Src Family Tyrosine Kinase Inhibitor Research Articles

Abstract 3099: Hnd-01: a potent inhibitor of wild type mutant BTK

Abstract Dasatinib is an oral dual BCR/ABL and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Based on the in vitro and in vivo characterization studies of dasatanib, several metabolites were identified. Dasatinib and its metabolite 20 were the major circulating species identified which represented 25.5% and 12.5% of total radioactivity in human plasma respectively. Here, we report the identification of HND-01, a small molecule inhibitor of Bruton’s tyrosine kinase (BTK) which was achieved by modifying metabolite 20 of dasatinib. Bruton’s tyrosine kinase (BTK) is a non-receptor kinase and a key regulator of the B-cell receptor (BCR) signaling pathway. BCR is critical for proliferation and survival of leukemic cells in many B cell malignancies, including chronic lymphocytic leukemia (CLL). BTK is a key target for the development of small molecule inhibitors in B cell malignancies. There are several covalent BTK inhibitors including ibrutinib that have been approved. These covalent inhibitors bind with high affinity to Cys-481 in the active site of BTK. However, some patients develop acquired resistance to ibrutinib due to Cys-481 mutation in the BTK binding site. The acquired resistance to covalent inhibitors can be overcome with the design of new reversible BTK inhibitors. The reversible BTK inhibitors will bind to different sites other than the cysteine residue of BTK and inactivate BTK. These drugs would potentially be active in the presence or absence of the cysteine-to-serine mutation. HND-01, a novel and potent reversible BTK inhibitor inhibits wild type & C481S mutant BTK with IC50 values of 0.157 nM for WTBTK and 0.032 nM for C481SBTK. HND-01 inhibits K562 cell proliferation (IC50 0.183 nM) and it is more stable than dasatinib in human liver microsomes (in vitro clearance; 142 mL/min/kg for HND-01 vs 211 mL/min/kg for dasatinib). In an established human lymphoma efficacy model (TMD-8 model), HND-01 showed stronger tumor inhibition (80.6%) than Ibrutinib (58%). These data demonstrates that HND-01 is a BTK inhibitor that is more potent with potential desirable ADME properties than ibrutinib. HND-01 was filled for international patents, China patent application # 202080030953.6, US patent application #17/636,476, EU application # EP 20853834.8, and Canada application # CA 3148436. Citation Format: Jing Zhang, Ahmed A. Samatar, Yue Tan, Shiqing Pi, Zhigang Zhou, Yan Xu, Daihong Yang. Hnd-01: a potent inhibitor of wild type mutant BTK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3099.

Non-tumorigenic epithelial breast cells and ionizing radiation cooperate in the enhancement of mesenchymal traits in tumorigenic breast cancer cells

AimsRadioresistance and recurrences are crucial hindrances in cancer radiotherapy. Fractionated irradiation can elicit a mesenchymal phenotype in irradiated surviving cells and a deep connection exists between epithelial mesenchymal transition, radioresistance, and metastasis. The aim of this study was to analyze the effect of the secretoma of irradiated non-tumorigenic mammary epithelial cells on surviving irradiated breast tumor cells regarding the gain of mesenchymal traits and migratory ability. Main methodsMDA-MB-231 and MCF-7 breast cancer cells, irradiated or not, were incubated with conditioned media from MCF-10A non-tumorigenic epithelial breast cells, irradiated or not. After five days, we evaluated the expression and localization of epithelial and mesenchymal markers (by western blot and indirect immunofluorescence), cell migration (using transwells) and metalloproteinases activity (by zymography). We also assessed TGF-β1 content in conditioned media by immunoblot, and the effect of A83-01 (a selective inhibitor of TGF-β receptor I) and PP2 (a Src-family tyrosine kinase inhibitor) on nuclear Slug and cell migration. Key findingsConditioned media from MCF-10A cells caused phenotypic changes in breast tumor cells with attainment or enhancement of mesenchymal traits mediated at least in part by the activation of the TGF-β type I receptor and a signaling pathway involving Src activation/phosphorylation. The effects were more pronounced mostly in irradiated tumor cells treated with conditioned media from irradiated MCF-10A. SignificanceOur results suggest that non-tumorigenic epithelial mammary cells included in the irradiation field could affect the response to irradiation of post-surgery residual cancer cells enhancing EMT progression and thus modifying radiotherapy efficacy.

Histamine Produced by Gram-Negative Bacteria Impairs Neutrophil’s Antimicrobial Response by Engaging the Histamine 2 Receptor

We found that histamine (10−9 M) did not have any effect on the in vitro capture of Escherichia coli by neutrophils but accelerated its intracellular killing. In contrast, histamine (10−6 M) delayed the capture of Escherichia coli by neutrophils and reduced the amounts of pHrodo zymosan particles inside acidic mature phagosomes. Histamine acted through the H4R and the H2R, which are coupled to the Src family tyrosine kinases or the cAMP/protein kinase A pathway, respectively. The protein kinase A inhibitor H-89 abrogated the delay in bacterial capture induced by histamine (10−6 M) and the Src family tyrosine kinase inhibitor PP2 blocked histamine (10−9 M) induced acceleration of bacterial intracellular killing and tyrosine phosphorylation of proteins. To investigate the role of histamine in pathogenicity, we designed an Acinetobacter baumannii strain deficient in histamine production (hdc::TOPO). Galleria mellonella larvae inoculated with the wild-type Acinetobacter baumannii ATCC 17978 strain (1.1 × 105 CFU) died rapidly (100% death within 40 h) but not when inoculated with the Acinetobacter baumannii hdc::TOPO mutant (10% mortality). The concentration of histamine rose in the larval haemolymph upon inoculation of the wild type but not the Acinetobacter baumannii hdc::TOPO mutant, such concentration of histamine blocks the ability of hemocytes from Galleria mellonella to capture Candida albicans in vitro. Thus, bacteria-producing histamine, by maintaining high levels of histamine, may impair neutrophil phagocytosis by hijacking the H2R.

PP2 protects from keratin mutation-associated liver injury and filament disruption via SRC kinase inhibition in male but not female mice.

Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury. We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury. Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice. PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Ser/Thr phosphorylation of K8/K18, which is phenocopied by RAF kinase inhibitor vemurafenib. The PP2/vemurafenib-associated findings, and their unique mechanisms of action, further support the potential role of select kinase inhibition as therapeutic opportunities for keratin and other IF-associated human diseases.

Activated ephrinA3/EphA4 forward signaling induces retinal ganglion cell apoptosis in experimental glaucoma

Previous studies have demonstrated that EphA4 participates in neuronal injury, and there is a strong interaction between ephrinA3 and EphA4. In this study, we showed that in a rat chronic ocular hypertension (COH) experimental glaucoma model, expression of EphA4 and ephrinA3 proteins was increased in retinal cells, including retinal ganglion cells (RGCs) and Müller cells, which may result in ephrinA3/EphA4 forward signaling activation on RGCs, as evidenced by increased p-EphA4/EphA4 ratio. Intravitreal injection of ephrinA3-Fc, an activator of EphA4, mimicked the effect of COH on p-EphA4/EphA4 and induced an increase in TUNEL-positive signals in normal retinas, which was accompanied by dendritic spine retraction and thinner dendrites in RGCs. Furthermore, Intravitreal injection of ephrinA3-Fc increased the levels of phosphorylated src and GluA2 (p-src and p-GluA2). Co-immunoprecipitation assay demonstrated interactions between EphA4, p-src and GluA2. Intravitreal injection of ephrinA3-Fc reduced the expression of GluA2 proteins on the surface of normal retinal cells, which was prevented by intravitreal injection of PP2, an inhibitor of src-family tyrosine kinases. Pre-injection of PP2 or the Ca2+-permeable GluA2-lacking AMPA receptor inhibitor Naspm significantly and partially reduced the number of TUNEL-positive RGCs in the ephrinA3-Fc-injected and COH retinas. Our results suggest that activated ephrinA3/EphA4 forward signaling promoted GluA2 endocytosis, then resulted in dendritic spine retraction of RGCs, thus contributing to RGC apoptosis in COH rats. Attenuation of the strength of ephrinA/EphA signaling in an appropriate manner may be an effective way for preventing the loss of RGCs in glaucoma.

Src Family Kinases as Therapeutic Targets in Advanced Solid Tumors: What We Have Learned So Far

Src is the prototypal member of Src Family tyrosine Kinases (SFKs), a large non-receptor kinase class that controls multiple signaling pathways in animal cells. SFKs activation is necessary for the mitogenic signal from many growth factors, but also for the acquisition of migratory and invasive phenotype. Indeed, oncogenic activation of SFKs has been demonstrated to play an important role in solid cancers; promoting tumor growth and formation of distant metastases. Several drugs targeting SFKs have been developed and tested in preclinical models and many of them have successfully reached clinical use in hematologic cancers. Although in solid tumors SFKs inhibitors have consistently confirmed their ability in blocking cancer cell progression in several experimental models; their utilization in clinical trials has unveiled unexpected complications against an effective utilization in patients. In this review, we summarize basic molecular mechanisms involving SFKs in cancer spreading and metastasization; and discuss preclinical and clinical data highlighting the main challenges for their future application as therapeutic targets in solid cancer progression

Antidepressant amitriptyline-induced matrix metalloproteinase-9 activation is mediated by Src family tyrosine kinase, which leads to glial cell line-derived neurotrophic factor mRNA expression in rat astroglial cells.

BackgroundAstrocytes have been implicated in the pathophysiology of mood disorders and in the mechanism of the pharmacological effects of antidepressant drugs by the production of neurotrophic/growth factors. Previous studies have identified astrocyte‐expressed Gαi/o‐coupled lysophosphatidic acid receptor 1 (LPAR1), as being involved in antidepressant‐induced production of glial cell line‐derived neurotrophic factor (GDNF) and matrix metalloproteinase‐9 (MMP‐9) activation, an important step in the production of GNDF. However, the precise mechanism of MMP‐9 activation by antidepressants has yet to be identified, in particular the intracellular signaling pathway between LPAR1/Gαi/o and MMP‐9.Methods and ResultsTreatment of rat C6 astroglial cells (C6 cells) with amitriptyline increased Src family tyrosine kinase phosphorylation in a time and concentration‐dependent manner. Amitriptyline‐induced GDNF mRNA expression was blocked by Src family tyrosine kinase inhibitors. In addition, inhibiting Src family tyrosine kinase blocked amitriptyline‐induced zymographic MMP‐9 activation in C6 cells. The amitriptyline‐induced zymographic MMP‐9 activity was completely blocked by selective inhibition of Gαi/o protein and LPAR1. Furthermore, the amitriptyline‐induced Src family tyrosine kinase phosphorylation was blocked by LPAR1, but not MMP‐9 inhibition, indicating that Src family tyrosine kinase involvement is downstream of LPAR1.ConclusionsThe current findings suggest that the pharmacological effect of antidepressant such as amitriptyline is mediated through an intracellular signaling pathway via the LPAR1/Gαi/o/Src family tyrosine kinase, which leads to MMP‐9 activation and GDNF production.

Abstract 1820: Overcoming drug resistance in NSCLC with SRC inhibitor

Abstract Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Overcoming drug resistance in NSCLC with SRC inhibitor [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 1820.

PDGF-induced migration of synthetic vascular smooth muscle cells through c-Src-activated L-type Ca2+ channels with full-length CaV1.2 C-terminus.

In atherosclerosis, vascular smooth muscle cells (VSMC) migrate from the media toward the intima of the arteries in response to cytokines, such as platelet-derived growth factor (PDGF). However, molecular mechanism underlying the PDGF-induced migration of VSMCs remains unclear. The migration of rat aorta-derived synthetic VSMCs, A7r5, in response to PDGF was potently inhibited by a CaV1.2 channel inhibitor, nifedipine, and a Src family tyrosine kinase (SFK)/Abl inhibitor, bosutinib, in a less-than-additive manner. PDGF significantly increased CaV1.2 channel currents without altering CaV1.2 protein expression levels in A7r5 cells. This reaction was inhibited by C-terminal Src kinase, a selective inhibitor of SFKs. In contractile VSMCs, the C-terminus of CaV1.2 is proteolytically cleaved into proximal and distal C-termini (PCT and DCT, respectively). Clipped DCT is noncovalently reassociated with PCT to autoinhibit the channel activity. Conversely, in synthetic A7r5 cells, full-length CaV1.2 (CaV1.2FL) is expressed much more abundantly than truncated CaV1.2. In a heterologous expression system, c-Src activated CaV1.2 channels composed of CaV1.2FL but not truncated CaV1.2 (CaV1.2Δ1763) or CaV1.2Δ1763 plus clipped DCT. Further, c-Src enhanced the coupling efficiency between the voltage-sensing domain and activation gate of CaV1.2FL channels by phosphorylating Tyr1709 and Tyr1758 in PCT. Compared with CaV1.2Δ1763, c-Src could more efficiently bind to and phosphorylate CaV1.2FL irrespective of the presence or absence of clipped DCT. Therefore, in atherosclerotic lesions, phenotypic switching of VSMCs may facilitate pro-migratory effects of PDGF on VSMCs by suppressing posttranslational CaV1.2 modifications.

Role of the Fyn-PKCδ signaling in SE-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy

Status epilepticus (SE) induces neuroinflammation and epileptogenesis, but the mechanisms are not yet fully delineated. The Fyn, a non-receptor Src family tyrosine kinase (SFK), and its immediate downstream target, PKCδ are emerging as potential mediators of neuroinflammation. In order to first determine the role of Fyn kinase signaling in SE, we tested the efficacy of a SFK inhibitor, saracatinib (25mg/kg, oral) in C57BL/6J mouse kainate model of acute seizures. Saracatinib pretreatment dampened SE severity and completely prevented mortality. We further utilized fyn−/− and fyn+/+ mice (wildtype control for the fyn−/− mice on same genetic background), and the rat kainate model, treated with saracatinib post-SE, to validate the role of Fyn/SFK in SE and epileptogenesis. We observed significant reduction in SE severity, epileptiform spikes, and electrographic non-convulsive seizures in fyn−/− mice when compared to fyn+/+ mice. Interestingly, significant reductions in phosphorylated pSrc-416 and PKCδ (pPKCδ-507) and naive PKCδ were observed in fyn−/− mice as compared to fyn+/+ mice suggesting that PKCδ signaling is a downstream mediator of Fyn in SE and epileptogenesis. Notably, fyn−/− mice also showed a reduction in key proinflammatory mediators TNF-α, IL-1β, and iNOS mRNA expression; serum IL-6 and IL-12 levels; and nitro-oxidative stress markers such as 4-HNE, gp91phox, and 3-NT in the hippocampus. Immunohistochemistry revealed a significant increase in reactive microgliosis and neurodegeneration in the hippocampus and hilus of dentate gyrus in fyn+/+ mice in contrast to fyn−/− mice. Interestingly, we did not observe upregulation of Fyn in pyramidal neurons of the hippocampus during post-SE in fyn+/+ mice, but it was upregulated in hilar neurons of the dentate gyrus when compared to naïve control. In reactive microglia, both Fyn and PKCδ were persistently upregulated during post-SE suggesting that Fyn-PKCδ may drive neuroinflammation during epileptogenesis. Since disabling the Fyn kinase prior to SE, either by treating with saracatinib or fyn gene knockout, suppressed seizures and the subsequent epileptogenic events, we further tested whether Fyn/SFK inhibition during post-SE modifies epileptogenesis. Telemetry-implanted, SE-induced, rats were treated with saracatinib and continuously monitored for a month. At 2h post-diazepam, the saracatinib (25mg/kg) or the vehicle was administered orally and repeated twice daily for first three days followed by a single dose/day for the next four days. The saracatinib post-treatment prevented epileptogenesis in >50% of the rats and significantly reduced spontaneous seizures and epileptiform spikes in the rest (one animal did not respond) when compared to the vehicle treated group, which had >24 seizures in a month. Collectively, the findings suggest that Fyn/SFK is a potential mediator of epileptogenesis and a therapeutic target to prevent/treat seizures and epileptogenesis.

Dasatinib related pericardial effusion requiring pericardial drainage

Introduction Dasatinib is an oral Bcr-Abl and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukaemia (ALL). Its common side effects include myelosuppression, oedema, diarrhea and nausea. It has also been associated with the formation of pleural and pericardial effusions. As a result, Dasatinib is to be avoided in patients with pre-existing effusions or predisposition to respiratory or cardiovascular disease. Case description A fit 62-year-old pilot with no relevant medical history was diagnosed with CML in 2014, and commenced on Dasatinib therapy (100mg OD). A subsequent trans-thoracic echocardiogram (TTE) revealed normal ventricles and cardiac valves. There was however a mild to moderate global pericardial effusion, without haemodynamic compromise. This was regularly monitored with TTEs and remained stable until May 2016, where it measured 2.1cm posteriorly around the LV and 1.0 cm around the RV. Due restrictions imposed by the Civil Aviation Authority in the UK, the patient was referred for pericardial window procedure, prior to being considered fit for flying. Conclusions Dasatinib is known to cause pleural and pericardial effusions. This has been reported in patients without any predisposing factors.(1) The link with pericardial effusions has been proven with robust statistical analysis.(2) No specific mechanism has been proposed but an immune mediated reaction or off target inhibition of growth factors may be involved.(3) Management includes dose interruption or reduction, and/or treatment with steroids.(3) Our case report re-enforces that Dasatinib is an important cause of pericardial effusion and TTE is the modality of choice for follow-up. Pericardial window and drainage may be needed in patients where this prohibits them from undertaking employment. Take-home message Dasatinib related pericardial effusions are a documented side effect of therapy. One should be vigilant in monitoring patients on the drug as effusions may progress over time and require intervention. TTE is the monitoring modality of choice. As far as we are aware this is the first case report for surgical intervention in a patient with Dasatinib induced pericardial effusion.

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2.

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT-PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild-type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto-ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G2 /M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G2 /M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.

Systematic drug sensitivity testing reveals synergistic growth inhibition by dasatinib or mTOR inhibitors with paclitaxel in ovarian granulosa cell tumor cells

ObjectiveResistance to standard chemotherapy poses a major clinical problem in the treatment of ovarian cancer patients. Adult-type granulosa cell tumor (AGCT) is a unique ovarian cancer subtype for which efficient treatment options are lacking in advanced disease. To this end, systematic drug response and transcriptomics profiling were performed to uncover new therapy options for AGCTs. MethodsThe responses of three primary and four recurrent AGCTs to 230 anticancer compounds were screened in vitro using a systematic drug sensitivity and resistance testing (DSRT) platform, coupled with mRNA sequencing. The responses of the AGCTs were compared with those of human granulosa luteal cells and bone marrow mononuclear cells. ResultsPatient-derived AGCT cells showed selective sensitivity to the Src family tyrosine kinase inhibitor dasatinib. A combination of either dasatinib or an mTOR-inhibitor everolimus with paclitaxel resulted in synergistic inhibition of AGCT cell viability. The key kinase targets of dasatinib and members of the mTOR pathway were constantly expressed at mRNA and protein levels, indicating multikinase signal addictions in the AGCT cells. Transcriptomic characterization of the tumors revealed no known oncogenic mutations, suggesting that the drug sensitivity of AGCTs was rather conveyed by selective target expression. ConclusionsWe used a systematic functional approach to reveal novel treatment options for a unique gynecological cancer. The selective synergy found between taxanes and dasatinib or mTOR inhibitors warrants further clinical investigations of these combinations in relapsed or aggressive AGCTs and demonstrate that high-throughput drug screening and molecular profiling can provide an effective approach to uncover new therapy options.

168 THE DEVELOPMENTAL POTENTIAL OF PARTHENOGENETIC EMBRYOS IS AFFECTED BY PROLACTIN DURING THE PROLONGED CULTURE OF BOVINE CUMULUS-ENCLOSED OOCYTES

The technique of somatic cell nuclear transfer includes a delayed activation of reconstituted oocytes (4–6 h after IVM). The prolonged culture of mammalian oocytes is known to be associated with aging of the ova leading to a decline in their quality and developmental capacity. The aim of the present research was to study effects of 2 closely related hormones, prolactin (PRL) and growth hormone (GH), during the prolonged culture of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-enclosed oocytes (CEO) were cultured for 16 h in TCM 199 containing 10% FCS, 10 μg mL−1 of porcine FSH, and 10 μg mL−1 of ovine LH. After 16 h of maturation, CEO or denuded oocytes were transferred to the fresh medium consisting of TCM 199 supplemented with 10% FCS and cultured for 12 h in the absence (Control) or in presence of 50 ng mL−1 of bovine PRL or 10 ng mL−1 of recombinant bovine GH or protein kinase inhibitors. The following inhibitors were used: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 10 mM), (2) triciribine (an inhibitor of Akt kinase, 25 μM), and (3) calphostin C (a protein kinase C inhibitor, 0.5 μM). After the prolonged culture for 12 h, oocytes were activated by sequential treatment with ionomycin (5 μM for 5 min) immediately followed by 6-dimethylaminopurine (2 mM for 4 h). Activated oocytes were cultured in CR1aa medium until Day 5 postactivation and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 7. The cleavage and blastocyst rates were assessed at Day 2 and 7, respectively. The data from 4 to 6 replicates (106–169 oocytes per treatment) were analysed by ANOVA. After the prolonged culture of CEO, the cleavage rate did not differ between the control and the hormone-treated groups, varying from 64.3 to 70.1%. Meanwhile, the blastocyst yield in the control group (12.5 ± 1.6%) was lower than in the PRL group (21.5 ± 2.4%, P < 0.05), but was similar to that in the GH group (13.9 ± 1.8%). Furthermore, the developmental capacity of cultured denuded oocytes was unaffected by both GH and PRL. The enhancing effect of PRL on the yield of parthenogenetic blastocysts derived from CEO was also observed in the presence of PP2 (26.8 ± 2.4 v. 15.7 ± 2.0%; P < 0.01). By contrast, calphostin C abolished this effect of PRL, although it did not affect the developmental potential of CEO in the control medium. At the same time triciribine reduced the blastocyst rate both in the control and PRL-treated groups (from 16.5 ± 1.8 to 8.1 ± 0.8% and from 24.3 ± 1.6 to 16.3 ± 2.4%, respectively; P < 0.05). Our data indicate that PRL can support the developmental potential of parthenogenetic embryos by affecting bovine oocytes during the prolonged culture, with the effect being mediated by cumulus cells and achieved via activation of protein kinase C. This research was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 15-08-99473).

Src family tyrosine kinase inhibitors suppress Nav1.1 expression in cultured rat spiral ganglion neurons.

Src family kinases regulate neuronal voltage-gated Na(+) channels, which generate action potentials. The mechanisms of action, however, remain poorly understood. The aim of the present study was to further elucidate the effects of Src family kinases on Nav1.1 mRNA and protein expression in spiral ganglion neurons. Immunofluorescence staining techniques detected Nav1.1 expression in the spiral ganglion neurons. Additionally, quantitative PCR and western blot techniques were used to analyze Nav1.1 mRNA and protein expression, respectively, in spiral ganglion neurons following exposure to Src family kinase inhibitors PP2 (1 and 10 μM) and SU6656 (0.1 and 1 μM) for different lengths of time (6 and 24 h). In the spiral ganglion neurons, Nav1.1 protein expression was detected in the somas and axons. The Src family kinase inhibitors PP2 and SU6665 significantly decreased Nav1.1 mRNA and protein expression (p < 0.05), respectively, in the spiral ganglion neurons, and changes in expression were not dependent on time or dose (p > 0.05).

Combination of Dasatinib with Conventional Chemotherapy Is Associated with a High Response Rate in High Risk Acute Myeloid Leukemia (AML)

Background: High risk AML patients (pts) have poor outcome with low complete remission (CR) rate and long term survival despite intensive chemotherapy.Tyrosine kinases play an important role in AML pathogenesis. Pre-clinical studies performed in our center (Blood. 2013 122:1900) have shown that Src family tyrosine kinases (SFK) including Lyn, Hck and Fgr are abnormally activated in AML compared to normal hematopoietic stem cell and progenitor cells. Other studies have shown that the c-Kit receptor is over-activated in a subset of AML pts and contributes to abnormal leukemia cell growth. We further show that the small molecule SFK and c-Kit inhibitor dasatinib reduces proliferation and survival of AML stem and progenitor cells. Importantly, dasatinib enhances the sensitivity of AML stem and progenitor cells to chemotherapeutic agents by inhibiting Akt signaling, increasing mdm2 phosphorylation and enhancing p53 activity in AML cells. Based on this data we conducted a phase I/II clinical study of the combination of dasatinib with conventional cytarabine-idarubicin ("7+3")-based induction chemotherapy in high-risk AML.Methods: Between September 20013 and July 2015, 18 adult AML pts were enrolled in the study. Eligibility criteria were high-risk AML, age >18 years, and suitability for intensive therapy. High risk AML was defined by one of the following criteria: older age (> 60 yrs), poor-risk cytogenetic and molecular abnormalities (ELN criteria), secondary disease (AML evolving from myelodysplasia or myeloproliferative neoplasm), therapy related (t-AML), or relapsed/refractory.This Phase I study used a 3 + 3 dose escalation design for dasatinib while keeping a fixed dose of cytarabine and idarubicin (cytarabine 200 mg/m2 CIV days 1-7, idarubicin 12 mg/m2 IV days 1-3). Dasatinib was started at a dose of 70 mg (dose level 1; DL-1) days 1-7, and escalated to 100 mg orally days 1-7 (DL 1). Pts who failed to achieve CR received second re-induction with the same regimen. Pts who achieved CR received consolidation high dose cytarabine and/or allogeneic stem cell transplantation (SCT) based on donor availability.Results: Of the 18 pts enrolled on the study, 7 pts (39%) had secondary AML, 5 pts (28%) relapsed AML, 2 pts (11%) t-AML, and 4 (22%) were newly diagnosed older AML pts (one with complex karyotype, one with trisomy 8 and one cytogenetically normal with FTL3-ITD). The median age of all pts was 62 yrs (range 27-73). Of the 18 pts, 13 pts are currently evaluable for response; of which 10 (77%) achieved CR/Cri. Of the 5 non-evaluable pts, one pt withdrew from study after 2 days of therapy, one pt was taken off study after 3 days due to cytarabine neurotoxicity, one pt died of intracranial hemorrhage before the day 14 bone marrow biopsy, one pt had therapy interrupted secondary to pneumonia and sepsis, and one is still receiving therapy. None of the pts required more than one induction to achieve CR. Of the 10 pts who achieved CR/Cri, 8 patients went on to receive allogeneic SCT. As expected, the most commonly reported grade 3 and 4 adverse events (AE) were anemia 50%, thrombocytopenia 44%, neutropenia 38%, and fatigue 27%. The most common grade 1-2 AE were GI toxicities 61% and rash 33%.Correlative studies performed on blood samples obtained from pts on day 3 after initiation of treatment (n=9) demonstrated significant decrease in of SRC activity (as indicated by reduction in phospho-SRC levels on immunoblotting, 0.52±0.11 of control, p=0.01), and increased expression of p53-target genes as evaluated by Q-RT-PCT [including Puma (16.2±6.9 fold, p=0.015), P21 (4.9±1.1 fold, p=0.004), DR5 (3.4±0.9, p=0.004), Bax (3.7±0.9 fold, p=0.001) and HDM2 (2.1±0.5, p=0.02)].Conclusion: Combination of dasatinib with conventional "7+3" induction chemotherapy is feasible in high-risk AML and leads to higher CR rate compared to historical data without increase in toxicity rate, allowing more pts to receive allogeneic transplantation. Correlative laboratory studies are consistent with pre-clinical studies suggesting that this combination is associated with significant inhibition of SRC activity and enhanced activation of p53-target genes. DisclosuresDos Santos:Amgen: Employment. Stein:Amgen: Speakers Bureau. Chen:Seattle Genetics: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Merck: Consultancy, Research Funding; genentech: Consultancy, Speakers Bureau. Khaled:Sequenom: Research Funding.

Prolactin and growth hormone affect metaphase-II chromosomes in aging oocytes via cumulus cells using similar signaling pathways.

General senescence of the adult organism is closely connected with reproductive one. Meanwhile, the age-related reduction in the female fertility is primarily associated with a decline in the gamete quality. Molecular and cellular changes in oocytes of old mammalian females are very similar to those occurring during aging of matured ova of their young counterparts, suggesting similarities in underlying mechanisms. The aim of the present work was to study actions of two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on age-associated modifications of metaphase-II (M-II) chromosomes in bovine oocytes using a model of the prolonged culture. We analyzed: (1) effects of PRL and GH on abnormal changes in the chromosome morphology in aging matured oocytes and the role of cumulus cells in these effects and (2) signaling pathways involved in the hormone actions. During the prolonged culture of oocytes, a gradual rise in the frequency of destructive modifications of M-II chromosomes was revealed. In the case of cumulus-enclosed oocytes (CEOs), PRL and GH exerted dose-dependent biphasic effects on the frequency of these modifications. Both PRL (50 ng/ml) and GH (10 ng/ml) decelerated the abnormal chromosome changes in CEOs, but did not affect the chromosome configuration in denuded oocytes. Concurrently, the presence of PRL and GH receptors in cumulus cells surrounding matured oocytes was demonstrated. Attenuating effects of both hormones on the chromosome modifications in aging CEOs were abolished by PP2 (an inhibitor of Src-family tyrosine kinases), triciribine (an inhibitor of Akt kinase), and calphostin C (a protein kinase C inhibitor). Our findings indicate that PRL and GH can exert the similar decelerating action on age-associated alterations in the M-II chromosome morphology in bovine ova, which is mediated by cumulus cells and may be related to activation of Src-family tyrosine kinases as well as Akt- and protein kinase C-dependent signal pathways.

Abstract 170: Mechanism of Cardiotoxicity of Tyrosine Kinase Inhibitors

Aim: It is becoming evident that many of the signaling elements driving cancer cell division, for which kinase inhibitors (KIs) are targeted, are the same as those necessary for cardiomyocyte viability. Adverse cardiac events have been reported for a number of KIs. Aim of this study was to identify KIs that induce toxicity to cardiomyocytes and to elucidate the central molecular mechanisms mediating the toxicity. Methods: 288 anti-cancer agents (123 are KIs) were screened for their ability to induce cardiotoxicity in cultured primary cardiomyocytes. Cell viability assay was done by measuring the ATP levels in cardiomyocytes after exposure of cells to a 3-log concentration range of each compound for 24 hours. Toxicity data was combined with kinase profiling data to identify protein kinases mediating the toxicity of KIs. In parallel, the molecular mechanism mediating the cardiomyocyte toxicity of dasatinib, a second generation Bcr-Abl and Src family tyrosine kinase inhibitor, was investigated. The role of Src kinase in regulation of cardiomyocyte viability was analyzed by siRNA-mediated Src knockdown. Overexpression of wild type Src and dasatinib- resistant Src in cardiomyocytes was performed to investigate the role of Src in regulating the cardiomyocyte toxicity of dasatinib. Western blotting was used to investigate for downstream signaling targets of Src. Results: Of the KIs, 70 compounds decreased the cardiomyocyte viability by 10-80 %. The kinase profiling data showed that IGF1R, MEK/ERK pathway, PI3K and Src kinases are the key kinases regulating cardiomyocyte viability. Dasatinib treatment dose-dependently increased cardiomyocyte death. Depletion of Src by siRNA also reduced cardiomyocyte viability. Dasatinib treatment attenuated FGF induced ERK phosphorylation. Overexpression of dasatinib-resistant mutant of Src, but not wild type Src, protected the cardiomyocyte from dasatinib toxicity and rescued the FGF-induced ERK phosphorylation. Conclusions: Cardiomyocyte toxicity of KIs can be attributed to inhibition of IGF1R, MEK/ERK pathway, PI3Ks and Src family kinases. Cardiomyocyte toxicity of dasatinib is mediated by Src kinase inhibition which in turn attenuates MEK/ERK pathway signalling.

Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis

Microgliosis is a major hallmark of Alzheimer’s disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD.

Effects of activation of Src-family tyrosine kinases on human LECs apoptosis and EMT under the high glucose environment

Background Diabetic cataract is one of the major ocular complications in diabetes mellitus, including cortical cataract, nuclear cataract, subcapsular cataract and mixed cataract, and different cataractogenesis may be associated with lens epithelial cells (LECs). Subcapsular cataract is one of diabetic cataracts.Studying the biological behavior of LECs in subcapsular cataract is crucial for prevention and treatment. Objective This study was to investigate the effects of Src-family tyrosine kinases (SFKs) on apoptosis and epithelial-mesenchymal transition (EMT) of human LECs cultured by high glucose. Methods Human LECs (HLE-B3) were cultured for 24 hours with DMEM containing 5.5 mmol/L glucose (normal control group), DMEM containing 35.5 mmol/L glucose (high glucose group) and DMEM containing 35.5 mmol/L glucose + 10 μmol/L PP1, a specific inhibitor of SFKs (PP1 group). In 3, 6, 12 and 24 hours after culture, the apoptosis of human LECs was detected by flow cytometry assay; morphological change of human LECs was observed under the inverted microscope, and the expressions of the markers of EMT, E-cadherin and α-smooth muscle actin (α-SMA), in the cells were detected by immnofluorescence staining.In addition, the alternations of p-Src418 (active c-Src), bcl-xl, survivin, caspase-3, E-cadherin and α-SMA proteins were assayed by Western blot analysis. Results An elevated expression level of p-Src418 was found in LECs in the high glucose group and peaked 6 hours after cultured.The expressions of p-Src418 (grey levels) were 0.125±0.036 in the high glucose group, and which was significantly higher than 0.042±0.011 in the normal control group and 0.035±0.018 in the PP1 group, respectively (both at P 0.05). The apoptotic rates of human LECs were(6.433±2.084)%, (10.333±2.610)% and (8.033±2.967)% in the PP1 group, which were higher than (3.233±1.320)%, (3.533±1.159)%, (5.733±0.230)% in the high glucose group and (3.133±1.170)%, (2.833±0.751)%, (3.333±1.201)% in the normal control group (all at P 0.05). In 6 hours and 12 hours after cell culture, the expression levels of bcl-xl and survivin (grey values) in human LECs were significantly declined, but the expression of caspase-3 was increased in the PP1 group compared with the the high glucose group and the normal control group (all at P<0.05). The LECs showed slender in shape 24 hours after culture in the high glucose group, but the cell shape was close to the normal in the PP1 group.Western blot and immunofluorescence assay revealed that the expression of E-cadherin in human LECs was significantly reduced and that of α-SMA was significantly increased 6 hours after culture in the high glucose group compared with the PP1 group and the normal control group (all at P<0.05). Conclusions High glucose activates c-Src kinase of LECs in high glucose environment and therefore induces EMT and inhibits apoptosis.However, PP1 impedes the biological process of EMT and apoptosis of LECs to maintain the epithelial characteristics even under the stress of high glucose. Key words: Glucose; Human; Epithelial cell, Lens; Apoptosis; Src-family tyrosine kinases; Epithelial-mesenchymal transition; Signaling pathway