Src Family Kinase Inhibitor Research Articles

Increased Src Family Kinase Activity Disrupts Excitatory Synaptic Transmission and Impairs Remote Fear Memory in Forebrain Shp2-Deficient Mice.

Src homolog domain-containing phosphatase 2 (Shp2) signals a variety of cellular and physiological functions including learning and memory. Dysregulation of ERK signaling is known to be responsible for the cognitive deficits associated with gain-of-function mutated Shp2 mimicking Noonan syndrome. However, here, we report that CaMKIIα-cre induced knockout (CaSKO) of Shp2 in hippocampal pyramidal neurons resulted in increased Src activity, upregulated phosphorylation of N-methyl-D-aspartate receptors (NMDARs) at Y1325 of GluN2A and at Y1472 of GluN2B, disrupted the balance of synaptic transmission, and impaired long-term potentiation and remote contextual fear memory. Administration of PP2, a specific Src family kinase inhibitor, reversed the tyrosine phosphorylation of NMDARs, restored basal synaptic transmission, and rescued the contextual fear memory deficit in CaSKO mice without altering the phospho-ERK level. Taken together, our results reveal a novel role of Shp2 in NMDAR-dependent synaptic function and fear memory via the Src signaling pathway rather than the ERK pathway, and suggest a complicated mechanism for Shp2-associated cognitive deficits.

LYN expression predicts the response to dasatinib in a subpopulation of lung adenocarcinoma patients.

Therapies targeting SRC family kinases (SFKs) have shown efficacy in treating non-small cell lung cancer (NSCLC). However, recent clinical trials have found that the SFK inhibitor dasatinib is ineffective in some patient cohorts. Regardless, dasatinib treatment may benefit some NSCLC patient subgroups. Here, we investigated whether expression of LYN, a member of the SFK family, is associated with patient survival, the efficacy of dasatinib, and/or NSCLC cell viability. LYN expression was associated with poor overall survival in a multivariate analysis, and this association was strongest in non-smoker female patients with adenocarcinoma (ADC). In lung ADC cells, LYN expression enhanced cell proliferation, migration, and invasion. Dasatinib inhibited LYN activity and decreased cell viability in LYN-positive ADC cell lines and xenografts. Additionally, we identified the SFKs SRC and YES as candidate dasatinib targets in LYN-negative ADC cell lines. Our findings suggest that LYN is a useful prognostic marker and a selective target of dasatinib therapy in the lung ADC subpopulation especially in female non-smokers with lung ADC.

Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis.

PurposeKX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo.Materials and MethodsThe antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects.ResultsKX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model.ConclusionKX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin

Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.

Regulation of Siglec-8-induced intracellular reactive oxygen species production and eosinophil cell death by Src family kinases

RationaleSiglec-8 is a surface receptor predominantly expressed on human eosinophils where its ligation induces reactive oxygen species (ROS) formation and cell death. Since Siglec-8 has intracellular tyrosine-based motifs, we hypothesized that Src family kinases (SFKs) are involved in ROS formation and cell death induced by Siglec-8 engagement. MethodsHuman peripheral blood eosinophils were purified and incubated with anti-Siglec-8 monoclonal antibodies (mAb, agonist), IL-5, and SFK pharmacological inhibitors. We focused on Siglec-8-induced cell death in short-term IL-5-activated cells leading to a regulated necrosis-type cell death. ROS production was determined by dihydrorhodamine (DHR) 123 labeling and flow cytometry, or by chemiluminescence using Amplex red. Activation of SFK was determined using phospholuminex and Western blotting. ResultsIn order to determine cellular localization of ROS production, we measured intra and extracellular ROS. While an ETosis stimulus (calcium ionophore A23187) led to extracellular ROS (ecROS) production, Siglec-8-engagement in short-term IL-5 activated cells led to intracellular ROS (icROS) accumulation. Consistently, inhibition of extracellular ROS by catalase inhibited ETosis, but not IL-5-primed Siglec-8-induced cell death. In order to determine signaling events for Siglec-8, we performed Western blotting and found SFK phosphorylation in lysates from eosinophils stimulated with anti-Siglec-8 mAb±IL-5. In order to identify which SFKs were involved, we used the phospholuminex assay and found increased levels of phosphorylated Fgr in the cytoplasmic fraction of cells co-stimulated with anti-Siglec-8 and IL-5 for 3 hours compared with cells stimulated with IL-5 alone. To test the involvement of SFKs in ROS production and cell death, we used SFK inhibitors PP2 and dasatinib, both of which completely inhibited eosinophil ROS production and cell death induced by anti-Siglec-8 and IL-5 co-stimulation. ConclusionSiglec-8 engagement in short-term IL-5-activated eosinophils causes icROS production and SKF phosphorylation, and both are essential in mediating Siglec-8-induced cell death.

Antitumor activity of 7RH, a discoidin domain receptor 1 inhibitor, alone or in combination with dasatinib exhibits antitumor effects in nasopharyngeal carcinoma cells.

Dysregulation of the discoidin domain receptors (DDRs) has been implicated in the development of numerous types of tumors, including head and neck cancer, and nasopharyngeal, breast, ovarian and esophageal carcinomas. Furthermore, agents that inhibit DDR1 activity are hypothesized to be useful for the treatment of nasopharyngeal carcinoma (NPC). The aim of the present study was to evaluate the effect of the DDR1 inhibitory (3-(2-(pyrazolo(1,5-a)pyrimidin-6-yl)-ethynyl)benzamide compound, 7RH, in NPC cells both in vitro and in vivo, and its effect when used in combination with dasatinib, a SRC family kinase (SFK) inhibitor. The effects of 7RH alone or in combination with dasatinib on cell viability were assessed using MTT assays and apoptosis was detected by flow cytometry. In addition, western blotting was performed to analyze the relative protein expression levels of cell cycle-associated genes in human NPC cell lines (CNE1, CNE2, HONE1 and SUNE1). Cell migration was also assessed using cell adhesion assays. Furthermore, tumor xenografts of CNE2 NPC cells were established in nude mice and the growth inhibitory effects of 7RH treatment alone or in combination with dasatinib were evaluated. Finally, knockdown of DDR1 protein expression was achieved by transfection of CNE2 cells with DDR1-specific small interfering RNA. Treatment with 7RH effectively suppressed the proliferation and induced the apoptosis of NPC cells. In addition, the Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT3) signaling pathway was downregulated by 7RH, whereas the activities of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways were upregulated in response to 7RH treatment. Furthermore, the expression levels of phosphorylated SRC were increased in NPC cells treated with 7RH; thus indicating that SRC exhibits a vital function in the resistance of NPC cells to 7RH via activation of the PI3K/AKT signaling pathway. The results of the present study indicate that DDR1 and SFK inhibition may present a potential therapeutic strategy for patients with NPC.

Peptide therapy in sepsis and inflammation: A novel strategy to suppress inflammation

Src family kinase (SFK) activation circumvents epidermal growth factor receptor (EGFR) targeting in head and neck squamous cell carcinoma (HNSCC); dual SFK-EGFR targeting could overcome cetuximab resistance.We conducted a Simon two-stage, phase II trial of the SFK inhibitor, dasatinib, and cetuximab in biomarker-unselected patients with cetuximab-resistant, recurrent/metastatic HNSCC. Pre- and post-treatment serum levels of interleukin-6 (IL6) were measured by ELISA. HNSCC cell lines were assessed for viability and effects of IL6 modulation following dasatinib-cetuximab treatment.In the first stage, 13 patients were evaluable for response: 7 had progressive and 6 had stable disease (SD). Enrollment was halted for futility, and biomarker analysis initiated. Low serum IL6 levels were associated with SD (raw p = 0.028, adjusted p = 0.14) and improved overall survival (p = 0.010). The IL6 classifier was validated in a separate trial of the same combination, but was unable to segregate survival risk in a clinical trial of cetuximab and bevacizumab suggesting serum IL6 may be specific for the dasatinib-cetuximab combination. Enhanced in vitro HNSCC cell death was observed with dasatinib-cetuximab versus single agent treatment; addition of IL6-containing media abrogated this effect.Clinical benefit and overall survival from the dasatinib-cetuximab combination were improved among patients with low serum IL6. Preclinical studies support IL6 as a modifier of dasatinib-cetuximab response. In the setting of clinical cetuximab resistance, serum IL6 is a candidate predictive marker specific for combined dasatinib-cetuximab. The trial was modified and redesigned as a biomarker-enriched Phase II study enrolling patients with undetectable IL6.

Targeting Src in endometriosis-associated ovarian cancer.

The SRC proto-oncogene is commonly overexpressed or activated during cancer development. Src family kinase inhibitors are approved for the treatment of certain leukemias, and are in clinical trials for the treatment of solid tumors. Src signaling is activated in endometriosis, a precursor of clear cell and endometrioid subtypes of epithelial ovarian cancers (OCs). We examined the expression of phosphorylated Src (Src-pY416) in 381 primary OC tissues. Thirty-six percent of OCs expressed Src-pY416. Src-pY416 expression was most common in endometriosis-associated OCs (EAOCs) (P=0.011), particularly in clear cell OCs where 58.5% of cases expressed Src-pY416. Src-pY416 expression was associated with shorter overall survival (log rank P=0.002). In vitro inhibition of Src signaling using 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) resulted in reduced anchorage-independent and -dependent growth, and in three-dimensional cell culture models PP2 disrupted aggregate formation in Src-pY416-positive but not in Src-pY416-negative cell lines. These data suggest that targeting active Src signaling could be a novel therapeutic opportunity for EAOCs, and support the further pre-clinical investigation of Src family kinase inhibitors for treating OCs expressing Src-pY416.

Regulation of paxillin-p130-PI3K-AKT signaling axis by Src and PTPRT impacts colon tumorigenesis.

Protein tyrosine phosphatase receptor T (PTPRT) is frequently mutated in a variety of human cancers including colorectal cancer. Here we report that PTPRT knockout increases the size of mouse colon tumors in the Apcmin+/− genetic background, suggesting that inactivation of PTPRT promotes tumor progression. We previously demonstrated that PTPRT dephosphorylates paxillin at tyrosine-Y88 residue. Consistently, phosphorylation of Y88 paxillin (pY88) is up-regulated in colon tumors derived from Apcmin+/− Ptprt−/− mice. An important downstream effector of pY88 paxillin is the oncogene Akt. Here, we show that pY88 paxillin impacts the Akt pathway by regulating the interaction between p130cas and the p85 regulatory subunit of PI3-Kinase. Additionally, while pY88 paxillin is a substrate of the tumor suppressor phosphatase PTPRT, the corresponding kinase has not been previously identified. In this study, we demonstrate that the oncogenic kinase Src directly phosphorylates paxillin at Y88. Moreover, colorectal cancer cells that express high levels of pY88 paxillin are sensitive to dasatinib treatment, suggesting that pY88 paxillin may serve as a predictive biomarker for Src family kinase inhibitors.

Abstract 177: Src family kinase Fyn promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer

Abstract FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PCa cells lines treated with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients. Citation Format: Karen Cavassani, Murali Gururajan, Margarit Sievert, Peng Duan, Michael Freeman, Gina Chia-Yi Chu, Neil Bhowmick, Leland Chung, Edward Posadas. Src family kinase Fyn promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 177.

Abstract CT060: Efficacy, safety and tolerability of dasatinib combined with afatinib: a phase I trial in patients with epidermal growth factor receptor mutant (EGFRm) advanced non-small-cell lung cancer (NSCLC) after acquired tyrosine kinase inhibitor (TKI) resistance

Abstract Background: Combining the Src family kinase inhibitor dasatinib and irreversible EGFR inhibitor afatinib results in enhanced antitumor activity in proliferation and apoptosis assays with EGFRm, TKI-resistant cell lines. Moreover, this combination results in higher tumor regression than either drug alone in PC9GR xenograft studies. We hypothesized that this drug combination would arrest progression in EGFRm NSCLC with acquired resistance. Therefore, we initiated a phase I open-label trial (NCT01999985) to evaluate dasatinib in combination with afatinib for patients (pts) with EGFRm advanced NSCLC who had progressed on previous EGFR TKIs. Methods: A phase I dose-escalation cohort with 3+3 design included patients (pts) with refractory NSCLC and ECOG performance status (PS) ? 1. Pts received continuous oral afatinib daily followed by addition of continuous oral dasatinib daily beginning on Day 8. After determination of maximum tolerated dose (MTD), a phase I cohort was expanded to include only pts with activating EGFR mutation who had acquired resistance to previous EGFR TKI. Safety, median progression-free survival (PFS), and tumor response were investigated. Plasma before and during treatment was collected for sequencing of circulating cell-free tumor DNA. Cox regression was performed. All statistical tests were 2-sided. Interim results are reported. Results: Of 31 pts screened, 25 were treated. Characteristics: 63% were female, median age was 66.8 years (range 53 - 81), and 72% were ECOG PS 1. Race: 75% Caucasian, 16% African-american. Median prior lines of therapy was 3. For EGFRm pts (n = 16), 56% had exon 19 deletion, 43% exon 21 mutation. In addition, 31% also had T790M detected in tumor prior to treatment. No significant interactions were observed with dasatinib and afatinib. Dose escalation halted at 40 mg afatinib and 100 mg dasatinib when 2 patients at this level experienced grade 2 or 3 diarrhea despite optimal supportive care. The most common drug-related adverse events of any grade from dose escalation included 63% diarrhea, 45% rash, 27% nausea, 18% oral mucositis, and fatigue. The MTD was 30 mg afatinib with 100 mg dasatinib. The PFS for EGFRm pts was 5.5 months (95% confidence interval 2.6 - 8.5). A trend toward more favorable PFS for T790M was observed (7.0 vs. 2.8 months, p = 0.06) in evaluable subjects. No confirmed partial or complete responses have been observed. Conclusions: Afatinib plus dasatinib was acceptably tolerated in pts with NSCLC in the escalation phase. Clinical benefit has been observed in the expansion phase, although without objective radiologic responses. Preliminary data support continued evaluation of afatinib 30 mg plus dasatinib 100 mg as the expansion phase dose in pts with EGFRm NSCLC with acquired TKI resistance. Citation Format: Ben Creelan, Jhanelle Gray, Diana Lima, Scott Antonia, Alberto Chiappori, Tawee Tanvetyanon, Rebecca DeVane, Charles Williams, Eric Haura. Efficacy, safety and tolerability of dasatinib combined with afatinib: a phase I trial in patients with epidermal growth factor receptor mutant (EGFRm) advanced non-small-cell lung cancer (NSCLC) after acquired tyrosine kinase inhibitor (TKI) resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT060.

Abstract 1127: KIT/D816V induces SRC-mediated tyrosine phosphorylation of MITF and altered transcription program in melanoma

Abstract The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KITD816V has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KITD816V induces tyrosine phosphorylation of the microphthalmia-associated transcription factor (MITF) through a triple protein complex formation between KIT, MITF and SRC family kinases. In turn, phosphorylated MITF activates target genes that are involved in melanoma proliferation, cell cycle progression, suppression of senescence, survival and invasion. By blocking the triple protein complex formation, thus preventing MITF phosphorylation, the cells become hypersensitive to SRC inhibitors. We have therefore delineated the mechanism behind the oncogenic effects of KITD816V in melanoma and provide a rationale for testing SRC family kinase inhibitors to treat KITD816V-transformed melanomas. Citation Format: Bengt Phung, Julhash U. Kazi, Alicia Lundby, Kristin Bergsteinsdottir, Jianmin Sun, Colin R. Goding, Göran Jönsson, Jesper V. Olsen, Eiríkur Steingrímsson, Lars Rönnstrand. KIT/D816V induces SRC-mediated tyrosine phosphorylation of MITF and altered transcription program in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1127.

Breast cancer cells evade paclitaxel-induced cell death by developing resistance to dasatinib.

Triple negative breast cancer (TNBC), which does not express the progesterone, estrogen, or HER2/neu receptor, is aggressive and difficult to treat. Paclitaxel, a tubulin stabilizing agent, is one of the most frequently prescribed anticancer agents for breast cancers, including TNBC. Residual disease that occurs due to resistance or partial resistance of cancer cells in a tumor against anticancer agents is the most important issue in oncology. In the present study, when MDA-MB-231 cells, a TNBC cell line, were treated with 30 µM paclitaxel, a slightly higher concentration than its GI50 value, for 6 days, a small number of cells with different morphologies survived. Among the surviving cells, small round cells were isolated, cloned, and named MDA-MB-231-JYJ cells. MDA-MB-231-JYJ cells were observed to be highly proliferative and tumorigenic. In addition, signal transduction molecules involved in proliferation, survival, malignancy, or stemness of cancer cells, such as c-Src, c-Met, Notch 1, c-Myc, Sox2, Oct3/4, Nanog, and E-cadherin were highly expressed or activated. While further study is required, MDA-MB-231-JYJ cells appear to have some of the characteristics of cancer precursor cells. Although MDA-MB-231-JYJ cells were isolated from the cells that survived in the continuous presence of paclitaxel, they were not resistant to paclitaxel but developed resistance to dasatinib, a Bcr-Abl and Src kinase family inhibitor. The activated state of Src and Notch 1, and the expression levels of c-Myc and cyclins in MDA-MB-231-JYJ cells were less affected than MDA-MB-231 cells by the treatment of dasatinib, which may explain the resistance of MDA-MB-231-JYJ cells to dasatinib. These results suggest that cancer cells that become resistant to dasatinib during the process of paclitaxel therapy in patients may appear, and caution is required in the design of clinical trials using these two agents.

Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis

Previous studies have demonstrated that cleaved high-molecular-weight kininogen (HKa) induces endothelial apoptosis and inhibits angiogenesis and have suggested that this occurs through inhibition of Src family kinases. This study assessed the role of tyrosine-protein kinase Lck (p56/Lck) in this pathway. We analyzed early events leading to apoptosis of human endothelial cells exposed to HKa. The role of p56/Lck was investigated using short interfering (si) RNA knockdown and lentivirus expression in assays of endothelial tube formation, sprouting of neovessels from murine aorta, and angiogenesis in Matrigel plugs. HKa stimulated expression and phosphorylation of p56/Lck. siRNA knockdown of p56/Lck promoted endothelial proliferation and blocked HKa-induced apoptosis and activation of p53, Bax, and Bak. Lentivirus expression of p56/Lck in endothelial cells induced apoptosis and blocked tube formation. Expression of p56/Lck in murine aortic rings blocked sprouting angiogenesis. Lentivirus expressing p56/Lck blocked angiogenesis in Matrigel plugs, while p56/Lck short hairpin RNA inhibited the antiangiogenic effect of HKa. Scrambled siRNAs and empty lentiviral vectors were used in all experiments. Apoptosis of proliferating endothelial cells and inhibition of angiogenesis by HKa requires p56/Lck. This suggests a novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.-Betapudi, V., Shukla, M., Alluri, R., Merkulov, S., McCrae, K. R. Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.

A phase 0 pharmacodynamic study of dasatinib in women with newly diagnosed endometrial cancer.

e23207Background: Dasatinib is a potent, oral inhibitor of Src family kinases, which are implicated in etiology and malignant behavior of various cancers. Direct measurement of dasatinib biologic e...

Lyn, a tyrosine kinase closely linked to the differentiation status of primary acute myeloid leukemia blasts, associates with negative regulation of all-trans retinoic acid (ATRA) and dihydroxyvitamin D3 (VD3)-induced HL-60 cells differentiation.

BackgroundLyn, an import member of Src family kinases (SFKs), is supposed to be implicated in acute myeloid leukemia (AML) pathogenesis and development by participation in AML differentiation, yet the details still remain incompletely understood. The expression status of Lyn and its correlation with multiple clinical parameters including cell differentiation degree, different cytogenetic risk classification, and the activity of myeloperoxidase (MPO) were thus investigated. To address the mechanisms underlying the involvement of Lyn in differentiation induction, the effects of dasatinib, an inhibitor for SFKs including Lyn, on the alterations of all-trans retinoic acid (ATRA)- or dihydroxyvitamin D3 (VD3)-induced differentiation, and c-Myc protein expression were investigated.MethodsPrimary AML blasts were obtained from 31 newly diagnosed AML patients with different French-American-British (FAB) subtypes. The expression of phosphorylated and total Lyn, c-Myc, and CD11b, CD11c and CD15 was analyzed by flow cytometry. The activation of Akt and Erk known to be involved in the regulation of c-Myc expression was investigated using western blotting.ResultsSignificant higher expression levels of total Lyn were observed in AML patients with favorable cytogenetics, higher MPO activity and FAB M2 subtype. A clear positive correlation between the expression levels of Lyn and differentiation status of primary AML blasts was observed. Dasatinib inhibited the expression of phosphorylated Lyn, and further enhanced the differentiation-inducing activity of ATRA and VD3 in HL-60 cells. Augmented downregulation of c-Myc protein expression was observed in the combination treatment with ATRA, VD3 and dasatinib compared to treatment with each reagent alone in HL-60 cells. The suppression of the activation of Akt and Erk was also observed concomitantly.ConclusionsThe expression level of total Lyn is closely linked to the differentiation status of AML blasts. The enhancement of differentiation-inducing activity of ATRA/VD3 by dasatinib suggested that Lyn was associated in the negative regulation of ATRA/VD3-induced HL-60 cells differentiation. The enhancement probably was attributed to the downregulation of c-Myc implicated with the suppression of the activation of Akt and Erk. These results provide novel insights into a possible combinational therapeutic approach by targeting Lyn for AML patients, and offer new possibilities for the combination therapy with VD3 and dasatinib.

Mechanisms and consequences of Siglec-8 internalization and toxic payload delivery in human eosinophils

Abstract Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell surface protein expressed selectively on human eosinophils, mast cells, and basophils that induces eosinophil apoptosis and inhibits mast cell mediator release. Hence, Siglec-8 is an ideal target for diseases involving these cells. However, the effective delivery of therapeutic agents to these cells through Siglec-8 requires an understanding of the dynamics of Siglec-8 surface expression, which have not been elucidated. We observed by flow cytometry that Siglec-8 endocytosis in eosinophils proceeds slowly and plateaus at 80% loss in response to antibody or polyvalent synthetic ligand. Surprisingly, based on sensitivity to pharmacological inhibition, Siglec-8 appears to be internalized in both a clathrin- and lipid raft-dependent manner. Actin cytoskeletal rearrangement and tyrosine kinase and PKC activity are necessary for endocytosis. Interestingly, Siglec-8 is shuttled to the cell surface in a manner dependent on actin rearrangement, microtubule polymerization, and the activities of tyrosine kinases, dynamin, and PI3K. Neither process is significantly affected by inhibitors of Src family kinases or PTPs. Internalized Siglec-8 localizes to the lysosome, and sialidase treatment of eosinophils enhances ligand binding, indicating the presence of masking sialylated cis ligands on the cell surface. Finally, conjugating the ribosome-inactivating protein saporin to an anti-Siglec-8 mAb enhanced cell death induction in unstimulated eosinophils. The dynamics of Siglec-8 surface expression therefore appear to be suitable for sustained targeting and may deliver drugs to effectively treat diseases involving these cell types, including malignancies.

Distinctive signalling in NK cells during cytotoxicity to the yeast Cryptococcus neoformans

Abstract Introduction Cryptococcus is a fungal pathogen that subverts the adaptive immune response. Fortunately, NK cells are capable of directly recognizing, triggering cytotoxicity and killing Cryptococcus. Although, NK cytotoxicity against tumor and virally infected targets are well studied, less is known about anti-fungal signaling. In this study, we investigated a novel NK cytotoxicity pathway involving Rac, Src family kinases (SFK), and integrins. Methods The involvement of Rac, SFK, integrin-linked kinase (ILK), and integrins were explored using small molecule inhibitors, siRNA knockdown, activated Rac pull down assays, and immunoblots. Results Our studies found that Cryptococcus activated both SFK and Rac signaling pathways. Small molecule inhibitors of SFK and Rac blocked Erk and PI3K activity. siRNA knockdown of Rac also inhibited PI3K activity, suggesting that Rac was an upstream non-canonical activator of PI3K. Moreover, individually Rac and SFK were not sufficient for activation of PI3K. Inhibition of Rac also resulted in a 90% loss of anti-cryptococcal cytotoxicity. Additionally, inhibitors of ILK prevented Rac activation and caused an 80% reduction in cryptococcal killing. Lastly, siRNA knockdown of beta-1 integrins caused an inhibition of ILK activation and 50% reduction in cryptococcal killing. Conclusion We found beta-1 integrins to be a fungal receptor and Rac an activator of PI3K. These findings build a signaling model of anti-fungal killing that is different from the canonical tumor killing model that involves beta-2 integrins and Rac downstream of PI3K. Our model provides various potential therapeutic targets that could enhance fungal clearance mediated by the innate immune system.

SRC Family Kinase Inhibition in Ewing Sarcoma Cells Induces p38 MAP Kinase-Mediated Cytotoxicity and Reduces Cell Migration.

Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, representing the second most common primary malignant bone tumor in children and adolescents. Although the development of a multimodal therapy, including both local control (surgery and/or radiation) and systemic multidrug chemotherapy, has determined a significant improvement in survival, patients with metastatic and recurrent disease still face a poor prognosis. Moreover, considering that ES primarily affects young patients, there are concerns about long-term adverse effects of the therapy. Therefore, more rational strategies, targeting specific molecular alterations underlying ES, are required. Recent studies suggest that SRC family kinases (SFKs), which are aberrantly activated in most cancer types, could represent key therapeutic targets also for ES. Here, we challenged ES cell lines with a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221), which was previously shown to be a valuable proapoptotic agent in other tumor types while not affecting normal cells. We observed that SI221 significantly reduced ES cell viability and proved to be more effective than the well-known SFK inhibitor PP2. SI221 was able to induce apoptosis in ES cells and also reduced ES cell clonogenic potential. Furthermore, SI221 was also able to reduce ES cell migration. At the molecular level, our data suggest that SFK inhibition through SI221 could reduce ES cell viability at least in part by hindering an SFK-NOTCH1 receptor-p38 mitogen-activated protein kinase (MAPK) axis. Overall, our study suggests a potential application of specific SFK inhibition in ES therapy. J. Cell. Physiol. 232: 129-135, 2017. © 2016 Wiley Periodicals, Inc.

Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology.

In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase Src as a host factor exploited by virulent M. tuberculosis for intracellular survival. We show that Src inhibition can effectively control tuberculosis in infected guinea pigs. Moreover, Src inhibition ameliorated TB-associated pathology in guinea pigs. Thus, Src inhibitors have strong potential to be developed as possible anti-TB drugs.