Growth Arrest-specific Gene Research Articles

Protective effects of estrogen against vascular calcification via estrogen receptor α-dependent growth arrest-specific gene 6 transactivation

Vascular calcification is one of the major complications of cardiovascular disease and is an independent risk factor for myocardial infarction and cardiac death. Postmenopausal women have a higher prevalence of vascular calcification compared with premenopausal women, suggesting protective effects of estrogen (E2). However, the underlying mechanisms of its beneficial effects remain unclear. In the present study, we examined the inhibitory effects of E2 on vascular smooth muscle cell (VSMC) calcification, and found that growth arrest-specific gene 6 (Gas6), a crucial molecule in vascular calcification, is transactivated by estrogen receptor α (ERα) in response to E2. In human aortic smooth muscle cells, physiological levels of E2 inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner. This inhibitory effect was significantly abolished by MPP, an ERα-selective antagonist, and ERα siRNA, but not by PHTPP, an ERβ-selective antagonist, and ERβ siRNA, implicating an ERα-dependent action. Apoptosis, an essential process for Pi-induced VSMC calcification, was inhibited by E2 in a concentration-dependent manner and further, MPP abolished this inhibition. Mechanistically, E2 restored the inhibited expression of Gas6 and phospho-Akt in Pi-induced apoptosis through ERα. Furthermore, E2 significantly activated Gas6 transcription, and MPP abrogated this E2-dependent Gas6 transactivation. E2-BSA failed to activate Gas6 transcription and to inhibit Ca deposition in VSMC, suggesting beneficial actions of genomic signaling by E2/nuclear ERα. Taken together, these results indicate that E2 exerts inhibitory effects on VSMC apoptosis and calcification through ERα-mediated Gas6 transactivation. These findings indicate a potential therapeutic strategy for the prevention of vascular calcification, especially in postmenopausal women.

Growth Arrest Specific 2 (GAS2) is a Critical Mediator of Germ Cell Cyst Breakdown and Folliculogenesis in Mice.

In the mouse ovary, the primordial follicle pool is established through a diverse array of signaling pathways and tissue remodeling events. Growth arrest specific gene two (GAS2) is a highly conserved cytoskeleton-associated protein whose in vivo function remains unclear. In Drosophila, loss of the GAS2 homolog, Pigs, results in infertility. We demonstrate herein that, in the mouse ovary, GAS2 is expressed in the stromal cells surrounding the oocyte cysts on 16.5 dpc, and in stromal cells surrounding growing follicles during juvenile and adult life. We have generated genetically engineered mice with inactivated Gas2. Gas2 homozygous mutant mice are viable but have severely impaired fertility in females, in which oocyte cyst breakdown is disrupted and follicle growth is impaired, with significantly reduced numbers of large antral follicles and corpora lutea. In these mutant mice, the organization of the basal lamina surrounding developing follicles is severely defective at multiple stages of folliculogenesis. We also found that Notch signaling activity was altered in ovaries from Gas2 null mice around the time of birth and during follicular development later in life. These results indicate that GAS2 is a critical and novel regulator of tissue remodeling in the ovary during oocyte cyst breakdown and folliculogenesis.

Effect of vitamin K in bone metabolism and vascular calcification: A review of mechanisms of action and evidences

ABSTRACTOsteoporosis is a public health concern associated with an increased risk of bone fractures and vascular calcification. Vitamin K presents unique benefits on these issues, although understudied. The two main forms of vitamin K are phylloquinone (vitamin K1) and menaquinone (vitamin K2). In this study, it was especially investigated the action of vitamin K2 in bones and vessels. Vitamin K2 has shown to stimulate bone formation by promoting osteoblast differentiation and carboxylation of osteocalcin, and increasing alkaline phosphatase, insulin-like growth factor-1, growth differentiation factor-15, and stanniocalcin 2 levels. Furthermore, vitamin K2 reduces the pro-apoptotic proteins Fas and Bax in osteoblasts, and decreases osteoclast differentiation by increasing osteoprotegerin and reducing the receptor activator of nuclear factor kappa-B ligand. In blood vessels, vitamin K2 reduces the formation of hydroxyapatite, through the carboxylation of matrix Gla protein and Gla rich protein, inhibits the apoptosis of vascular smooth muscle cells, by increasing growth arrest-specific gene 6, and reduces the transdifferentiation of vascular smooth muscle cells to osteoblasts. The commonly used dosage of vitamin K2 in human studies is 45 mg/day and its application can be an interesting strategy in benefitting bone and vascular health, especially to osteoporotic post-menopausal women.

Growth arrest specific gene 7 is associated with schizophrenia and regulates neuronal migration and morphogenesis.

BackgroundSchizophrenia is a highly heritable chronic mental disorder with significant abnormalities in brain function. The neurodevelopmental hypothesis proposes that schizophrenia originates in the prenatal period due to impairments in neuronal developmental processes such as migration and arborization, leading to abnormal brain maturation. Previous studies have identified multiple promising candidate genes that drive functions in neurodevelopment and are associated with schizophrenia. However, the molecular mechanisms of how they exert effects on the pathophysiology of schizophrenia remain largely unknown.ResultsIn our research, we identified growth arrest specific gene 7 (GAS7) as a schizophrenia risk gene in two independent Han Chinese populations using a two-stage association study. Functional experiments were done to further explore the underlying mechanisms of the role of Gas7 in cortical development. In vitro, we discovered that Gas7 contributed to neurite outgrowth through the F-BAR domain. In vivo, overexpression of Gas7 arrested neuronal migration by increasing leading process branching, while suppression of Gas7 could inhibit neuronal migration by lengthening leading processes. Through a series of behavioral tests, we also found that Gas7-deficient mice showed sensorimotor gating deficits.ConclusionsOur results demonstrate GAS7 as a susceptibility gene for schizophrenia. Gas7 might participate in the pathogenesis of schizophrenia by regulating neurite outgrowth and neuronal migration through its C-terminal F-BAR domain. The impaired pre-pulse inhibition (PPI) of Gas7-deficient mice might mirror the disease-related behavior in schizophrenia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0238-y) contains supplementary material, which is available to authorized users.

MiR-184 Regulates Proliferation in Nucleus Pulposus Cells by Targeting GAS1

The precise mechanism of nucleus pulposus proliferation in the degeneration of the intervertebral disk pathogenesis remains to be implicated. MicroRNAs (MiRNAs) are a class of 18-22 nucleotides, which are small, noncoding RNAs that inhibit protein translation by binding to the 3'-UTR of target gene. Recent studies have shown that miRNAs play a crucial role in various cell biologies such as cell proliferation, invasion, migration, and cell cycle. However, the role of miR-184 in nucleus pulposus proliferation is still unknown. qRT-PCR was performed to measure the expression of miR-184. CCK-8 assay, qRT-PCR, and Western blot were used to measure the functional role of miR-184 in nucleus pulposus (NP) cells. Western blot and Luciferase assays were done to find the miR-184 target gene. We demonstrated that expression of miR-184 was upregulated in degenerative NP tissues compared with that in the control NP tissues, and the expression of miR-184 was positively correlated with disk degeneration grade. We identified Growth Arrest Specific Gene 1 (GAS1) as a direct target gene of miR-184 in NP cells, and ectopic expression of miR-184 promoted NP cells proliferation. In addition, we found that GAS1 expression was downregulated in degenerative NP tissues compared with that in the control NP tissues and the GAS1 expression was inversely correlated with the grade of disk degeneration. Moreover, we demonstrated that miR-184 overexpression could induce AKT phosphorylation and ectopic expression of GAS1 decreased the miR-184 overexpressing NP cells proliferation. These results demonstrated that miR-184 and the GAS1/Akt pathway may be a potential therapeutic target for intervertebral disc degeneration.

Genetic variations in the growth arrest-specific 6 protein gene in patients with acute coronary syndrome

Growth arrest-specific gene 6 (GAS6) encodes a vitamin K-dependent protein that regulates inflammation, angiogenesis, and atherosclerotic plaque formation. The level of GAS6 expression is associated with plaque stability and stroke. The role of GAS6 in cardiovascular disease, particularly in acute coronary syndrome (ACS), was explored. The study investigated the role of the single nucleotide polymorphism (c.834 + 7G >A) of GAS6 in ACS. The genotype frequencies for GG, AG, and AA, respectively, in patients with ACS were 57.1% (16/28), 28.6% (8/28), and 14.3% (4/28) and were 20% (4/20), 40% (8/20), and 40% (8/20) in the control group. The AA genotype and A allele were less frequent in patients with ACS than in controls (P < 0.05). Our study indicates that the AA genotype and A allele of the GAS6 gene relate to ACS and may have a protective role against ACS.

The roles of TAM receptor tyrosine kinases in the mammalian testis and immunoprivileged sites.

Three members of a receptor tyrosine kinase family, including Tyro3, Axl, and Mer, are collectively called as TAM receptors. TAM receptors have two common ligands, namely, growth arrest specific gene 6 (Gas6) and protein S (ProS). The TAM-Gas6/ProS system is essential for phagocytic removal of apoptotic cells, and plays critical roles in regulating immune response. Genetic studies have shown that TAM receptors are essential regulators of the tissue homeostasis in immunoprivileged sites, including the testis, retina and brain. The mechanisms by which the TAM-Gas6/ProS system regulates the tissue homeostasis in immunoprivileged sites are emerging. The roles of the TAM-Gas6/ProS system in regulating the immune privilege were intensively investigated in the mouse testis, and several studies were performed in the eye and brain. This review summarizes our current understanding of TAM signaling in the testis and other immunoprivileged tissues, as well as highlights topics that are worthy of further investigation.

Gas6-Tyro3 signaling is required for Schwann cell myelination and possible remyelination.

Myelin plays important roles in vertebrates, ensuring the rapid propagation of action potentials and the long-term integrity of axons, but the molecular mechanisms of myelin formation remain poorly understood. Recent studies have demonstrated that myelination is regulated by the TYRO3, AXL (also known as UFO) and MERTK (TAM) family of enzymes, which consists of Tyro3 (also known as Brt, Dtk, Rse, Sky, and Tif), Axl (also known as Ark, Tyro7, and Ufo), and Mer (also known as Eyk, Nym, and Tyro12) along with its ligand growth arrest-specific gene 6 (Gas6) (Binder et al., 2011; Tsiperson et al., 2011; Miyamoto et al., 2015). The three TAMs are also expressed in the brain during postnatal development, specifically, in the white matter, which consists of myelinated axons (Prieto et al., 2000). These studies are helping to identify the signals that are associated with myelination in oligodendrocytes and Schwann cells.

Role of Protein S and Gas6 in the Development of Purpura Fulminans

Protein S (PS) homozygous deficiency causes purpura fulminans (PF) thought to result from the imbalance between procoagulant and anticoagulant factors.PS shares similarities with Growth Arrest Specific gene 6 (Gas6) but Gas6-/- mice display an antithrombotic phenotype whereas complete PS deficient (ProS1-/-) mice have a thrombotic phenotype. Beyond their role in coagulation both Gas6 and PS have cellular functions through tyrosine kinase receptors TAM. Gas6 plays also a role in erythropoiesis and previous data suggest that ProS1-/- embryos are anemic.To mimic severe acquired PS deficiency and thus monitor PF development in adult mice, we induced Pros1 gene silencing via poly I:C-inducible Mx1-Cre+ mice. Once induced, ProS1lox/loxMx1 Cre+ and ProS1lox/- Mx1Cre+ mice showed a marked reduction of plasma PS levels compared with ProS1lox/lox mice (47.7±6% ProS1lox/loxMx1 Cre+; 14.5±7% ProS1lox/-Mx1 Cre+, p<0.001). Although spontaneous thromboses were found in lungs and liver sections from both ProS1lox/loxMx1 Cre+ and ProS1lox/- Mx1Cre+ mice, no PF lesions were observed within a 2-month observation period, suggesting that circulating PS level was still too high to induce PF.As a second strategy to reduce PS, we treated ProS1+/- and ProS1+/+ adult mice with warfarin (0.8mg/day, during 5 days), and the treatment induced 75% mortality in ProS1+/- and 5% in WT mice (p<0.001). Interestingly, only a few ProS1+/- mice developed lesions compatible with PF. Early lesions showed vascular engorgement, intradermal edema and rare intravascular thrombosis. In advanced lesions, we noticed massive red blood cell (RBC) extravasation, several intra-epidermal hemorrhagic blisters and necrotic areas.Because Gas6-/- mice are protected against thrombosis, our third strategy was to generate a mouse model combining ProS1 and Gas6 deficiency to overcome the ProS1-/- lethal phenotype. Surprisingly, embryonic mortality, due to massive thrombosis with fibrin deposition, occurred earlier in ProS1-/- Gas6-/- embryosthan in ProS1-/- embryos.RBC extravasation observed during warfarin treatment pointed to a vascular wall involvement during PF. Consequently, we investigated the vasculature using E15 embryonic dorsal skin. Staining with anti-VE-Cadherin and anti-Ter119 antibodies (Ab) confirmed RBC extravasation while anti-CD31 Ab showed areas with underdeveloped and less dense vascular networks, collapsed vessels and less vessels branch points in ProS1-/- andProS1-/-Gas6-/- than in WT mice. Massively enlarged lymphatic vessels and increased macrophages infiltration were observed in ProS1-/- andProS1-/-Gas6 -/- embryos by anti-Lyve1 and F4/80 Ab.Histology of blood vessels and liver from ProS1-/- and ProS1-/- Gas6-/- embryos revealed a high number of circulating immature RBC compatible with increased erythropoiesis because of severe bleeding due to consumption coagulopathy and vascular damage. In addition, numerous isolated erythroblast nuclei were found in these embryos indicating altered phagocytosis. Moreover, preliminary data showed higher cytokine levels in ProS1-/- than in WT embryos. Exclusively in ProS1-/- Gas6-/-, we found a very high level of hypochromic erythrocytes suggesting troubled iron metabolism linked to inflammation and iron recycling.To investigate potential erythropoietic defects, fetal liver (E14.5) single-cell suspensions were cultured for colony forming assays. No difference was found in BFU-E colonies from ProS1-/- and WT embryos. In contrast, BFU-E colonies were 2-fold reduced in ProS1-/-Gas6-/- compared toProS1+/+ Gas6-/- embryos. These data were consistent with our previous observations on the role of Gas6 in erythropoiesis. Both ProS1-/- and ProS1-/-Gas6-/- displayed 50% less CFU-E colonies than WT embryos, pointing to a blunted response to erythropoietin (EPO) in the context of inflammation promoted by PF. These results were confirmed by flow cytometry.In conclusion, with 14.5% plasmaticPS, no PF lesions were observed in adult mice. Gas6 deficiency did not overcome lethality in mice homozygous for PS deficiency. PF developed during warfarin treatment and in ProS1-/- andProS1-/-Gas6-/- embryos which showed thrombosis occurring together with vascular wall damage and inflammatory processes that appeared to impact iron metabolism and response to EPO. DisclosuresNo relevant conflicts of interest to declare.

Autocrine and Paracrine Regulatory Mechanisms of Growth Arrest-Specific Gene 6 Contribute to Disease Progression of Multiple Myeloma

Abstract Multiple myeloma (MM) is a lethal hematologic neoplasm characterized by proliferation of malignant plasma cells in part due to abnormal surrounding microenvironment, but the mechanism of disease progression remains largely unknown in MM. Thus, we screened molecules differentially expressed in bone marrow (BM) cells of MM patients using microarray analysis, which revealed significant upregulation of Gas6 (growth arrest-specific gene 6), a vitamin K-dependent protein with a structural homology with protein S. Since upregulation of Gas6 and its tyrosine kinase receptors have been reported to associate with various malignancies, we hypothesized that Gas6 contributed to the pathogenesis of MM. Gas6 was overexpressed in the BM of MM patients and in various MM cell lines. We found that the serum levels of Gas6 protein were markedly increased in 18 symptomatic MM patients compared to 16 healthy volunteers using human Gas6 ELISA. High levels of soluble Gas6 were detected in the supernatants of in vitro cultured MM cell lines RPMI-8226 and AMO-1 using Gas6 ELIZA. Analysis of apoptosis by flow-cytometry (FCM) with annexin V showed that exogenous Gas6 (100-200 ng/mL) inhibited the apoptosis of MM cells. MTT cell proliferation assay showed that exogenous Gas6 induced cell proliferation of MM cells with increased extracellular signal regulated kinase (ERK) phosphorylation. These results suggest that the presence of an autocrine loop of Gas6 and its receptor with ERK signaling enhanced the proliferative and anti-apoptotic effects on MM cells. Bone marrow stromal cell (BMSC) line HS-5 was found to secrete high levels of soluble Gas6 into culture supernatants using Gas6 ELIZA. Reduction of MM cell apoptosis by HS-5 cell-conditioned medium (CM) was suppressed by Gas6 neutralizing antibody (20 microg/mL) using FCM with annexin V. HS-5 cell-CM induced a marked increase in cell proliferation of MM cells, which was completely inhibited by Gas6 neutralizing antibody (0.2-20 microg/mL) using MTT cell proliferation assay. Our results showed a critical role of Gas6 in the pathology of apoptosis inhibition and cell proliferation of MM cells through paracrine mechanisms. Interleukin (IL)-6 is identified as a major growth and anti-apoptotic factor of MM cells through autocrine mechanisms. Conversely, BMSCs provide efficient support for MM cell survival by paracrine IL-6 stimulation. As determined by western blotting and real-time PCR, HS-5 cell-CM induced Gas6 upregulation, which was suppressed by IL-6 neutralizing antibody (1-10 microg/mL in a dose-dependent manner in MM cells. Meanwhile, HS-5 cell-CM induced the increased IL-6 expression, which was reversed by Gas6 neutralizing antibody in MM cells. Our findings indicate that Gas6 possessed both autocrine and paracrine functions similar to IL-6 between MM cells and BMSCs. Intercellular cell adhesion molecule-1 (ICAM-1) enhanced adhesion of MM cells to BMSCs and subsequently resulted in MM disease progression. Our immnoblotts showed that HS-5 cell-CM induced ICAM-1upregulation, and the ICAM-1 upregulation was reversed by Gas6 neutralization antibody, indicating that Gas6 stimulation increased ICAM-1 synthesis and subsequently accelerated the proliferation of MM cells. The present study offers new insights into autocrine and paracrine actions of Gas6 in concert with IL-6 between MM cells and BMSCs on apoptosis inhibition and cell proliferation of MM cells. We suggest that Gas6-related signaling pathways may be an attractive therapeutic target for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Protective Effects of Growth Arrest-Specific Protein 6 (Gas6) on Sepsis-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a serious complication of sepsis, which has a high mortality rate. Growth arrest-specific protein 6 (Gas6), the protein product of the growth arrest specific gene 6, has been shown to have an anti-apoptotic effect as well as pro-survival capability. Here, we investigated the effects of Gas6 on sepsis-associated AKI in mice subjected to cecal ligation and puncture (CLP). We found that the administration of rmGas6 significantly reduced serum urea nitrogen and creatinine and improved the survival of septic mice. Furthermore, the renal pathological damage induced by CLP was attenuated by rmGas6 treatment. Finally, rmGas6 reduced the renal tissue apoptotic index and the expression of Bax, while it upregulated the expression of Bcl-2. The data suggest that rmGas6 might be used as a potential therapeutic agent for sepsis-induced AKI.

Relationship Between Increased Expression of the Axl/Gas6 Signal Cascade and Prognosis of Patients with Upper Tract Urothelial Carcinoma.

Axl, which is in the TAM family of receptor tyrosine kinases, and its ligand, growth arrest-specific gene 6 (Gas6), have been associated with worse prognoses after the surgical treatment of some types of cancers. We herein investigated the biological significance of the protein expression of Axl and Gas6 on the outcomes of patients with upper tract urothelial carcinoma (UTUC). The protein expression of Axl and Gas6 was evaluated by immunohistochemistry, and their relationships with clinicopathological features were investigated in surgical specimens obtained from 161 patients who had been surgically treated for UTUC. Axl labeling was strong in 67 of 161 (42 %) cases, while Gas6 labeling was strong in 72 of 161 (45 %) cases. The strong expression of Axl correlated with that of Gas6. A high pathological stage (p = 0.009), strong expression of Gas6 (p = 0.038), and strong expression of Axl (p = 0.016) were independent factors for predicting worse cancer-specific survival (CSS). In a subgroup analysis of patients with pT < 2 (N = 53), no significant difference in CSS was observed between patients weakly and strongly expressing Axl/Gas6. In contrast, a subgroup analysis of patients with pT ≥ 2 (N = 108) revealed that the expression levels of Axl and Gas6 correlated with CSS. The protein expression of Axl and its ligand Gas6 may be a useful indicator for a worse clinical outcome in UTUC patients, especially patients with pT ≥ 2, who underwent radical nephroureterectomy.

Gas1 is a receptor for sonic hedgehog to repel enteric axons

The myenteric plexus of the enteric nervous system controls the movement of smooth muscles in the gastrointestinal system. They extend their axons between two peripheral smooth muscle layers to form a tubular meshwork arborizing the gut wall. How a tubular axonal meshwork becomes established without invading centrally toward the gut epithelium has not been addressed. We provide evidence here that sonic hedgehog (Shh) secreted from the gut epithelium prevents central projections of enteric axons, thereby forcing their peripheral tubular distribution. Exclusion of enteric central projections by Shh requires its binding partner growth arrest specific gene 1 (Gas1) and its signaling component smoothened (Smo) in enteric neurons. Using enteric neurons differentiated from neurospheres in vitro, we show that enteric axon growth is not inhibited by Shh. Rather, when Shh is presented as a point source, enteric axons turn away from it in a Gas1-dependent manner. Of the Gαi proteins that can couple with Smo, G protein α Z (Gnaz) is found in enteric axons. Knockdown and dominant negative inhibition of Gnaz dampen the axon-repulsive response to Shh, and Gnaz mutant intestines contain centrally projected enteric axons. Together, our data uncover a previously unsuspected mechanism underlying development of centrifugal tubular organization and identify a previously unidentified effector of Shh in axon guidance.

TAM receptor deficiency affects adult hippocampal neurogenesis.

The Tyro3, Axl and Mertk (TAM) subfamily of receptor protein tyrosine kinases functions in cell growth, differentiation, survival, and most recently found, in the regulation of immune responses and phagocytosis. All three receptors and their ligands, Gas6 (growth arrest-specific gene 6) and protein S, are expressed in the central nervous system (CNS). TAM receptors play pivotal roles in adult hippocampal neurogenesis. Loss of these receptors causes a comprised neurogenesis in the dentate gyrus of adult hippocampus. TAM receptors have a negative regulatory effect on microglia and peripheral antigen-presenting cells, and play a critical role in preventing overproduction of pro-inflammatory cytokines detrimental to the proliferation, differentiation, and survival of adult neuronal stem cells (NSCs). Besides, these receptors also play an intrinsic trophic function in supporting NSC survival, proliferation, and differentiation into immature neurons. All these events collectively ensure a sustained neurogenesis in adult hippocampus.

The Linker Protein GAS7 Negatively Regulates Pre-B Cell Differentiation and Amplifies Proliferation and Survival Signals in Acute Lymphoblastic Leukemia

Background:Growth arrest-specific gene 7 (Gas7) functions as an adaptor for SH2- and SH3-containing proteins, in particular in cells that undergo growth arrest. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Interestingly, MLL-GAS7 fusion molecules resulting from the t(11;17)(q23;p13) chromosomal translocation have been reported in treatment-related acute myeloid leukemia (AML; Megonigal et al., 2000) and in a pediatric acute lymphoblastic leukemia (ALL). While the function of MLL has been extensively studied, the role of its fusion partner GAS7 in normal hematopoiesis and leukemia has not been elucidated.Results: Studying gene expression changes during normal B cell development, we identified Gas7 as the gene with the strongest relative increase at the pre-B cell receptor checkpoint. At the transition from IL7-dependent Fraction C’ to IL7-independent small resting pre-B cells (Fraction D), GAS7 mRNA levels were upregulated by >13-fold in both human and mouse B cell progenitors. Withdrawal of IL7 cytokine signaling and Cre-mediated conditional deletion of Stat5ab recapitulated the strong increase of GAS7 expression under cell culture conditions. These finding suggest that GAS7 is part of an adaptive response of differentiating pre-B cells to attenuation of cytokine/Stat5 signaling. Consistent with this scenario, we found that Gas7-/-pre-B cells undergo accelerated differentiation, including spontaneous Ig κ light chain gene recombination and loss of Stat5-signaling. Conversely, overexpression of GAS7, reduced responsiveness of pre-B cells to normal differentiation stimuli. These findings suggest that the linker molecule GAS7 is a negative regulator of pre-B cell differentiation.Likewise, we found that tyrosine kinase inhibitor treatment of human Ph+ ALL cells resulted in a strong increased of GAS7 expression, in parallel with loss of Stat5 function. To elucidate the function of Gas7 in B cell lineage leukemia, we transformed bone marrow pre-B cells from Gas7-/- mice with BCR-ABL1. Gas7 deficient Ph+ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis. In agreement with effects of Stat5 on the sensitivity of Ph+ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph+ ALL cells showed massively increased susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 caused loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Co-immunoprecipitation experiments with flag tagged GAS7 in patient-derived Ph+ALL cells revealed that GAS7 physically interacts with STAT5 and retains STAT5-Y694 in an active conformation.Thereby, GAS7 can propagate even weak Stat5 activity and maintain residual cytokine or BCR-ABL1 oncogenic signaling in normal and malignant pre-B cells.Conclusions: Here show that GAS7 functions as an important positive regulator of Stat5 downstream of cytokine receptors in normal pre-B cells and downstream of BCR-ABL1 and other oncogenes in leukemia. Owing to the GAS7-dependent reinforcement of Stat5-dependent survival and proliferation signaling, normal and leukemic pre-B cells can survive periods of reduced cytokine/oncogene signaling. These findings suggest that the interaction interface between GAS7 and Stat5 represents a potential target for small molecule scaffolds and peptides. DisclosuresNo relevant conflicts of interest to declare.

Development of an Axl/Mer Dual Inhibitor, ONO-9330547: Promising Single Agent Activity in an Acute Myeloid Leukemia (AML) Model

Purpose: Axl and Mer belong to the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases and play a role in regulating cell proliferation, survival, migration and cytokine production. Axl/Mer are over-expressed in various types of haematological and solid tumour cancers and appear to play a key role in epithelial-to-mesenchymal transitions (EMTs), known to be important in tumour metastases and drug resistance. AML is a clonal disease of haematopoietic progenitors characterised by acquired heterogeneous genetic changes that alter cell proliferation and differentiation and Axl is an independent prognostic marker and therapeutic target in AML. Recent studies revealed that AML cells induce expression and secretion of the Axl ligand growth arrest specific gene 6 (Gas6) by bone marrow derived stromal cells (BMDSCs). Gas6 mediates proliferation, survival and chemoresistance of Axl-expressing AML cells, giving rise to a chemoprotective tumour niche environment. Standard chemo-treatment for AML affects not only AML cells but healthy cells which cause severe side effects. In addition, only 40-45% of patients <65 years reach long-term survival with current treatment options and only 10% of patients >65 can be cured. Therefore, there is still a high, unmet medical need for new therapies. ONO-9330547 is a small molecule inhibitor that binds to Axl and Mer kinase with a potency (IC50) of 2.2 and 0.4 nM respectively, therefore, the anti-tumour activity of ONO-9330547 in AML cells was evaluated.Methods: AML cell lines were implanted subcutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 100-200 mm3. ONO-9330547 was administered orally at doses up to 3 mg/kg twice daily (BD) for 24 days. Tumour volumes were measured twice a week after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumours reached a maximum volume of 2,000 mm3. Phosphorylation of Axl (P-Axl) and Mer (P-Mer) were detected by Western blotting or Flow cytometry.Results: In AML cell lines assays, ONO-9330547 strongly inhibited both P-Axl and P-Mer with an IC50in the nM range, which resulted in growth inhibitions and inductions of apoptosis. In the MV-4-11 xenograft model, tumour growth inhibition at the final treatment day was 40% in the 0.3 mg/kg BD group and 78% in the 1 mg/kg BD and 100% in the 3 mg/kg BD groups respectively (All treatment groups: P<0.001 vs Vehicle). Specifically, the treatment with ONO-9330547 1 or 3 mg/kg BD resulted in higher and sustained P-Axl inhibition throughout the study, and treatment of 3 mg kg BD resulted in complete remission in all treated mice. Interestingly, up-regulation of Axl and Mer were observed in AML cell lines after the treatment of doxorubicin or Ara-C. The combination of ONO-9330547 with Ara-C resulted in significant anti-tumour effect compared with respective monotherapy.Conclusion: ONO-9330547 is a highly potent dual Axl/Mer inhibitor with evidence of efficacy in AML cells. Moreover, up-regulation of Axl/Mer after chemotherapy indicates the potential combination of ONO-9330547 with standard therapy to overcome treatment resistance in AML. Additional work to investigate and clarify the involvement of Axl/Mer in other types of leukemia are currently underway. DisclosuresYasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Yoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Fujikawa:ONO Pharmaceutical Co., Ltd.: Employment. Tanaka:ONO Pharmaceutical Co., Ltd.: Employment. Koike:ONO Pharmaceutical Co., Ltd.: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.

Gas6'ing the innate immune response during experimental asthma.

Growth arrest-specific gene 6 (Gas6) binds Tyro3, Axl, and Mertk (TAM) receptors and exerts prominent effects in many diseases, but little is known about its role in asthma. Herein, we examined the role of Gas6 and TAM receptors differentially in an experimental asthma model driven by Aspergillus fumigatus. A. fumigatus-sensitized mice were challenged with live A. fumigatus conidia, and airway hyperresponsiveness and airway remodeling were determined 28 days later. When administered to mice from Days 14 to 28 after conidia challenge, anti-Axl monoclonal antibody, but not anti-Mertk monoclonal antibody, treatment significantly inhibited airway hyperresponsiveness and airway remodeling compared with the appropriate control IgG group. These results demonstrate that Gas6 has modulatory functions in fungal asthma via Axl receptor activation in immune and nonimmune cells.

Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice.

Growth arrest-specific gene (Gas)6 is a secreted vitamin K-dependent protein with pleiotropic effects via activation of receptor tyrosine kinase Tyro3, Axl, and Mertk receptors, but little is known about its role in allergic airway disease. We investigated the role of Gas6 in the development of fungal allergic airway disease in mice. The immune response was evaluated in Gas6-deficient (Gas6-/-) and wild-type (WT) mice and in recombinant Gas6-treated WT mice during Aspergillus fumigatus-induced allergic airway disease. Gas6 plasma levels were significantly elevated in adult clinical asthma of all severities compared with subjects without asthma. In a murine model of fungal allergic airway disease, increased protein expression of Axl and Mertk were observed in the lung. Airway hyperresponsiveness (AHR), whole lung Th2 cytokine levels, goblet cell metaplasia, and peribronchial fibrosis were ameliorated in Gas6-/- mice compared with WT mice with fungal allergic airway disease. Intranasal Gas6 administration into WT mice had a divergent effect on airway inflammation and AHR. Specifically, a total dose of 2 μg of exogenous Gas6 (i.e., low dose) significantly increased whole lung Th2 cytokine levels and subsequent AHR, whereas a total dose of 7 μg of exogenous Gas6 (i.e., high dose) significantly suppressed Th1 and Th2 cytokines and AHR compared with appropriate control groups. Mechanistically, Gas6 promoted Th2 activation via its highest affinity receptor Axl expressed by myeloid DCs. Intranasal administration of Gas6 consistently exacerbated airway remodeling compared with control WT groups. These results demonstrate that Gas6 enhances several features of fungal allergic airway disease.

Receptor Tyrosine Kinases, TYRO3, AXL, and MER, Demonstrate Distinct Patterns and Complex Regulation of Ligand-induced Activation

TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs.

Role of Growth arrest-specific gene 6-Mer axis in multiple myeloma.

Multiple myeloma is a mostly incurable malignancy characterized by the expansion of a malignant plasma cell (PC) clone in the human bone marrow (BM). Myeloma cells closely interact with the BM stroma, which secretes soluble factors that foster myeloma progression and therapy resistance. Growth arrest-specific gene 6 (Gas6) is produced by BM-derived stroma cells and can promote malignancy. However, the role of Gas6 and its receptors Axl, Tyro3 and Mer (TAM receptors) in myeloma is unknown. We therefore investigated their expression in myeloma cell lines and in the BM of myeloma patients and healthy donors. Gas6 showed increased expression in sorted BMPCs of myeloma patients compared with healthy controls. The fraction of Mer(+) BMPCs was increased in myeloma patients in comparison with healthy controls whereas Axl and Tyro3 were not expressed by BMPCs in the majority of patients. Downregulation of Gas6 and Mer inhibited the proliferation of different myeloma cell lines, whereas knocking down Axl or Tyro3 had no effect. Inhibition of the Gas6 receptor Mer or therapeutic targeting of Gas6 by warfarin reduced myeloma burden and improved survival in a systemic model of myeloma. Thus, the Gas6-Mer axis represents a novel candidate for therapeutic intervention in this incurable malignancy.