition of JAK2 and JAK3 activity, allowing for more intensive dosing and greater combinability with other agents, particularly those with hematological toxicities. We have developed highly selective inhibitors of JAK1 and used them to address the hypothesis that JAK1 is the primary driver of STAT3 mediated signaling in cancer cells. The compounds are highly selective in enzyme assays, with 1000-fold selectivity for JAK1 vs. other JAK family members, and minimal activity against other kinases at 1uM. In cell lines, they are over 500-fold selective for JAK1 vs. JAK2, measured using assays specific for JAK1-mediated STAT3 vs. JAK2-mediated STAT5 phosphorylation. In vivo, the compounds are also highly selective for JAK1 mediated signaling, measured by comparing inhibition of STAT3 phosphorylation in H1975 subcutaneous xenografts vs. inhibition of STAT5 phosphorylation in a disseminated TEL-JAK2 transformed BaF3 model. At doses where inhibition of STAT3 phosphorylation was greater than 90%, only minimal inhibition of STAT5 phosphorylation was observed. In a wide variety of human tumor lines (CRC, NSCLC, pancreatic, DLBCL) with a variety of genetic backgrounds (K-ras, b-raf, PI3K, EGFR mutant, PTEN null), complete knockdown of pSTAT3 (Y705) was observed with JAK1 selective compounds, at concentrations achievable in vivo. These effects are consistent with experiments where inhibition of Y705 phosphorylation of STAT3 was achieved with a JAK1-specific siRNA. At doses where in vivo activity was observed, the compounds had only minimal effect in an erythropoietin-driven mouse model of polycythemia and extramedullary erythropoiesis. These data support the hypotheses that in a variety of tumor types, pSTAT3-mediated signaling is dependent on JAK1 kinase activity, and that a selective JAK1 inhibitor can suppress STAT3 activation at doses that avoid the hematological toxicity associated with JAK2 and JAK3 inhibition. Citation Format: Richard Woessner, Geraldine Bebernitz, Kirsten Bell, Robert Caccese, Nakpangi Johnson, Jason Kettle, Caroline Rivard, Patricia Schroeder, Qibin Su, Haixia Wang, Minwei Ye, Weijia Zheng, Michael Zinda, Paul Lyne, Melissa Vasbinder, Dennis Huszar, Claudio Chuaqui. Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2013-931

Temporal Expression of Cytokines and Signal Transducer and Activator of Transcription Factor 3 Activation after Neonatal Hypoxia/Ischemia in Mice

Hypoxia/ischemia (HI) is a prevalent reason for neonatal brain injury with inflammation being an inevitable phenomenon following such injury; but there is a scarcity of data regarding the signaling pathway involved and the effector molecules. The signal transducer and activator of transcription factor 3 (STAT3) is known to modulate injury following imbalance between pro- and anti-inflammatory cytokines in peripheral and central nervous system injury making it a potential molecule for study. The current study investigates the temporal expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, IL-1ra, IL-4, IL-10, IL-13 and phosphorylated STAT3 (pSTAT3) after carotid occlusion and hypoxia (8% O₂, 55 min) in postnatal day 7 C57BL/6 mice from 3 h to 21 days after hypoxia. Protein array illustrated notable changes in cytokines expressed in both hemispheres in a time-dependent manner. The major pro-inflammatory cytokines showing immediate changes between ipsi- and contralateral hemispheres were IL-6 and IL-1β. The anti-inflammatory cytokines IL-4 and IL-13 demonstrated a delayed augmentation with no prominent differences between hemispheres, while IL-1ra showed two distinct peaks of expression spread over time. We also illustrate for the first time the spatiotemporal activation of pSTAT3 (Y705 phosphorylation) after a neonatal HI in mice brain. The main regions expressing pSTAT3 were the hippocampus and the corpus callosum. pSTAT3+ cells were mostly a subpopulation of activated astrocytes (GFAP+) and microglia/macrophages (F4/80+) seen only in the ipsilateral hemisphere at most time points studied (till 7 days after hypoxia). The highest expression of pSTAT3+ cells was observed to be around 24-48 h, where the presence of pSTAT3+ astrocytes and pSTAT3+ microglia/macrophages was seen by confocal micrographs. In conclusion, our study highlights a synchronized expression of some pro- and anti-inflammatory cytokines, especially in the long term not previously defined. It also points towards a significant role of STAT3 signaling following micro- and astrogliosis in the pathophysiology of neonatal HI-related brain injury. In the study, a shift from pro-inflammatory to anti-inflammatory cytokine profile was also noted as the injury progressed. We suggest that while designing efficient neuroprotective therapies using inflammatory molecules, the time of intervention and balance between the pro- and anti-inflammatory cytokines must be considered.

Active SHP2 protects the pulmonary endothelium through a FAK‐mediated mechanism

Enhanced protein tyrosine phosphorylation is associated with increased vascular permeability. We have previously shown that chemical and molecular inhibition of the protein tyrosine phosphatase, SHP2, promotes edema formation in the pulmonary endothelium (EC) in vitro and in vivo; thus implicating SHP2 in a protective role of pulmonary EC barrier function. To study whether this function of SHP2 translates to therapeutic value, we assessed the effect of molecular activation of SHP2 on EC barrier function in response to edemagenic agents, lipopolysaccharide (LPS) and thrombin. Both LPS and thrombin reduced SHP2 activity, which correlated with an elevation in focal adhesion kinase (FAK) Y397 phosphorylation and decrease in SHP2 protein‐protein associations with FAK, p120RasGAP, and p190RhoGAP. In contrast, the barrier protective agent S1P elevated SHP2 activity, enhanced FAK Y576 phosphorylation and increased FAK‐SHP2 association. In vivo, overexpression of constitutively active SHP2 (caSHP2) enhanced EC monolayer resistance at basal conditions and attenuated LPS and thrombin‐induced permeability, however had no effect on s1P‐induced barrier protection. In vivo, overexpression of caSHP2 completely attenuated LPS‐induced pulmonary edema formation. Our data demonstrated that the protective role for SHP2 in maintaining pulmonary EC barrier function may be mediated via FAK.

CD44‐mediated Src signaling in endothelial barrier dysfunction

CD44 is a cell‐surface receptor for hyaluronan, a major glycosaminoglycan component of extracellular matrix. The objective of this study was to define the role of CD44 in endothelial barrier function and its downstream signal. Using cultured pulmonary microvascular endothelial cells, we found that blocking CD44 with high molecular weight HA (HMW‐HA) increased electric resistance in hydrogen peroxide‐stimulated endothelial monolayers, indicating a protective role of blocking endothelial CD44 signaling in the regulation of microvascular permeability. To confirm this finding in vivo, intravital microscopy was conducted in the rat mesentery. Our result showed that intravenous administration of HMW‐HA prevented plasma leak caused by the inflammatory mediator PAF. Western blot analysis indicated that oxidative stress and CD44 activator (i.e., CD44 antibody IM‐7) caused comparable Src activation (Y416 phosphorylation) in endothelial cells. The protective effect of HMW‐HA on endothelial barrier function was associated with inhibition of Src phosphorylation at Y416. Inhibition of Src activity by PP2, a selective inhibitor for Src‐family kinases, blocked CD44‐induced barrier dysfunction. Taken together, these results suggest that activation of endothelial CD44 increases microvascular permeability through Src phosphorylation. Supported by NIH RO1 HL061507, HL070752, GM097270 and HL096640.

Prostaglandin E2 receptor EP1-mediated phosphorylation of focal adhesion kinase enhances cell adhesion and migration in hepatocellular carcinoma cells

The prostaglandin E₂ (PGE₂) EP1 receptor has been implicated in hepatocellular carcinoma (HCC) cell invasion. However, little is known about the mechanisms of EP1 receptor-mediated cell adhesion and migration. We previously showed that PGE₂ promotes cell adhesion and migration by activating focal adhesion kinase (FAK). The present study was designed to elucidate the association between the EP1 receptor and FAK activation in HCC cells and to investigate the related signaling pathways. The effects of PGE₂, EP1 agonist 17-phenyl trinor-PGE₂ (17-PT-PGE₂), PKC and EGFR inhibitors on FAK activation were investigated by treatment of Huh-7 cells. Phosphorylation of FAK Y397 and c-Src Y416 was investigated by western blotting. Cell adhesion and migration were analyzed by WST and transwell assays, respectively. Protein kinase C (PKC) activity was measured with a PKC assay kit. The results showed that 17-PT-PGE₂ (3 µM) increased FAK Y397 phosphorylation by more than 2-fold and promoted cell adhesion and migration in Huh-7 cells. In transfected 293 cells, expression of the EP1 receptor was confirmed to upregulate FAK phosphorylation, while the EP1 receptor antagonist sc-19220 decreased PGE₂-mediated FAK activation. PKC activity and c-Src Y416 phosphorylation were enhanced after 17-PT-PGE₂ treatment. Both PKC and c-Src inhibitor suppressed the 17-PT-PGE₂-upregulated FAK phosphorylation, as well as 17-PT-PGE₂-induced cell adhesion and migration. In addition, exogenous epidermal growth factor (EGF) treatment increased FAK phosphorylation. The EGF receptor (EGFR) inhibitor also suppressed 17-PT-PGE₂-upregulated FAK phosphorylation. Our study suggests that the PGE₂ EP1 receptor regulates FAK phosphorylation by activating the PKC/c-Src and EGFR signal pathways, which may coordinately regulate adhesion and migration in HCC.

Autocrine role for Gas6 with Tyro3 and Axl in leiomyosarcomas

Leiomyosarcoma (LMS) represent 15 % of adult sarcomas. The aim of this work was to identify novel altered pathways in LMS, which may be of therapeutic value for patients. Thirteen fresh frozen samples of soft tissue and visceral LMS were analyzed and compared with normal smooth muscle uterine tissue (NSM) for phosphoproteomic profile. Four proteins were found differentially expressed including Tyro3. The functional role of Tyro3 and its ligand Gas6 was investigated in two LMS cell lines, SK-LMS-1 and CNIO-AA. Four proteins and phosphoproteins were differentially expressed in LMS samples vs NSM: A loss of FAK Y397 phosphorylation was observed in all LMSs, while Tyro3, MSH2 and PKC theta were consistently overexpressed. Gas6, the major ligand of Tyro3, was expressed in 8 of the 13 LMS samples, and Gas6 expression highly correlated to Akt Y473 phosphorylation and to a lesser extent to Erk1/2 phosphorylation. SK-LMS-1 and CNIO-AA LMS expressed Tyro3, Axl and Gas6 at high level in CNIO-AA while at low levels in SK-LMS-1. Exposure of both cell lines to foretinib, a tyrosine kinase inhibitor of Met, Axl and Tyro3, reduced cell viability and induced caspase 3/7 activation. Transfection of CNIO-AA with small interfering RNA directed against Tyro3 and Axl genes induced a reduction of the expression of the specific proteins and, when combined, significantly reduced CNIO-AA cell viability. Leiomyosarcomas overexpress Tyro3. Gas6, a ligand of Tyro3, exerts an autocrine activities though Tyro3 and Axl in a subgroup of LMS.

Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression.

Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the peri-ovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer.

Targeting the LYN/HS1 Signaling Axis in Chronic Lymphocytic Leukemia

AbstractAbstract 928B-cell receptor (BCR) signaling and cytoskeletal activation represent critical steps in the pathogenesis of Chronic Lymphocytic Leukemia (CLL). We previously demonstrated that Hematopoietic cell specific Lyn substrate 1 (HS1) i) is a potential prognostic marker as CLL patients whose leukemic cells carry hypo-phosphorylated (hypo-p) HS1 have a better prognosis than patients with the hyper-phosphorylated (hyper-p) form; ii) is a central interactor of several cytoskeletal components and iii) has a profound effect on the development and progression of CLL in the Eμ-TCL1 transgenic mouse suggesting that the hyper-phosphorylation of the molecule may be responsible of HS1 inactivation rather than its activation.To better define the role of HS1 phosphorylation in CLL cell biology we dissected the BCR signaling pathway in cells with differential phosphorylation of the HS1 protein. Taking advantage of the CLL cell line MEC1 where we silenced the expression of HS1, we observed that the phosphorylation status of several BCR signalling molecules, including LYN kinase, is reduced in the absence of HS1. A similar pattern of modifications was confirmed in primary cells from 62 patients with different phosphorylation status of HS1 (Figure A n=35 HS1hypo-p vs n=27 HS1hyper-p, p=0,01), thereby identifying a distinct phospho-signature associated to HS1. LYN phosphorylation correlates with clinical parameters as phosphoLYN+ patients showed significantly increased (Figure B n=28 phosphoLYN+ vs n=28 phosphoLYN− p=0,0002) Progression-free survival and Treatment-free survival (Figure C n=27 phosphoLYN+ vs n=26 phosphoLYN− p=0,0002) as compared to phosphoLYN−patients.We further investigated the relationship between HS1 and LYN phosphorylations and showed that LYN kinase activation- Tyrosine (Y)396 phosphorylation- is associated with the phosphorylation of the active site Y397 on HS1 protein (Spearman correlation n=40, p<0,0001). Patients may thus be categorized into two subgroups based on the phosphorylation status of the two molecules as “active-HS1” - HS1 hypo-p/phosphoLYN+ and “inactive-HS1” - HS1 hyper-p/phosphoLYN−-.These different patterns correlated with distinct cytoskeletal functionality in terms of migration (active-HS1 n=12 vs inactive-HS1 n=9, p= 0,009), spontaneous adhesion (active-HS1 n=12 vs inactive-HS1 n=9, p=0,03), adhesion to an HS5 stromal cell layer (active-HS1 n=9 vs inactive-HS1 n=10 p=0,006) and F-actin polymerization (active-HS1 n=12 vs inactive-HS1 n=11, p=0,001). Targeting the LYN/HS1 axis in vitro with the tyrosine kinase inhibitor Dasatinib significantly affected primary CLL cell viability with a direct correlation with basal LYN phosphorylation (Sperman correlation n=32, p=0,04) and interfered with cytoskeleton activity, with a preference for patients carrying the “active-HS1” signature. Accordingly, 100nM Dasatinib markedly reduced migration of active-HS1 samples (active-HS1 n=10, p=0,003 vs inactive-HS1 n=7, p>0,05), with no significant effect on inactive-HS1 samples. F-actin polymerization (active-HS1 n=9 p=0,003, vs inactive-HS1 n=8 p=0,01) was reduced in both groups by Dasatinib treatment, while spontaneous (active-HS1 n=7, p=0,03 vs inactive-HS1 p>0,05) and HS5-mediated cell adhesion (active-HS1 n=8, p=0,03 vs inactive-HS1 n=8 p>0,05) were affected by the treatment only in the active-HS1 group.In the adoptive transfer mouse model based on the EmTCL1 transgenic mouse, a 4-week Dasatinib treatment reduced the percentage of CD19+CD5+Igk+ CLL cells in the blood as soon as one week after treatment initiation and throughout the whole treatment period (p(Day6)=0,007, p(Day13)=0,005, p(Day20)=0,004, p(day of killing)=0,01). At sacrifice, a significant reduction in the absolute number of leukemic cells in the spleen (p=0,05), bone marrow (p=0,03) and lymph nodes (p=0,04) was observed in treated mice as compared to controls. Consistent with the biochemical action of Dasatinib, significant reduction in SRC-Y416 phosphorylation was observed after intracellular staining of leukemic B cells (p=0,04) at the end of treatment.These observations suggest that the phosphorylations status of LYN and HS1 as a biological indicator of respectively distinct signaling pathway and cytoskeletal-related features that can be selectively targeted for therapeutic intervention in a sizable fraction of CLL patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Impact of Shear Stress On the Interaction of Human Platelet Integrin αIIbβ3 with Fibrinogen.

AbstractAbstract 2167 Objectives:Shear stress can activate platelets resulting in subsequent platelet aggregation. The so-called “shear-induced platelet aggregation” (SIPA) contributes to various vascular diseases (Speich et al., Am J Physiol Cell Physiol 2008). Several signaling pathways were proposed to be involved in this process, e.g., αIIbβ3-mediated signaling (Feng et al., Am J Physiol Cell Physiol 2006). We investigated the impact of shear stress on the αIIbβ3–ligand interaction in human platelets adherent onto fibrinogen. Platelets on immobilized fibrinogen were exposed to various shear rates and signaling of Src and FAK tyrosine kinases, both essential in the integrin downstream signaling pathways, were examined. Specifically, we analyzed the role of αIIbβ3 in shear-induced platelet signaling (i) by comparing the Src Y418 and FAK Y397 phosphorylation activities between platelets on immobilized fibrinogen and platelets on BSA matrix in response to shear stress, and (ii) by performing experiments in the presence of the αIIbβ3 antagonist abciximab. Methods:Human washed platelets were incubated on immobilized fibrinogen 100 μg/ml or 1% BSA either under static conditions or exposed to shear rates of 500 s−1 or 5000 s−1, respectively. Specific phosphorylation of Src (pY418) and FAK (pY397) was determined by Western blot and quantified densitometrically. Experiments under flow conditions were performed in a cone-plate viscometer. Results:Both Src and FAK exhibited phosphorylation under static conditions on immobilized fibrinogen after 2 min of adhesion. A shear rates of 500 s−1 did not increase the phosphorylation activities. By contrast, high shear rates (5000 s−1) significantly enhanced both Src and FAK phosphorylations in fibrinogen-adherent platelets (3-fold increase each, p<0.05). In the absence of immobilized fibrinogen, platelets incubated with BSA matrix did not show any Src activation under static conditions and only a very low Y418 phosphorylation activity in response to a shear rate of 500 s−1. A shear rate of 5000 s−1 considerably induced Src pY418 activity compared to platelets exposed to physiological shear stress (10-fold increase, p< 0.01). In response to shear rates of 500 s−1 or 5000 s−1, we detected a significantly higher Src activation in platelets adherent onto fibrinogen (500 s−1: 10-fold higher, p<0.01; 5000 s−1: 2-fold higher, p<0.05) than in platelets incubated over a BSA matrix indicating a ligand-dependent signaling. When platelets over BSA were exposed to a shear rate of 5000 s−1, FAK also exhibited a significant elevation of pY397 activity (9-fold increase, p<0.05). By contrast to Src, in platelets exposed to a shear rate of 500 s−1 or 5000 s−1, we observed approximately equal FAK pY397 activation, independent of the presence or absence of immobilized fibrinogen. In platelets incubated for 10 min on a fibrinogen matrix under static conditions, we did not detect any change in the Src activation compared to 2 min incubation. The activity of FAK pY397, however, was time-dependent and showed a 3-fold higher phosphorylation extent after 10 min than after 2 min adhesion (p<0.05). In response to a shear rate of 500 s−1 both Src Y418 and FAK Y397 phosphorylations exhibited a considerable time-dependent enhancement (comparing the phosphorylation activities after incubation for 2 or 10 min). This enhancement could be seen both in platelets adherent onto fibrinogen and in platelets over BSA (3 to 6-fold increase, p<0.05). In platelets exposed to a shear rate of 5000 s−1 for 10 min, the Src and FAK phosphorylation activities were similar to platelets after 2 min. Abciximab inhibited the Src and FAK signaling in platelets exposed to 5000 s−1 on immobilized fibrinogen. The same inhibition was seen in platelets exposed to 5000 s−1 over BSA (p<0.05). Conclusions:Exposure of platelets to high shear rates induces a significant increase of both Src and FAK signaling compared to platelets under static conditions. Whereas Src activation remains predominantly ligand-dependent in fibrinogen-adherent platelets even under shear stress, FAK signaling appears to be shear-induced. The finding that, abciximab inhibits the activation of both Src and FAK in the absence of fibrinogen, emphasizes the role of integrin αIIβ3 in the shear-induced platelet signaling. Disclosures:No relevant conflicts of interest to declare.