STAT5B Research Articles

Human signal transducer and activator of transcription 5b (STAT5b) mutation causes dysregulated human natural killer cell maturation and impaired lytic function

Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent infections, and combined immune deficiency. In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b-/- mice. We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b-/- mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function. This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b-/- mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation. Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.

Regulation of murine skeletal muscle growth by STAT5B is age- and sex-specific

BackgroundSexually dimorphic growth has been attributed to the growth hormone (GH)/insulin-like growth factor 1 (IGF1) axis, particularly GH-induced activation of the intracellular signal transducer and activator of transcription 5B (STAT5B), because deletion of STAT5B reduces body mass and the mass of skeletal muscles in male mice to that in female mice. However, it remains unclear why these effects are sex- and species-specific, because the loss of STAT5B retards growth in girls, but not in male mice. Our objectives were to determine whether sexually dimorphic growth of skeletal muscle persisted in STAT5B−/− mice and investigate the mechanisms by which STAT5B regulates sexually dimorphic growth.MethodsBlood and skeletal muscle were harvested from male and female STAT5B−/− mice and their wild-type littermates from the onset of puberty to adulthood.ResultsGrowth of the skeleton and skeletal muscles was retarded in both sexes of STAT5B−/− mice, but more so in males. Although reduced, sexually dimorphic growth of skeletal muscle persisted in STAT5B−/− mice with an oxidative shift in the composition of myofibres in both sexes. Concentrations of IGF1 in blood and skeletal muscle were reduced in male STAT5B−/− mice at all ages, but only in female STAT5B−/− mice at the onset of puberty. Expression of androgen receptor (AR) and oestrogen receptor alpha (ERα) mRNA and protein was reduced in skeletal muscles of male and female STAT5B−/− mice, respectively. Loss of STAT5B abolished the sexually dimorphic expression of myostatin protein and Igf1, Ar, Erα, suppressor of cytokine signalling 2 (Socs2), and cytokine-inducible SH2-containing protein (Cis) mRNA in skeletal muscle.ConclusionsSTAT5B appears to mediate GH signalling in skeletal muscles of male mice at all ages, but only until puberty in female mice. STAT5B also appears to mediate the actions of androgens and oestrogens in both male and female mice, but sexually dimorphic growth persists in STAT5B−/− mice.

PF362 A FIRST HIGH-DEFINITION LANDSCAPE OF SOMATIC MUTATIONS IN CHRONIC LYMPHOPROLIFERATIVE DISORDER OF NK CELLS

Background: Chronic Lymphoproliferative Disorder of NK cells (CLPD-NK) is included in the 2017 WHO classification of mature T- and NK-cell neoplasms as provisional entity. Similarly to T-cell large granular lymphocytes leukemia (T-LGLL), CLPD-NK is characterized by an indolent clinical course and is clearly distinguished from the aggressive NK cell leukemia (ANKL), this latter behaving as an acute severe disease. CLPD-NK and T-LGLL patients mostly present cytopenias when symptomatic. Accumulating evidence on the genomic landscape of T-LGLL pointed to a key role of somatic mutations within JAK-STAT (mostly in STAT3 and STAT5B genes) and RAS-MAPK pathways (Coppe et al. Leukemia 2017) in this form. Recent data highlighted more heterogeneous molecular features in ANKL, involving JAK2 and STAT3 mutations, lesions of epigenetic modifiers, tumour suppressors and a putative role of Epstein Barr Virus (Dufva et al. Nat Comm. 2018). Aims: In our cohort of 57 CLPD-NK patients the frequency of STAT mutations has been found significantly lower (<10% of all cases) compared to literature data (Barilà et al. Blood Cancer J. 2018). The genomic landscape of CLPD-NK patients negative for STAT mutations was thus investigated to better elucidate the molecular basis of the disease. Methods: Ten CLPD-NK patients were recruited according to negativity for STAT mutations and typical immunophenotype (CD16+/CD57±/CD56±). WES profiling (Agilent SureSelect 60 Mbp, Illumina sequencing, paired end reads) of immunomagnetically purified leukemic clone (>95% purity) and of normal granulocytes, as control, was obtained for each patient. Sequencing data were analyzed by an in-house bioinformatic pipeline encompassing data cleaning, read mapping against the reference genome and somatic variant calling with complementary approaches (MuTect, MuTect2 and Strelka2). Variants were next filtered according to population allele frequency (gnomAD <5%), clinical significance (COSMIC and ClinVar) and predicted functional impact on proteins (SnpEff, MetaSVM and MetaLR). Results: High sequencing depth was obtained with a mean coverage of 150 reads. From over 6,000 somatic variants detected in the cohort, 869 SNPs and 14 indels, rare and of high predicted impact were prioritized. Number of variants (88 per patient in average) and mutation spectra were very homogeneous in the cohort. Of note, the high discovery power of our settings disclosed an extensive subclonality of somatic variants, with over 78% with an allele variant frequency (VAF) lower than 5% in the leukemic clone (Figure 1A). Nevertheless, 20 deleterious and high VAF somatic variants occurred in each patient in average (Figure 1B). Very low recurrence of mutated genes in CLPD-NK patients was found. Pathway-derived mutation network analysis (Lovisa et al. Haematologica 2019) revealed functional connections among genes mutated in different patients. A major involvement of JAK-STAT pathway is likely to be ruled out, but all cases presented deleterious high VAF mutations of genes and pathways potentially impacting on cell survival, proliferation and chromatin-remodelling. In addition, somatic lesions in proteins involved in innate immunity and NK-cells activation seem particularly interesting.Summary/Conclusion: Ongoing comparison of genes and pathways mutated in CLPD-NK with available data for T-LGLL and ANKL is providing new clues on biological similarities and differences of these clinical entities. This study contributes to a better definition of the molecular mechanisms accounting for CLPD-NK, thus opening new areas of investigation.

The Signal Transducer and Activator of Transcription 5B (STAT5B) Gene Promotes Proliferation and Drug Resistance of Human Mantle Cell Lymphoma Cells by Activating the Akt Signaling Pathway.

BackgroundMantle cell lymphoma (MCL) is a high-grade B-cell lymphoma with poor prognosis. Fludarabine is used alone or in combination for relapsed and advanced-stage MCL. The expression of the signal transducer and activator of transcription 5B (STAT5B) gene is associated with tumorigenesis in solid tumors, but its role in MCL remains unknown. The aims of this study were to investigate the role of STAT5B in GRANTA-519 human mantle cell lymphoma cells and drug resistance.Material/MethodsGRANTA-519 human mantle cell lymphoma cells were cultured with and without 10 μM fludarabine dephosphorylated 9-β-D-arabinofuranosyl-2-fluoroadenine, (2-F-araA) or 10 μM 4-hydroperoxycyclophosphamide (4-HC). The MTT assay assessed cell proliferation. Flow cytometry was used to investigate the cell cycle in MCL cells treated with the specific inhibitor of the Akt pathway, LY294002, and assessed cell cycle and cell apoptosis. Western blot was used to detect the expression levels of p-Akt/Akt and STAT5B/p-STAT5B. The gene expression profiles of lymph node (LN)-derived MCL cells were compared with peripheral blood (PB)-derived lymphocytes using bioinformatics and hierarchical cluster analysis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to determine the expression of the marker of proliferation Ki-67 (MKI67) gene.ResultsSTAT5B was significantly upregulated in LN-derived MCL cells compared with PB lymphocytes. Increased expression of STAT5B was associated with increased MCL cell proliferation and reduced cell apoptosis and was associated with drug resistance and activation of Akt.ConclusionsSTAT5B promoted cell proliferation and drug resistance in human MCL cells by activating the Akt signaling pathway.

Influence of IL-1β, STAT3 & 5 and TLR-5 gene polymorphisms on rheumatic heart disease susceptibility in north Indian population

BackgroundRheumatic heart disease (RHD) is the most serious complication of heart that comprises inflammatory reactions in heart valves. Many studies have demonstrated the contribution of host genetic factors in susceptibility to RHD and many cytokine gene variants have been linked with susceptibility to RHD. We sought to determine the role of genetic variants in IL-1β, STAT3, STAT5B and TLR5 genes in conferring risk of RHD in two cohorts of RHD patients. MethodsThe study included 400 echocardiography confirmed RHD patients and 300 controls from North Indian Population. We categorized RHD patients into two sub-groups based on involvement of heart valves, mitral valve lesion alone (MVL), and combined valve lesions including mitral valve (CVL). Genotyping for all the polymorphisms was done using TaqMan /PCR-RFLP methods. ResultsOur results showed that the genotypic frequencies of IL-1β, STAT3, STAT5B andTLR5 genes polymorphisms were significantly associated with RHD risk. To validate our results, we performed a replication study in additional 200 cases with similar clinical characteristics and results again confirmed consistent findings with RHD risk. In subgroup analysis, STAT3 polymorphism remained significant with MVL in RHD patients. ConclusionIL-1β, STAT3, STAT5B and TLR5 genes polymorphism may be useful markers for the identification of individuals with high risk of RHD in the susceptible population.

Single nucleotide polymorphisms in candidate genes are significantly associated with resistance to Haemonchus contortus infection in goats

BackgroundHaemonchosis is a major economic problem in goat production in humid, tropical and subtropical regions. The disease is caused by an abomasal nematode, Haemonchus contortus, which is highly pathogenic in small ruminants. The aim of this study was to identifying single-nucleotide polymorphisms (SNP) that were associated with fecal egg counts (FEC) and could be used as markers to identify resistance to H. contortus in goats.ResultsTen novel variants in the CIITA, ATP2A3, HSPA8, STAT5B, ESYT1, and SERPING1 genes were associated with FEC in goats with a nominal significance level of P < 0.05. Two missense mutation in the exon region of the caprine CIITA gene resulted in replacement of arginine with cysteine at position 9473550 (R9473550C) and aspartic acid with glutamic acid at position 9473870 (D9473870E). Chinese goat breeds had significantly higher FEC than Bangladeshi goat breeds within their respective genotypes. Polymorphism information content (PIC), effective allele number (Ne), and heterozygosity (He) were greatest for the STAT5B_197_A > G SNP locus in all goat breeds. Pairwise coefficients of linkage disequilibrium (D´, r2) revealed complete LD (r2 = 1) between significant SNP polymorphisms in CIITA and SERPING1 and strong LD (r2 = 0.93 and 0.98) between polymorphisms in HSPA8 and ATP2A3, respectively. Correlation coefficient (r) between FEC and body weight (BW) was significantly positive (r = 0.56***, P < 0.001) but that between FEC and packed cell volume (PCV) was negatively significant (r = − 0.47**, P < 0.01) in the total population of goats. On the other hand, correlation coefficient (r) between BW and PCV was not significant in total population of goats. Association analysis revealed that haplotypes within ATP2A3, HSPA8, and SERPING1 were significantly associated with FEC. Quantitative real-time PCR revealed that the relative expression of mRNA was higher (P < 0.001) for resistant, compared to susceptible, groups of goats for all candidate genes except CIITA.ConclusionsThis study identified SNP markers that can potentially be used in marker-assisted selection programs to develop goat breeds that are resistant to H. contortus.

Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic β-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress. In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic β-cell line we have assayed nitric oxide and its end products, superoxide levels, H₂O₂ levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting. Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic β-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H₂O₂ production. H₂O₂ metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H₂O₂. Stat5b silencing finally reduces AKT expression, a prosurvival signal. Our study enables us to conclude that β-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H₂O₂ accumulation and the ensuing oxidative stress enhances β-cell damage.

Cloning and functional characterization of STAT5a and STAT5b genes in buffalo mammary epithelial cells

Signal transducer and activator 5 (STAT5) plays important roles in regulating mammary glandular cell proliferation and milk-protein gene expression. However, the functions of STAT5a and STAT5b genes in lactation of buffalo remain uninvestigated. In this study, full-length STAT5a (2502 bp) and STAT5b (2515 bp) coding sequences were isolated for the first time. The highest STAT5a gene expression was found in buffalo mammary glands and the highest STAT5b gene expression was found in buffalo brains and mammary glands. H&E staining indicated that STAT5a and STAT5b are mainly expressed in epithelial cells of buffalo mammary glands. Next, we investigated the functions of STAT5 by knocking down and overexpressing STAT5 in buffalo mammary epithelial cells (BuMECs). According to our results, STAT5 knockdown resulted in inhibited G1/S transition of BuMECs and significantly lower expression of milk-protein genes, whereas overexpression of STAT5 resulted in significantly higher expression of milk-protein genes. In summary, our results demonstrate that STAT5 can regulate the cell cycle transition of BuMECs and affect the expression of milk-protein genes. Our research lays a foundation for further study of the role of STAT5 in mammary gland development and lactation.

Molecular cloning and bioactivity of an IL-2 homologue in large yellow croaker (Larimichthys crocea)

Interleukin-2 (IL-2), an important immunomodulatory cytokine, plays a crucial role in promoting the proliferation, activation and differentiation of T cells. Here, the cDNA of an IL-2 homologue (LcIL-2) in large yellow croaker (Larimichthys crocea) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcIL-2 gene is 426 bp long and encoded a precursor protein of 141 amino acids (aa), with a 20-aa signal peptide and a 121-aa mature peptide containing two putative N-glycosylation sites at Asn77 and Asn101. The LcIL-2 is preferentially expressed in lymphocytes-rich tissues, such as spleen and blood, and is increased in head kidney and spleen upon inactivated trivalent bacterial vaccine or poly(I:C) stimulation. LcIL-2 expression could also be detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM) and primary head kidney granulocytes (PKG), with the highest level in PKL. In addition, the expression level of LcIL-2 in PKL was slightly induced by LPS or poly(I:C), while markedly induced by PHA or Con-A. The recombinant LcIL-2 protein produced in Pichia pastoris could increase the expression of genes involved in Th1 (IL-2, IFN-γ and T-bet) and Th2 (IL-4/13A, IL-4/13B and GATA3) development and differentiation, and of the IL-2 downstream transcription factor STAT5B gene, but inhibit the expression of genes related to Th17 (IL-17A/F2 and IL-17A/F3) development and differentiation. Taken together, our results indicated that LcIL-2 possesses similar structural and functional characteristics to other vertebrate IL-2s, and may play a role in T cell development and differentiation.

RGS4 Regulates Neurite Outgrowth And Cell Proliferation Mediated by STAT5B Transcriptional Responses

Regulators of G proteins signaling (RGS) serve several cellular functions, despite their initial role as GTPase activating proteins. Recent evidence suggests that RGS proteins interact with transcription factors to mediate transcriptional responses. RGS4, a member of the B/R4 family of RGS proteins, is a multitask protein highly expressed in developing neurons, that has been shown to be a key regulator of opioid receptor signaling (ORs) by confering selectivity for G protein coupling, inhibiting OR‐mediated ERK1,2 phosphorylation and accelerating receptor internalization (Leontiadis et al. 2009; Georgoussi et al. 2006). We have previously shown that δ‐OR forms a multiprotein signaling complex, consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neurite outgrowth upon DSLET‐δ‐OR activation (Georganta et al. 2010;2013). Based on these findings and the fact that RGS4 directly interacts with STAT5B to negatively regulate STAT5B activation (Pallaki et al. 2017) we wondered whether RGS4‐STAT5B interplay could be involved in cellular mechanisms controlling neurogenesis.Primary neuronal cortical cultures from RGS4−/− mice, lacking a functional RGS4, exhibited differential neuronal sprouting compared to wild types, as assessed by an increased axonal length and branch number, upon DSLET administration. Additional studies in brain extracts from adult RGS4−/− mice demonstrated increased levels of phospho‐STAT5B while exhibited altered expression levels of STAT5B inducible genes Snx9 and Ptk2 which are both highly implicated in axonogenesis. This result indicates that RGS4 is implicated in neurite outgrowth via a STAT5B‐mediated transcriptional regulation. On the other hand, Neuro2A cells stably expressing RGS4 exhibited a significant reduction in the cell number and proliferation rate whereas isolated RGS4−/− neural stem cells exhibited elevated proliferative properties with a concomitant increase of the mRNA levels of the STAT5B anti‐apoptotic target genes, Bcl‐2 and Bcl‐xl. The same expression pattern was also confirmed in RGS4−/− mature adult brain tissues. Collectively, these studies demonstrate for the first time a novel selective role of RGS4 in STAT5B mediated‐transcriptional responses providing insights into the role of RGS4 involvement in neuronal development and synaptic signaling upon δ‐opioid administration.Support or Funding InformationThis work was supported by grants NORMOLIFE (LSHC‐CT037733) and the GSRT Excellence II, 3722 ‐”NO‐ALGOS” to Z.G. The facilities of the Hellenic Research Infrastructure OPENSCREEN‐GR were used for protein expression.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of age and gender on the genetic regulation of serum 25-hydroxyvitamin D - the FIN-D2D population-based study

In addition to sunlight and dietary sources, several genes in the metabolic pathway of vitamin D affect serum 25-hydroxyvitamin D (25OHD) concentration. It is not known whether this genetic regulation is influenced by host characteristics. We investigated the effect of age and gender on the genetic regulation of serum 25OHD concentration.In total, 2868 Finnish men and women aged 45–74 years participated in FIN-D2D population-based health survey in 2007. Of the 2822 participants that had serum 25OHD concentration available, 2757 were successfully genotyped. Age and gender-dependent association of SNPs with serum 25OHD concentration was studied in 10 SNPs with previously found association with vitamin D metabolites.Associations of 3 SNPs with serum 25OHD concentration were dependent on age with greater effects on younger (≤60 y) than older (>60 y) adults (rs10783219 in VDR, rs12512631 in GC and rs3794060 in NADSYN1/DHCR7; pinteraction = 0.03, 0.02 and 0.01, respectively). The results suggested a novel association between serum 25OHD concentration and rs8082391 in STAT5B gene in men but not in women (pinteraction = 0.01). After multiple testing correction with false discovery rate method, two age-dependent interactions (rs3794060 in NADSYN1/DHCR7 gene and rs12512631 in GC gene) remained statistically significant.This is the first study to suggest that genetic regulation of serum 25OHD concentration is age-dependent. Our results also indicated a novel association between serum 25OHD concentration and SNP in STAT5B gene in men. The results need to be confirmed in future studies preferably in a larger sample.

DNA and RNA-sequence based GWAS highlights membrane-transport genes as key modulators of milk lactose content

BackgroundLactose provides an easily-digested energy source for neonates, and is the primary carbohydrate in milk in most species. Bovine lactose is also a key component of many human food products. However, compared to analyses of other milk components, the genetic control of lactose has been little studied. Here we present the first GWAS focussed on analysis of milk lactose traits.ResultsUsing a discovery population of 12,000 taurine dairy cattle, we detail 27 QTL for lactose concentration and yield, and subsequently validate the effects of 26 of these loci in a distinct population of 18,000 cows. We next present data implicating causative genes and variants for these QTL. Fine mapping of these regions using imputed, whole genome sequence-resolution genotypes reveals protein-coding candidate causative variants affecting the ABCG2, DGAT1, STAT5B, KCNH4, NPFFR2 and RNF214 genes. Eleven of the remaining QTL appear to be driven by regulatory effects, suggested by the presence of co-locating, co-segregating eQTL discovered using mammary RNA sequence data from a population of 357 lactating cows. Pathway analysis of genes representing all lactose-associated loci shows significant enrichment of genes located in the endoplasmic reticulum, with functions related to ion channel activity mediated through the LRRC8C, P2RX4, KCNJ2 and ANKH genes. A number of the validated QTL are also found to be associated with additional milk volume, fat and protein phenotypes.ConclusionsOverall, these findings highlight novel candidate genes and variants involved in milk lactose regulation, whose impacts on membrane transport mechanisms reinforce the key osmo-regulatory roles of lactose in milk.

Essential roles of stat5.1/stat5b in controlling fish somatic growth

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of growth hormone (GH) signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-of-function mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally, we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

Fusion of the genes ataxin 2 like,ATXN2L, and Janus kinase 2,JAK2, in cutaneous CD4 positive T-cell lymphoma

Acquired mutations were recently described in cutaneous T-cell lymphomas for the JAK1, JAK3, STAT3, and STAT5B genes of the JAK-STAT pathway. In the present study, RNA-sequencing of a primary cutaneous CD4 positive T-cell lymphoma carrying a three-way t(9;13;16)(p24;q34;p11) chromosome translocation showed that JAK2 from chromosome band 9p24 was rearranged and fused to a novel partner gene, ATXN2L, from 16p11. RT-PCR together with Sanger sequencing verified the presence of the ATXN2L-JAK2 fusion transcript. The ATXN2L-JAK2 fusion gene would code for a chimeric protein containing all domains of ATXN2L and the catalytic domain of the JAK2 tyrosine kinase. The ATXN2L-JAK2 chimeric protein could lead to constitutive activation of the downstream JAK-STAT signaling pathway in a manner similar to that seen for other JAK2 fusion proteins.

Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B.

We previously reported that EphA4, a member of the Eph family of receptor tyrosine kinases, is an important modulator of growth hormone (GH) signaling, leading to augmented synthesis of insulin-like growth factor 1 (IGF1) for postnatal body growth. In the present study, we report the molecular interactions of EphA4, GH receptor (GHR), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 5B (STAT5B). EphA4 binds to GHR at both its extracellular and intracellular domains and phosphorylates GHR when stimulated with a ligand. The cytoplasmic domain of EphA4 binds to the carboxy-terminus of JAK2 in contrast to the known binding of GHR to the amino-terminus. STAT5B binds to the amino-terminal kinase domain of EphA4. Ligand-activated EphA4 and JAK2 phosphorylate each other and STAT5B, but JAK2 does not appear to phosphorylate EphA4-bound STAT5B. Ligand-activated EphA4 induces the nuclear translocation of STAT5B in a JAK2-independent manner. GHR expression is required for the activation of STAT5B signaling, even via the JAK2-independent pathway. Various ephrins that have affinity for EphA4 induce STAT5B phosphorylation. These findings suggest the molecular mechanisms by which ephrin/EphA4 signaling enhances the canonical GH-IGF1 axis.

Hypothyroidism decreases JAK/STAT signaling pathway in lactating rat mammary gland

Thyroid pathologies have deleterious effects on lactation. Especially hypothyroidism (HypoT) induces premature mammary involution at the end of lactation and decreases milk production and quality in mid lactation. Milk synthesis is controlled by JAK2/STAT5 signaling pathway and prolactin (PRL), which activates the pathway. In this work we analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT on PRL signaling pathway on mammary glands from rats on lactation (L) days 2, 7 and 14. HypoT decreased prolactin receptor expression, and expression and activation of Stat5a/b protein. Expression of members of the SOCS-CIS family, inhibitors of the JAK-STAT pathway, decreased in L2 and L7, possibly as a compensatory response of the mammary cells to maintain PRL responsiveness. However, on L14, the level of these inhibitors was normal and the transcription of α-lactoalbumin (lalba), a target gene of the PRL pathway, decreased by half. HypoT altered the transcriptional capacity of the cell and decreased mRNA levels of Prlr and Stat5b on L14. Stat5b gene has functional thyroid hormone response elements in the regulatory regions, that bind thyroid hormone receptor β (TRβ) differentially and in a thyroid hormone dependent manner. The overall decrease in the PRL signaling pathway and consequently in target gene (lalba) mRNA transcription explain the profound negative impact of HypoT on mammary function through lactation.

A novel regulatory role of RGS4 in STAT5B activation, neurite outgrowth and neuronal differentiation

The Regulator of G protein Signalling 4 (RGS4) is a multitask protein that interacts with and negatively modulates opioid receptor signalling. Previously, we showed that the δ-opioid receptor (δ-OR) forms a multiprotein signalling complex consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neuronal differentiation and neurite outgrowth upon δ-ΟR activation. Here, we investigated whether RGS4 could participate in signalling pathways to regulate neurotropic events. We demonstrate that RGS4 interacts directly with STAT5B independently of δ-ΟR presence both in vitro and in living cells. This interaction involves the N-terminal portion of RGS4 and the DNA-binding SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ-OR and/or erythropoietin receptor results in inhibition of [D-Ser2, Leu5, Thr6]-enkephalin (DSLET)-and erythropoietin-dependent STAT5B phosphorylation and subsequent transcriptional activation. DSLET-dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knockout mice (RGS4-/-) exhibit enhanced neuronal sprouting after δ-OR activation. Additional studies in adult brain extracts from RGS4-/- mice revealed increased levels of p-STAT5B. Finally, neuronal progenitor cultures from RGS4-/- mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti-apoptotic STAT5B target genes bcl2 and bcl-xl. These observations suggest that RGS4 is implicated in opioid dependent neuronal differentiation and neurite outgrowth via a “non-canonical” signaling pathway regulating STAT5B-directed responses.

Novel Mutations in NOTCH and Altered Wnt/β-Catenin Pathway Indicate a Role of Embryonic Signals in the Pathogenesis of T-Cell Prolymphocytic Leukemia

Background:Recurrent mutations in ATM, JAK1/3 and STAT5B genes have been identified in T-cell prolymphocytic leukemia (T-PLL), a mature T cell leukemia with poor survival. However, much remains to be understood regarding the dysregulated pathways involved in the pathogenesis of this aggressive disease. Here, we integrated the analyses of whole exome sequencing (WES) and transcriptomes of a cohort of 12 T-PLL samples to gain further insights into molecular mechanisms of this malignancy.Methods: Leukemic cells were isolated from peripheral blood or bone marrow samples of T-PLL patients. Tumor DNA and RNA, and germline DNA were extracted from purified leukemic T cells and bone marrow derived stromal cells, respectively. WES (n=12) and transcriptome analysis (n=10) were performed and data analyzed with published standard methods (Robinson et al. 2010 and DePristo et al. 2011)Results: 7 paired tumor and germline DNA and 5 tumor-only DNA samples were used for WES analysis. Mutations in ATM and TET2 were detected in 4 (33%) and 1 (8%) patients, respectively. JAK3 (p.M511I, p.Q501H and p.Q503H) or STAT5B (2 p.T628S and 1 p.N642H) mutations each occurred in 3 (25%) cases and were mutually exclusive, consistent with prior reports. Importantly, we found several novel mutations in genes with regulatory roles in the NOTCH signaling pathway. Specifically, 2 somatic mutations p.A3V and p.A3S in NOTCH2 and 2 other mutations (p.S244L and p.A1414V) in NOTCH4 were identified in 4 (33%) patients. One mutation was in the EGF- like and another one in the NODP domain critical for NOTCH-mediated cellular differentiation. Two (16.7%) novel missense mutations (p.G174D and p.V836I) were found in MAML2. Three missense mutations (2 p.S583N and 1 p.R190H) occurred in MAML1. Both MAML2 and MAML1 are essential cofactors of the NOTCH signaling pathway. In addition, a somatic mutation in the NOTCH signal regulator DTX2 (p.G268D) was detected. Altogether 7 (58.3%) patients harbored mutations in genes with a regulatory role in the NOTCH pathway. Among the epigenetic modifiers, we found deletions and point mutations in KDM6B and KDM6A in 4 (30%) patients including 2 codon deletions in KDM6B (p.X751Val) and 1 missense mutation each in KDM6B (p.R1016Q) and KDM6A (p.V510G). KDM6A and KDM6B are histone H3 lysine 27 demethylases known to be involved in regulation of DNA repair and play an important role in embryonic differentiation.Transcriptome analyses of 10 T-PLL cases versus normal CD3+ T cells from 5 healthy donors revealed 706 up-regulated and 655 down-regulated genes. Consistent with prior data, TCL1A and TCL1B were significantly overexpressed (23.7 and 13.1 fold, respectively). Using Ingenuity Pathway Analysis, we found PTEN Signaling, glutamate receptor, and axonal guidance signaling to be among the top 3 upregulated pathways. The top upstream regulators implicated for the up-regulated signals were FGF, Wnt/β-catenin and TNF. The top 3 down-regulated pathways were granulocyte adhesion and diapedesis, IL-17A in psoriasis and IL-10 signaling. The top regulators for the down-regulated pathways were LPS, TNF and IFNg. Wnt/β-catenin and Human embryonic stem cell pluripotency signaling were among the top 20 up-regulated pathways. Many genes involved in the regulation of Wnt signal pathway were included in the above pathways, including Wnt ligands (Wnt9A, Wnt4), transmembrane receptors (CDH1, LRP5, FZD6, FZD7, SMO), cytoplasmic factor (AXIN2), and nuclear factors (MYC, SOX12, SOX8, SOX18, CCND1).Summary: Our study identified recurrent mutations in the NOTCH signaling pathway as well as dysregulated embryonic developmental pathway, e.g. Wnt/NOTCH as newly implicated molecular mechanisms in T-PLL pathogenesis for further functional studies. Additionally, novel (KDM6A and KDM6B) and known mutations (ATM, TET2) in chromatin regulatory genes underscore the importance of epigenetic dysregulation in the tumorigenesis of T-PLL. DisclosuresParikh:Pharmacyclics: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ding:Merck: Research Funding.

Subset-Specific Recurrence of Mutations and Identification of Functional Modules Provides New Clues about the Pathogenesis of Large Granular Lymphocyte Leukemia

Background: Large granular lymphocyte (LGL) leukemia is a rare disease characterized by a clonal persistence of cytotoxic T cells or natural killer (NK) cells. Patients usually suffer from cytopenias and other organ-related autoimmune phenomena. These are putatively mediated by the cytotoxic LGL cells constitutively activated following an antigen-driven immune response. In addition to gain-of-function mutations in the STAT3 gene, which occur in 40-50% of patients, recurrent alterations only in the STAT5b and TNFAIP3 tumor suppressor genes have been described thus far. However, based on gene expression analyses, JAK/STAT pathway activation and deregulation of several pro-apoptotic (sphingolipid and FAS/FAS ligand) and pro-survival signaling pathways (PI3K/AKT and RAS) are common features of LGL leukemia.In this project, we aimed to characterize the genomic landscape of LGL leukemia using exome sequencing and systems genetics approaches in a patient cohort including both T- and NK-LGL cases and patients without known STAT mutations.Methods: The study cohort included 19 patients diagnosed with LGL leukemia that underwent exome sequencing analysis with matched germline controls. 13 patients had CD8+ T LGL and 3 patients CD4+ T LGL phenotype and 3 patients were NK LGL cases. From the T LGL leukemia cases CD8+ or CD4+ T cells were sorted (according to the dominant phenotype) and used as the tumor sample. In NK LGL leukemias, sorted CD3neg,CD16/56+ NK cells constituted the tumor fraction that underwent exome sequencing. Polyclonal blood lymphocytes depleted from LGL cells were used as germline controls. The exome was captured with the Nimblegen SeqCap EZ Exome Library v2.0 and the sequencing was performed with the Illumina HiSeq2000 sequencing platform. All bioinformatics steps were carried out using a custom bioinformatics pipeline. Putative somatic variants were identified by subtracting, for each patient, the ones called in the normal samples from those found in the tumor sample. After filtering by call quality and allele frequency in ExAC database, somatic variants were prioritized according to the predicted impact from the SnpEff software. Genes hit by variants putatively altering their function were finally mapped to Kegg and Reactome to generate pathway-derived meta gene networks for the identification of affected functional components.Results: 4 patients had STAT3 mutations and 4 additional cases had STAT5B mutations. In addition to STAT mutations, a number of novel somatic variants, which were recurrently mutated were discovered. These included the tumor suppressor gene FAT4, the epigenetic regulator KMT2D, as well as genes involved in the control of cell proliferation (CDC27 and ARL13B).With the systems genetics approach based on integration of pathway-derived mutated gene network topologies for identification of connected components we were able to discover affected functional modules. The main network component included key genes, which either directly interact (such as the FLT3 tyrosine kinase) or are functionally connected (such as ADCY3, ANGPT2, CD40LG, PRKCD, PTK2, KRAS, and RAB12 genes) with STAT proteins. Additional modules with putative pathogenetic relevance in LGL leukemia and mutated in the absence of STAT mutations were cell cycle control (CDC27, PLK1, CDC25B, RAD21), Notch signaling (NOTCH2, NOTCH3 and MAML3) and epigenetic regulation through histone-lysine methyltransferase activity (KMT2D and ASH1L).The comparison of various LGL leukemia subtypes revealed that the mutation burden was especially high among the CD4+ T LGL leukemia cases. Part of the genes and modules affected were shared between the different subtypes of LGL leukemia, but for example KIR2DL1 mutations were only found in CD8+ and NK LGL leukemia cases.Conclusions: With the exome sequencing and systems genetic approach we were able to discover specific gene networks, which are recurrently mutated in LGL leukemia and particularly in patients without STAT mutations. As several mutated genes are directly or indirectly connected with the STAT pathway, the data strengthen the key role of JAK/STAT signaling in LGL leukemia. The novel identified pathway modules beyond STAT networks provide intriguing insights into the pathobiology of LGL leukemia. DisclosuresMaciejewski:Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding.

STAT5B Gene Polymorphisms are Associated with Egg Production and Egg Quality Traits in Laying Hens

STAT5B Gene Polymorphisms are Associated with Egg Production and Egg Quality Traits in Laying Hens