Src Family Kinase Inhibitor Research Articles

Abstract 1821: Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer

Abstract Background: Almost 10% of colon cancers harbor the canonical activating V600 BRAF mutation that in melanoma cancers has been shown to dramatically sensitize cells to targeted therapy and prolong survival. In the colon, such tumors are associated with worse survival. Recent clinical trials of combination therapies targeting the EGFR and MAPK pathways in these tumors have demonstrated impressive response rates. However, the clinical benefit has been limited by the rapid emergence of drug resistance. Identifying and targeting the genes complicit in this process and thus re-sensitize resistant cancer cells may be of clinical benefit. The landscape of drug resistance in cancer patients is composed of (a) loss-of-function (LoF), (b) gain-of-function (GoF) and (c) point mutation-mediated perturbations of genes, with often no single mechanism being predominant. Therefore, capturing the full breadth of resistance genes for any drug would require multiple genetic screens to be executed in parallel. Methods: We performed 3 genome-wide genetic screens in parallel in BRAF mutant colon cancer cells treated with a BRAF/MEK/EGFR inhibitor combination, to capture all of the resistance mechanisms described above. To screen for LoF events we used a genome-wide CRISPR/Cas9 sgRNA library. For GoF resistance events, a lentiviral-based insertional mutagenesis vector was used to randomly integrate the SFFV enhancer/promoter sequence throughout the genome. Finally, to saturate the genome of each cell line with point mutations we used N-Ethyl-N-nitrosourea (ENU) to randomly mutagenize every base in the genome. Each genetic screen was analysed separately to identify the relevant genes and pathways that confer drug resistance before aggregating the outputs of all three screens for a more comprehensive view of the drug resistance landscape. Results: The most recurrent and strongest hit from the CRISPR knockout screen was the inhibitor of Src family kinases, CSK. A Gaussian Kernal Convolution analysis for recurrent integrations detected in resistant clones following the insertional mutagenesis screen identified five genes as likely resistance candidates - BRAF, SOS1, MET, FRS2 and KRAS. Finally, Illumina exome sequencing of resistant clones following ENU mutagenesis revealed statistical enrichment for non-synonymous point mutations in three genes in the MAPK pathway, namely MAP2K1, NRAS and KRAS. Of note, loss of CSK was also shown to confer resistance to targeted therapies in other cancer types. Conclusion: Genome-wide genetic screens identified genes in the MAPK pathway likely to cause resistance clinically, and many of which are amenable to therapeutic targeting. Of note, the resistance as a result of loss of CSK instead activates Src signalling. It can be overcome through the addition of a Src inhibitor, and may be relevant across a range of cancer types as a resistance mechanism. Citation Format: Jonathan Brammeld, Marco Ranzani, Elizabeth A. Coker, Stacey Price, Theodoros I. Roumeliotis, Barbara Lupo, Mi Petljak, Steven P. Williams, Francesco Iorio, Francesco Sassi, Nanne Aben, Kosuke Yusa, Livio Trusolino, Lodewyk Wessels, David J. Adams, Mathew Garnett, Bissan Al-Lazikani, Jyoti S. Choudhary, Andrea Bertotti, Ultan McDermott. Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1821.

Abstract 4126: DNA methyltransferase inhibitors target a YES1-YAP1 signaling axis in childhood rhabdomyosarcoma

Abstract We have previously shown that the SRC family kinase (SFK) member, YES1, is highly expressed and rapidly activated upon IGF-1R blockade in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children and adolescents. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors where it functions as an oncoprotein when the upstream Hippo tumor suppressor pathway is silenced, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. One activator of the Hippo pathway, RASSF1a, is epigenetically silenced in many cancer types including sarcomas. Here we show a loss of RASSF1a expression in RMS cell lines via promoter methylation. Treatment with DNA methyltransferase inhibitors (DNMTis) can re-activate RASSF1a expression, resulting in YAP phosphorylation and inactivation. DNMTi treatment also significantly inhibits RMS cell growth, which can be rescued by overexpression of a constitutively active YAPS127A. In the alveolar variant (aRMS) in particular, DNMTis increase apoptosis and decrease both YES1 expression and activation. This suggests DNMTi treatment can inhibit both YAP1 and YES1 signaling. To further investigate the connection between YES1 and YAP1 we performed proximity ligation assays and discovered that YES1 and YAP1 interact in the nucleus of RMS cell lines. Furthermore, dasatinib treatment, a known SFK inhibitor, prevents YAP1 nuclear accumulation suggesting a role for YES1 in regulating YAP1 cellular localization. YAP1 is also tyrosine phosphorylated, which may be one way YES1 regulates YAP1. Lastly, cells expressing a constitutively active YAPS127A have increased activation of SFK suggesting a positive feedback loop whereby YAP1 activity can further activate YES1 through an unknown mechanism. These data uncover a signaling axis between the YES1 and YAP1 oncoproteins in RMS and suggest an alternative way to silence YES1 and YAP1 through use of DNMTis. We will further analyze this signaling through shRNA knockdown of YES1 and evaluate combination treatment of a DNMTi and dasatinib in vitro and in vivo. Citation Format: Katherine Slemmons, Choh Yeung, Josh Baumgart, Amy McCalla-Martin, Lee Helman. DNA methyltransferase inhibitors target a YES1-YAP1 signaling axis in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4126.

Abstract 2441: Effects of hypoxia on Src family members' activity in prostate cancer cells

Abstract Metastasis is the major reason of disease progression and poor prognosis in prostate cancer. Src family kinases (SFKs), including c-Src, Lyn and Fyn, are non-receptor tyrosine kinases that have been shown to play an essential role in castration resistance and metastasis in prostate cancer. Hypoxia, a typical hallmark of solid malignancies, is able to promote metastasis-associated functions. However, whether SFK members are upregulated under hypoxia is unclear. The current project will detect the impact of hypoxia on cell functions and molecular features in prostate cancer cells. For hypoxic exposure, prostate cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h). To test cell migration, “wound-healing” assay was used. Cell invasion was detected by seeding cells into Matrigel-coated transwell chamber and cells on the bottom membranes were counted after treatment. Clonogenic assay was performed to test cell survival. At the molecular level, SFKs' phosphorylation in cancer cells and patient tissues was detected by Western blotting, and gene knockdown was accomplished by siRNA transfection. To detect the effects of Src inhibitors, cells were treated with saracatinib for indicated time periods. Phosphorylation of all three typical SFKs, e.g., c-Src, Lyn and Fyn, were highly expressed in prostate cancer patient tissues. At the cellular level, while short term hypoxic exposure (2-6 h) induced greater effects than prolonged hypoxia (24 h) in PC-3ML, these cellular functions were increased with 24 h hypoxic exposure in C4-2B cells. Further, hypoxia enhanced SFK phosphorylation in the pattern that was consistent with cell functions in both cell lines. Knockdown SRC, but not LYN or FYN, abolished hypoxia-induced invasion and p-SFK expression. Lastly, SFK inhibitor saracatinib showed stronger inhibition on functional behaviors facilitated under hypoxia than normal conditions. Our data show that hypoxia is able to enhance metastatic phenotypes by activating SFKs in prostate cancer cells. Interestingly, c-Src may be the most important signaling molecule for hypoxia-mediated behaviors. More importantly, SFK inhibitors are able to impair hypoxia-induced cell functions, suggesting their therapeutic potential by suppressing tumor metastasis that is driven under hypoxia in prostate cancer. Citation Format: Yao Dai, Dietmar Siemann. Effects of hypoxia on Src family members' activity in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2441.

PO-462 Investigating the potential anti-cancer properties of a novel src family kinase inhibitor in triple negative breast cancer

IntroductionThe Src Family Kinases (SFKs) are key mediators in various oncogenic pathways and are commonly activated in many forms of cancer. Of the nine members of SFKs, increasing reports suggest aberrant activation of Lyn and Fyn in breast cancer. Bioinformatics analysis reveals that Lyn and Fyn expression are strongly associated with the claudin-low TNBC patient group. Due to the current lack of targeted therapy for the TNBC subtype, it becomes worthwhile to investigate the potential of Lyn and Fyn as novel therapeutic targets. While preclinical studies involving current SFK inhibitors were promising, subsequent clinical trials have shown limited clinical benefits. In this project, we developed a novel in-house SFK inhibitor (Drug X) that specifically targets Lyn and Fyn kinases.Material and methodsPatient data analysis, Ligand-based computation, Proteome Profiler Array, in vivo mouse Pharmacokinetics and Pharmacodynamics studies, Bioinformatics analysis, Affymetrix Microarray, Secretome Analysis, Cell viability assays, Flow cytometry, Western blot, qPCR, Immunofluorescence, Transwell assays, 3D Matrigel Assays, and Tube-formation assay.Results and discussionsProteome Profiler Array results validated the ligand-based target prediction analyses of Drug X in vitro. Mouse studies show that Drug X is well-tolerated and exhibited significant tumour burden load reduction. Pharmacokinetics of Drug X was characterised. Overall, Drug X showed moderate systemic clearance, high volume of distribution and moderate oral bioavailability. Downstream functional studies were performed, and Drug X was found to induce apoptosis via the intrinsic pathway. Also, Drug X could down-regulate the mesenchymal properties of mesenchymal-like TNBC cell lines via the modulation of key EMT-related genes. Preliminary in vitro and in vivo rat aortic ring assay data also suggested that Drug X could harbour potent anti-angiogenic effects.ConclusionBased on in vitro and in vivo experiments, preliminary findings have supported that Drug X could potentially be a promising novel drug candidate for TNBC treatment and on-going efforts are being made to further elucidate the underlying mechanisms of Drug X in modulating the key oncogenic processes – namely from the angle of apoptosis, EMT and angiogenesis.

Abstract 2371: Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines

Abstract Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase, is an important mediator of immune receptor signaling in B cells, mast cells, neutrophils, and macrophages. SYK is expressed in 90% of AML blasts and has been reported to be is constitutively activated (Hahn et al., Cancer Cell 2009). High expression of phosphorylated SYK (pSYK) is associated with unfavorable outcomes, independent of other AML prognostic predictors such as age, cytogenetics, and white blood cell count (Boros et al., Oncotarget 2015). Entospletinib (ENTO) is a potent and selective inhibitor of SYK currently in clinical trials for the treatment of AML as monotherapy and in combination with standard-of-care therapies. We investigated the effects of ENTO on SYK phosphorylation and signaling in FLT3-ITD mutant AML cells. We found that ENTO blocked constitutive phosphorylation of pSYKY352, pSTAT5Y694, and MYC protein expression in FLT3-ITD-driven AML cell lines, MV4-11 and MOLM14. The concentration of ENTO required to inhibit pSYKY352 and pSTAT5Y694 correlated with inhibition of cell viability. Phosphorylation at these sites was inhibited by the selective FLT3 inhibitor, quizartinib; but not inhibited by Src family kinase inhibitor (dasatinib) or JAK inhibitors (ruxolitinib or tofacitinib). Further, a second-generation, highly selective SYK inhibitor, GS-9876, failed to inhibit the constitutive phosphorylation of these sites. Comparison of the biochemical and cellular kinase activities of the SYK and FLT3 inhibitors suggests that the observed inhibition of constitutive phosphorylation of pSYKY352 and pSTAT5Y694 and MYC expression was mediated by the direct activity of ENTO against FLT3-ITD, rather than SYK. Activation of FcγRI has been previously shown to activate SYK (Oellerich et al., Blood 2013). We demonstrate SYK activation at pSYKY348 and pSYKY323 after stimulation of the FCγRI with human IgG in the FLT3-ITD mutant cell line, MV4-11. Downstream signaling molecules pERK1/2T202/Y204, pVAV1Y160, and pLYNY507 were also activated by FCγR stimulation. ENTO inhibited phosphorylation of pSYKY348, pSYKY323 and pVAV1 Y160. The second-generation SYK inhibitor, GS-9876, also inhibited FCγRI-mediated activation of pSYKY348,pSYKY323 and pVAV1 Y160, suggesting that these phosphorylation events were mediated by SYK activity. In conclusion, ENTO inhibited constitutive and FCγRI-activated SYK phosphorylation and signaling pathways in FLT3-ITD mutant cell lines, resulting in inhibition of cell viability. Inhibition of constitutive SYK phosphorylation is mediated by direct activity of ENTO against FLT3-ITD; however, inhibition of FCγRI activated SYK results from direct inhibition of SYK. Citation Format: Stacey L. Tannheimer, Adam Kashishian, Rick Sorensen, Kathleen S. Keegan. Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2371.

Abstract 144: Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells

Abstract Many patients with triple negative breast cancer (TNBC) exhibit poor prognosis and are at high risk of tumor relapse and metastasis due to the resistance to conventional chemotherapy. These tumor recurrences have been attributed to the presence of breast cancer stem cells (BCSCs), a subpopulation of breast cancer cells that possesses stem-like properties and displays resistance to chemotherapy. Therefore, targeting BCSCs is a priority to overcoming chemotherapy failure in TNBCs. We generated chemotherapy-resistant TNBC cells through cyclic treatments with paclitaxel (pac). The pac-resistant cells displayed increased self-renewal potential compared to the parental TNBC cells, higher percentage BCSCs and increased phosphorylation of Src kinase at Tyr416. After performing a kinase library screening on BCSCs in chemotherapy-resistant cells, we identified dasatinib, a Src kinase family inhibitor, potently reduced BCSC self-renewal and two BCSC populations (ALDH+ and CD24lowCD44high) as well as phosphor-Src. We also demonstrated dasatinib to block pac-induced BCSC enrichment and Src activation in the parental TNBC cells. Interestingly, we found that dasatinib induced an epithelial differentiation of pac-resistant cells, resulting in their enhanced sensitivity to paclitaxel. The combination treatment of dasatinib and paclitaxel not only decreased the proportion of BCSCs and their self-renewal capacity but also synergistically reduced cell viability of pac-resistant cells. In vivo studies further demonstrated the effectiveness of dasatinib/paclitaxel combination treatment in inhibiting breast tumor growth. All these data suggest that dasatinib is a promising anti-BCSC agent and can be used in combination with paclitaxel to overcome chemotherapy resistance in TNBCs. Citation Format: Jun Tian, Chieh Lo, Fatmah Al Raffa, Meiou Dai, Jean-Jacques Lebrun. Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 144.

Withdrawal from spinal application of remifentanil induces long-term potentiation of c-fiber-evoked field potentials by activation of Src family kinases in spinal microglia.

It is well known that remifentanil, a widely used intravenous anesthesia drug, can paradoxically induce hyperalgesia. The underlying mechanisms are still not clear despite the wide investigations. The present study demonstrated that withdrawal from spinal application of remifentanil could dose-dependently induce long term potentiation (LTP) of C-fiber evoked field potentials. Remifentanil withdrawal could activate Src family kinases (SFKs) in microglia, and upregulate the expression of tumor necrosis factor alpha (TNFα) in spinal dorsal horn. Furthermore, pretreatment with either microglia inhibitor Minocycline, SFKs inhibitor PP2 or TNF αneutralization antibody could block remifentanil withdrawal induced spinal LTP, whereas supplement of recombinant rat TNFα to the spinal cord could reverse the inhibitory effect of Minocycline or PP2 on remifentanil withdrawal induced LTP. Our results suggested that TNFαrelease following SFKs activation in microglia is involved in the induction of LTP induced by remifentanil withdrawal.

Inhibition of SRC family kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous cell carcinoma.

The immune system plays a critical role in the establishment, development, and progression of head and neck squamous cell carcinoma (HNSCC). As treatment with single-immune checkpoint agent results in a lower response rate in patients, it is important to investigate new strategies to maintain favorable anti-tumor immune response. Herein, the combination immunotherapeutic value of CTLA4 blockade and SFKs inhibition was assessed in transgenic HNSCC mouse model. Our present work showed that tumor growth was not entirely controlled when HNSCC model mice were administered anti-CTLA4 chemotherapeutic treatment. Moreover, it was observed that Src family kinases (SFKs) were hyper-activated and lack of anti-tumor immune responses following anti-CTLA4 chemotherapeutic treatment. We hypothesized that activation of SFKs is a mechanism of anti-CTLA4 immunotherapy resistance. We, therefore, carried out combined drug therapy using anti-CTLA4 mAbs and an SFKs' inhibitor, dasatinib. As expected, dasatinib and anti-CTLA4 synergistically inhibited tumor growth in Tgfbr1/Pten 2cKO mice. Furthermore, dasatinib and anti-CTLA4 combined to reduce the number of myeloid-derived suppressor cells and Tregs, increasing the CD8+ T cell-to-Tregs ratio. We also found that combining dasatinib with anti-CTLA4 therapy significantly attenuated the expression of p-STAT3Y705 and Ki67 in tumoral environment. These results suggest that combination therapy with SFKs inhibitors may be a useful therapeutic approach to increase the efficacy of anti-CTLA4 immunotherapy in HNSCC.

Dasatinib sensitises KRAS-mutant cancer cells to mitogen-activated protein kinase kinase inhibitor via inhibition of TAZ activity

PurposeOncogenic KRAS mutations occur frequently in solid tumours, but no clinically applicable targeted strategy is yet available for treating human cancers with mutant KRAS. Here we aimed to identify a strategy for the treatment of KRAS-driven cancers. Experimental DesignCell viability and colony forming assays were used to assess the in vitro effect of dasatinib and trametinib as single agents or in combination. Western blot was used to analyse the phosphorylated protein and total protein levels. Xenograft models were used to evaluate the in vivo effect of drug combination on KRAS-driven tumour growth. ResultsHere, we report the discovery of a synergistic interaction between dasatinib (ABL and SRC family kinase inhibitor) and the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in KRAS-mutant cancer cells. We demonstrated that dasatinib enhanced the antitumour effect of trametinib against the KRAS-mutant cancer models both in vitro and in vivo, and the combination resulted in a significant reduction of cytoplasmic and nucleic TAZ protein level, and therefore decreased downstream protein levels of YAP/TAZ signalling pathway. Furthermore, direct knockdown of TAZ by small interfering RNA was able to increase the sensitivity of KRAS-mutant cells to trametinib treatment. ConclusionThese results indicate that dasatinib enhances the antitumour activity of MEK inhibitor through inhibition of TAZ activity and identify dasatinib and trametinib combination as a potential strategy for the treatment of KRAS-driven cancers.

Targeting autophagy sensitises lung cancer cells to Src family kinase inhibitors.

Lung cancer is the main cancer killer in both men and women, mostly due to the rapid development of drug resistant metastatic disease. Here, we evaluate the potential involvement of SRC family kinases (SFK) in lung cancer biology and assess the possible benefits of their inhibition as a therapeutic approach. We demonstrated that various SRC family members, including LYN and LCK, normally expressed solely in hematopoietic cells and neural tissues, are overexpressed and activated in a panel of SCLC and NSCLC cell lines. This was clinically relevant as LYN and FYN are also overexpressed in lung cancer clinical specimens. Moreover, LYN overexpression correlated with decreased patient survival on univariate and multivariate analysis. Dasatinib (BMS-354825), a SRC/ABL inhibitor, effectively blocked SFK activation at nanomolar concentrations which correlated with a significant decrease in cell numbers of multiple lung cancer cell lines. This effect was matched by a decrease in DNA synthesis, but only moderate induction of apoptosis. Indeed, dasatinib as well as PP2, another SFK inhibitor, strongly induced autophagy that likely prevented apoptosis. However, inhibition of this autophagic response induced robust apoptosis and sensitised lung cancer cells to dasatinib in vitro and in vivo. Our results provide an explanation for why dasatinib failed in NSCLC clinical trials. Furthermore, our data suggest that combining SFK inhibitors with autophagy inhibitors could provide a novel therapeutic approach in this disease.

Selective inhibition of Src family kinases by SU6656 increases bone mass by uncoupling bone formation from resorption in mice

Mice deficient in the non-receptor tyrosine kinase Src exhibit high bone mass due to impaired bone resorption and increased bone formation. Although several Src family kinase inhibitors inhibit bone resorption in vivo, they display variable effects on bone formation. SU6656 is a selective Src family kinase inhibitor with weaker activity towards the non-receptor tyrosine kinase Abl and receptor tyrosine kinases which are required for appropriate osteoblast proliferation, differentiation and function. Therefore, we sought to determine whether SU6656 could increase bone mass by inhibiting bone resorption and by stimulating bone formation, and to explore its mechanisms of action. Four-month-old female C57Bl/6J mice received intraperitoneal injections of either 25 mg/kg SU6656 or its vehicle every other day for 12 weeks. SU6656-treated mice exhibited increased bone mineral density, cortical thickness, cancellous bone volume and trabecular thickness. SU6656 inhibited bone resorption in mice as shown by reduced osteoclast number, and diminished expressions of Oscar, Trap5b and CtsK. SU6656 did not affect Rankl or Opg expressions. However, it blocked c-fms signaling, osteoclastogenesis and matrix resorption, and induced osteoclast apoptosis in vitro. In addition, SU6656 stimulated bone formation rates at trabecular, endosteal and periosteal bone envelopes, and increased osteoblast number in trabecular bone. SU6656 did not affect expressions of clastokines favoring bone formation in mice. However, it stimulated osteoblast differentiation and matrix mineralization by specifically facilitating BMP-SMAD signaling pathway in vitro. Knockdown of Src and Yes mimicked the stimulatory effect of SU6656 on osteoblast differentiation. In conclusion, SU6656 uncouples bone formation from resorption by inhibiting osteoclast development, function and survival, and by enhancing BMP-mediated osteoblast differentiation.

C-Src kinase is involved in the tyrosine phosphorylation and activity of SLC11A1 in differentiating macrophages.

Studies have demonstrated that the solute carrier family 11 member 1 (SLC11A1) is heavily glycosylated and phosphorylated in macrophages. However, the mechanisms of SLC11A1 phosphorylation, and the effects of phosphorylation on SLC11A1 activity remain largely unknown. Here, the tyrosine phosphorylation of SLC11A1 is observed in SLC11A1-expressing U937 cells when differentiated into macrophages by phorbol myristate acetate (PMA). The phosphorylation of SLC11A1 is almost completely blocked by treatment with PP2, a selective inhibitor of Src family kinases. Furthermore, we found that SLC11A1 is a direct substrate for active c-Src kinase and siRNA-mediated knockdown of cellular Src (c-Src) expression results in a significant decrease in tyrosine phosphorylation. We found that PMA induces the interaction of SLC11A1 with c-Src kinase. We demonstrated that SLC11A1 is phosphorylated by Src family kinases at tyrosine 15 and this type of phosphorylation is required for SLC11A1-mediated modulation of NF-κB activation and nitric oxide (NO) production induced by LPS. Our results demonstrate important roles for c-Src tyrosine kinase in phosphorylation and activation of SLC11A1 in macrophages.

Overexpression of Tpl2 is linked to imatinib resistance and activation of MEK-ERK and NF-κB pathways in a model of chronic myeloid leukemia.

The introduction of tyrosine kinase inhibitors (TKI) has transformed chronic myeloid leukemia (CML) into a chronic disease with long‐term survival exceeding 85%. However, resistance of CML stem cells to TKI may contribute to the 50% relapse rate observed after TKI discontinuation in molecular remission. We previously described a model of resistance to imatinib mesylate (IM), in which K562 cells cultured in high concentrations of imatinib mesylate showed reduced Bcr‐Abl1 protein and activity levels while maintaining proliferative potential. Using quantitative phosphoproteomic analysis of these IM‐resistant cells, we have now identified significant upregulation of tumor progression locus (Tpl2), also known as cancer Osaka thyroid (COT1) kinase or Map3k8. Overexpression of Tpl2 in IM‐resistant cells was accompanied by elevated activities of Src family kinases (SFKs) and NF‐κB, MEK‐ERK signaling. CD34+ cells isolated from the bone marrow of patients with CML and exposed to IM in vitro showed increased MAP3K8 transcript levels. Dasatinib (SFK inhibitor), U0126 (MEK inhibitor), and PS‐1145 (IκB kinase (IKK) inhibitor) used in combination resulted in elimination of 65% of IM‐resistant cells and reduction in the colony‐forming capacity of CML CD34+ cells in methylcellulose assays by 80%. In addition, CML CD34+ cells cultured with the combination of inhibitors showed reduced MAP3K8 transcript levels. Overall, our data indicate that elevated Tpl2 protein and transcript levels are associated with resistance to IM and that combined inhibition of SFK, MEK, and NF‐κB signaling attenuates the survival of IM‐resistant CML cells and CML CD34+ cells. Therefore, combination of SFK, MEK, and NF‐κB inhibitors may offer a new therapeutic approach to overcome TKI resistance in CML patients.

MP45-11 NEW STRATEGIES FOR INHIBITION OF NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION BY PHARMACOLOGIC INTERVENTION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2018MP45-11 NEW STRATEGIES FOR INHIBITION OF NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION BY PHARMACOLOGIC INTERVENTION Qingfeng Yu, Christian Gratzke, Paul Kuppermann, Annika Herlemann, Yiming Wang, Frank Strittmatter, Raphaela Waidelich, Christian Stief, and Martin Hennenberg Qingfeng YuQingfeng Yu More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Paul KuppermannPaul Kuppermann More articles by this author , Annika HerlemannAnnika Herlemann More articles by this author , Yiming WangYiming Wang More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1450AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inhibition of prostate smooth muscle contraction by a1-adrenoceptor antagonists (a1-blockers) is the first option for medical treatment of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH). In fact, increased prostate smooth muscle tone may drive urethral obstruction and LUTS. However, the efficacy of α1-blockers is limited to 50%, because non-adrenergic mediators including endothelin-1 and thromboxane A2 (TXA2) increase prostate smooth muscle tension in parallel to α1-adrenoceptors and may therefore keep urethral obstruction despite therapy with α1-blockers. Consequently, future options with highest efficacy need to target α1-adrenergic and non-adrenergic contractions at once. Recently, several compounds have been reported to inhibit adrenergic prostate contraction, but their effects on non-adrenergic contraction are still unknown. Here, we examined effects of Rac-GTPase inhibitors and of a Src family kinase (SFK) inhibitor on non-adrenergic prostate contractions. METHODS Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, where they were induced by the α1-adrenoceptor agonist methoxamine, the TXA2 analog U46619, or by endothelin-1. RESULTS Methoxamine, U46619, and endothelin-1 induced concentration-dependent contractions of prostate strips. Inhibitory effects of the Rac inhibitors EHT1864 (100 µM) and NSC23766 (100 µM) and of the SFK inhibitor AZM475721 (10 µM) on α1-adrenergic contraction were confirmed by inhibition of methoxamine-induced contractions by all three inhibitors. EHT1864 caused significant inhibition of endothelin-1- and U46619-induced contractions, which was observed at 0.1-3 μM of endothelin-1 (p<0.001 between EHT1864 and control group), and at 1-30 μM of U46619 (p<0.001 between EHT1864 and control group). NSC23766 caused significant inhibition of U46619-induced contractions, which was observed at 3-10 µM of U46619 (p<0.001 between NSC23766 and control group). NSC23766 did not change endothelin-1-induced contractions. AZM475721 did not change U46619- or endothelin-1-induced contractions. CONCLUSIONS From the three examined inhibitors, EHT1864 is the most promising from a translational view, as it inhibited TXA2- and endothelin-1-induced besides α1-adrenergic prostate contractions. This reflects divergent pharmacologic profiles of EHT1864 and NSC23766, although both are Rac-GTPase inhibitors. In vivo, urodynamic effects of EHT1864, but not of AZM475721 may exceed those of α1-blockers. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e601-e602 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Qingfeng Yu More articles by this author Christian Gratzke More articles by this author Paul Kuppermann More articles by this author Annika Herlemann More articles by this author Yiming Wang More articles by this author Frank Strittmatter More articles by this author Raphaela Waidelich More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mutations in Lyn Kinase Causes Changes in Neutrophil Function and Migration

The Src Family Kinase (SFK) Lyn plays important regulatory roles in immune cells including neutrophils. As a signaling protein, Lyn has been shown to regulate neutrophil migration and endothelial adhesion, contributing to inflammation. Lyn contains a tyrosine kinase domain as well as SH2 and SH3 domains allowing for adaptor functions. A set of patients with systemic neutrophil‐induced inflammation have been shown to have de novo germline mutations in Lyn, Y508* and Y508F, affecting the negative regulatory tyrosine residue of Lyn. The purpose of this study is to evaluate the physiological, cellular, and biochemical effects of this mutation, and further understand the role of Lyn Kinase in neutrophil function. Patients were found to have a gain‐of‐function mutation and presented with skin vasculitis that improved with administration of the SFK inhibitor, dasatinib. We generated stable expression lines of WT, Y508*, and Y508F pMX‐Lyn‐GFP in neutrophil‐like PLB‐985 cells. Kinase activity assays show increased phosphorylation of SFK substrate, SAM68, and overall tyrosine phosphorylation with both Y508*and Y508F expression compared to WT, indicating higher kinase activity in the mutants. Further experiments using kinase‐dead mutants indicate that the higher kinase activity is caused by increases in Lyn's kinase activity and not its adaptor function. In addition, we evaluated the chemotaxis of PLB‐985 cells towards the chemoattractant fmlp using transwell migration assays and microfluidic devices that allow for the generation of chemoattractant gradients. Y508* and Y508F PLB‐985 cells were found to have significantly increased chemotaxis towards 10 mM fmlp (p&lt;0.05) than WT Lyn. Our data supports the role of Lyn as a positive regulator of migration, demonstrating that disruption in the regulatory domain of Lyn can cause cellular and physiological changes. Furthermore, it highlights the role of the de novo germline mutations in Lyn as a cause of human inflammatory disease.Support or Funding InformationSupported by the Hilldale Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Signaling Bias and Antagonism of Pilocarpine for M3 Muscarinic Acetylcholine Receptor

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth since the 19th century and classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with its main therapeutic target ‐ muscarinic M3 receptor (M3R). We found that pilocarpine was 1,000 times less potent in stimulating mouse eye pupil constriction than muscarinic agonists oxotremorin‐M (Oxo‐M) or carbachol (CCh), even though all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo‐M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in HEK293T or mouse insulinoma MIN6 cells. Pilocarpine also fails to stimulate insulin secretion, and instead, antagonizes insulinotropic effect of Oxo‐M and CCh‐induced Ca2+ upregulation. However, in HEK293T or CHO‐K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another Gq‐coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in PIP2. In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R, but not M3R. Moreover, pilocarpine blocked CCh‐stimulated PIP2 hydrolysis in M3R‐overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates ERK1/2 in MIN6 cells. The stimulatory effect on ERK1/2 was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward b‐arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level and signaling pathway downstream of this receptor.Support or Funding InformationThis work was supported by the National Institutes of Health Grants RO1DK105427 and RO1DK111538This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impedance-based analysis of Natural Killer cell stimulation

The use of impedance-based label free cell analysis is increasingly popular and has many different applications. Here, we report that a real-time cell analyzer (RTCA) can be used to study the stimulation of Natural Killer (NK) cells. Engagement of NK cells via plate-bound antibodies directed against different activating surface receptors could be measured in real time using the label-free detection of impedance. The change in impedance was dependent on early signal transduction events in the NK cells as it was blocked by inhibitors of Src-family kinases and by inhibiting actin polymerization. While CD16 was the only receptor that could induce a strong change in impedance in primary NK cells, several activating receptors induced changes in impedance in expanded NK cells. Using PBMCs we could detect T cell receptor-mediated T cell activation and CD16-mediated NK cell activation in the same sample. Performing a dose-response analysis for the Src-family kinases inhibitor PP1 we show that T cells are more sensitive to inhibition compared to NK cells. Our data demonstrate that the RTCA can be used to detect physiological activation events in NK cells in a label-free and real-time fashion.

Tyrosine kinase Fyn promotes osteoarthritis by activating the β-catenin pathway

ObjectivesTo investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice.MethodsCartilage samples...

Dasatinib-induced pulmonary arterial hypertension.

Drug-induced (group 1) pulmonary hypertension (PH) is an important subgroup of PH involving dasatinib as a likely related agent, which is a second-generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukaemia (CML). The mechanism of dasatinib-induced pulmonary arterial hypertension (PAH) is unclear. However, the occurrence of PAH with late onset in CML patients suggests a chronic pathological mechanism with an insidious onset rather than an acute inflammatory or cardiac aetiology. Dasatinib has a broader effect than other TKIs; the major known difference between dasatinib and other TKIs is the additional inhibition of Src family kinases. Therefore, Src inhibition was thought to play a role in the development of dasatinib-induced PAH. However, recently, it was also speculated that chronic dasatinib therapy may cause pulmonary endothelial damage, attenuate hypoxic pulmonary vasoconstriction responses and increase susceptibility to PAH independently of the Src family kinase-induced mechanism. Dasatinib-induced PAH usually seems to be reversible with the cessation of the drug, and sometimes with PAH-specific treatment strategies. Transthoracic echocardiography can be recommended as a routine screening prior to dasatinib initiation, and this non-invasive procedure can be utilized in patients having signs and symptoms attributable to PAH during dasatinib treatment.

A thermodynamic-based approach for the resolution and prediction of protein network structures

The rapid accumulation of omics data from biological specimens has revolutionized the field of cancer research. The generation of computational techniques attempting to study these masses of data and extract the significant signals is at the forefront.We suggest studying cancer from a thermodynamic-based point of view. We hypothesize that by modelling biological systems based on physico-chemical laws, highly complex systems can be reduced to a few parameters, and their behavior under varying conditions, including response to therapy, can be predicted.Here we validate the predictive power of our thermodynamic-based approach, by uncovering the protein network structure that emerges in MCF10a human mammary cells upon exposure to epidermal growth factor (EGF), and anticipating the consequences of treating the cells with the Src family kinase inhibitor, dasatinib.