Src Family Of Nonreceptor Tyrosine Kinases Research Articles

Enhanced Fyn-tau and NR2B-PSD95 interactions in epileptic foci in experimental models and human epilepsy

Abstract Epilepsy and Alzheimer’s Disease (AD) share some common pathologies such as neurodegeneration, seizures, and impaired cognition. However, the molecular mechanisms of these changes are still largely unknown. Fyn, a Src-family non-receptor tyrosine kinase (SFK), and its interaction with tau in mediating brain pathology in epilepsy and AD can be a potential therapeutic target for disease modification. Although Fyn and tau pathology occurs in both AD and epilepsy, the dynamics of Fyn-tau and PSD95-NR2B interactions affected by seizures and their impact on brain pathology in epilepsy have not been investigated. In this study, we demonstrate a significant increase of Fyn-tau interactions following seizure induction by kainate in both acute and chronic rodent models, and in human epilepsy. In the early phase of epileptogenesis, we show increased Fyn/tau/NR2B/PSD95/neuronal nitric oxide synthase (nNOS) complexes after status epilepticus and a post-synaptic increase of phosphorylated tau (pY18 and AT8), Fyn (pSFK-Y416), and NMDAR (pNR2B-Y1472), and nNOS. Hippocampal Proximity Ligation Assay and co-immunoprecipitation revealed a sustained increase of Fyn-tau and NR2B-PSD95 complexes/binding in rat chronic epilepsy at three months post-status epilepticus. Enhanced Fyn-tau complexes strongly correlated with the frequency of spontaneously recurring convulsive seizures and epileptiform spikes in the chronic epilepsy model. In human epileptic brains, we also identified increased Fyn-tau and NR2B-PSD95 complexes, tau phosphorylation (pY18 and AT8), and Fyn activation (pSFK-Y416), implying the translational and therapeutic potential of these molecular interactions. In tau knockout mice and in rats treated with a Fyn/SFK inhibitor saracatinib, we found a significant reduction of phosphorylated Fyn, tau (AT8 in saracatinib-treated), NR2B, and nNOS, and their interactions (Fyn-tau and NR2B-PSD95 in saracatinib-treated group; NR2B-PSD95 in tau knockout group). The reduction of Fyn-tau and NR2B-PSD95 interactions in the saracatinib-treated group, in contrast to the vehicle-treated group, correlated with the modification in seizure progression in the rat chronic epilepsy model. These findings from animal models and human epilepsy provide evidence for the role of Fyn-tau and NR2B-PSD95 interactions in seizure-induced brain pathology and suggest that blocking such interactions could modify the progression of epilepsy.

Membrane lipid rafts are required for AMPA receptor tyrosine phosphorylation.

Membrane lipid rafts are sphingolipids and cholesterol-enriched membrane microdomains, which form a center for the interaction or assembly of palmitoylated signaling molecules, including Src family non-receptor type protein tyrosine kinases. Lipid rafts abundantly exist in neurons and function in the maintenance of synapses. Excitatory synaptic strength is largely controlled by the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the mammalian brain. AMPA receptor endocytosis from the synaptic surface is regulated by phosphorylation of the GluA2 subunit at tyrosine 876 by Src family kinases. Here, I revealed that tyrosine phosphorylated GluA2 is concentrated in the lipid rafts fraction. Furthermore, stimulation-induced upregulation of GluA2 tyrosine phosphorylation is disrupted by the treatment of neurons with a cholesterol-depleting compound, filipin III. These results indicate the importance of lipid rafts as enzymatic reactive sites for AMPA receptor tyrosine phosphorylation and subsequent AMPA receptor internalization from the synaptic surface.

Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID‐19 patients (Treon et al., Blood, 2020). Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases (Ashleigh et al., Oncotarget,2015). Previous report shows that binding of curcumin to active conformation of DYRK2 at sub nanomolar range makes it an excellent DYRK‐2 inhibitor(Banerjee et al., PNAS,2018), but curcumin's fate is tainted by its instability in the cellular environment and its nonspecific binding nature(Kathryn et al., J. Med. Chem.,2017). Besides, the planar symmetry makes curcumin less druggable toward asymmetric binding pockets such as kinases (Knight et al., Chem. Biol. 2005). small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity (Hanson et al., Chembiol., 2019). Here, we show that functionalization of the 4‐arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor which binds specifically to the inactive conformation of Hck, similar to type‐II kinase inhibitors that are less promiscuous.Moreover, the lead compound showed no inhibitory effect on two other kinases (DYRK2 and Abl) which can be explained by difference in p‐loop dynamics between Abl and src family kinases. We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signalling pathway in triple‐negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodelling its target affinity and cellular stability.

Discovery of selective irreversible inhibitors of B-Lymphoid tyrosine kinase (BLK)

B-lymphoid tyrosine kinase (BLK), a member of the SRC family nonreceptor tyrosine kinase, is involved in the B-cell receptor (BCR) signaling pathway and B cell development and function. Dysregulation of BLK is associated with autoimmune diseases and cancer. However, there is an absence of good tool compounds for BLK, and the molecular mechanisms by which BLK mediates physiological and pathological processes are poorly understood. Herein, we present the discovery of a novel series of selective and irreversible inhibitors of BLK with nanomolar potency against BLK in biochemical and cellular assays. Compound 25 demonstrated potent antiproliferative activities against several B cell lymphoma cell lines. These compounds constitute the first series of selective inhibitors developed for BLK and could help expedite the exploration of BLK functions.

Sublethal doxorubicin promotes migration and invasion of breast cancer cells: role of Src Family non-receptor tyrosine kinases

BackgroundDoxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed.MethodsEffects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox.ResultsThe results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK—including Fyn, Yes, and Src—partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells.ConclusionsOverall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.

Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.

Fgr contributes to hemorrhage-induced thalamic pain by activating NF-κB/ERK1/2 pathways.

Thalamic pain, a type of central poststroke pain, frequently occurs following ischemia/hemorrhage in the thalamus. Current treatment of this disorder is often ineffective, at least in part due to largely unknown mechanisms that underlie thalamic pain genesis. Here, we report that hemorrhage caused by microinjection of type IV collagenase or autologous whole blood into unilateral ventral posterior lateral nucleus and ventral posterior medial nucleus of the thalamus increased the expression of Fgr, a member of the Src family nonreceptor tyrosine kinases, at both mRNA and protein levels in thalamic microglia. Pharmacological inhibition or genetic knockdown of thalamic Fgr attenuated the hemorrhage-induced thalamic injury on the ipsilateral side and the development and maintenance of mechanical, heat, and cold pain hypersensitivities on the contralateral side. Mechanistically, the increased Fgr participated in hemorrhage-induced microglial activation and subsequent production of TNF-α likely through activation of both NF-κB and ERK1/2 pathways in thalamic microglia. Our findings suggest that Fgr is a key player in thalamic pain and a potential target for the therapeutic management of this disorder.

Fyn depletion ameliorates tauP301L-induced neuropathology

The Src family non-receptor tyrosine kinase Fyn has been implicated in neurodegeneration of Alzheimer’s disease through interaction with amyloid β (Aβ). However, the role of Fyn in the pathogenesis of primary tauopathies such as FTDP-17, where Aβ plaques are absent, is poorly understood. In the current study, we used AAV2/8 vectors to deliver tauP301L to the brains of WT and Fyn KO mice, generating somatic transgenic tauopathy models with the presence or absence of Fyn. Although both genotypes developed tau pathology, Fyn KO developed fewer neurofibrillary tangles on Bielschowsky and Thioflavin S stained sections and showed lower levels of phosphorylated tau. In addition, tauP301L-induced behavior abnormalities and depletion of synaptic proteins were not observed in the Fyn KO model. Our work provides evidence for Fyn being a critical protein in the disease pathogenesis of FTDP-17.

Lonidamine-ethyl ester-mediated remodelling of the Sertoli cell cytoskeleton induces phosphorylation of plakoglobin and promotes its interaction with α-catenin at the blood-testis barrier.

Several compounds affect male fertility by disrupting the adhesion of germ cells to Sertoli cells, which results in the release of undeveloped germ cells into the seminiferous tubule lumen that are incapable of fertilising the ovum. Indazole carboxylic acids are one class of compounds exhibiting such effects and they have been investigated as non-hormonal contraceptives for potential human use. The aims of this study were to investigate the effects of lonidamine-ethyl ester, an indazole carboxylic acid, on spermatogenesis and cell junctions, in particular, desmosomes. We found two doses of lonidamine-ethyl ester at 50mg kg-1 to disrupt Sertoli-germ cell adhesion. By light and fluorescent microscopy, pronounced changes were observed in the distribution of actin microfilaments and intermediate filaments, as well as in the localisation of plakoglobin, a protein with structural and signalling roles at the desmosome and adherens junction at the blood-testis barrier. Furthermore, immunoblotting and immunoprecipitation experiments using testis lysates revealed a significant upregulation (P<0.01) of plakoglobin and Tyr-phosphorylated plakoglobin. Co-immunoprecipitation experiments showed an increase in the interaction between plakoglobin and fyn proto-oncogene, an Src family non-receptor tyrosine kinase, after treatment, as well as an increase in the interaction between plakoglobin and α-catenin. Taken collectively, these data indicate that a disruption of Sertoli cell and spermatocyte-spermatid adhesion in the seminiferous epithelium by lonidamine-ethyl ester results in the phosphorylation of plakoglobin, thereby promoting its interaction with α-catenin at the blood-testis barrier.

Abstract 2590: KD019: Blood brain barrier penetrant HER2/neu, Src, and EGFR inhibitor

Abstract KD019 is an orally bioavailable small molecule inhibitor of molecular drivers of cancer growth and progression, including HER2/neu, Src family nonreceptor tyrosine kinases (Src) and EGFR. This profile of activity underlies the robust activity of KD019 in animal models of cancer. Although alternative agents targeting these tyrosine kinase receptors are available, only KD019 targets all of these pathways without the need for a combination strategy. A more important shortcoming of alternative agents is a lack of blood brain barrier (BBB) penetration (Brain/Plasma concentration ratio&amp;lt;&amp;lt;1), significantly limiting their ability to target oncogenic molecular pathways in intracranial tumors. The BBB permeability of KD019 was heretofore untested. In the present work, KD019 is demonstrated to achieve concentrations in the brain equivalent to that in blood utilizing quantitative whole body autoradiography (QWBA). Lister Hooded partially pigmented rats were administered a single dose of 14C labeled KD019 and tissue radioactivity was evaluated utilizing phosphor-storage imaging plates and a Fuji FLA-5100 fluorescent image analyzing system (Quotient Bioresearch, Rushden, UK). Brain/blood radioactivity ratio was approximately 1 at 6-24 hours after dosing indicating significant brain penetration of KD019. BBB penetrance was further supported by a study performed in CD1 mice utilizing LC/MS/MS to determine tissue KD019 concentrations following a single oral dose of 100 mg free base/kg unlabeled drug (Pharmaron, Beijing, China). Brain/plasma KD019 ratios were 2.3-4.4 from 1-24 hours after dosing with brain concentrations of 2137-8253 ng/g tissue, compared to 0.03-0.07 for the EGFR/HER2 inhibitor lapatinib with brain concentrations of 0-286 ng/g tissue. To demonstrate that the penetration of KD019 into the brain parenchyma would translate into anticancer effects, KD019 efficacy was tested in a GL261 orthotopic syngeneic model of glioma (Molecular Imaging, Michigan, USA). GL261-luc2 luciferase expressing cells were implanted intracranially (2 mm right lateral and 1 mm anterior from Bregma, 2-3 mm down from burr hole) into C57BL/6 albino mice. KD019 dosed orally once daily at 75 mg base/kg on Days 8-12 and 15-19 after intracranial implantation significantly reduced the intracranial tumor growth as evaluated by bioluminescence, and extended median survival time by 20%. In conclusion, KD019 is a BBB penetrant TKI with an important and novel profile of kinase activity that, based on the data presented, will be advanced further in nonclinical and clinical experiments aiming at benefiting patients with intracranial tumors, a significant unmet clinical need. KD019 is currently being tested in combination with trastuzumab in patients with HER2-positive breast cancer and brain metastases. Citation Format: James R. Tonra, Masha Poyurovsky, Kevin G. Liu, Jeegar Patel, Nishta Rao, Robert Tilton, John L. Ryan, Mark S. Berger, Larry Witte, Ji-In Kim, Samuel D. Waksal. KD019: Blood brain barrier penetrant HER2/neu, Src, and EGFR inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2015-2590

Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

Hck inhibitors as potential therapeutic agents in cancer and HIV infection.

Hematopoietic cell kinase (Hck) is a member of the Src-family of non-receptor tyrosine kinases, which plays many roles in signalling pathways involved in the regulation of cell processes. Hck is expressed in cells of hematopoietic origin, specifically myelomonocytic cells and B lymphocytes. It participates in phagocytosis, adhesion, migration, regulation of protrusion formation on cell membrane, lysosome exocytosis, podosome formation and actin polymerization. More importantly from a medicinal chemistry point of view, high levels of Hck are involved in chronic myeloid leukemia and other hematologic tumors. Furthermore, Hck activity has been associated with virus infections including HIV-1. In particular, Hck is activated by the HIV-1 accessory protein Nef, a multifunctional HIV-1 protein that accelerates progression to AIDS and enhances the infectivity of progeny viruses. Nef binding to Hck leads to kinase activation which is important in AIDS pathogenesis. For these reasons, Hck represents a potentially good therapeutic target for the treatment of both specific cancers and HIV infection. This article summarizes Hck biological activities connected with malignancies and HIV infection, many of which have been only recently reported, and presents an overview of the compounds endowed with Hck inhibitory activity, especially focusing on the medicinal chemistry aspect.

Hematopoietic Cell Kinase (HCK) Is Involved in Myelodysplastic Syndromes and Acute Leukemia Pathophysiology By Modulating PI3K Pathway and Enhancing Erythroid Differentiation

Introduction: Src family non-receptor tyrosine kinase regulates diverse cellular responses mainly by activating PI3K signaling, an important pathway that contributes to many cancers pathology. HCK, a Src family member, is restricted expressed in hematopoietic cells; however, high levels of HCK have been reported in solid tumors and hematologic neoplasms and have been associated with a worse prognosis. Identification of unbalanced pathways and upregulated proteins and a better knowledge of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) pathogenesis could help identify potential druggable targets and new prognostic biomarkers. The clinical and biological role of HCK in MDS and AML pathophysiology has not yet been elucidated and may be a novel therapeutic approach.Aims: To evaluate HCK expression in hematopoietic stem cells (HSC/CD34+) and in total bone marrow (BM) cells from healthy donors and MDS and AML patients. We further attempted to analyze the HCK knockdown and drug inhibition on differentiation, proliferation, apoptosis and migration in HSC/CD34+ and in a panel of human leukemia cell lines (KG1a, U937, HL60, P39).Materials and Methods: A total of 34 healthy donors and 139 patients at diagnosis (MDS=75 [low-risk=46, high-risk=29], AML with Myelodysplastic related changes (AML-MRC)=21 and de novo AML=43), were included in the study. HCK gene and protein expression was analyzed by qPCR and Western blot. Results are expressed as median (minimum–maximum) after appropriate statistical analysis. For functional analysis, HCK was inhibited with specific lentiviral vector system in HSC/CD34+ (isolated from umbilical cord blood units and BM), KG1a, U937, HL60 and P39 cells. Apoptosis was evaluated by Annexin-V/PI, cell growth by CellTiter assay, migration by Transwell, CXCR4 expression and actin polymerization by flow cytometry. We also verified the effects of a HCK specific inhibitor, ASN05260065, kindly provided by ASINEX and Dr Maurizzio Bottas.Results: HCK mRNA were significantly increased in HSC/CD34+ from patients with MDS (4.87 [0.03-14.75]; P=.003) and AML (8.36 [0.70-24.47]; P=.003) compared to healthy donors (1.19 [0.06-3.02]). When patients were stratified according to WHO classification, HCK expression was significantly higher in low-risk MDS compared to high-risk and AML-MRC (8.10 [2.30–14.75] vs 3.22 [0.03–6.33] and 1.02 [0.70–2.78], P<.001). Similar results were found in BM cells: HCK transcripts were significantly increased in MDS compared to healthy donors, and in low-risk compared to high-risk MDS (P<.0001). Interestingly, 5 of 5 MDS patients showed a significant decrease in HCK expression after disease progression. Also, a negative correlation was observed between BM blast percentage and HCK expression in MDS patients. In HSC/CD34+ cells, BFU-E colony number was significantly lower after HCK inhibition. HCK depletion reduced cell death in HSC/CD34+, P39 and KG1a cells and diminished proliferation in KG1a and U937 cells. Moreover, HCK knockdown decreased the phosphorylation of AKT, mTOR (PI3K pathway members) and ERK and treatment of HCK silenced cells with PI3K inhibitors had no synergic effect. Confirming the HCK lentivirus silencing results, HCK specific inhibitor treatment downregulated PI3K and ERK signaling. In addition, HCK inhibition significantly reduced migration towards CXCL12 chemoattractive stimuli by regulating CXCR4 expression and F-actin polymerization. Attempts have been made to transduct CD34+ from low-risk MDS patients however more than 80% cells were apoptotic and impossible to perform further assays.Conclusions:HCK is overexpressed in HSC/CD34+ and BM cells from MDS, mainly in low-risk MDS, and de novo AML. During MDS progression, HCK expression reduction correlates with increasing blast percentage and could indicate less dependence on microenvironment. HCK inhibition reduced BFU-E colony formation and our previous results showed that HCK expression increases during erythroid differentiation of non-transduced low-risk MDS CD34+ cells. Thus, HCK seems to play a role as a downstream effector in erythropoietin (Epo) signaling which could explain the higher HCK expression in low-risk MDS. Besides, HCK is an upstream regulator of PI3K and MAPK pathway that enhances cell apoptosis, survival and proliferation. Our study described new insights to the role of HCK in Epo/PI3K signaling and in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1827: Inhibition of Src, HER2, and EGFR by the multi-kinase inhibitor KD019 overcomes trastuzumab resistance in breast cancer

Abstract KD019 (previously EXEL-7647) is an orally bioavailable spectrum selective small molecule kinase inhibitor of Src family nonreceptor tyrosine kinases (Src) as well as HER2, EGFR, and VEGFR2/KDR. Because of its complement of targets KD019 is able to disrupt multiple components of tumor pathology and, in earlier studies, KD019 demonstrated robust activity in multiple in vivo tumor models. In recent years Src has emerged as a critical convergent node downstream of multiple signaling cascades initiated by transmembrane receptor tyrosine kinases (RTKs), most notably HER2, EGFR and IGF-1R. Crosstalk between Src and RTKs facilitates RTK-dependent tumor growth and, in patients with HER2-amplified (HER2+) breast cancer, where Src is upregulated, may confer resistance to anti-HER2 therapies. Based on the target specificity of KD019, including Src, we hypothesized that KD019 may prove to be an effective treatment in overcoming trastuzumab resistance in HER2+ breast cancer. Here we present data showing that KD019 effectively inhibited growth of several trastuzumab resistant cell lines with IC50 of 0.3 uM in vitro. Not only did KD019 potently inhibit HER2 and EGFR phosphorylation in these cells, but it also efficiently inhibited the phosphorylation of c-Met through receptor hetero-oligomerization mechanism, thus downregulating another pathway by which tumors may escape HER2 targeted therapies. Further, we demonstrate that once daily dosing of KD019 strongly inhibited tumor growth in xenograft models of trastuzumab resistance and that Src activity was inhibited in KD019 treated tumors even 24 hrs after drug administration. The significance of inhibiting the specific combination of targets is underscored by the fact that KD019 was 10 fold more potent when compared to saracatinib (a selective Src inhibitor) in inhibiting growth of trastuzumab resistant cells, which are also insensitive to blockade of individual RTKs. Together, these results provide a rational for clinical investigation of KD019 in patients whose tumors are resistant to anti-HER2 therapies. Citation Format: Rigen Mo, Nishta Rao, James Tonra, Samuel Waksal, Masha Poyurovsky. Inhibition of Src, HER2, and EGFR by the multi-kinase inhibitor KD019 overcomes trastuzumab resistance in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1827. doi:10.1158/1538-7445.AM2014-1827

Regulation of Src and Csk Nonreceptor Tyrosine Kinases in the Filasterean Ministeria vibrans

The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2–kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals.

Abstract B84: Activated c-Src enhances hypoxia in human glioblastoma multiforme and promotes tumor angiogenesis

Abstract Exhibiting substantial vascularization and propensity to infiltrate surrounding brain tissue, glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Even with aggressive intervention afflicted individuals uniformly receive poor prognosis. Growth factor receptor tyrosine kinases (RTKs) are frequently amplified, overexpressed, and constitutively activated in malignant gliomas, and because targeting these molecules has produced promising results in the treatment of several human neoplasms, RTKs are emerging as increasingly viable targets in effective treatment of GBM. Functional ambiguity between normal and constitutively activated RTK signalling compounds the challenges of identifying suitable targets, however. Likewise, the functions of many reported downstream signalling molecules, namely Src family non-receptor tyrosine kinases (SFKs), remain cryptic. Our lab has previously developed a clinically relevant in vivo model that reliably maintains the genomic profiles of corresponding patient tumors, and employing this model, we are able to accurately investigate mechanisms of RTK signalling in human GBM. Ongoing studies suggest that tumor overexpression and activation of the epidermal growth factor receptor (EGFR) promotes the invasive phenotype in human GBM potentially via c-Src dependent signal transduction. In fact, we have observed SFK activation downstream of EGFR. To further investigate this, we implanted GBM patient tumor cells from two individuals with distinct EGFR protein expression, tumor phenotype, and c-Src activity into the brains of nude rats. Minimally expressing EGFR tumor cells from a patient displaying an angiogenic in vivo phenotype were transduced to stably overexpress activated c-Src. Likewise, EGFR-overexpressing tumor cells from a patient displaying an invasive, non angiogenic in vivo phenotype were transduced to stably overexpress kinase-inactive-dominant-negative c-Src. To our surprise, compared with corresponding xenograft control tumors, xenograft tumors overexpressing constitutively activated c-Src showed substantially increased contrast enhancement on MRI, enhanced histological features of angiogenesis, and revealed marked increases in molecular markers of tumor hypoxia and angiogenesis on western blot. Meanwhile, compared with corresponding xenograft control tumors, xenograft tumors overexpressing kinase-inactive-dominant-negative c-Src did not show increased contrast enhancement on MRI, showed no histological evidence of angiogenesis, and maintained a non-angiogenic molecular profile on western blot. We hereby report that activation of the SFK c-Src enhances tumor hypoxia, promotes the aggressive angiogenic tumor phenotype, and is subsequently not likely a primary downstream SFK substrate of overexpressed EGFR in human GBM. Overexpression of wild type EGFR may, instead, signal through a different SFK to promote the invasive phenotype. These results suggest that, more so than invasion, c-Src activation may be a valuable diagnostic and prognostic indicator of malignant progression. Furthermore, these results indicate that c-Src activation may be a strategic target in the therapeutic intervention of human malignant glioma. Citation Format: Eskil Eskilsson, Krishna M. Talasila, Gro V. Røsland, Lina Leiss, Narve Brekkå, Per Øystein Sakariassen, Per Øyvind Enger, Rolf Bjerkvig, Hrvoje Miletic. Activated c-Src enhances hypoxia in human glioblastoma multiforme and promotes tumor angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B84.

Heterogeneous Nuclear Ribonucleoprotein (hnRNP) F Is a Novel Component of Oligodendroglial RNA Transport Granules Contributing to Regulation of Myelin Basic Protein (MBP) Synthesis

Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3'UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis.

Src family protein tyrosine kinases modulate L-type calcium current in human atrial myocytes

In the heart, L-type voltage dependent calcium channels (L-VDCC) provide Ca 2+ for the activation of contractile apparatus. The best described pathway for L-type Ca 2+ current (I Ca,L) modulation is the phosphorylation of calcium channels by cAMP-dependent protein kinase A (PKA), the activity of which is predominantly regulated in opposite manner by β-adrenergic (β-ARs) and muscarinic receptors. The role of other kinases is controversial and often depends on tissues and species used in the studies. In different studies the inhibitors of tyrosine kinases have been shown either to stimulate or inhibit, or even have a biphasic effect on I Ca,L. Moreover, there is no clear picture about the route of activation and the site of action of cardiac Src family nonreceptor tyrosine kinases (Src-nPTKs). In the present study we used PP1, a selective inhibitor of Src-nPTKs, alone and together with different activators of I Ca,L, and demonstrated that in human atrial myocytes (HAMs): (i) Src-nPTKs are activated concomitantly with activation of cAMP-signaling cascade; (ii) Src-nPTKs attenuate PKA-dependent stimulation of I Ca,L by inhibiting PKA activity; (iii) Gα s are not involved in the direct activation of Src-nPTKs. In this way, Src-nPTKs may provide a protecting mechanism against myocardial overload under conditions of increased sympathetic activity.

Simultaneous exposure of transformed cells to SRC family inhibitors and CHK1 inhibitors causes cell death

The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01 (7-hydroxystaurosporine), AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor -induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK -/- transformed fibroblasts and suppressed by over expression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor -induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133+ cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular sensitivity to anticancer treatments. These findings may provide pivotal insights in the context of fractionated radiation-based therapeutic interventions in brain cancer.